scholarly journals Screening for Residual Disease in Pediatric Burkitt Lymphoma Using Consensus Primer Pools

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Melissa Agsalda ◽  
Ian Kusao ◽  
David Troelstrup ◽  
Bruce Shiramizu
Keyword(s):  
Burkitt Lymphoma ◽  
Assessment Tool ◽  
Residual Disease ◽  
Patient Specific ◽  
Consensus Primer ◽  
Specific Primers ◽  
Original Tumor ◽  
Non Hodgkin Lymphoma ◽  
Lymphoma Therapy

Assessing molecular persistent or minimal residual disease (PD/MRD) in childhood Burkitt lymphoma (BL) is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs). Because BL is characterized by rearranged immunoglobulin heavy chain (IgVH) genes,IgVHprimer pools fromIgVH1–IgVH7regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of usingIgVHprimer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD usingIgVHprimer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy.

Exploring the Perth Hamstring Assessment Tool and Lower Extremity Functional Scale in a Proximal Hamstring Avulsion Cohort: A Cross-sectional Study

2021 ◽  
pp. 036354652110085
Author(s):  
Elsa Pihl ◽  
Kenneth B. Jonsson ◽  
Mida Berglöf ◽  
Nina Brodin ◽  
Olof Sköldenberg ◽  
...  
Keyword(s):  
Lower Extremity ◽  
Assessment Tool ◽  
Patient Specific ◽  
Activity Limitations ◽  
Functional Tests ◽  
Functional Scale ◽  
Proximal Hamstring Avulsion ◽  
Proximal Hamstring ◽  
Lower Extremity Functional Scale

Background: The goal of treatment for a proximal hamstring avulsion (PHA) is an objectively restored muscle and a subjectively satisfied, pain-free patient at follow-up. Different self-reported and performance-based outcome measures have been used to evaluate recovery, but their validity is poorly investigated. Purpose: To investigate 1) the correlation between the commonly used self-reported outcome measurements, the Perth Hamstring Assessment Tool (PHAT) and the Lower Extremity Functional Scale (LEFS); 2) to what extent these scores can be explained by physical dysfunction as measured by performance-based tests; 3) whether performance-based tests can discriminate between the injured and uninjured extremity; and 4) which activity limitations are perceived by patients several years after the injury. Study design: Cohort study (Diagnosis); Level of evidence, 3. Methods: We included a consecutive series of patients treated for or diagnosed with PHA in our department between 2007 and 2016 having at least 2 tendons avulsed from the ischial tuberosity. Participants attended 2 study visits, answered questionnaires (PHAT, LEFS, and Patient-Specific Functional Scale [PSFS]), and performed physical performance–based tests (single-leg hop tests, single–step down test, and isometric and isokinetic strength tests). Results: A total of 50 patients were included (26 men [52%], 24 women [48%]; mean age, 50.9 years [SD, 9.8 years]). The mean follow-up time was 5.5 years (SD, 2.7 years), and 74% had been surgically treated. The correlation between PHAT and LEFS was strong ( r = 0.832) and statistically significant ( P < .001). Seven of the performance-based tests exhibited a statistically significant but weak correlation with LEFS (0.340-0.488) and 3 of the tests to PHAT (–0.304 to 0.406). However, only peak torque could significantly discriminate between the extremities. The activity limitation most commonly mentioned in PSFS was running (16 patients [32%]). Conclusion: Although PHAT and LEFS correlated strongly, the correlations between functional tests and the patient-reported outcome scores were weak, and most functional tests failed to discriminate between the injured and uninjured lower extremity in patients with PHA 5 years after injury. In general, patients alleged few activity limitations, but running difficulty was a common sequela after PHA.

Impact of Minimal Residual Disease (MRD) on Prognosis in Children with Acute Lymphoblastic Leukemia (ALL) According to WBC Count, Age and TEL/AML1 Status at Diagnosis. Results of the AIEOP-BFM ALL 2000 Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 544-544 ◽  
Author(s):  
Valentino Conter ◽  
André Schrauder ◽  
Helmut Gadner ◽  
Maria Grazia Valsecchi ◽  
Martin Zimmermann ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Patient Specific ◽  
Specific Gene ◽  
Childhood All ◽  
Historical Factors ◽  
Wbc Count ◽  
Minimal Residual

Abstract Minimal residual disease (MRD), the most sensitive method to evaluate treatment response, has been adopted to stratify patients in study AIEOP-BFM ALL 2000. To assess whether PCR-MRD levels discriminate outcome in patients classified by WBC count, age at diagnosis, NCI criteria (Standard Risk, SR: WBC < 50,000/cmm and age 1–9 years; High Risk, HR: all others) and TEL/AML1 status. Between 07–2000 and 07–2006, 4,730 Ph-negative patients were enrolled in AIEOP-BFM ALL 2000 study. They were treated with BFM Induction (protocol IA) consolidation (protocol IB), extra-compartment/intensified consolidation (HD-MTX in non-HR patients, blocks in HR patients), reinduction therapy (one or more Protocols II or III), followed by maintainance. BM samples obtained at weeks 5 (Time Point 1, TP1) and 12 (TP2) of induction/consolidation therapy were used for PCR-based MRD analysis of patient specific gene targets. At least 2 sensitive markers (≥ 1 x 10−4) could be determined in 3,707 (78.4%) patients. SR was defined by MRD− at both TP1 and TP2; HR by MRD ≥1x10−3 at TP2; Intermediate Risk (IR): all others. Median follow-up was 3 years; 5-year percent EFS (SE) estimates are given.Patients at MRD-SR, IR or HR had, respectively, an EFS of 93.0 (1.0), 80.5 (1.5) and 43.4 (6.0) in patients with WBC <50,000/cmm vs 90.4 (2.6), 72.4 (3.0) and 47.0 (5.1) in patients with WBC ≥50,000/cmm. Patients at MRD SR, IR or HR had, respectively, EFS of 93.6 (1.0), 80.3 (1.5) and 44.1 (5.4) if aged 1–9 years vs 87.2 (3.4), 73.9 (3.0) and 49.3 (5.2) if aged ≥10 years. Patients at SR by NCI criteria [N= 2,355, EFS of 85.3 (1.0)] were stratified by PCR-MRD as SR (N=1046; 44.4%), IR (N=1198; 50.9%), or HR (N=111; 4.7%). EFS in these subgroups was 93.9 (1.0), 81.3 (1.6) and 43.9 (7.2), respectively (p<0.001). In patients at HR by NCI criteria [N=1,352, EFS of 75.6 (1.6)], 403 (29.8%), 774 (57.3%) and 175 (12.9%) respectively were at SR IR and HR by MRD. EFS was 89.4 (2.2) in MRD SR, 74.7 (2.3) in MRD IR and 47.9 (4.2) in MRD HR patients (p<0.001). Of 3,707 study patients, 3,410 were investigated for TEL/AML1 status: 771 (22.6%) were positive and 2,639 were negative. TEL/AML1+ patients were at SR (N=444; 57.6%) or IR (N=317; 41.1%) or HR (N=10; 1.3%) by PCR-MRD; EFS in this subgroup was 94.4% (1.5), 80% (3.7) and 60% (18.4), respectively (p<0.001). TEL/AML1− patients at SR (N=887; 33.6%) or IR (N=1497; 56.7%) or HR (N=255; 9.7%) had an EFS of 91.6% (1.3), 78.5% (1.4) and 45.7% (4.5), respectively (p<0.001). PCR-MRD in patients treated with BFM-oriented therapy overcomes the prognostic value of “historical” factors such as WBC count, age, NCI criteria or TEL/AML1 status, as it markedly discriminates prognosis within each subgroup defined by these variables. Study design for contemporary risk-directed therapy of childhood ALL should incorporate a technique for MRD determination.

Applicability of DHPLC for Low Sensitive Residual Disease Detection in RUNX1 and CEBPA Mutated AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3067-3067
Author(s):  
Susanne Schnittger ◽  
Frank Dicker ◽  
Christiane Eder ◽  
Jana Sundermann ◽  
Alexandra Spiel ◽  
...  
Keyword(s):  
Residual Disease ◽  
Mutation Screening ◽  
Patient Specific ◽  
Wave System ◽  
Sample Number ◽  
Available N ◽  
Mutational Hotspots ◽  
Equity Ownership ◽  
The Impact

Abstract Abstract 3067 Poster Board III-4 During the last years it has been shown that PCR based detection of minimal residual disease (MRD) has high relevance for early detection of relapse and overall prognostication. This has been proven for several fusion transcripts but also for NPM1 as a target in normal karyotype AML (NK-AML). Other mutations frequently found in NK-AML are RUNX1 mutations (8-10%) and CEBPA mutations (10-15%). However, these mutations are distributed throughout the entire coding reading frames of CEBPA and RUNX1 making mutation analyses more laborious compared to analysis of genes with mutational hotspots like NPM1. In addition, it is nearly impossible to establish high sensitive real time PCR assays for every patient specific mutation. In contrast, DHPLC (denaturing high performance liquid chromatography) is a method that effectively can detect unknown mutations and for known mutations has a sensitivity of up to 1%. Therefore we analyzed the impact of DHPLC analysis for the applicability and value as predictive MRD analysis. At diagnosis mutation screening by DHPLC was performed first. Both genes were amplified each by four different PCR reactions that were subsequently analysed on a WAVE system (Transgenomic, Inc., Omaha, USA). Positive reactions were further characterized by sequencing. The respective fragment or fragments containing the mutations was/were subsequently also analyzed in the follow up samples. The sensitivity was dependent on the kind of the mutation and its position within the PCR fragment and was between 1 and 10% as estimated by limited dilution experiments. Paired diagnostic and relapse samples were available in 15 cases (12 RUNX1 and 3 CEPBA). The respective mutations were retained at relapse in all cases indicating the stability of both markers, rendering them eligible for follow up evaluation. Next, we analysed 30 patients with CEBPA mutation detected at diagnosis and further investigated 91 samples during follow up. 12 of these cases had two different mutations that were localized on two different DHPLC fragments and thus could be analysed in parallel. For RUNX1 we analysed 144 follow up samples of 60 patients that revealed one (n=51) or two (n=9) RUNX1 mutations at diagnosis. Six of the CEBPA mutated and 13 of the RUNX1 mutated cases had an FLT3-ITD in addition. The median follow up sample number per patient was 3 (range 2-13) and the median follow up time was 339 days (range: 57-3001 days). In the subsequent analysis both cohorts were combined. The follow up samples were simply rated as negative or positive. According to previous studies in fusion gene positive patients and NPM1 mutated patients the impact of the DHPLC results on survival was analysed for defined time intervals after start of treatment: interval 1 (days 18-60), interval 2 (days 61-120), interval 3 (day 121-365), interval 4 (days >365). DHPLC results within these intervals were as follows: interval 1 (positive: n=16; negative n=17), interval 2 (positive: n=14; negative n=19) interval 3 (positive: n=38; negative n=65), interval 4 (positive: n=22; negative n=38). Whenever in the follow up samples two different mutations were available (n=99), the results were shown to be concordant. The impact of the results was analysed by Kaplan Meier analysis. For overall survival a trend for significance was found for interval 1 (medians not reached; p=0.157) and interval 2 (medians not reached; p=0.090) and a significant impact for interval 3 (median: not reached vs. 981 days; p=0.015) and interval 4 (median not reached: vs. 285 days; p=0.048), demonstrating that negative DHPLC results correlate with longer OS. This effect could even more clearly be shown for event free survival with respective results for interval 1 (median: 463 vs 731; p=0.048), interval 2 (median: 499 vs 731 days; p=0.109) and interval 3 (p<0.001) (too few samples for interval 4). As neither age, WBC or pretreatment FLT3 status were significantly associated with outcome in this cohort a multivariate analysis could not be performed. These data clearly show that in the absence of sensitive markers for RQ-PCR low sensitive PCR can be very useful for follow up controls at least in RUNX1 and CEBPA mutated AML. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Lab: Employment. Sundermann:MLL Munich Leukemia Lab: Employment. Spiel:MLL Munich Leukemia Lab: Employment. Wendland:MLL Munich Leukemia Lab: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Kern:MLL Munich Leukemia Lab: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Cell-Free DNA for Minimal Residual Disease Monitoring in Multiple Myeloma Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3423-3423 ◽  
Author(s):  
Lenka Kubiczkova-Besse ◽  
Daniela Drandi ◽  
Lenka Sedlarikova ◽  
Stefania Oliva ◽  
Manuela Gambella ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Residual Disease ◽  
Patient Specific ◽  
Cell Free Dna ◽  
Qrt Pcr ◽  
Free Dna ◽  
Diagnostic Samples ◽  
Igh Rearrangement ◽  
Minimal Residual

Abstract Background Circulating nucleic acids, such as cell-free DNA (cf-DNA), are becoming a promising minimally-invasive diagnostic tool for cancer detection. Recent studies demonstrated that tumor-derived cf-DNA can be used to monitor tumor burden and response to treatment in patients (pts) with solid tumors as well as hematological malignancies (Dawson et al, 2013, Armand et al, 2013). In this study we investigated the clinical utility of cf-DNA in the monitoring of minimal residual disease (MRD) of pts with multiple myeloma (MM) carrying the tumor specific immunoglobulin (IGH) rearrangement. Methods Cf-DNA was extracted from 1 ml of serum sample from 13 MM patients enrolled in Italian CRD/MEL-200 and EMN-02 protocols. The total amount of cf-DNA was estimated by fluorometric measurement (median 560 ng, range 15-5158 ng) and the length of fragments was evaluated by high sensitivity dsDNA chips (Agilent). Patient specific clonal IGH rearrangement was identified at the time of diagnosis from bone marrow (BM) genomic DNA (gDNA) as previously reported (Ladetto et al, 2000). For each patient, MRD in BM and peripheral blood (PB) was estimated by real time quantitative PCR (qPCR) using ASO-specific primers and the quantification was based on serial 10-fold dilution standard curves from plasmid carrying the patient specific IGH rearrangement. The amount of IGH rearrangement in cf-DNA (cf-IGH) was estimated by qPCR and droplet digital PCR (ddPCR) (Bio-Rad) on diagnostic and follow up samples and was expressed as the amount of copies per 1 µg of total cf-DNA. qPCR and ddPCR results were interpreted according to the Euro-MRD guidelines (van der Velden et al, 2003). Results Overall, 54 cf-DNA samples from MM serum (13 diagnostic, 41 follow-up samples) were analyzed for the presence of patient specific IGH rearrangement. The most abundant fraction of cf-DNA was 180-220bp, than 350-400bp and 700-10000bp (in 100%, 85% and 68% of samples respectively), whereas longer fragments more often appeared in follow-up samples. By qPCR, cf-IGH at diagnosis were observed in 11/13 diagnostic samples. Only 3/13 pts were quantifiable (116, 85, 187 copies/1 µg of cfDNA) and 8/13 pts were positive but not quantifiable (PNQ) cf-IGH. By ddPCR, levels of cf-IGH at diagnosis were observed in 9/13 pts. 6/13 pts were quantifiable (246, 195, 96, 88, 184, 25 copies/1µg of cfDNA), and only 3/13 pts were PNQ. In follow-up samples, levels of cf-IGH were undetectable by qRT-PCR; however in 5 samples they were PNQ by ddPCR. Interestingly, in one available relapse sample, cf-IGH reappeared again to quantifiable level (61 copies by qRT-PCR and 190 copies by ddPCR). The levels of cf-IGH are quantifiable in samples with higher amount of tumor specific IGH rearrangements in BM or PB; however, no association was observed between cf-IGH level at diagnosis and disease burden estimated by the PCs infiltration in BM or the monoclonal immunoglobulin concentration in blood/urine. Conclusions These data show the potential utility of cf-IGH monitoring in MM pts. Although by qPCR, cf-IGH were detected in 11/13 pts, they were quantifiable only in 3/13 pts and ddPCR was more precise as it was able to quantify cf-IGH in 6/13 pts. Since cf-IGH copies were quantifiable only in diagnostic samples and in 1 available sample at the relapse, we conclude that higher amounts of serum are necessary to overcome the limitation of assay sensitivity. Potential advantages and predictive value, for monitoring tumor marker in a non-invasive manner, need to be further validated on larger cohort of samples using increased amount of cf-DNA. Work was supported by IGA grants NT12130, NT14575. This work is funded by a Black Swan Research Initiative grant by the International Myeloma Foundation "Dynamics of microRNA and cell-free DNA profiles during multiple myeloma progression“. Disclosures Boccadoro: Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Array BioPharma: Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Sanofi Aventis: Honoraria.

Monitoring Minimal Residual Disease in AML By Patient Specific Mutational Fingerprint Using Multiplex PCR and Deep Sequencing

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1715-1715
Author(s):  
Christina Orsmark Pietras ◽  
Henrik Lilljebjörn ◽  
Vladimir Lazarevic ◽  
Marianne Rissler ◽  
Mats Ehinger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Complete Remission ◽  
Multiplex Pcr ◽  
Residual Disease ◽  
Patient Specific ◽  
Mutation Pattern ◽  
Pcr Assays ◽  
Minimal Residual ◽  
Risk Of Relapse

Abstract Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of abnormal hematopoietic progenitor cells. With induction chemotherapy, patients attain a high rate of complete remission as measured by cytogenetic and flow cytometry markers; however, the majority eventually experience relapse. Accurate monitoring of minimal residual disease (MRD) can provide important information for relapse prediction, but current techniques rely on single somatic mutations or a small number flow cytometry markers. It was recently shown that subsets of leukemia-associated mutations can persist after treatment, even if standard clinical evaluation suggests complete remission. Such patients have an increased risk of relapse and reduced overall survival. It is however difficult to foresee which mutations at diagnosis that will persist and contribute to the leukemic relapse. To reliably monitor MRD status, a compelling strategy would be to ascertain as many mutations as possible. We here demonstrate how this can be achieved using an automated design of multiplex PCR primers followed by deep sequencing of the PCR products, enabling monitoring of MRD and mutation pattern based on each patients initial unique mutational fingerprint. Methods: We selected five patients with AML or high risk myelodysplastic syndrome (MDS) with whole exome sequencing (WES) data available from a diagnostic bone marrow or a peripheral blood sample together with a matched skin biopsy for identification of somatic variants. All five cases had material available from presentation, at least one follow up time point, and at relapse. All somatic coding mutations with a variant allele frequency (VAF) above 5% from the WES that passed the sequencing quality threshold were included, constituting the patients mutational fingerprint. The number of mutations ranged from 9 to 33 per patient. Individualized multiplex PCR assays (1-2 multiplex PCR assays/patient) were designed towards all fingerprint mutations using in-house software together with MPprimer. The multiplex PCRs were performed using Qiagen multiplex PCR kit (Qiagen). Each patient specific fingerprint analysis was performed on paired diagnosis, follow up and relapse samples. Sequencing libraries were generated using Nextera XT DNA library prep kit (Illumina) and sequencing was performed on a NextSeq500 (Illumina). Variant recalling was performed using freebayes and only variants with a VAF>5% and coverage above 100X in the diagnostic sample were considered successful MRD markers. Results: Automatic primer design was possible for 84 out of the total 88 mutations (95%). 75 of the targets (89%) were regarded successfully amplified in the multiplex PCR (sequencing coverage above 100X) and had a median coverage of 6566X. The error rate was estimated to around 1%. This multiplex PCR and sequencing approach allowed us to track each patient's unique mutation pattern in the follow up samples and at relapse. We could identify three patients in which all mutations were cleared in the follow up samples prior to relapse (Fig. 1a, b, c) and two patients in which not all mutations were cleared in the follow up samples (Fig. 1d, e). We could also identify which of the mutations at diagnosis that were present also at relapse (Fig. 1a-e). Hence, this approach is a relatively cost effective, fast and reliable assay for monitoring the disease-causing AML clone during a follow up. Conclusions: Traditional MRD monitoring by detection of single mutations or aberrant expression of flow cytometry markers is a proven and powerful method for identifying patients with a higher risk of relapse. However, a known problem with this approach is the risk that some markers are lost at relapse. We here describe a straight forward method allowing the diagnostic patient specific mutational fingerprint to be followed, which should serve as a more stable disease marker of the aberrant clone. In the five patients investigated, we could track mutations that were cleared, persisted despite clinical signs of remission, and mutations that were retained or lost at relapse. In a clinical setting, following an initial screen for somatic variants by WES, the individual multiplex PCR MRD assay could easily and at a relatively low cost be performed in any small NGS lab, thus allowing improved risk stratification and follow up of patents diagnosed with AML and other malignant hematologic disorders. Disclosures Fioretos: Cantargia: Equity Ownership.

scholarly journals Quantitative Analysis of MLL Fusion Transcripts By Droplet Digital PCR to Monitor Minimal Residual Disease in MLL-Rearranged Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2746-2746
Author(s):  
Sadia Afrin ◽  
Christine RC Zhang ◽  
Claus Meyer ◽  
Mellissa Maybury ◽  
Thy Pham ◽  
...  
Keyword(s):  
Cell Line ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Digital Pcr ◽  
Patient Specific ◽  
Fusion Transcripts ◽  
Highly Sensitive ◽  
A Cell

Abstract Rearrangements of the mixed lineage leukemia gene (MLL, re-named KMT2A) result in aggressive leukemia. Current risk stratification of MLL-rearranged (MLL-r) leukemia is directed by the fusion partner gene and, increasingly, by minimal residual disease (MRD) assessment after induction therapy. The clinical significance of quantifying fusion transcript levels in leukemia patients is firmly established in chronic myeloid leukemia and acute promyelocytic leukemia but is less well studied in MLL-r patients. Real-time quantitative PCR (RQ-PCR) is the standardized assay for molecular MRD monitoring in patients with MLL-rearranged leukemia. However, this method is less precise when few leukemic cells are present, thus limiting its application for highly sensitive MRD monitoring. Droplet digital PCR (ddPCR) allows for absolute quantification of fusion transcripts when multiple copies of fusion transcripts are present per cell. Therefore, we aimed to evaluate whether determining MLL fusion transcript levels by ddPCR could improve the sensitivity of MRD monitoring in MLL-r leukemia. A total of 44 diagnostic and follow-up samples obtained from paediatric MLL-r leukemia patients (26 ALL, 18 AML) were subjected to targeted next-generation sequencing to obtain patient-specific fusion sequences. MLL fusion transcripts were quantified by ddPCR in a total of 17 samples obtained from 4 paediatric AML patients with MLL fusions involving MLLT3 (n = 3) and MLLT10 (n = 1). Fusion-specific probe assays were designed from each of the patient specific fusion sequences for MRD assessment by ddPCR. To determine the detection limit of this method in quantifying MLL fusion transcripts, two MLL-r AML cell lines (MV4-11 and THP-1), and one MLL-wt cell line (Kasumi-1) were used. MLL fusion transcript level of detection of ddPCR was determined by serially diluting MLL-r cDNA into MLL-wt cDNA (Kasumi-1). Using 20ng of MLL-r cDNA in 200ng diluent as the highest concentration, a 10-fold dilution series was performed to make concentrations ranging from 10−2 to 10−7. Each ddPCR reaction mixture contained 11ul of cDNA mix as template with 1X Supermix no dUTP (Bio-Rad), 500 nM of both F/R primers and 250 nM of 5'-FAM labelled probe (IDT). Droplets were generated using a QX200 Droplet Generator (Bio-Rad). A general thermal cycler protocol with annealing at 61°C for 1 minute was performed and positive fluorescence droplets were read using QX200 Droplet Reader (Bio-Rad). MRD of patient samples, derived from ddPCR, was then compared to MRD derived from DNA-based RQ-PCR, following the guidelines established by the EuroMRD group. Our ddPCR method showed high reliability and sensitivity, with the detection limit determined to be 10-5 for a cell line with low MLL fusion transcript expression (THP-1), and 10-6 for a cell line with high MLL fusion transcript expression (MV4-11). Comparison of results obtained by RQ- PCR and ddPCR in a total of 17 diagnostic and follow-up samples from 4 AML patients showed excellent/good concordance between methods for 13 samples with moderate MRD levels. The 4 samples with low levels of MRD (10-4 to 10-5) below the quantitative range as defined by EuroMRD for RQ-PCR were all detectable by ddPCR, highlighting that ddPCR could provide robust and highly sensitive MRD assays compared to the standardized RQ-PCR assays. In conclusion, ddPCR is a promising technique that can reproducibly and reliably quantify MLL-r transcripts for MRD monitoring of MLL-r leukemia. Highly sensitive and robust molecular MRD monitoring by ddPCR holds promise for improving response-based therapeutic stratification and prediction of molecular relapse before overt hematological relapse. Disclosures No relevant conflicts of interest to declare.

Applicability of FLT3-Length Mutations in Exon 14 as a Follow up Marker in AML: An Experience of 148 Cases and 517 Follow up Samples Using Conventional and Real Time PCR.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2014-2014
Author(s):  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Gudrun Mellert ◽  
Wolfgang Hiddemann ◽  
Claudia Schoch ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Residual Disease ◽  
Therapy Response ◽  
Normal Karyotype ◽  
Patient Specific ◽  
Clinical Relapse ◽  
Time Points ◽  
Length Mutations

Abstract Quantification of minimal residual disease (MRD) is becoming increasingly important to guide therapy in patients with acute myeloid leukemia (AML). As only 25% of all AML have fusion genes that have been proven useful as MRD-markers, new targets are urgently needed to make high sensitive PCR studies possible in more AML cases. As length mutations in exon 14 of the FLT3 gene (FLT3-LM) are the most frequent genetic aberrations detected in AML and are predominantly found in AML with normal karyotype this is a highly promissing marker to assess prognosis predicted by MRD in this prognostically intermediate group. Recently, FLT3-LM as a follow up marker was discussed controversly because of possible instabillity. To further evaluate this issue we analyzed 62 paired samples at diagnosis and at relapse that were positive at both or only one of these two time points. 53 were positive at both time points. In these cases the mutation (mt) to wildtype (wt) ratio was increasing in 25 and decreasing in 6 cases. Five cases were mt- at diagnosis and gained the mutation at relapse. Four cases (6.5%) lost the mt at relapse. However, these cases also had a change in cytogenetics (n=2), or immunophenotype (n=2), and FAB subtype (n=4). Thus, these cases may be regarded as different leukemias. In addition, we analyzed 517 follow up samples (day 16 – day 1548) of 148 different patients with FLT3-LM at diagnosis by conventional RT-PCR (sensitivity: up to 10−3). First, all follow up samples were rated positive (n=281) or negative (n=236) for the FLT3-LM as determined at any time point. The negative group had a longer OS (median not reached vs 902 days; p=0.0008) and EFS (413 vs. 279 days, p=0.0004). In a next step, five different follow up checkpoints were defined: 1 (up to day 21 after start of therapy), 2 (day 22–60), 3 (day 61–120), 4 (day 121–365), and 5 (after day 365). Survival was best predicted by interval 4 for OS (median not reached vs. 580 days, p=0.0031) and EFS: (406 vs. 221 days; p=0.0001) in 79 negative and 57 positive samples analyzed at this checkpoint. Also prognostically relevant for OS were checkpoints 2 (31 negatives, 16 positivs, 614 vs. 325 days, p=0.0213), and checkpoint 3 (56 negatives and 23 positives, 614 vs. 325 days, p=0.0215). Thus, sole FLT3-LM status during follow up is a highly predictive parameter for prognosis. In 13 cases that were positive for a second marker 72 samples were analyzed by quantitative real time PCR in parallel (PML-RARA: n=5, MLL-PTD: n=5, DEK-CAN: n=2, MLL-AF6: n=1). A high correlation of the results was found (p=0.01). In addition, 10 cases were 8–15 available follow up samples including at least one relapse were selected for real time PCR. In these cases patient specific primers spanning parts of the individual mutation were designed. With these PCRs a high sensitivity of 10e5–10e7 was riched. The data obtained clearly indicate the relapses 3–4 months before clinical relapse. In conclusion, these data show that 1) FLT3-LM is a very powerful marker for the analysis of therapy response and early detection of relapse. 2) Instability is a minor problem especially because the instabilily in most cases is in the direction of accumulation of the mutation.

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

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

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

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