Identification of Novel Molecular Markers for the Follow-up of Minimal Residual Disease in Hematooncological Disorders

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4905-4905
Author(s):  
David Warren Hardekopf ◽  
Tereza Jancuskova ◽  
Marie Jarosova ◽  
Lucie Krutilkova ◽  
Radek Plachy ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Long Range ◽  
Residual Disease ◽  
Molecular Techniques ◽  
Molecular Diagnostic ◽  
Multicolor Fish ◽  
Bac Clones ◽  
Long Range Pcr ◽  
Minimal Residual

Abstract Abstract 4905 Modern molecular diagnostic techniques allow the detection of minimal residual disease (MRD) in hematological malignancies with high sensitivity, allowing patient-specific assays of MRD levels over the course of treatment. One challenge for these techniques is finding appropriate MRD marker targets. For instance, PCR-based MRD analysis requires unique sequence-specific information for the malignant clone of interest. In many acute leukemias, such detailed information is lacking, either because no abnormality is detected (e.g. in 40–50% of adult Acute myeloid leukemia-AML), or the resolution of cytogenetic methods is too low to precisely define newly-found abnormalities. We have used multicolor-FISH (mFISH) to perform karyotyping of 141 patients. Using this approach we have identified chromosomal abnormalities in about 35% of analyzed cases. The aberrations found involved the recurrent aberrations t(15;17), t(8;21), inv(16), t(9;22),−5q, −7, +8 but also an array of unique abnormalities, such as der(15)t(5;15)(q15;q24), t(6;12)(p22;q13), and der(17)t(12;17)(q14;pter), which may serve as possible targets for molecular MRD follow-up. To further characterize such targets, we seek to bridge the gap from cytogenetic resolution down to higher-resolution molecular techniques. For this, we first employ the mBAND technique, followed by multicolor FISH using DNA probes with known cytogenetic location. These probes are drawn from the Human Minimal Genomic Clone Set (Version 1.0, Source Bioscience LifeSciences) of over 25,000 bacterial artificial chromosome (BAC) clones with human DNA inserts. These are tiling clones covering almost 100% of the genome, and therefore allow us to develop FISH probes to regions containing almost any conceivable target breakpoint. Starting with breakpoint information from mFISH/mBAND, we perform consecutive multi-color/multi-BAC hybridizations around the region of interest, looking for breakpoint-spanning probes. Using this system, we are able to move from a resolution of around 2 Mbp (the limit of mBAND) to the resolution of individual BAC clones (average 150 kbp) in three hybridizations. To achieve further resolution, long-range PCR products are designed within the region defined by breakpoint-spanning BACs, with labeling and FISH-mapping of these probes. At present, we are starting to perform these hybridizations on stretched DNA (fiber-FISH or combed DNA), where one micrometer of a stretched DNA molecule represents about 2000 base pairs, allowing visualization of probe distance and spacing, and even higher-resolution breakpoint analysis. Chromosome microdissection followed by high-throughput sequencing (Roche GS Junior) is also being pursued to identify novel breakpoints by rapidly sequencing large chromosomal regions on both sides of a translocation. The final goal is to map genomic abnormalities to a resolution amenable to long-range PCR, yielding specific targets for MRD detection, and allowing clone-specific Real-Time PCR assays for sensitive and specific monitoring of MRD in hematooncological patients. Disclosures: Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

Assessment Of Minimal Residual Disease In Acute Myeloblastic Leukemia In Multiparameter Flow Cytometry

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2613-2613 ◽  
Author(s):  
Francis Lacombe ◽  
Kaoutar Allou ◽  
Christine Arnoulet ◽  
Lydia Campos ◽  
Adrienne de Labarthe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Internal Control ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Molecular Techniques ◽  
Multiparameter Flow Cytometry ◽  
Minimal Residual

Abstract Acute myeloid leukemias (AML) represent a vast and complex group of diseases where numerous molecular lesions have been and keep being described. From the immunophenotypic point of view, probably because of the variety of cells in the myeloid lineage, a rather large variety also exists. The detection of minimal residual disease (MRD) in such conditions therefore meets the challenge of tracking the proper anomaly and correctly separate leukemic cells from their normal counterparts. In an oligocentric project initiated in France in late 2006, ten cytometry platforms and six molecular biology laboratories collaborated to detect MRD concomitantly in flow and with molecular techniques. The flow cytometry part of this work is reported here. A total of 307 consecutive patients were tested at diagnosis with a comprehensive common panel allowing for the detection of immature markers and potential leukemia-associated immunophenotypic patterns. Follow-up samples were planned to be obtained after induction and at the end of treatment, with an optional control before the second consolidation. In fine, 274 patients had at least one point of follow up. All samples were tested in a technique of whole bone marrow lysis no wash, avoiding any cell loss. The flow cytometry panel comprised ten five-colors tubes, all containing CD45 as gating marker. A newly developed strategy was devised to analyse MRD data. The approach of the GTLLF (Arnoulet, Cytometry part B, 2011) was first applied to properly identify mature polymorphonuclears, monocytes and lymphocytes, allowing for a negative Boolean selection of immature cells in the region dubbed “bermudes” by this group. Focusing on this area, each combination of the four markers tested together with CD45 was then displayed in a total of six biparametric histograms. For each of them, on the diagnosis sample, quadrant gates were constructed so that the lower left one contained no blast cells. A Boolean operation was then designed to exclude all these six areas, thereby combining the positive blasts present in the three other parts of each quadrant. The resulting population was visualized on a CD45/side scatter biparametric histogram to check that the cells appeared as a focused cluster at a precise position. The same strategy was then applied for each patient’s consecutive samples, always checking whether any cells identified with this protocol displayed the scattered pattern of cells engaged in maturation (no MRD) or constituted a focused population without maturation (positive MRD). The amount of MRD was then calculated taking into account as denominator the whole population of nucleated cells in the sample (excluding debris on a live gate). As internal control a specific feature of the Kaluza software was used to merge samples obtained for a given patient in order to display on the same worksheet the diagnosis and follow up samples using the principal component separation provided by the “radar” tool of this software. This original method proved to be easily applicable and provide a consistent help for MRD interpretation. All patients could be assessed for MRD with only two of the ten tubes used. These contained the following combinations : CD15, CD13, CD33, CD34, CD45 and CD7, CD117, CD33, CD34, Cd45. At diagnosis, any combination of expression of CD13, CD33, CD117 and CD34 could be observed, the percentage of positive cases for each of these antigens being respectively 86%, 89%, 81% and 58%. As a whole, 93% of the follow-up samples (MRD) tested contained less than 5% of cells with an immunophenotype comparable to that of diagnosis. This figure was 77% for less than 1% and 43% for less than 0.1%. The strategy devised for files analysis was easily applicable for all patients except those with myelomonocytic leukemia. For some of them, separation of the blasts from the monocytic compartment could be problematic in regenerating bone marrow samples. In conclusion, the flow cytometry part of this multicenter study allowed to establish that the combination of CD45 with CD13, CD33, CD117 and CD34 with the additional information provided by CD5 and CD7 represents a quasi-universal panel, now easy to implement on instruments with 8 or 10 detectors, for the detection of MRD in multiparameter in flow cytometry. Moreover, a powerful strategy of listmodes analysis was developed allowing for the direct comparison of several samples from the same patients and/or of a given sample and normal (control) bone marrow. Disclosures: No relevant conflicts of interest to declare.

Depth of response and minimal residual disease status in ultra high-risk multiple myeloma and plasma cell leukemia treated with daratumumab, bortezomib, lenalidomide, cyclophosphamide and dexamethasone (Dara-CVRd): Results of the UK optimum/MUKnine trial.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 8001-8001
Author(s):  
Martin F. Kaiser ◽  
Andrew Hall ◽  
Katrina Walker ◽  
Ruth De Tute ◽  
Sadie Roberts ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Residual Disease ◽  
Cell Leukemia ◽  
Ultra High Risk ◽  
Grade 3 ◽  
Minimal Residual

8001 Background: Patients with ultra high-risk (UHiR) newly diagnosed multiple myeloma (NDMM) and patients with plasma cell leukemia (PCL) continue to have dismal outcomes and are underrepresented in clinical trials. Recently, improved responses with anti-CD38 monoclonal antibody combination therapy have been reported for NDMM patients. We report here outcomes for NDMM UHiR and PCL patients treated in the OPTIMUM/MUKnine (NCT03188172) trial with daratumumab, cyclophosphamide, bortezomib, lenalidomide, dexamethasone (Dara-CVRd) induction, augmented high-dose melphalan (HDMEL) and ASCT. With final analysis follow-up surpassed in Feb 2021, we report here early protocol defined endpoints from induction to day 100 post ASCT. Methods: Between Sep 2017 and Jul 2019, 107 patients with UHiR NDMM by central trial genetic (≥2 high risk lesions: t(4;14), t(14;16), t(14;20), gain(1q), del(1p), del(17p)) or gene expression SKY92 (SkylineDx) profiling, or with PCL (circulating plasmablasts > 20%) were included in OPTIMUM across 39 UK hospitals. Patients received up to 6 cycles of Dara-CVRd induction, HDMEL and ASCT augmented with bortezomib, followed by Dara-VR(d) consolidation for 18 cycles and Dara-R maintenance. Primary trial endpoints are minimal residual disease (MRD) status post ASCT and progression-free survival. Secondary endpoints include response, safety and quality of life. Data is complete but subject to further data cleaning prior to conference. Results: Median follow-up for the 107 patients in the safety population was 22.2 months (95% CI: 20.6 – 23.9). Two patients died during induction due to infection. Bone marrow aspirates suitable for MRD assessment by flow cytometry (10-5 sensitivity) were available for 81% of patients at end of induction and 78% at D100 post ASCT. Responses in the intention to treat population at end of induction were 94% ORR with 22% CR, 58% VGPR, 15% PR, 1% PD, 5% timepoint not reached (TNR; withdrew, became ineligible or died) and at D100 post ASCT 83% ORR with 47% CR, 32% VGPR, 5% PR, 7% PD, 10% TNR. MRD status was 41% MRDneg, 40% MRDpos and 19% not evaluable post induction and 64% MRDneg, 14% MRDpos and 22% not evaluable at D100 post ASCT. Responses at D100 post ASCT were lower in PCL with 22% CR, 22% VGPR, 22% PR, 22% PD, 12% TNR. Most frequent grade 3/4 AEs during induction were neutropenia (21%), thrombocytopenia (12%) and infection (12%). Grade 3 neuropathy rate was 3.7%. Conclusions: This is to our knowledge the first report on a trial for UHiR NDMM and PCL investigating Dara-CVRd induction and augmented ASCT. Response rates were high in this difficult-to-treat patient population, with toxicity comparable to other induction regimens. However, some early progressions highlight the need for innovative approaches to UHiR NDMM. Clinical trial information: NCT03188172.

scholarly journals Minimal residual disease may predict bone marrow relapse in patients with hairy cell leukemia treated with 2-chlorodeoxyadenosine

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1556-1560 ◽  
Author(s):  
S Wheaton ◽  
MS Tallman ◽  
D Hakimian ◽  
L Peterson
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Hairy Cell Leukemia ◽  
Residual Disease ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Hematoxylin And Eosin ◽  
Anti Cd20 ◽  
Minimal Residual

Minimal residual disease (MRD) can be detected in bone marrow core biopsies of patients with hairy cell leukemia (HCL) after treatment with 2-chlorodeoxyadenosine (2-CdA) using immunohistochemical (IHC) techniques. The purpose of this study was to determine whether the presence of MRD predicts bone marrow relapse. We studied paraffin- embedded bone marrow core biopsies from 39 patients with HCL in complete remission (CR) 3 months after a single cycle of 2-CdA. Biopsies performed 3 months posttherapy and annually thereafter were examined by routine hematoxylin and eosin (H&E) staining and IHC using the monoclonal antibodies (MoAbs) anti-CD45RO, anti-CD20, and DBA.44. At 3 months after therapy, 5 of 39 (13%) patients had MRD detectable by IHC that was not evident by routine H&E staining. Two of the five patients (40%) with MRD at 3 months have relapsed, whereas only 2 of 27 (7%) patients with no MRD and at least 1 year of follow up relapsed (P = .11). Over the 3-year follow-up period, two additional patients developed MRD. Overall, three of six (50%) patients with MRD detected at any time after therapy have relapsed, whereas only 1 of 25 (4%) patients without MRD has relapsed (P = .016). These data suggest that the presence of MRD after treatment with 2-CdA may predict relapse.

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.

Co-Existence of Various Sub-Clones in TEL-AML1 Positive Acute Lymphoblastic Leukemia in Association with Sub-Microscopic Deletion of AML1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1096-1096
Author(s):  
Amos Toren ◽  
Rachel Rothman ◽  
Bella Bielorai ◽  
Malka Reichart ◽  
Ninette Amariglio ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Gene Rearrangement ◽  
Chromosomal Abnormalities ◽  
Residual Disease ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Submicroscopic Deletion ◽  
Minimal Residual

Abstract The TEL/AML1 fusion gene is the most common gene rearrangement in pediatric acute lymphoblastic leukemia (ALL). Although considered to be a low risk leukemia it has a 20% risk of late relapse. The coexistence of different sub clones at diagnosis, based on polymerase chain reaction (PCR) studies of Ig/TCR gene rearrangement, was recently reported in this subtype of ALL. Their different response to chemotherapy may explain the emergence of certain sub clones at relapse, and may serve as a marker for minimal residual disease follow-up. Several chromosomal rearrangements such as t(9;22), t(8;21), inv(16) and rearrangements of the MLL gene are frequently associated with submicroscopic deletions and some of them have prognostic significance. Such deletions were not reported in t(12;21) positive ALL. Bone marrow cells from 76 pediatric patients with ALL at diagnosis were analyzed for the presence of the TEL/AML1 fusion gene by interphase fluorescence in situ hybridization (FISH). We used a new system of combined analysis enabling a very large-scale study of the cells of interest with regard to morphology, FISH and immunophenotyping. Fourteen patients were positive for the translocation. Four of them had several sub clones associated with various combinations of additional chromosomal abnormalities. The most striking was an atypical and unexpected hybridization pattern consistent with a submicroscopic deletion of the 5′ region of the AML1 breakpoint (intron2) not previously reported. We describe the use of a larger probe for AML1 (AML1/ETO) to exclude the possibility of insertion of TEL into the AML1 region without breakage and to reduce the false positivity due to optical fusion. This may enable a better monitoring of minimal residual disease in cases with submicroscopic deletion. All patients had some sub-clones with TEL deletion. Other abnormalities included trisomy and tetrasomy 21 as well as double TEL-AML1 fusion. The analysis of numerous sub-clones at presentation in these patients suggests clonal evolution at an early stage of the disease. These sub-clones may have different sensitivities to chemotherapy, and some of them may reappear at relapse. The frequency of AML1 deletion in t(12;21) in addition to other chromosomal abnormalities, is unknown. The involvement of these findings in the generation of leukemic sub clones, their prognostic significance and role in minimal residual disease follow-up deserves further studies in a large number of patients and a longer follow-up.

Sequential Quantitative Minimal Residual Disease in BCR-ABL Positive Acute Lymphoblastic Leukaemia: Differential Observations between Major and Minor Fusion Gene Rearrangements and Variation Due to Source Material.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1093-1093
Author(s):  
Gareth Gerrard ◽  
Wayne Mitchell ◽  
Anthony Goldstone ◽  
Raj Chopra ◽  
Georgina Buck ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Minimal Residual Disease ◽  
Lymphoblastic Leukaemia ◽  
Residual Disease ◽  
Fluorescent Detection ◽  
Sample Type ◽  
Transcript Levels ◽  
Qrt Pcr ◽  
Minimal Residual

Abstract Background: Minimal Residual Disease (MRD) monitoring in BCR-ABL+ acute lymphoblastic leukaemia (ALL) is a valuable tool in the management of Philadelphia positive adult ALL due to the markedly poor prognosis engendered by this leukemia subtype. As part of the UKALLXII trial we received samples from 104 de novo adult ALL patients, 26 of which (25%) were found to be BCR-ABL+. BM and PB received at presentation and during chemotherapy or post SCT were tested for MRD as part of a modified UKALLXII trial protocol which aimed at assessing the efficacy of combined chemotherapy and Imatinib treatment and SCT. Aims: 1) To establish which sample source offers the highest level of sensitivity; 2) To determine if differences in disease level is associated with the BCR-Abl rearrangement exhibited. Methods: BM and/or PB samples were received at presentation and at monthly follow-up points and were tested by quantitative real-time PCR (QRT-PCR) using a Roche LightCycler 1.3 with the SYBR-green fluorescent detection system. The BCR-ABL transcript levels were normalised as a ratio against Abl levels. Operating procedures were adapted from the recommendations of the European Study Group for MRD in ALL. Results: 138 samples (65 PB, 73 BM) were analysed from 26 BCR-ABL+ patients: 15M, 11F; median age 43y (range 17y to 58y). 17 (65%) were found to be minor and nine (35%) were major. Ten patients have received Imatinib and nine have undergone transplant (7 allo-SCT, 2 autograft). 2 patients have relapsed and 2 have died (1 following relapse). Paired t-tests between PB and BM sample BCR-Abl transcript levels show that BM offers a significantly higher level of sensitivity with a median BCR-ABL level of 2.93 x 10−4 against 1.8 x 10−4 for PB (p= 0.0056, n=46). Baseline ABL levels show the converse (1.49 x 104 BM against 1.77 x 104 PB, p= 0.016, n=46) suggesting that the generally greater quantity of material associated with PB samples may result in higher quality RNA. BCR-Abl levels between major and minor patients show that patients exhibiting the major form have significantly higher levels of disease in both PB (median 3.1 x 10−2 major (n= 24) against 3.3x10−4 in patients exhibiting minor BCR-Abl (n=41), (p< 0.0001) and BM samples (median 2.0 x 10−2 major (n= 28) against 3.3 x 10−4 minor (n=45), (p= 0.0009). Paired sequential analysis for 11 patients with 2 or more follow-up samples confirms this observation (median 3.5 x 10−2 major (n=4) against 3.5 x 10−4 minor (n=7), (p= 0.008). Conclusions: In the QRT-PCR MRD monitoring of adult ALL BM samples offer a small but significantly higher level of sensitivity than PB samples. Patients exhibiting the major BCR-ABL rearrangement have significantly higher levels of disease than patients with the minor form. These data describing differential disease status within the BCR-ABL+ subgroup and variance due to sample type may be important in providing guidelines for patient management.

Quantification of Minimal Residual Disease (MRD) in MLL-PTD Positive AML Can Be Used for Prognostication in AML with Normal Karyotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2026-2026
Author(s):  
Martin Weisser ◽  
Claudia Schoch ◽  
Wolfgang Kern ◽  
Wolfgang Hiddemann ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Early Detection ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Normal Karyotype ◽  
Significant Prognostic Factor ◽  
Expression Levels ◽  
Log Reduction ◽  
Minimal Residual

Abstract Partial tandem duplications of the MLL gene (MLL-PTD) occur with the same frequency of approximately 6% as PML-RARA, AML1-ETO, and CBFB-MYH11 that have previously been shown to be relevant markers for detection of minimal residual disease (MRD). MLL-PTD has a frequency of 11% in the normal karyotype and here it is associated with an unfavourable prognosis. In the normal karyotype AML PCR-based MRD detection was not applicable so far, and thus MLL-PTD may be a promising MRD marker for this large intermediate AML subtype. So far, the clinical significance of the expression level of MLL-PTD at diagnosis or MRD during the course of therapy have not been investigated. Using real time RT-PCR MLL-PTD expression levels were quantified relative to the control gene ABL. Quantitative data were available from 143 patients at diagnosis. The range of the MLL-PTD/ABL ratios at diagnosis was 9.6–1255 (median: 149). The levels of MLL-PTD expression in 50 healthy control samples were between 0 and 0.08 (median 0.02). In total 47 of these patients with a median follow up sample number of 4 per patient (range 2–17) were evaluated for MRD during and after therapy. Twenty-one patients were evaluable for MRD analysis after 2 months after start of therapy. Thirty-one patients were evaluable for MRD analysis after 4 and 6 months after start of therapy. A 2-log reduction of MLL-PTD expression after 2 months, 4 months and 6 months was a significant prognostic factor for overall survival (OS: p=0.0179, p=0.035, p=0.048, respectively) as well as for event free survival (EFS: p=0.0047, p=0.037, p=0.020, respectively). There is no evidence that a 3-log reduction after 2 and 4 months improves OS significantly (p=0.62 and 0.24 respectively). This was mainly due to the low number of patients that achieved a 3 log decrease (6/21 and 10/31 respectively). In contrast, at 6 months a 3-log decrease had a significant impact on OS and EFS (p=0.049 and p=0.030, respectively). Neither the expression levels at diagnosis nor fusion type (involved MLL exons) showed prognostic significance. However, age above 60 years and leukocytosis above 50 G/l were associated with a worse prognosis (p=0.028 and p=0.0035, respectively). Overall, the MLL-PTD expression levels correlated well to the course of the disease and a molecular relapse was detected before clinical manifestation in 2 patients based on increasing expression ratios. Another 15 cases were assessed at clinical relapse when expression levels were again in the range of the diagnostic sample. Here the median interval between the last follow up and relapse was too long (9.2 months) for early detection of relapse. Thus, more frequent follow up analyses at least every three months should improve the early detection rate. In conclusion, this analysis confirms the significance of MRD levels as a prognostic factor in this AML subtype. 1) MLL-PTD can be applied in 11% of intermediate risk group AML. 2) Patients with an apriori high risk for relapse can be identified on the basis of a 2 log reduction. 3) Relapses can be early detected based on increasing expression ratios.

Monitoring of Minimal Residual Disease in NPM1 Mutated Acute Myeloid Leukemia (AML) – a Single Center Experience in 27 Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4672-4672
Author(s):  
Dana Dvorakova ◽  
Zdenek Racil ◽  
Ivo Palasek ◽  
Marketa Protivankova ◽  
Ivana Jeziskova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Quantitative Polymerase Chain Reaction ◽  
Npm1 Mutation ◽  
Mgb Probe ◽  
Minimal Residual

Abstract Abstract 4672 Background Mutations within NPM1 gene occurs in about 60% of adult cytogenetic normal AML (CN-AML) and represent the single most frequent molecular aberration in this subgroups of patients. These mutations usually occur at exon 12 and induce most frequently a net insertion of four base pairs. Aims To examine the applicability and sensitivity of DNA-based real-time quantitative polymerase chain reaction (RQ-PCR) with mutation-specific reverse primers and common minor groove binding (MGB) probe and to evaluate whether minimal residual disease levels are of prognostic relevance in CN-AML patients with NPM1 mutations. Methods Patients were treated within different AML trials and follow-up samples of peripheral blood or bone marrow were referred to perform an RQ-PCR. Samples were analysed at diagnosis, during, and after therapy. The NPM1 mutations were A (17 pts), B (1 pt), D (2 pts) and 7 patients with individual rare types. For all cases, levels of minimal residual disease were determined by DNA-based RQ-PCR with mutation-specific reverse primer, one common forward primer and one common MGB probe. The NPM1 mutation value was normalized on the number of albumin gene copies and expressed as the number of NPM1 mutations every 106 genomic equivalents. This assay is highly specific as no wildtype NPM1 could be detected. Maximal reproducible sensitivity was 10 plasmide molecules per reaction. Results A total of 950 samples of bone marrow and/or peripheral blood from 27 patients have been analyzed. Twenty of 27 patients (74%) achieved molecular remission (MR), twenty-six of 27 patients (96%) achieved hematological remission (HR). 6 of 27 (22%) patients achieved HR without MR and one patient failed therapy. 8 of 20 patients (40%) with MR after treatment relapsed at molecular level and except one in all these patients hematological relaps occured (one patient is still in HR with bone marrow blast present, but < 5%). Considering relapsed patients, time from molecular to hematological relapse was 1 to 5 months (median: 3 months). Considering all 14 patients with HR without MR (6 pts) or with molecular relapse (8 pts), in 11 of them hematological relaps occured (79%) and molecular positivity anticipating hematological relaps with median of 3,5 month (1-7 months). 3 of these 14 patients are still in HR. Conclusions Mutations within NPM1 gene are a sensitive marker for monitoring minimal residual disease in CN-AML patients. RQ-PCR using a MGB probe is an efficient approach to long-term follow-up of residual leukemia cells and frequent quantitative monitoring is useful for reliably predicting hematological relapse. Achievement of negativity appears to predict favorable clinical outcome. This work was partially supported by research grant No. MSM0021622430 Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document