scholarly journals NGS-Based Monitoring of Measurable Residual Disease in FLT3-ITD Positive Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2778-2778
Author(s):  
Tamara J. Blätte ◽  
Laura K. Schmalbrock ◽  
Sabrina Skambraks ◽  
Sibylle Cocciardi ◽  
Anna Dolnik ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
High Sensitivity ◽  
Analysis Program ◽  
Advisory Committees ◽  
High Coverage ◽  
Measurable Residual Disease

Abstract Background: In acute myeloid leukemia (AML), most patients respond to chemotherapy initially, but the risk of relapse remains high. Continued monitoring of measurable residual disease (MRD) following therapy onset enables early assessment of treatment response and clinical intervention. However, while appropriate assays exist for several genetic markers, some of the most common aberrations in AML, FLT3 internal tandem duplications (FLT3-ITDs), have remained a challenging target due to insertion site and length heterogeneity. The established diagnostic assay based on PCR and fragment analysis (FA), on the other hand, lacks sufficient sensitivity. In consequence, extensive monitoring of FLT3-ITD MRD in AML patients over time and treatment is not yet available. At the same time, with the approval of the FLT3-targeting tyrosine kinase inhibitor (TKI) midostaurin, the need for detailed monitoring of FLT3-ITD clones has increased. Aims: We have developed a new next-generation sequencing (NGS) based assay and analysis program to detect FLT3-ITDs with high sensitivity and specificity. We aimed to demonstrate our method's applicability to MRD monitoring and evaluated the progression of FLT3-ITD MRD in patients treated with midostaurin within the AMLSG 16-10 trial. Methods: FLT3 exons 14 and 15 were PCR-amplified and sequenced on the Illumina MiSeq to generate at least 1 million 250 bp paired-end reads. ITDs were detected using our novel analysis program that performs all steps of the analysis, does not require any manual filtering and reports ITDs extensively annotated to the user. To assess our assay's concordance with established methods, FLT3-ITDs in all diagnosis and relapse samples were characterized by FA and Sanger sequencing (SSeq). Results: Assay specificity was confirmed with 0 ITDs reported in four FLT3-ITD⁻ control samples (healthy volunteers n=2, AML patient n=1, AML cell line HL60 n=1). To assess assay sensitivity, we serially diluted DNA of the ITD⁺ AML cell line MOLM14 in DNA of the ITD⁻ AML cell line HL60. The expected 21 bp ITD was detected in all four dilutions, down to a variant allele frequency (VAF) of 6.7x10⁻⁵ (0.0067 %). We further processed matched diagnosis and relapse samples from 9 ITD⁺ AML patients at low coverage (mean: 430000 paired-end reads) and confirmed concordance of ITD site, length (range 21-198 bp) and VAF with results from FA/SSeq. For 4/9 patients, we independently sequenced a total of 12 matched diagnosis, remission and relapse samples to our target coverage of 1.5 million paired-end reads. In all of these, ITDs detected by FA were confirmed and ITD site, length and VAF were again concordant between FA/SSeq and our NGS-based approach; results from the diagnosis and relapse samples sequenced to both low and high coverage were highly reproducible. In 11/12 samples sequenced to high coverage, our assay identified additional ITD clones that were not identified by FA: The mean number of ITDs detected was 4.8 at diagnosis (1.5 by FA), 2.0 at clinical remission (not measured by FA) and 1.5 at relapse (0.5 by FA). VAFs of these ITDs ranged from 0.007-38 % (mean 5.3 %); ITDs that were solely detected by our NGS-assay were present at lower VAFs than those also identified by FA/SSeq (NGS-assay only: mean 0.21 %, range 0.007-2 %; also detected by FA/SSeq: mean 21.4 %, range 2-38 %). About half of the ITDs detected by NGS at diagnosis (11/19 ITD clones) were not detected at any of the later time points and thus presumably eradicated by treatment. Conversely, 8/19 ITD clones persisted at remission (6/19) or reoccurred at relapse (2/19). In fact, nearly all of the ITD clones present at relapse were already identified at an earlier time point. Conclusion: In sum, we could demonstrate that our NGS-based assay and analysis program achieve high accuracy and precision and enable MRD monitoring in FLT3-ITD positive AML patients. Its high sensitivity enables the detection of additional ITD clones occurring at low allelic frequencies not only at remission but also at diagnosis and relapse. Further evaluation of MRD monitoring in FLT3-ITD positive AML patients treated with midostaurin within the AMLSG 16-10 trial is currently ongoing. Disclosures Bullinger: Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau.

scholarly journals Venetoclax and Azacitidine for Older Newly Diagnosed Patients with Acute Myeloid Leukemia: A Single-Institution Pilot Study Using Measurable Residual Disease to Guide Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2638-2638 ◽  
Author(s):  
Amanda Winters ◽  
Jonathan A Gutman ◽  
Enkhtsetseg Purev ◽  
Brett M. Stevens ◽  
Shanshan Pei ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Refractory Disease ◽  
Advisory Committees ◽  
Ras Pathway ◽  
Measurable Residual Disease ◽  
Count Recovery

Background: Venetoclax (ven) was approved for older untreated acute myeloid leukemia (AML) patients due to high response rates and durable remissions. As a participating site in the dose escalation study, we observed deeper/more durable responses in some who received >400mg ven. We also noted 16/33 discontinued azacitidine (aza) after achieving a response; 9 relapsed and 7 remained in long term remission on ven only. Based on these observations, we designed a study that hypothesized: A)Higher initial doses of ven would allow deeper/more durable responses, and B)Multi modality high sensitivity measurable residual disease (MRD) testing could identify patients able to discontinue aza and remain on maintenance ven. Methods: This is an ongoing phase 2 study (NCT03466294) of 42 untreated AML patients ≥60 who decline/are ineligible for induction. Patients have adequate organ function and white blood cell counts <25x109/L (hydrea permitted). In cycle 1, patients receive aza 75mg/m2 on days (d) 1-7 and ven, escalated from 100 to 200 to 400 to 600mg on d 1-4. Ven continues at 600mg d 5-28 and bone marrow biopsies (BMBXs) are performed on d 8 and 28. Patients who achieve morphologic remission without count recovery have up to 14 days off therapy before subsequent cycles, with growth factor support; "upgraded" responses are recorded if count recovery occurs. Non responders discontinue or receive up to two additional cycles of aza and ven 600mg. Responders who remain MRD+ by multiparameter flow cytometry (MPFC, Hematologics) and/or digital droplet PCR (ddPCR) for as many identifiable diagnostic genes as possible also receive up to 2 additional cycles of aza and ven 600mg. MRD+ responders after 3 cycles continue aza and ven 400mg until toxicity/progression. Patients who experience MRD- responses at any time stop aza and continue ven 400mg daily (Fig 1). Results: 30 patients enrolled between May 2018 and July 2019; median age is 71 (60-88), 10% evolved from MDS and 10% and 73% had intermediate and unfavorable risk disease by ELN, respectively (Table 1). 732 adverse events (AEs) occurred; 46 (6%) were serious, the most common were neutropenic fever (37%) and pneumonia (13%). The most common >grade 2 related AEs were leukopenia (53%), thrombocytopenia (44%) and neutropenia (35%); there were no related grade 5 AEs. The overall response rate was 70% (21/30; CR=19, MLFS=2). Median number of cycles to achieve best response was 1. Significant blast reductions were seen on day 8; of the 28 with interpretable day 8 BMBXs, 10 achieved MLFS on day 8. 4 completed ≥1 cycle and were refractory. An additional 4 did not complete cycle 1: 1 died of disease and 3 elected to come off therapy (all subsequently died of disease). Four (19%) responders relapsed, after a median 180 days (27-279). With median follow up of 214 days, median response duration has not been reached. 10 patients died, after a median 65 days (29-256); 1/30 died within 30 days. Median overall survival has not been reached. Of the 26 who completed ≥1 cycle, 19 were MRD- by MPFC, including 18/19 who achieved CR. Of these 26, 3 were not monitored by ddPCR: for 2 patients this was due to the absence of detectable baseline mutations and for 1 patient it was due to refractory disease. The remaining 23 had ddPCR monitoring; 3 became MRD- by this modality (Fig 2). All 3 were also MRD- by MPFC and per protocol discontinued aza and initiated ven maintenance (Fig 1). MRD negativity by both parameters occurred after cycles 1, 2 and 3, respectively. One MRD- patient relapsed after 216 days; two remain in remission after 301 and 124 days. An additional 4 who achieved MRD+ responses discontinued aza at their insistence (and in violation of the protocol); 1 relapsed after 279 days, and 3 remain in ongoing remission. Univariate predictors of refractory disease were FAB M0/M1 (OR 0.070, p=0.02) and RAS pathway mutations (OR 14.25, p=0.02). Conclusions: Higher initial doses of ven are tolerated in this population. Blast reduction occurs quickly in many patients (day 8), for this low intensity regimen. Response rates are consistent with lower doses of ven. Very deep responses, as measured by highly sensitive MRD methods (MPFC and ddPCR are capable of sensitivity up to 0.02%), are attainable. Longer follow up time will determine if higher ven doses and MRD-driven decisions related to continuation of aza result in more durable responses. Increased maturation of blasts and RAS pathway mutations are predictors for refractory disease. Disclosures Lyle: Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo Incyte: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Pollyea:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Diachii Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty-Seven: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals A Prospective Phase 2 Study of Venetoclax and Low Dose Ara-C (VALDAC) to Target Rising Molecular Measurable Residual Disease and Early Relapse in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1261-1261
Author(s):  
Ing S Tiong ◽  
Sun Loo ◽  
Emad Uddin Abro ◽  
Devendra Hiwase ◽  
Shaun Fleming ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Phase 2 ◽  
Advisory Committees ◽  
Early Relapse ◽  
Phase 2 Study ◽  
Measurable Residual Disease ◽  
Acute Myeloid

Abstract Introduction Rising molecular measurable residual disease (MRD) is an arbiter of clinical relapse in acute myeloid leukemia (AML). Venetoclax (VEN) is active against IDH and NPM1 mutant (mt) AML as monotherapy (Konopleva et al, 2016 and Chua et al, 2020) and can yield MRD negative remission when combined with low dose ara-C (LDAC) in patients unfit for intensive chemotherapy (DiNardo and Tiong et al, 2020). In a retrospective study, we showed that VEN in combination with hypomethylating agents or LDAC could erase rising NPM1mt MRD in 6/7 cases (Tiong et al, 2020). We now present a prospective phase 2 study of VEN and LDAC in patients with molecular MRD failure or oligoblastic AML relapse. Methods This multicenter phase 2 study stratified patients into oligoblastic relapse (marrow blasts 5-15%; Group A), or molecular MRD failure (Group B) as defined by the European LeukemiaNet (ELN) recommendations (failure confirmed by 2 interval samples) (Schuurhuis et al, 2018). Patients received VEN 600 mg (days 1-28) and LDAC 20 mg/m 2 (days 1-10). Primary objectives were morphologic or MRD response (≥1 log reduction) in groups A and B, respectively. Key secondary objectives were allogeneic hematopoietic cell transplantation (allo-HCT) realization and relapse-free (RFS) and overall survival (OS). The study had Alfred Health ethics approval (196/19). NPM1mt and other fusion transcript levels (per 10 5 ABL) from bone marrow were analyzed by RT-qPCR, IDH1 and IDH2 by Bio-Rad TM droplet digital PCR. Results The study enrolled 32 patients, with 29 evaluable (cut-off date 15/7/21). The median age of the study population was 62 years; 79% had intermediate cytogenetic risk, 66% NPM1mt, 11% FLT3-ITD and 37% IDH1/IDH2 mt. Most received prior intensive chemotherapy (93%) and 2 (7%) allo-HCT in first remission. Median interval from AML diagnosis to study entry was 12.6 months (Table 1). After a median follow-up of 7.9 months, patients had received a median of 3 cycles (range 1-14) of VEN-LDAC, with 13 patients ongoing. The main reasons for treatment cessation were allo-HCT (n=10; 34%) or donor lymphocyte infusion (n=2; 7%), treatment failure (n=3) or an adverse event (n=1). Hematologic complete/incomplete response (CR/CRi) among 11 patients with oligoblastic relapse (group A) was 73% and included: CR (n=5, 45%) or CRi (n=3, 27%), with an additional patient with morphologic leukemia-free state and 2 patients with stable disease. Overall, across both groups, median RFS and OS were not reached, estimated at 78% and 91% at 1 year, respectively. Among 18 patients with molecular MRD failure (group B) treated with VEN+LDAC, molecular response (≥1 log reduction) was achieved in 72%, and the RFS and OS were estimated at 83% and 87% at 1 year, respectively. Analysis of a sub-group of patients with NPM1mt (n=18); 6 and 12 from Groups A and B, respectively revealed the median NPM1mt transcript level at study entry to be 8985 copies (IQR 826, 94,431). A molecular response was achieved in 14 (78%) patients, including 9 (50%) with complete molecular remission (CR MRD-), with most responses achieved within 2 cycles of therapy (Figure B). Treatment with VEN-LDAC was generally well tolerated, with 15 serious adverse events reported within the first 2 cycles, including infection (n=6; 19%) and febrile neutropenia (n=3; 9%). Only one subject discontinued treatment due to stroke. Conclusions In this prospective study, in patients with first oligoblastic relapse or MRD failure, VEN in combination with LDAC induced a high rate of molecular MRD remission that was rapidly achieved, resulting in a high rate of survival at 12-months (&gt;90%) and with low toxicity. Follow-up is ongoing to determine the durability of response. Treatment of patients with MRD or early clinical failure may represent an attractive clinical trial setting for investigation of novel, non-intensive AML therapies. This approach will be investigated in a future multi-arm, precision-based platform trial called INTERCEPT (Investigating Novel Therapy to Target Early Relapse and Clonal Evolution as Pre-emptive Therapy in AML). Figure 1 Figure 1. Disclosures Tiong: Servier: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Pfizer: Consultancy. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Fleming: Amgen Inc: Research Funding. Bajel: Amgen: Speakers Bureau; Abbvie, Amgen, Novartis, Pfizer: Honoraria. Fong: Amgen, BMS: Speakers Bureau; Amgen: Research Funding; AbbVie, Amgen, Novartis, Pfizer, Astellas: Honoraria. Wei: Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: This presentation will discuss the use of venetoclax in targeting measurable residual disease and early relapse of acute myeloid leukemia.

scholarly journals Comparison of Acute Myeloid Leukemia Measurable Residual Disease Detection By Flow Cytometry in Peripheral Blood and Bone Marrow

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2729-2729
Author(s):  
Colin D. Godwin ◽  
Yi Zhou ◽  
Megan Othus ◽  
Carole M. Shaw ◽  
Kelda M. Gardner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Correlation Coefficient ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Spearman Correlation ◽  
Advisory Committees ◽  
Measurable Residual Disease ◽  
Acute Myeloid

BACKGROUND: The current recommendation against the need for bone marrow aspiration (BMA) in routine follow-of persons with acute myeloid leukemia (AML) in remission preceded the recognition that multiparameter flow cytometry (MFC) is a sensitive and specific means to detect imminent morphologic relapse. Given this recognition, we wondered whether BMA is now necessary, or if concordance between MFC results in peripheral blood (PB) and BMA is such as to make BMA unnecessary, at least for evaluation of measurable residual disease (MRD) by MFC. Previous studies have demonstrated a strong correlation between disease detection by MFC in PB and BMA. Here we examined 724 paired PB and BMA samples from 482 patients to further examine the concordance between PB and BMA blast detection by MFC, particularly among patients in morphologic remission. PATIENTS AND METHODS: We included adults in our institutional AML database, covering 2008-2018. Our Hematopathology database was queried to identify PB and BMA MFC sample pairs with samples considered "paired" if measured within one week of each other. If an individual had multiple pairs, all were included unless otherwise specified. Ten-color MFC was performed routinely on BMA aspirates with a panel of three antibody combinations, with the same antibody combinations applied to PB samples. When identified, the abnormal population was quantified as a percentage of the total CD45+ white cell events. Any level of residual disease was considered positive. Complete remission (CR) and relapse were defined according to the European LeukemiaNet 2017 classification. Relationship between PB and BMA blast % was measured using Spearman's Rank-Order Correlation. Relationship between PB and BMA samples identified as positive or negative is illustrated using 2 X 2 tables (Table 1). RESULTS: Considering all 724 sample pairs, the Spearman correlation coefficient between PB and BMA blast percentage was 0.93, and was 0.91 considering only the first sample pair for each individual patient (n= 482). 315 sample pairs were positive by PB, 97% of which were also positive by BMA while 95% of 409 pairs negative by PB were also negative by BMA. Similar results were seen considering only a patient's first pair. Restricting analysis to patients with pairs obtained between the dates of CR and relapse, the Spearman correlation coefficient was 0.82 with 91% of 35 cases positive in PB also positive in BMA; 93% of 114 pairs negative in PB were also negative in marrow. As a complementary means to compare pairs when AML burden was low, we examined only pairs where the BMA MFC showed <5% blasts. Here, the Spearman correlation coefficient between PB and BMA blasts was 0.83. 90% of 70 positive PB cases were also positive by BMA while 95% of 295 negative PB cases were also negative by BMA. Examining pairs taken from patients in morphologic remission immediately prior to undergoing hematopoietic cell transplant yielded a Spearman correlation coefficient of 0.92, with all 9 PB positive cases also being positive in BMA and 96% of PB negative cases being negative in BMA. CONCLUSIONS: This is the largest cohort of AML PB and BMA sample pairs analyzed by MFC to-date. The percentages of blasts measured in PB and BMA are strongly correlated. In the 365 pairs from patients with MRD-level disease, the predictive value of PB MFC positivity for BMA positivity was 90% (63/70) while the predictive value PB MFC negativity for BMA negativity was 95%. Disclosures Othus: Glycomimetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Gardner:Abbvie: Speakers Bureau. Walter:BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Kite Pharma: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy; Seattle Genetics: Research Funding; Argenx BVBA: Consultancy; Aptevo Therapeutics: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership.

scholarly journals Clonal Hematopoiesis and Its Implications for Flow Cytometric Assessment of Measurable Residual Disease in Patients with NPM1-mutated Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 38-39
Author(s):  
Sanam Loghavi ◽  
Courtney D. DiNardo ◽  
Koichi Takahashi ◽  
Rashmi Kanagal-Shamanna ◽  
Tomoyuki Tanaka ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Advisory Committees ◽  
Clonal Hematopoiesis ◽  
Hla Dr ◽  
Measurable Residual Disease ◽  
Acute Myeloid ◽  
Myeloid Progenitors

INTRODUCTION: NPM1 mutations (NPM1mut) occur in ∼30% of acute myeloid leukemia (AML) and frequently co-occur with mutations in other genes including those attributed to clonal hematopoiesis (CH) including DNMT3A and TET2, among others. CH mutations may persist beyond attaining NPM1mut-negative remission. Persistent CH may be associated with immunophenotypic alterations in myeloid progenitors detected by flow cytometry (FC) which poses an interpretive challenge in assessment of measurable residual disease (MRD) by FC. The aim of this study was to characterize the immunophenotypic alterations associated with persistent CH in the setting of NPM1mut clearance and to determine their possible clinical or biologic significance. METHODS: The cohort included 67 consecutive patients (pts) with NPM1mut AML treated at our institution between 01/2017 and 11/2019. FC assessment for MRD was performed an eight-color panel using FACSCanto II instruments (BD Biosciences, San Diego, CA) with a sensitivity of 10-3 to 10-4. Whole bone marrow (BM) DNA was interrogated for mutations with an 81-gene myeloid next-generation sequencing (NGS) panel using an Illumina MiSeq sequencer (Illumina, Inc., San Diego, CA, USA) with a sensitivity: 1% variant allelic frequency (VAF). RESULTS: Pts included 26 men and 41 women with a median age of 64 (range, 19-84) years with newly diagnosed NPM1mut AML. AML blasts had the following immunophenotype at baseline: promyelocytic-like phenotype (CD34-, CD117+, HLA-DR-) in 18 (27%), aberrant myeloid CD34-/CD117+/HLA-DR+ in 15 (22%), aberrant myeloid CD34+ in 13 (19%), myelomonocytic in 11 (16%), and monocytic in 10 (15%) cases. All pts had additional co-mutations at baseline (Fig 1). The most frequently co-mutated genes were DNMT3A (58%) FLT3 (51%), TET2 (27%), IDH2 (24%), PTPN11 (19%), IDH1 (18%), NRAS (16%), and SRSF2 (12%). Pts were treated with intensive (35;52%) and non-intensive induction regimens (32; 48%) (Fig 1); 22 (33%) received an allogeneic hematopoietic stem cell transplant as post-remission consolidation. We compared FC and NGS results in follow-up samples in pts achieving NPM1mut negative remission with adequate data available for comparison (n=50). 13 (26%) pts cleared all mutations whereas 37 (74%) had persistent CH. The most common mutations in the setting of residual CH involved DNMT3A (70%), TET2 (27%), IDH2 (19%) and IDH1 (11%). Among 37 pts with residual CH, 19 (51%) had no phenotypic alterations detected by FC while 17 (49%) had myeloid progenitors with alterations in intensity of antigen expression (increased CD13, CD123, CD117 and/or decreased CD38) or deviation from normal maturation but not diagnostic for AML MRD (herein referred to as pre-leukemic (PL) phenotype); 1 sample was MRD+ by FC. Mutation VAF of ≥5% was significantly more common (p=0.008) in cases with FC PL+ (100%) vs cases with normal FC phenotype (63%). IDH2 and SRSF2 mutation were exclusively observed in PL+CH+ cases with the former being statistically significant when compared with the FC-normal group (p=0.016). PL phenotype by FC did not correlate with intensity of induction therapy (41% treated with intensive regimens vs 59% non-intensive). The CH+/PL+ cohort had more pts ≥60 yrs old (67%) but the difference was not significant. There was no significant association between PL+ and residual mutation count. Presence of PL+ phenotype was not associated with a shorter relapse-free survival (RFS) (median not reached for both groups). CONCLUSIONS: Post-remission clonal hematopoiesis in the setting of NPM1mut clearance is common, and may result in immunophenotypic changes in myeloid progenitors, posing interpretive challenges for MRD assessment by FC. These alterations may be attributable to specific CH characteristics, such as IDH2 and SRSF2 mutations and VAF, but are not associated with a shorter RFS and thus should not be interpreted as residual AML or considered a high-risk attribute. Additional studies in other AML subtypes are warranted to further delineate these changes and their clinical significance. Figure 1 Disclosures DiNardo: Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Takeda: Honoraria; Jazz: Honoraria; ImmuneOnc: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Syros: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; MedImmune: Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Short:AstraZeneca: Consultancy; Takeda Oncology: Consultancy, Honoraria, Research Funding; Amgen: Honoraria; Astellas: Research Funding. Kadia:JAZZ: Honoraria, Research Funding; Ascentage: Research Funding; Astra Zeneca: Research Funding; Incyte: Research Funding; Celgene: Research Funding; Novartis: Honoraria; Cyclacel: Research Funding; Genentech: Honoraria, Research Funding; Amgen: Research Funding; Abbvie: Honoraria, Research Funding; Cellenkos: Research Funding; Pulmotec: Research Funding; Astellas: Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Konopleva:Sanofi: Research Funding; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Rafael Pharmaceutical: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ascentage: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; Agios: Research Funding; Ablynx: Research Funding. Kantarjian:Adaptive biotechnologies: Honoraria; Aptitute Health: Honoraria; BioAscend: Honoraria; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria; Ascentage: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; BMS: Research Funding; Immunogen: Research Funding; Jazz: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Sanofi: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Ravandi:Macrogenics: Research Funding; Celgene: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; Xencor: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding.

scholarly journals Assessment of Minimal/Measurable Residual Disease Testing in Acute Myeloid Leukaemia By Molecular Methods in an Interlaboratory Study

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3461-3461
Author(s):  
Stuart Scott ◽  
Richard Dillon ◽  
Christian Thiede ◽  
Sadia Sadiq ◽  
Ashley Cartwright ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Residual Disease ◽  
Standard Of Care ◽  
Negative Sample ◽  
Research Support ◽  
Advisory Committees ◽  
Log Reduction ◽  
Measurable Residual Disease

Abstract Background Minimal/measurable residual disease (MRD) testing is increasingly utilised and accepted as standard of care to manage a range of different haematological malignancies. It's use as a surrogate outcome in clinical trials of new therapies is being explored, where it has the potential to accelerate drug assessment and approval. The phenotypic and genetic heterogeneity of acute myeloid leukaemia (AML) has limited the use of MRD in this context; however, the European LeukaemiaNet (ELN) MRD working group have recently published consensus guidelines to standardise both flow cytometric and molecular genetic MRD testing. To assess the accuracy of testing and concordance between laboratories, crucial to patient safety, external quality assessment (EQA)/proficiency testing (PT) is required. Aims To determine the performance of molecular methods for measuring of MRD using the t(8;21)(q22:q22) RUNX1-RUNX1T1, inv(16)(p13q22) CBFB-MYH11, t(15;17)(q24.1;q21.2) PML-RARA and NPM1 Type A markers in an international interlaboratory study. Methods A total of 12 batches of lyophilised EQA material were manufactured. These consisted of three batches of samples for each marker all containing 9x10^6 cells: an MRD 'high' sample; an MRD 'low' sample; and an MRD 'negative' sample. The t(8;21)(q22:q22) RUNX1-RUNX1T1 positive samples were manufactured using the KASUMI-1 cell line, the inv(16)(p13q22) CBFB-MYH11 positive samples using the ME-1 cell line; t(15;17)(q24.1;q21.2 PML-RARA positive samples using the NB4 cell line and the NPM1 Type A (NM_002520.6:c.860_863dup) positive samples using the OCI-AML3 cell line. MRD positive samples were diluted with HL60 cells to achieve the desired MRD level. MRD negative samples were manufactured using the HL60 cell line. The samples were shipped at ambient temperature to the 29 laboratories in 12 countries. Participants were asked to test the blinded samples with their in-house assay and report % normalised ratio of the relevant marker alongside additional methodological and technical data. Results For t(8;21) RUNX1-RUNX1T1, all participants who returned results (n=23) classified the MRD 'high' and MRD 'low' samples as positive and the MRD 'negative' sample as negative. The robust mean log reduction between the MRD 'high' and MRD low sample was 2.7 (range 2.5-2.9). For inv(16) CBFB-MYH11, all participants who returned results (n=22) classified the MRD 'high' sample as positive, 21/22 (95.5%) classified the MRD 'low' sample as positive and 21/22 (95.5%) classified the MRD negative sample as negative. The robust mean log reduction between the MRD 'high' and MRD 'low' sample was 3.16 (range 2.8-4.2). For t(15;17) PML-RARA, all participants who returned results (n=22) classified the MRD 'high' sample as positive, 21/22 (95.5%) classified the MRD 'low' sample as positive and 21/22 (95.5%) classified the MRD negative sample as negative. The robust mean log reduction between the MRD 'high' and MRD 'low' sample was 2.1 (range 1.4-2.4). For NPM1, all participants who returned results (n=23) classified the MRD 'high' as positive, 21/23 (91.3%) classified the MRD 'low' sample as positive and, 17/23 (73.4%) classified the MRD negative sample as negative. The robust mean log reduction between the MRD 'high' and MRD 'low' sample was 3.8 (range 3.2-4.2). Summary/Conclusion The majority of participants in this study were able to detect and accurately quantify MRD when assessing the t(8;21)(q22:q22) RUNX1-RUNX1T1, inv(16)(p13q22) CBFB-MYH11, t(15;17)(q24.1;q21.2) PML-RARA and NPM1 markers, at levels that would be expected within a clinical trial or standard of care setting. A high proportion of participants reported false positive results in the NPM1 marker negative sample. This would have significant consequences clinically, with NPM1 marker false-positivity potentially committing patients to unneeded additional chemotherapy and/or transplant with the attendant risk of morbidity and mortality which highlights the need for ongoing EQA in this area. UK NEQAS LI will work with laboratories advocating they undertake a root cause analysis process to identify the source(s) of error contributing to false positive NPM1 marker results and support their subsequent corrective actions; sharing any educational findings with all participants. Figure 1 Figure 1. Disclosures Scott: Novartis: Research Funding; Biorad: Research Funding. Dillon: Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events; Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Jazz: Other: Education events; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees. Whitby: Roche: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Other: Teaching.

scholarly journals Prognostic Role of Multiparameter Flow Cytometry-Based Measurable Residual Disease Assessment in Patients with Acute Myeloid Leukemia Harboring DNMT3A/TET2/ASXL1 Mutation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 8-9
Author(s):  
Muhned Alhumaid ◽  
Georgina S. Daher-Reyes ◽  
Aaron D Schimmer ◽  
Andre C. Schuh ◽  
Anne Tierens ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Statistical Significance ◽  
Multiparameter Flow Cytometry ◽  
Long Term Outcomes ◽  
Advisory Committees ◽  
Measurable Residual Disease

BACKGROUND: Multiparameter flow cytometry (MFC) has increasingly been used for measurable residual disease (MRD) assessment in patients with acute myeloid leukemia (AML), while next-generation sequencing (NGS)-based MRD monitoring tool is in clinical development for its application. Clonal hematopoiesis (CH), in which leukemia-associated somatic mutations gene are present in individuals with no apparent hematologic disease, adds a challenge in the detection of MRD. In patients with AML, CH could be potentially pre-leukemic, while persistent mutations in DNMT3A, TET2 orASXL1 (DTA) in remission marrow are usually removed from the analysis of residual leukemic cells. However, reports suggest that persistent DTA mutations in remission may be correlated with an increased relapse risk. In the patients with DTA mutations, the use of NGS for MRD monitoring is limited or modified due to the presence of CH clone in the remission marrow. We evaluated whether MFC-MRD can be adjunctive to predict the risk of AML relapse in this population of 221 patients with DTA mutation (DNMT3A (n=123), ASXL1 (n=56) or TET2 (n=100). METHODS: The present study evaluated long-term outcomes in AML patients who achieved first complete remission (CR1) and compared outcomes according to MFC-based MRD status (was defined as negative if patients achieved 0.1 or less) assessed at the time of CR1. A total of 435 patients diagnosed with AML and treated with induction chemotherapy between 2015 and 2018 were included. MFC-MRD was assessed in 336 patients in CR1 (77%). NGS was performed using samples obtained at the time of initial diagnosis and used for mutational subgroup classification. Overall survival (OS) was calculated as the date of CR1 to the date of death and censored on the date of the last follow-up. Relapse-free survival (RFS) was defined as the time from the date of CR1 to the date of relapse or death from any cause. Cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated considering competing risk. The Kaplan-Meier method using a log-rank test and a multivariate Cox proportional hazard model was used for analyses of time-to-event endpoints. For CIR and NRM, Gray test was performed for the risk factors and the Fine-Gray model was adopted for the multivariate model. RESULTS: According to the MFC-MRD status, i.e., the group with positive MRD (MRDpos; n=118, 35%) vs. those with negative MRD (MRDneg; n=218, 65%), we evaluated OS, RFS, and CIR. The MFC-MRDneg group showed better OS at 2 years 67.0% than the MFC-MRDpos group 40.7% (p&lt;0.001). The MFC-MRDneg group also showed a higher RFS rate at 2 years (58.7%) than the MFC-MRDpos group (40.6%) (p=0.001). The CIR was higher in the MFC-MRDpos group, 26.9%, than in the MFC-MRDneg group 21.1%, but with borderline statistical significance (p=0.083). NRM was slightly higher in the MFC-MRDpos group, 32.5%, than in the MFC-MRDneg group, 20.2%, but with borderline statistical significance (p=0.057). We divided the groups according to the number of induction treatment courses, AML type, cytogenetics risk, and age (&lt;60 vs ≥60), and compared OS, RFS, CIR and NRM between MFC-MRDpos vs MRDneg groups, which showed that MFC-MRD is relevant for risk stratification regardless of above-mentioned clinical variables Tab1. Also, we evaluated MFC-MRD status at CR by mutational profile subgroup. Long-term outcomes such as OS, RFS, CIR or NRM were compared by the mutational subgroup. It consistently showed a trend of superior OS, RFS and lower risk of CIR in patients with MFC-MRDneg compared to MFC-MRDposTab1. Of interest, in the subgroup of patients carrying any DTA mutations (n=221), those with MFC-MRDneg (n=103) showed better OS (HR 1.61 [1.01-2.55%]; p=0.042), RFS (HR 1.66 [1.06-2.61%]; p=0.026) and CIR (HR 1.99[1.03-3.83%]; p=0.04) compared to those MFC-MRDpos (n=64; Fig 1). Multivariate analysis confirmed that the MFC-MRDneg is an independent prognostic factor in patients with DTAmutwith respect to OS: MFC-MRDpos (HR 1.63, p=0.04) and age (≥60; HR 2.04, p=0.008) for OS; for RFS, MFC-MRDpos (HR 1.71, p=0.02) and age (≥60; HR 2.32, p= 0.001); for CIR, MFC-MRDpos (HR 2.31, p=0.01) and HCT (HR 0.14, p=&lt;0.001). Conclusion: These findings suggest that in AML patients with DTAmut, MFC-MRD status at the time of remission assessment can be a tool for MRD assessment when NGS-based MRD assessment is limited. Further study is strongly warranted to reach a clearer conclusion with multiple cohorts. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock . Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. McNamara:Novartis: Honoraria. Maze:Pfizer: Consultancy; Novartis: Honoraria; Takeda: Research Funding. Gupta:Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding.

scholarly journals Day 14 Measurable Residual Disease As a Predictor of Post-Induction Response in Patients with Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1287-1287
Author(s):  
Reyes María Martín-Rojas ◽  
Jon Badiola ◽  
Pablo Silva De Tena ◽  
Ana Pérez-Corral ◽  
Ignacio Gómez-Centurión ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Bone Marrow Aspiration ◽  
Advisory Committees ◽  
Post Induction ◽  
Measurable Residual Disease ◽  
Acute Myeloid

Abstract INTRODUCTION Several studies have shown that morphological remission at day 14 is a predictor of post-induction response in patients with acute myeloid leukemia (AML) undergoing an intensive treatment. However, the role of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) at day 14 remains unknown. The aim of our study is to explore the role of MRD at day 14 and its association with outcomes of patients with AML undergoing an intensive treatment. METHODS We conducted a retrospective study in adult patients with newly a diagnosed AML in our center between 2007 and 2020. Adult patients who received intensive chemotherapy, excluding those with an acute promyelocytic leukemia, were included. Bone marrow aspiration was performed at day 14 after induction to assess morphological response and MRD by MFC. Early blast clearance (EBC) was defined as &lt;5% of blasts and negative MRD was defined as &lt;0.1% abnormal cells within mononucleated cells by MFC. Day 14 aspiration findings were compared with clinical data. This study was approved by our Institutional Ethics Committee. Data were analyzed using IBM SPSS Statistics version 24. RESULTS A total of 131 patients were analyzed. Median age was 55.6 years (IQR 42.3-64.2). The most frequent AML subtype was AML with myelodysplasia-related changes (34.4%), followed by NPM1-mutated AML (32.1%). The most commonly used induction regimen was "7+3" (96.2%) (Table 1). On day 14 bone marrow aspiration, median cellularity was 0.5/5 (IQR 0.5-1). 107 patients (81.7%) showed a blast reduction &gt;50% compared to diagnosis and 87 patients (66.4%) had less than 5% of blasts. In this latter group, 28.6% of patients had a positive MRD and 71.4% had a negative MRD. NPM1-mutated AML showed the highest EBC rates while AML with myelodysplasia-related changes had the lowest rates (83.3% versus 55.5%; p=0.04). Furthermore, there were statistically significant differences in EBC rates based on the 2017 European Leukemia Net risk stratification, with 80% of EBC in low risk, 66.6% in intermediate risk and 53.4% in high risk AML (p=0.038). No differences were observed in MRD at day 14 based on AML subtypes or risk stratification. We subsequently analyzed the negative (NPV) and positive predictive values (PPV) of day 14 bone marrow aspiration results by morphology and MFC to predict post-induction results. As a predictor of post-induction CR, day 14 EBC had a NPV of 82% and a PPV of 69%, while day 14 MRD had a NPV of 86% and a PPV of 49%. However, for predicting post-induction MRD, day 14 EBC had a NPV of 49% and a PPV of 15%, while day 14 MRD had a NPV of 71% and PPV of 74%. The correlation between day 14 and post-induction bone marrow aspiration is shown in Table 2. Bivariate analysis showed that achieving CR with negative MRD in post-induction bone marrow aspiration was associated with EBC (p&lt;0.001) and negative MRD (p=0.04) at day 14 bone marrow aspiration. No statistically significances were observed based on marrow cellularity. A multivariate analysis using logistic regression showed that negative MRD by MFC at day 14 was the only independent predictor variable to achieve post-induction CR with negative MRD (OR 4.95% CI 1.0-15.9; p=0.04). CONCLUSION Patients showing EBC with negative MRD on day 14 bone marrow aspiration are more likely to achieve post-induction CR with negative MRD, with day 14 MRD by MFC being the only independent factor able to predict post-induction CR with negative MRD in our cohort. However, further prospective studies are needed to confirm our findings. Figure 1 Figure 1. Disclosures Martín-Rojas: Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Kwon: Novartis, Celgene, Gilead, Pfizer: Consultancy, Honoraria.

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

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

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

scholarly journals Duplex Sequencing for Ultra-Low Frequency Measurable Residual Disease Detection in Adult Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3475-3475
Author(s):  
Jacob Higgins ◽  
Megan Othus ◽  
Laura W. Dillon ◽  
Thomas H. Smith ◽  
Elizabeth Schmidt ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Advisory Committees ◽  
Diagnostic Samples ◽  
Duplex Sequencing ◽  
Measurable Residual Disease ◽  
Acute Myeloid ◽  
Time Point

Abstract Background: The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). However, &gt; 20% of MRD negative cases (as assessed by flow cytometry) subsequently relapse. We sought to determine if ultrasensitive Duplex Sequencing (DS), which relies on double-stranded consensus-making to achieve an error rate below one-in-ten-million, yields better prognostic performance via molecular MRD detection. Methods: Retrospective targeted DNA sequencing of 29 genes recurrently mutated in adult AML was performed on paired diagnostic and remission bone marrow samples from patients enrolled on the SWOG trial S0106 (randomized 7+3 versus 7+3 + gemtuzumab ozogamicin (GO)). Patients were selected if they had remission samples with flow cytometry results (n=67). Non-error corrected sequencing was performed on diagnostic samples (average depth 279x) and DS was performed on remission samples (average duplex molecular depth 27,002x). For each patient, potential germline variants were identified and excluded from the analysis if the variant allele fraction (VAF) was ≥ 35% at both diagnosis and remission, or ≥ 40% at either time point and a gnomAD allele frequency ≥ 0.05. Somatic variants present at diagnosis were classified as potentially deleterious if computationally predicted as such and with a VAF ≥ 5% (≥ 1% for FLT3-ITD/NPM1 insertions). For analysis of residual disease in remission, we evaluated the following outcomes (events): overall survival (OS; death), relapse-free survival (RFS) and time to relapse (TTR; relapse with death a competing event). All outcomes were measured from date of morphologic remission to date of event, with patients without event censored at date of last contact. Associations between residual disease and outcomes were assessed using Cox regression models (cause-specific model for TTR). Results: The median age was 48 years (range 8-60). 32 patients were randomized to 7+3 and 30 to 7+3+GO. A total of 172 potentially deleterious variants were identified in the diagnostic samples. Variants had an average VAF of 31% (range 1.4-91.5%) at diagnosis and were detected in 23 of the 29 genes, with FLT3 being the most frequently mutated. Of the 67 patients analyzed, 93% (n=62) had at least one variant detected at diagnosis (median 2, range 0-9) and 68% (n=42) had at least one residual diagnostic variant also found in the remission sample. We defined the presence of DS MRD as non-DTA (DNMT3A, TET2, ASXL1) time-of-diagnosis mutations identified at the remission time point with a VAF &gt; 0.1% and/or an NPM1 VAF &gt; 0.01% (PMID:31860405). DS MRD was strongly associated with all outcomes, with hazard ratios (and 95% CI) for TTR: 7.1 (2.7-18.9); RFS: 4.9 (2.2-10.9) and OS: 5.1 (2.1-12.3). As a comparator, we correlated treatment outcomes with the results of a flow cytometry MRD assay previously carried out on the same samples during the S0106 trial. The prognostic association of flow MRD with TTR, RFS and OS was less strong (i.e., a smaller hazard ratio) than DS MRD, with TTR: 2.5 (0.9-6.7); RFS: 2.2 (0.9-5.4) and OS: 2.4 (1.0-6.1). RFS and TTR for DS MRD and flow cytometry are plotted below for the 62 patients with a variant detected at diagnosis. Comparing DS MRD with flow cytometry, discordance was found in 20 cases: 15 cases where DS was positive and flow negative, and 5 cases in the opposite direction. Among the 15 discordant cases with DS positive and flow negative, 9 relapsed and 2 died without relapse and 4 were alive without relapse at last contact. Among the 5 discordant cases with DS negative and flow positive, 1 relapsed, 1 died without relapse, and 3 were alive without relapse at last contact. Conclusions: Among the 67 patients evaluated in this prospectively collected study, the presence of MRD defined by DS was strongly associated with adverse disease outcomes. The vast majority of patients had at least one time-of-diagnosis mutation that could be tracked as a measure of MRD. When compared with flow cytometry, DS exhibited superior negative and positive predictive values for foretelling future relapse, although this could potentially reflect the historical version of the flow cytometry assay used. These findings suggest that DS is a powerful tool that could be used in patient management and for early treatment assessment in clinical trials. Figure 1 Figure 1. Disclosures Hourigan: Sellas: Research Funding. Radich: Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Clearance of Somatic Gene Mutations in Patients with Acute Myeloid Leukemia Prior to Allogeneic Hematopoietic Cell Transplantation (HCT) Predicts Outcome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4621-4621
Author(s):  
Onyee Chan ◽  
Chetasi Talati ◽  
Hannah Asghari ◽  
Nelli Bejanyan ◽  
Hany Elmariah ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hematopoietic Cell Transplantation ◽  
Residual Disease ◽  
Conditioning Regimen ◽  
Gene Mutations ◽  
Advisory Committees ◽  
Somatic Gene ◽  
Line Of Therapy

Background: In recent years, genomic studies have uncovered a number of driver gene mutations in acute myeloid leukemia (AML). There is great interest in leveraging residual disease detection methods including next-generation sequencing (NGS) to predict outcomes, especially in the setting of allogeneic hematopoietic cell transplantation (HCT). One study showed measurable minimal residual disease (MRD) at the time of HCT increases the risk of relapse in patients who received a reduced-intensity conditioning (RIC) regimen (Hourigan et al. 2019). In this study, we evaluate the prognostic impact of somatic mutation clearance using NGS prior to HCT in patients with AML. Methods: We identified a total of 139 patients with AML who underwent HCT at the Moffitt Cancer Center (2013-2018). Using European LeukemiaNet (ELN) criteria, patients were included if at the time of HCT they were adverse risk in complete remission (CR)1, intermediate risk in CR1, favorable risk in CR1 if indication for transplant present, or favorable risk in CR2 with at least one time point when NGS was performed before and after HCT. We utilized clinical data captured by BMT Research and Analysis Information Network (BRAIN). Molecular testing via NGS included 54-gene TruSight Myeloid panel tested on Illumina sequencers with a lower limit of detection of 5%. Positive persistent detectable disease (PDD) was defined as presence of detectable mutations on NGS at HCT. Univariate and multivariate analyses were conducted using log-rank and Cox regression, respectively. Kaplan-Meier analysis was used to estimate overall survival (OS) and relapse free survival (RFS) from the time of diagnosis. Cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated by the Fine and Gray model. Results: Of the 139 patients (74 males/65 females), 59% were PDD positive at HCT and 41% PDD negative at HCT. Median age at HCT was 59 years. More patients were in ELN-defined adverse risk (46.8%) in comparison to intermediate risk (35.3%) or favorable risk (18%). In both cohorts, majority of the patients had 1 line of therapy prior to HCT. Overall, 57.6% of patients received myeloablative conditioning regimen (MAC) with the remaining receiving RIC. More patients received MAC in both PDD positive at HCT and PPD negative at HCT groups (Table 1). There were 35 patients (25.2%) who relapsed after HCT, and 17 had NGS available at diagnosis, at the time of HCT, and at relapse. The mutation frequencies and changes over time are shown in Figure 1. Univariate analysis showed inferior OS in patients who are PDD positive at HCT compared to PDD negative at HCT (HR 1.98, 95% CI 1.06-3.72, p=0.032). After adjusting for ELN risk and PDD status, the patients who received more than 1 line of therapy prior to HCT had significantly worse OS (p=0.005). Patients with negative PDD at HCT had a significantly better OS at 2-year compared to PDD positive at HCT patients, 78.7% vs. 62.4% (p=0.029) with a median follow up of 29.9 months (Figure 2A). The RFS at 2-year were 72.6% for PDD negative at HCT patients and 51.8% for PDD positive at HCT patients (p=0.090). There was no difference in NRM or CIR between these two groups (p=0.605 and p=0.136, respectively). Further subgroup analysis did not find a significant difference between PDD status and different types of conditioning regimen (Figure 2B). Conclusions: In this study, we report that clearance of somatic gene mutations in AML patients prior to HCT confers better outcomes compared to those with measurable PDD at HCT. There is a survival advantage in patients who received fewer lines of treatment prior to HCT. Larger cohort and greater depth of NGS coverage is needed to better clarify the impact of conditioning regimen in this population. Disclosures Talati: Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Daiichi-Sankyo: Honoraria; Astellas: Honoraria, Speakers Bureau; Pfizer: Honoraria; Celgene: Honoraria; Agios: Honoraria. Bejanyan:Kiadis Pharma: Other: advisory board. Komrokji:JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy; Novartis: Speakers Bureau; JAZZ: Speakers Bureau. Kuykendall:Janssen: Consultancy; Incyte: Honoraria, Speakers Bureau; Abbvie: Honoraria; Celgene: Honoraria. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nishihori:Novartis: Research Funding; Karyopharm: Research Funding. Sallman:Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Research Funding; Incyte: Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding, Speakers Bureau. Sweet:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Pfizer: Consultancy; Incyte: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Stemline: Consultancy; Jazz: Speakers Bureau.

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