scholarly journals Monitoring Molecular Response, Minimal Residual Disease and Clonal Structures in Polycythemia Vera Patients Treated with Interferon Alpha

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1944-1944
Author(s):  
Roland Jäger ◽  
Edith Bogner ◽  
Kerstin Trenker ◽  
Michael Gurbisz ◽  
Bettina Gisslinger ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Minimal Residual Disease ◽  
Interferon Alpha ◽  
Research Funding ◽  
Mutant Allele ◽  
Residual Disease ◽  
Jak2 V617f ◽  
Mutational Burden ◽  
Paired Analysis ◽  
Minimal Residual

Abstract Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) primarily characterized by an elevated red blood cell count, consequently to clonal expansion of myeloid progenitor cells driven almost exclusively by a mutation of a specific single nucleotide in JAK2 exon 14 (JAK2-V617F). JAK2-V617F mutational burden therefore represents a surrogate measure for the size of the "liquid tumor" underlying PV, i.e. the amount of malignant cells in the peripheral blood. We previously reported on sustained molecular responses (MR) in PV patients on interferon alpha (IFNa) based therapies. Monitoring MR is crucial for understanding the therapeutic potential of IFNa and can be an important parameter in future PV patient care. Thus, highly sensitive laboratory techniques providing most accurate quantification at lowest limit of detection (LOD) are required. Here we evaluate four different methods for JAK2-V617F mutational burden monitoring, which are digital droplet PCR (ddPCR), next-generation sequencing (NGS), quantitative PCR (qPCR) and allele-specific PCR (AS-PCR). Moreover, using an NGS-based approach we investigate the patients' clonal composition by targeted re-sequencing involving 54 genes in longitudinal patient samples. To determine the characteristics of the different methods for burden quantification, we benchmarked a ddPCR assay (Assay ID dHsaCP2000061/dHsaCP2000062, Bio-Rad, Hercules, CA) using serial dilutions of JAK2-V617F positive in JAK2-V617F negative gDNA. The assay showed an LOD of 0.01% JAK2-V617F with a false positivity rate of 0 mutation-positive droplets in 8 independent measurements of healthy donor gDNA. Next we quantified the JAK2-V617F burden in baseline and follow-up (FU) gDNAs from a PV patient cohort on IFNa-based therapy (n=51) using ddPCR, a qPCR-based assay (ipsogen MutaQuant, Qiagen, Hilden, Germany), an NGS-based approach (TruSight Myeloid Sequencing Panel, Illumina, San Diego, CA) and an AS-PCR assay described previously (Kralovics et al, Blood, 2006). Covering the full spectrum of allelic burden values, correlations with ddPCR-derived values were best for NGS (R2=0.998) followed by qPCR (R2=0.976) and AS-PCR (R2=0.951). While mutational burden values >10% JAK2-V617F showed high concordance between methods, JAK2-V617F burdens <10% exhibited significant deviations from the benchmarked ddPCR assay, suggesting that ddPCR is most suitable for monitoring residual disease in PV. Therefore, applying ddPCR, we next re-evaluated two PV patients with baseline JAK2-V617F burdens of 46% and 28%, respectively, both previously classified as complete MR based on JAK2-V617F undetectable by AS-PCR. For those two patients, ddPCR revealed an actual residual disease of 3.02% and 2.39% mutant allele burden at 138 and 68 weeks post initiation of IFNa therapy, respectively. Further monitoring of these patients showed a sustained decrease in mutant allele burden to 1.53% and 0.20% at weeks 217 and 171, respectively. In addition to JAK2-V617F burden quantification, our NGS approach allowed for quantitative evaluation of mutations in 54 genes implicated in myeloid diseases. After somatic variant calling, paired analysis of baseline and FU samples (n=96) from 48 PV patients revealed an average of 2.8 variants per patient in a total of 17 genes. Besides JAK2, recurrently affected genes were TET2, PHF6, ASXL1 and CEBPA. The paired analysis allowed for tracking changes in clonal structures upon therapy. Most notably, TET2-JAK2 double-positive clones present in two patients showed mutant burden decreases of 13% and 59% respectively, arguing against an implication of TET2 mutations in IFNa resistance. Furthermore, PHF6 positive clones present in three patients showed ambiguity upon therapy, shrinking concordantly with the JAK2 positive clone in two patients while expanding as a JAK2-independent clone in the third patient. In summary, while larger studies will be required to assign statistical significance to mutations implicated in IFNa-based therapies, targeted re-sequencing of serial samples in MPN patient cohorts has the potential to deconvolute the impact of the patients' clonal architecture on therapeutic success. Moreover, as medications such as IFNa-based drugs have been shown to induce deep MR in a substantial fraction of PV patients, the use of highly accurate assays such as ddPCR will be crucial for monitoring minimal residual disease. Disclosures Klade: AOP Orphan: Employment. Gisslinger:Baxalta: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria. Hoermann:Novartis: Honoraria; Ariad: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Impact of Minimal Residual Disease in Transplant Ineligible Myeloma Patients: Results from the UK NCRI Myeloma XI Trial

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 245-245 ◽  
Author(s):  
Ruth Mary de Tute ◽  
Andy C Rawstron ◽  
David A Cairns ◽  
Charlotte Pawlyn ◽  
Faith E Davies ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Research Funding ◽  
Residual Disease ◽  
Prognostic Significance ◽  
Continuous Variable ◽  
Powerful Predictor ◽  
Qualitative Variable ◽  
The Impact ◽  
Minimal Residual ◽  
Support Research

Abstract Introduction. Minimal residual disease (MRD) is a powerful predictor of outcome in multiple myeloma (MM). We have previously demonstrated, in transplant eligible patients, that the level of MRD as a continuous variable independently predicts both PFS and OS, with approximately a one year median OS benefit per log depletion (J Clin Oncol 2013; 31:2540-7 and Blood 2015; 125:1932-5). The impact of MRD also appears to be independent of therapy received. There is more limited data on the applicability of MRD assessment in transplant ineligible patients, largely as a consequence of low rates of CR historically within this patient cohort. Patients and Methods. In this analysis we have assessed the impact of MRD on PFS amongst patients treated within the non-intensive arm of the NCRI Myeloma XI trial. Patients were randomised between thalidomide (CTDa) and lenalidomide (RCDa) based induction therapies with responding patients being subsequently randomised to maintenance with lenalidomide monotherapy, or no further therapy. Bone marrow aspirates were obtained at the end of induction and this analysis represents a subset of 297 patients (median age 74 years). MRD was assessed using flow cytometry (sensitivity 10-4) with a minimum of 500,000 cells evaluated with six-colour antibody combinations including CD138/CD38/CD45/CD19 with CD56/CD27 in all cases and CD81/CD117 in additional cases as required. Results. Overall MRD-negativity was demonstrated in 41/297 (13.8%). When considered according to induction therapy received 25/154 (16.0%) of patients randomized to RCDa were MRD-negative compared to 16/143 (10.8%) of those randomized to CTDa (p=0.24; Fisher's exact test). MRD-negativity was associated with a significant outcome advantage as the median PFS was 34 months versus 18 months for MRD-positive patients (p<0.0001, HR 0.44 [95% confidence interval (CI 0.29-0.67)]). This effect was noted in both RCDa (median PFS 17m v 32m; p=0.001, HR 0.41 [95%CI 0.23-0.69]) and CTDa (median PFS 19m v 34m; p=0.03, HR 0.49 [95%CI 0.26-0.95]). When the impact of MRD was assessed according to induction regimen the outcome of MRD-negative and MRD-positive patients was similar with both regimens (see figure). The impact of MRD was also assessed as a continuous variable across 5 logs of residual disease. Sequential improvements in outcome with each log reduction were demonstrable. Median PFS for the following disease levels; <0.01%, 0.01 - <0.1%, 0.1% - <1%, 1% - <10% and >/=10% were 34, 26, 16, 14 and 9 months respectively (p<0.0001). This pattern was demonstrable in both RCDa and CTDa treated patients (p<0.0001 for both). Multivariate analysis confirmed the independent predictive value of MRD both as a qualitative and continuous quantitative variable (p<0.0001 for both). In both instances achieving an immunofixation-negative CR was not a significant prognostic variable when included in the model with MRD. Conclusions. We would conclude that MRD is a powerful predictor of outcome in transplant ineligible patients and is a meaningful therapeutic goal in this patient group. In contrast to conventional CR it retains independent prognostic significance both as a quantitative and qualitative variable. This data further supports the role of MRD as a primary endpoint and surrogate marker for survival in future clinical trials. Figure. Figure. Disclosures Rawstron: Janssen: Research Funding; BD Biosciences: Other: Remuneration; Gilead: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Genzyme: Honoraria; AbbVie: Honoraria; Roche: Honoraria; Celegene: Honoraria. Pawlyn:Celgene: Consultancy, Honoraria, Other: Travel Support; Takeda Oncology: Consultancy. Davies:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Kaiser:Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Takeda: Consultancy; Bristol-Myers Squibb: Consultancy, Other: Travel support; Chugai: Consultancy. Jones:Celgene: Honoraria, Research Funding. Cook:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Glycomimetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Jenner:Janssen: Consultancy, Honoraria, Other: Travel support, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel support; Takeda: Consultancy, Honoraria, Other: Travel support. Drayson:Abingdon Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jackson:MSD: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment. Owen:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Other: Travel support; Janssen: Consultancy, Other: Travel support.

scholarly journals High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia

2019 ◽  
Vol 94 (8) ◽  
pp. 921-928 ◽  
Author(s):  
Sanjay S. Patel ◽  
Geraldine S. Pinkus ◽  
Lauren L. Ritterhouse ◽  
Jeremy P. Segal ◽  
Paola Dal Cin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Mutant Allele ◽  
De Novo ◽  
Residual Disease ◽  
Allele Burden ◽  
First Remission ◽  
Minimal Residual ◽  
Acute Myeloid

High Percentage of JAK2 exon 12 Mutation in Polycythemia Vera and Essential Thrombocythemia Among Populations of Qatar

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5064-5064
Author(s):  
Mohamed A. Yassin ◽  
Hanadi Rafii El-Ayoubi ◽  
Nader Al-Dewik
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Genomic Dna ◽  
Research Funding ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Research Fund ◽  
Idiopathic Myelofibrosis ◽  
Essential Thrombocytosis ◽  
High Incidence

Abstract Abstract 5064 The chronic myeloproliferative Neoplasm (NPM) are clonal hematopoietic stem cell malignancies with 3 main subtypes: polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis. PV is characterized by increased RBC proliferation in the absence of erythropoietin and proliferation of myeloid lineages usually is noted, A gain-of-function mutation of Janus kinase 2 (JAK2) V617F, is identified in about 95% of patients with PV and about 50% of patients with essential thrombocytosis and idiopathic myelofibrosis. It has been shown that JAK2 exon 12 mutations can activate erythropoietin signaling pathways while these findings have been confirmed by many studies from Western countries, there are no reports from Asian countries in general and Arab countries in particular about the prevalence of the JAK2 exon 12 mutation in patients with PV and ET. In the present study, we determined the prevalence of JAK2V617F and JAK2 exon 12 mutations in patients with PV and ET in Qatar. Materials and Methods We enrolled patients with a diagnosis of PV and ET at National Centre for Cancer Care and Research in Qatar from January till June 2012. The diagnosis of PV and ET was established according to the 2008 World Health Organization criteria. The study included 82 patients. Clinical information and the CBC data at diagnosis were obtained from medical records. Pretreatment serum erythropoietin levels. Total DNA was isolated from buffy coat cells taken from peripheral blood using a kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Allele-specific polymerase chain reaction (PCR) was performed using 80 ng of genomic DNA as the template in a35-cycle PCR reaction at an annealing temperature of 58°C, as previously described. The mutant allele yields a 203-base-pair (bp) PCR product (sensitivity of mutant allele detection <1%). For exon 12 mutation screening, 80 ng of genomic DNA was amplified by specific primers designed to amplify a region of 453 bp containing the 128 bp of the exon 12 sequence of JAK2. PCR products were directly sequenced in both directions on an ABI 3730 DNA Analyzer using the BigDye Terminator Sequencing kit. Results We examined the occurrence of JAK2V617F and JAK2 exon 12 mutations in a clinical cohort of 82 patients with polycythemia vera (PV) and Essential thrombocythemia (ET) Of which 42 patients had PV aged 25 to 53, 13 (31%) females and 29 (69 %) males and V617F mutation was detected in all of them exon 12 mutation was detected in 38 (90. 47%) patients. We found 2 different exon 12 mutations:3 N542-E543del, 1 F537-K539delinsL, and among 40 ET patients aged 25 to 59, 22 (55 %) males and 18 (45%) females, 35 patients (87. 5%) were JAK2 V617F and JAK 2 exon12 positive and 5 (12. 5%) were JAK2V617F as well as exon 12 negative patients with V617F and exon 12 mutations showed significantly higher WBC and platelet counts at diagnosis than patients with exon V617F mutation alone (P =. 021 and P =. 038, respectively). We report a surprisingly high incidence of exon 12 mutations in MPN patients with PVand ET in Qatar, a result quite different from reports in the Western literature (P =. 001). Conclusion Our data suggest that exon 12 mutation of JAK2 in patients with PV and ET may have an uneven geographic distribution. A clinical laboratory providing the V617F test alone may risk missing a substantial number of patients with PV in areas with a high incidence of exon 12 mutation. the importance of such associations may need further studies and evaluations. Disclosures: Yassin: Qatar National Research Fund: Patents & Royalties, Research Funding. Rafii El-Ayoubi:Qatar National Research Fund: Patents & Royalties, Research Funding. Al-Dewik:Qatar National Research Fund: Patents & Royalties, Research Funding.

Switching to Nilotinib Is Associated with Continued Deeper Molecular Responses in CML-CP Patients with Minimal Residual Disease After ≥ 2 Years On Imatinib: Enestcmr 2-Year Follow-up Results

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 694-694 ◽  
Author(s):  
Timothy P. Hughes ◽  
Jeffrey H. Lipton ◽  
Nelson Spector ◽  
Brian Leber ◽  
Ricardo Pasquini ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Minimal Residual Disease ◽  
Research Funding ◽  
Residual Disease ◽  
Molecular Response ◽  
Advisory Committees ◽  
Molecular Responses ◽  
The Difference ◽  
Minimal Residual

Abstract Abstract 694 Background: Superior rates of deeper molecular responses were achieved with nilotinib vs imatinib in patients newly diagnosed with Philadelphia chromosome–positive (Ph+) chronic myeloid leukemia in chronic phase (CML-CP) in the Evaluating Nilotinib Efficacy and Safety in Clinical Trials—newly diagnosed patients (ENESTnd) trial. In addition, the 12-month (mo) analysis of the ENEST—complete molecular response (ENESTcmr) study demonstrated that switching to nilotinib after a minimum of 2 years on imatinib led to increased rates of major molecular response (MMR) and deeper molecular responses vs remaining on imatinib. Results from ENESTcmr are presented here with minimum 24 mo of patient follow-up. Methods: Patients with Ph+ CML-CP who had achieved complete cytogenetic responses but still had persistent BCR-ABL positivity by real-time quantitative polymerase chain reaction (RQ-PCR) after ≥ 2 years on imatinib were eligible. Patients (n = 207) were randomized to switch to nilotinib 400 mg twice daily (BID; n = 104) or to continue on the same dose of imatinib (400 or 600 mg once daily [QD]; n = 103). Rates of MMR, MR4 (BCR-ABL ≤ 0.01% according to the International Scale [IS], corresponding to a 4-log reduction), MR4.5 (BCR-ABL ≤ 0.0032%IS, corresponding to 4.5-log reduction), and undetectable BCR-ABL via RQ-PCR with ≥ 4.5-log sensitivity were measured. Results: Among all randomized patients (intent-to-treat population), significantly more patients treated with nilotinib continued to achieve undetectable BCR-ABL by 24 mo (32.7% on nilotinib vs 16.5% on imatinib; P =.005; Table).The difference between the arms in achievement of this endpoint increased between 1 and 2 years (from 12.4% to 16.2%). The median time to MR4.5 and undetectable BCR-ABL was also significantly faster on nilotinib than on imatinib (P = .005 and .003, respectively). Cumulative rates of MR4.5 and undetectable BCR-ABL continued to be higher with nilotinib in patients without those responses at baseline, and the difference between arms appeared to increase over time. The safety profiles for nilotinib and imatinib were consistent with prior studies. By 24 mo, no patients in either arm progressed to accelerated phase/blast crisis. No patients on nilotinib died since the 12-mo analysis; 1 patient on imatinib died from metastatic prostate cancer in follow-up after discontinuation from the study. Conclusions: Switching to nilotinib led to significantly faster, deeper molecular responses in patients with minimal residual disease on long-term imatinib therapy. Since the 12-mo analysis, rates of deep molecular response (MR4.5 and undetectable BCR-ABL) have remained significantly higher in patients who did not have the response at baseline and were switched to nilotinib (vs those remaining on imatinib). In fact, the difference in favor of nilotinib increased between 1 and 2 years. These results suggest that switching to the more potent, selective tyrosine kinase inhibitor nilotinib is beneficial in patients with minimal residual disease after long-term imatinib therapy. Achievement of these deeper molecular responses (MR4.5 and undetectable BCR-ABL) after switching to nilotinib may enable a greater proportion of CML-CP patients to be eligible for future discontinuation studies. Cumulative rates of confirmed undetectable BCR-ABL by 24 mo will be presented as the confirmation assessments for several responders were not available at the time of this analysis. Disclosures: Hughes: Novartis Pharmaceuticals Corp: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Ariad: Consultancy; CSL: Research Funding. Lipton:Novartis: Consultancy, Research Funding, Speakers Bureau. Spector:Novarits: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy. Leber:Novartis: Advisory Board Other, Honoraria, Speakers Bureau. Schwarer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Etienne:Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding. Purkayastha:Novartis Pharmaceuticals Corp: Employment. Collins:Novartis Pharmaceuticals Corp: Employment. Szczudlo:Novartis Pharmaceuticals Corp: Employment. Cervantes:Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Teva Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Minimal Residual Disease (MRD) in Myeloma: Independent Outcome Prediction and Sequential Survival Benefits per Log Tumour Reduction

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3416-3416 ◽  
Author(s):  
Andy C Rawstron ◽  
Walter Gregory ◽  
Ruth M de Tute ◽  
Faith E Davies ◽  
Susan E Bell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Clinical Trials ◽  
Minimal Residual Disease ◽  
Research Funding ◽  
Outcome Prediction ◽  
Residual Disease ◽  
Surrogate Endpoint ◽  
Cell Transplant ◽  
Minimal Residual

Abstract Minimal residual disease (MRD), as assessed by flow cytometry is a powerful predictor of outcome in multiple myeloma (MM). We and others have previously demonstrated that such analyses are informative in patients treated with autologous stem cell transplant (ASCT) and non-transplant regimens. It predicts outcome in patients in conventional CR and is applicable to patients with standard and adverse risk cytogenetics. As a consequence MRD assessment is under consideration as a surrogate endpoint for clinical trials. This is urgently needed in MM as >5yrs follow-up is typically required to demonstrate survival differences in trials of upfront therapy. If surrogate end points are to be used in clinical trials it is essential that a reproducible effect is demonstrable using multivariate models. Previous studies have confirmed the effect of MRD on PFS but a consistent effect on OS has been not been definitively shown. This may in part be due to the availability of effective salvage therapy but it is also possible that the traditional threshold of 10-4 for analysis and the categorization of patients as MRD-postive or negative is suboptimal. Flow cytometry does provide a quantitative assessment of residual tumour over a large range and the degree of tumour depletion may be more informative than a positive-negative analysis. 397 patients from the MRC Myeloma IX trial were included in this analysis. Patients were randomly assigned to CTD (cyclophosphamide, thalidomide, and dexamethasone) or CVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone) induction for 4-6 cycles followed by standard high-dose melphalan (HDM) ASCT. BM aspirates were obtained at day 100 for MRD analysis. 500,000 cells were evaluated with six-colour antibody combinations including CD138/CD38/CD45/CD19 with CD56/CD27 in all cases and CD81/CD117 in additional cases as required. PFS and OS data analysis was landmarked from the date of the MRD assesment. Of the 397 patients with MRD data available at day 100 after ASCT, 247/397 (62.2%) achieved <0.01% MRD. The level of residual disease varied across four logs in MRD-positive patients (0.01-<0.1% in 49/397, 0.1-<1% in 72/397, 1-<10% in 26/397 and ≥10% in 3/397). The PFS and OS for individuals with ≥1% residual disease was comparable to individuals with a PR/MR/SD confirming that MRD assessment is most relevant in CR. The level of MRD correlated with outcome. The median PFS for patients with ≥10% MRD at day 100 after ASCT was 0.8 years, with 1-<10% MRD was 1.7 years, with 0.1-<1% MRD was 1.9 years, with 0.01-<0.1% MRD was 2.7 years and for patients with <0.01% MRD was 3.1 years (P<0.001). The median OS for these groups was 1 yr, 4 yrs, 5.9 yrs, 6.8 yrs and for patients with <0.01% MRD not reached with >7.5 yrs median follow-up (P<0.001, see figure). A Cox proportional hazards model was used to further evaluate factors influencing outcome. B2M and MRD were log-transformed and along with age were considered as continuous variables. ISS, haemoglobin (<115g/l), platelets (<150x10^9/l) and cytogenetics were used as stratification factors. Cytogenetic groups were classified as unfavourable for patients with gain(1q), del(1p32), t(4;14), t(14;20), t(14;16), and del(17p), or favourable for hyperdiploidy, t(11;14) and t(6;14), or unknown/inevaluable. MRD assessment (χ2 11.8, P=0.0006) and cytogenetics (χ2 35.5, P=<0.0001) were the only factors that retained significance in this multivariate model. Conventional categorical response, ISS and B2M were not predictive of OS (p=0.99, 0.16 and 0.56 respectively). We would conclude that MRD quantitation is more informative than a positive or negative categorization with a 10-4 threshold and independently predicts outcome. In this analysis we were able to demonstrate an approximate 1 year survival benefit per log tumour depletion. A lower cutpoint for predicting improved outcome was not reached and more sensitive assays will likely improve outcome prediction further. This data strongly supports the role of MRD assessment as a surrogate endpoint in clinical trials. Figure 1 Figure 1. Disclosures Rawstron: Celgene: Consultancy; BD Biosciences: Consultancy, Intrasure Patents & Royalties. Gregory:Celgene: Consultancy. Davies:Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Novartis: Consultancy. Cook:Celgene: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria. Jackson:Celgene: Honoraria; Janssen-Cilag: Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Owen:Celgene: Consultancy, Honoraria, Research Funding.

scholarly journals Final Results of the AML12 Trial of the Spanish Cetlam Group in Adults with Acute Myeloid Leukemia (AML) up to the Age of 70 Years: Risk Adapted Post-Remission Allocation Based on Genetic Data and Minimal Residual Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 289-289
Author(s):  
Jorge Sierra ◽  
Ana Garrido ◽  
Marina Diaz Beya ◽  
Montserrat Hoyos ◽  
Marisa Calabuig ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Research Funding ◽  
Residual Disease ◽  
High Dose ◽  
Severe Toxicity ◽  
Group Setting ◽  
Npm1 Mutation ◽  
Allogeneic Hct ◽  
Molecular Features ◽  
Minimal Residual

BACKGROUND: AML risk classification is based on genetics (cytogenetics and molecular features) and more recently also on minimal residual disease (MRD) after chemotherapy. These two aspects allow predicting relapse and supporting or not the most anti-leukemia treatment that remains allogeneic hematopoietic cell transplantation (HCT). We prospectively investigated the combined use of the two predictive markers to allocate post-remission therapy with or without HCT. Objectives of the study were testing: a) if this approach was feasible in a multicenter setting; b) the proportion of patients who were allocated to an allogeneic HCT and finally received the procedure; c) the final distribution into the risk categories and their outcome; d) to analyze the outcome of patients with favorable or intermediate genetics moved to the high risk category because of positive MRD. METHODS: Adult patients with primary AML treated at 15 academic hospitals were included between February 2012 and December 2018. Induction chemotherapy consisted of idarubicin 12 mg/m2 days 1-2-3 and cytarabine 200 mg/m2 days 1 to 7. Consolidation courses were high-dose cytarabine (3 g/m2 or 1.5 g/m2 if ≥60 y/o). The number of consolidation courses was based on genetic risk: 3 in favorable genetics category (FGC) (CBF, NPM1mut/FLT3-ITDwild or ratio&lt;0.5, and CEBPA biallelic mutation); and one in the intermediate genetics category (IGC), including intermediate cytogenetics without favorable or unfavorable (FLT3-ITD, MLL, EVI1) molecular features, as well as in adverse genetics category (AGC). Following, the mandatory option was allogeneic HCT in the AGC and in the other genetic categories when MRD was positive. In the IGC without MRD autologous or HLA preferentially matched allogeneic HCT was a center decision. MRD was assessed by flow (positive &gt;0.1%) and/or quantitative PCR of the specific transcripts (RUNX1/RUNX1T1, CBFβ/MYH11 and NPM1). RESULTS: Seven hundred forty-five patients (median age: 55, range18-70 y/o, 51% male) were enrolled. Cytogenetics according the revised MRC classification in 707 informative cases was: CBF AML 12%, intermediate 65% (75% of them normal karyotype), and adverse 23%. FLT3-ITD was detected in 28% of patients with intermediate risk cytogenetics and NPM1 mutation in the same group was present in the 48%. Complete remission (CR) was achieved in 81% (n=603) of patients, 82% and 80% in patients up to and above 60 yrs, respectively. Induction death occurred in 9% of patients, 7% and 11% the two age groups, and 10% of patients had refractory leukemia; 542 (90%) of the 603 CR patients completed the consolidation phase and were risk allocated taking into account genetics and MRD. The remaining CR patients were not allocated because of early relapse (n=22), death in CR (n=5), severe toxicity (n=22) or others (n=12). After risk allocation, 208 (38%) patients were in the genetics-MRD combined favorable group (CFG), 103 (19%) in combined intermediate group (CIG) and 231 (43%) in the combined adverse group (CAG). In the latter, 185 (80%) of patients received an allogeneic HCT in first CR. Fifty-seven patients (11%) moved from the genetically FGC or IGC to the CAG because of high MRD at the end of consolidations. Median follow-up in survivors was 25 months. Overall 4-years survival (OS) of the whole series is 48±2%; event-free survival (EFS) is 77+3% in the CFG group, 45+6% in the CIG and 34+4% in the CAG (p&lt;0.001) due to difference in the cumulative relapse incidence (19%, 38% and 45%, respectively, p&lt;0.001 ). In the 57 patients who were MRD positive at the end of consolidation (FGC and IGC) had an OS of 53±8% and EFS of 45±7% at 4 years. CONCLUSION Risk adapted therapy for primary AML based on genetics and MRD is feasible in a cooperative group setting. The proportion of CR was high (&gt;80%) even in patients older than 60 y/o. MRD assessment at the end of consolidation moved 57 patients with favorable or intermediate genetics to the CAG. Avoiding HCT in first CR in the FGC patients associated to EFS above 75% at 4 years. Allogeneic transplantation feasibility was 80% when this was the intended treatment because of adverse genetics and/or MRD positivity. Risk assessment based on genetics and MRD continues separating three groups of patients with different outcomes. Since relapses remain frequent when adverse AML features are present, further approaches after transplantation, such as targeted agents and immune therapies deserve investigation. Disclosures Sierra: Astellas: Honoraria; Pfizer: Honoraria; Daiichi-Sankyo: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau. Salamero:Daichii Sankyo: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Esteve:Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Daiichi Sankyo: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Long-Term Complete Molecular Remissions in Untreated Symptomatic Follicular Lymphoma Treated with Rituximab as Single Agent and in Combination with Interferon-alpha-2a: Analysis of Minimal Residual Disease in the Randomized Phase II Study M39035.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1393-1393
Author(s):  
Jesper Jurlander ◽  
Christian Geisler ◽  
Hans Hagberg ◽  
Harald Holte ◽  
Tuula Lehtinen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Interferon Alpha ◽  
Phase Ii ◽  
Residual Disease ◽  
Phase Ii Study ◽  
First Year ◽  
Minimal Residual

Abstract From 9/98 to 11/99, 126 patients with symptomatic previously untreated or first relapse (&lt; 6 months of chlorambucil and/or local radiotherapy) CD20+ low-grade lymphoma, were included in a multicenter randomised phase II study. The treatment consisted of a first cycle of rituximab 375 mg/sqm q wk x 4. Pts in CR at week 14 were observed with no further treatment until symptomatic relapse, while pts with SD or PD went off study. Pts with PR or minor response were randomised to receive either a second cycle of rituximab 375 mg/sqm q wk x 4 or interferon-alpha-2a (IFN) 3 MIU/day sc (wk 1), 4,5 MIU/day (wk 2–5) in combination with rituximab 375 mg/sqm q wk (w 3–6). The clinical data from this study has previously been reported (Kimby E, et al. Ann Oncol2002;13 (Suppl 2):85). 38 patients (30%) fulfilled the criteria for CR, and were eligible for analysis of minimal residual disease (MRD). 14 more patients achieved CR at a time point later than first follow up after end of treatment. Per protocol, these patients are not included in the present analysis. By standard DNA-based PCR, presence of either a t(14;18) fusion transcript (MCR/mbr) or a clonal rearrangement of the Ig heavy chain (CDR3) could be detected in the diagnostic bone marrow and blood sample from 23 patients. These patients have now been studied for MRD, with a median follow-up time of 62 months. In dilution experiments the sensitivity of the assays was between 1:10−3 and 1:10−4. A given sample was considered negative if the PCR reaction was negative in three independent experiments, using up to 2 μg of template DNA. Patients were tested in blood and bone marrow at 10–16 weeks, 38–40 weeks and 52 weeks following treatment. A total of 175 samples, including 49 samples from patients in continued CR up to 5 years after treatment, have been analysed. Of 72 paired blood and bone marrow samples, only three showed inconsistency between blood and bone marrow, all three being positive in bone marrow and negative in blood. The frequency of MRD negativity 10–16 weeks after treatment was 4/9 (44%) in patients who received 1 cycle of rituximab, 3/5 (66%) in patients who received two cycles of rituximab and 7/9 (77%) in patients who received two cycles of rituximab with IFN priming. This trend towards a dose-response relation was however not significant, due to the small number of patients in each treatment group. The median duration of CR in patients who were negative at all three timepoints during the first year (n=14) was 62 months, compared to 21 months in patients (n=9) with one or more positive samples (p&lt;0,005). At a median follow up of 62 months 9/14 patients who were MRD negative through the first year remain in complete molecular remission, compared to 1/9 patients who had one or more positive blood or bone marrow samples during the first year (p&lt;0,03). Thus, sustained long-term complete molecular remissions are achievable with rituximab alone or in combination with IFN, and predictable by MRD status during the first year post treatment. Whether the quality of response is related to the dose of rituximab or the combination with IFN, and whether the response can be predicted using blood samples alone, must await the results of the ongoing ML16865 randomised phase III trial of rituximab vs IFN/rituximab in the same group of patients.

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

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

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

Combined ROR1 and CD160 Detection For Improved Minimal Residual Disease In Patients With Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2572-2572 ◽  
Author(s):  
Timothy W. Farren ◽  
Fengting Liu ◽  
Marion G. Macey ◽  
Thomas J. Kipps ◽  
Noel Warner ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Flow Cytometric Assay ◽  
Single Tube ◽  
Flow Cytometric ◽  
Sequential Gating ◽  
Minimal Residual

Abstract Introduction Over the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). Objective To compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. Methods Between October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at <0.05%. Results To establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from <0.01% to 38.59%, of which 37% of samples had levels below <0.01%. Analyzing all flow cytometric methods, a highly significant correlation was observed between all three: CD160FCA vs ERIC: Spearman R=0.96 (95%CI: 0.93 - 0.97, p<0.001); CD160FCA vs ROR-160FCA: Spearman R=0.97 (95%CI: 0.96 – 0.99, p<0.001); ROR-160FCA vsERIC: Spearman R=0.97 (95%CI: 0.94 – 0.98, p<0.001). 54 samples had levels of disease <1%. Bland-Altman assay comparison in these patients again demonstrated significant associations between the assays (CD160FCA vs ERIC: mean 0.08 ±0.15; CD160FCA vs ROR-160FCA: mean 0.04 ±0.19; ROR-160FCA vs ERIC: mean 0.03 ±0.25). Light chain restriction was detectable in 24 patients (size of the restricted population ranged from 0.2% to 47% of all cellular events). This sub-group of patients also demonstrated excellent correlation between level of LCR and detectable disease by CD160FCA (Spearman R=0.96, 95%CI: 0.92-0.98, p<0.001), ERIC (R=0.95, 95%CI: 0.92-0.98, p<0.01) and ROR-160FCA (R=0.97, 95%CI 0.93-0.98, p<0.001). Conclusion Monitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods. References Farren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183. Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures: Farren: BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

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