scholarly journals Clinical Relevance of- Limit of Detection (LOD) - Limit of Quantification (LOQ) - Based Flow Cytometry Approach for Measurable Residual Disease (MRD) Assessment in Acute Myeloid Leukemia (AML)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-38
Author(s):  
Raffaele Palmieri ◽  
Alfonso Piciocchi ◽  
Valentina Arena ◽  
Luca Maurillo ◽  
Maria Ilaria Del Principe ◽  
...  
Keyword(s):  
Health Care ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Limit Of Detection ◽  
Leukemic Cells ◽  
For Profit ◽  
Health Care Company ◽  
Measurable Residual Disease ◽  
Acute Myeloid ◽  
Care Company

Background: In Acute Myeloid Leukemia (AML), identification of measurable residual disease (MRD) thresholds with clinical significance is still a matter of debate. For this purpose, multiparametric flow cytometry (MFC) is extensively employed for MRD quantification, due to high sensitivity (down to 1:10-3/10-5 cells) and wide applicability (up to 90% of cases). The identification of 20 clustered residual leukemic cells seems sufficient for the recognition of MRD presence (lower limit of detection [LOD]), whereas a cluster of 50 events may be the minimum threshold for the quantification of a cell population (lower limit of quantitation [LOQ]), provided a sufficient denominator of relevant events (500'000-1'000'000) is acquired. Methods: Using a MFC assay, we assessed the predictive power of a threshold calculated applying the criteria of LOD and LOQ on 261 intensively treated AML patients enrolled in the GIMEMA AML1310 prospective trial.According to the protocol design, patients with a bone marrow residual leukemic cells count (RLCc) equal or above 0.035% of the total no. of mononuclear (MNC) cells qualified as MRDpos,, whileusing LOD and LOQ, we selected the following categories of patients: 1) LODneg if RLCc was below LOD (20x100/total no. of events); 2) LODpos-LOQneg if RLCc was between LOD and LOQ; and 3) LOQpos if RLCc was above LOQ (50x100/total no. of events). Results: The ELN target of 500'000 events was reached in 182/261 (69.6%) patients. Overall, using the predefined AML1310 protocol MRD threshold, 154 (59%) and 107 (41%) were MRDneg and MRDpos, respectively, whereas 74 (28.4%), 43 (16.5%) and 144 (54.4%) patients were classified as LODneg, LODpos-LOQneg and LOQpos, respectively. Two-year overall survival (OS) was 75.4% vs. 79.8% vs. 66.4% for LODneg, LODpos-LOQneg and LODpos, respectively (p=0.1197), and 74.5% vs. 66.4% according to AML1310 protocol 0.035% threshold for MRDneg and MRDpos patients, respectively (p=0.3521). Due to superimposable outcome, LOD-LOQneg and LODpos-LOQneg categories were combined. Accordingly, LODneg/LODpos-LOQneg and LOQpos groups clearly differed in terms of OS (77% vs. 66.4%, p=0.0437) [FIGURE 1A]. Such a figure was challenged in multivariate analysis (p=0.048, HR 0.628, 95% CI 0.396-0.997) that confirmed the independent role of LOD-LOQ approach in influencing OS. To enhance the predictivity of LOD-LOQ estimate, we then focused on samples acquisition of which passed the 500'000 events, according to ELN guidelines. Among 182/261 (69.7%) cases with > 500'000 MNC events as denominator, LODneg/LODpos-LOQneg and LOQpos subgroups were clearly distinct in terms of OS (2-years OS of 83.5% vs. 69.4%, p=0.009). [FIGURE 1B] Similarly, also when selecting those patients (158/261 [60.5%]) whose acquisition passed 500'000 CD45+ events, LODneg/LODpos-LOQneg and LOQpos showed a different behavior with 2-years OS of 86.7% vs. 69.0%, respectively (p=0.004). [FIGURE 1C] Finally, when considering the interaction of the 3 LOD-LOQ categories with possible post-remissional strategies, LODneg/LODpos-LOQneg patients submitted to autologous stem cell transplant showed the best 2-years OS (88.9%) as compared to all the other categories (allogeneic stem cells transplant and no graft-based treatments) (p=0.026). Summary/Conclusion: In conclusion, the use of LOD-LOQ method results in a more sensitive detection of MRD that, in turn, translates in a more accurate recognition of patients with different prognosis. Actually, such an approach allowed to dissect even further the category of patients called MRDneg according to the AML1310 protocol definition, since MRDneg subjects who belonged to a "true negative" LOD-LOQ sub-group [LODneg/LODpos-LOQneg] had a better outcome than the other MRDneg ones. This MRD approach could serve as a useful tool to personalize post-remission strategy in intensively treated AML patients, through selection of high-quality remission patients who may benefit from less intensive post-consolidation therapies. Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Venditti:Novartis: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Pfizer: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; Amgen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Jazz: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company).

scholarly journals Epidemiology of Acute Myeloid Leukaemia in New Zealand: A National Cancer Registry Analysis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
Henry Chan ◽  
Alana Cavadino ◽  
Clinton Lewis
Keyword(s):  
Health Care ◽  
Overall Survival ◽  
New Zealand ◽  
Pacific Islanders ◽  
European Descent ◽  
For Profit ◽  
Economically Deprived ◽  
Health Care Company ◽  
Acute Myeloid ◽  
Care Company

Background: Acute myeloid leukaemia (AML) is a blood cancer characterised by the expansion of a malignant myeloid progenitor. The estimated age-standardised incidence rate in many western countries has remained static over the last 2 decades at 3-4 per 100,000, whilst long-term survival has improved, especially for the younger individuals. However, disparities remain for the older individuals, people of ethnic minority background, and those who are more socio-economically deprived. Previous evaluation of population data from New Zealand has shown a similar pattern, but a more recent analysis has not been done. Here we present the incidence and long-term survival of patients with AML in New Zealand (NZ), using the New Zealand Cancer Registry (NZCR). Method: The NZCR was established in 1948 and it became mandatory by law to report all new cases of malignancy by 1994. We extracted all AML cases from the registry between 1 January 1997 and 31 December 2016. Cases with an ICD-10-CM code for acute myeloid leukaemia and its subtypes including acute promyelocytic leukaemia (e.g. C92.0) were included. Individuals residing overseas or without an address were excluded, and individuals with a diagnosis of acute promyelocytic leukaemia (APML) were analyzed separately. The socio-economic status of the individual was estimated based on their domicile area using the New Zealand 2013 Index of Deprivation (NZDep2013) which is a geographically based composite measure of deprivation. Overall survival was calculated from the date of diagnosis to the date of death or last follow-up (31 December 2016). Multivariable Cox-proportional hazard models were used to evaluate potential associations with survival time in NZ AML cases. Results: During this 20-year period, 154 cases of APML and 2876 cases of AML (excluding APML) were reported to the registry on individuals residing in New Zealand. Of the AML cases, 53% were male and the median age at the time of diagnosis was 67 (IQR 52-77), with a small positive correlation between year of diagnosis and age at diagnosis (Spearman's rho=0.05, p=0.009). The majority of cases (77%) were of European descent, 12% were New Zealand Maori, and 6% were Pacific Islanders. Individuals of European descent were significantly older at diagnosis compared to other ethnicities (median of 70 vs 51 for Maori, 56 for Pacific Islanders, and 58 for all other ethnicities, p<0.001). AML appeared to disproportionally affect those more socio-economically deprived, with 23% of cases reported in the most deprived 20% of the population, compared with only 16% of the cases in the least deprived 20%. The annual crude incidence remained stable during this period at an average of 3.42 per 100,000 (ranging from 2.57 to 4.29, figure 1), and was significantly higher in the older adults (figure 2). Age-standardised rates were lower (figure 1), with an average of 2.6 (range 1.9 to 3.4) cases per 100,000, and a small but significant average annual decrease over the study period. The estimated 1, 2, and 5-year survival for the entire cohort was 38%, 27%, and 22%, respectively. Age at diagnosis was a significant predictor of inferior survival, with a hazard ratio (HR) for all-cause mortality of 2.06, 3.95, 6.39 and 10.84 for the 50-59, 60-69, 70-79 and >80 age groups, respectively, compared to those aged <50. Shorter overall survival was also noted in individuals in the more socio-economically deprived 50% of the population (HR 1.13, 95% CI 1.03-1.23). Conclusion The incidence of AML in New Zealand has remained static in the last 2 decades, consistent with data from other western countries. Lower age-standardised rates and the small decrease in these observed over the study period are likely to reflect the increasingly and comparatively older population in NZ. Maori and Pacific Islanders appeared to present at a younger age than individuals of European descent. Age at diagnosis and socio-economic deprivation were shown to be an adverse prognostic factor for overall survival. Further in-depth analysis is required to determine the cause of these observations at a population level. Disclosures Chan: AbbVie:Membership on an entity's Board of Directors or advisory committees;Janssen:Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau;Celgene:Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company);Amgen:Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company);Roche:Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company).

Post-remission clonal hematopoiesis; Practical implications for measurable residual disease assessment in acute myeloid leukemia (AML).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 7529-7529
Author(s):  
Sanam Loghavi ◽  
Tomoyuki Tanaka ◽  
Ken Furudate ◽  
Sa A Wang ◽  
Koichi Takahashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Disease Status ◽  
Leukemic Cells ◽  
Disease Assessment ◽  
Individual Gene ◽  
Clonal Hematopoiesis ◽  
Measurable Residual Disease ◽  
Acute Myeloid

7529 Background: Clonal Hematopoiesis may persist following complete remission (CR) in patients with acute myeloid leukemia (AML) but does not necessarily indicate residual AML and may represent persistence of pre-leukemic stem cells. Post-remission CH identified by NGS has not been systemically studied in parallel with measurable residual disease (MRD) detection by flow cytometric immunophenotyping (FCI). Methods: We studied bone marrow sample from AML patients at baseline and CR by targeted deep NGS of 295 genes (median 403x depth) and compared the results to FCI. Measurable residual disease (MRD) detection by FCI was performed by comparing the phenotype at CR to baseline and by detection of leukemia associated immunophenotype (LAIP) and derivation from normal (DFN) (sensitivity: 0.1%). Post-CR CH was defined as presence of mutations originally detected in AML with variant allele frequency > 2.5%. FCI results were categorized into 4 groups: a) AML MRD negative by LAIP or DFN b) AML MRD+ (similar to baseline) c) AML MRD+ (different from baseline), d) Negative for AML MRD, but aberrant phenotype suggestive of pre-leukemic cells. We correlated FCI and NGS results. Results: 101 patients were included in the study. 45 (45%) had persistent post-CR clonal hematopoiesis; 23 (51%) had phenotypic alterations detected by FCI including AML MRD+ in 18 (40%) and pre-leukemic cells in 5 (10%). Among patient with no detectable mutations by NGS (n = 56; 55%), 14 (25%) had FCI aberrancies including AML MRD+ in 4 (7%) and pre-leukemic cells in 10 (18%). CH was significantly more common in samples with residual phenotypic aberrancies detected by FCI (p = 0.004). There was no significant correlation between FCI group d and persistent CH (p = 0.4). Persistent ASXL1 (p = 0.024, OR = 7.2 ) and RUNX1 (p = 0.016; OR = 17.3) mutations were significantly associated with FCI abnormalities. The correlation coefficient between FCI abnormalities and RUNX1 mutations inferred from a Bayesian network structure was 0.66. Conclusions: NGS and FCI are complementary in evaluating post treatment disease status in AML. Post CR-CH is associated with phenotypic abnormalities that either represent residual AML or pre-leukemic cells. The latter may not have the same prognostic implications as AML MRD; however, the association with outcome needs to be elucidated. Single cell DNA sequencing technologies may be helpful in more accurately deciphering the association of individual gene mutations and their contribution to phenotypic aberrations.

scholarly journals Measurable Residual Disease in Acute Myeloid Leukemia Using Flow Cytometry: A Review of Where We Are and Where We Are Going

2020 ◽  
Vol 9 (6) ◽  
pp. 1714
Author(s):  
Caroline Dix ◽  
Tsun-Ho Lo ◽  
Georgina Clark ◽  
Edward Abadir
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Hematologic Malignancies ◽  
Leukemic Cells ◽  
Prognostic Models ◽  
Measurable Residual Disease ◽  
Acute Myeloid

The detection of measurable residual disease (MRD) has become a key investigation that plays a role in the prognostication and management of several hematologic malignancies. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the role of MRD in AML is still emerging. Prognostic markers are complex, largely based upon genetic and cytogenetic aberrations. MRD is now being incorporated into prognostic models and is a powerful predictor of relapse. While PCR-based MRD methods are sensitive and specific, many patients do not have an identifiable molecular marker. Immunophenotypic MRD methods using multiparametric flow cytometry (MFC) are widely applicable, and are based on the identification of surface marker combinations that are present on leukemic cells but not normal hematopoietic cells. Current techniques include a “different from normal” and/or a “leukemia-associated immunophenotype” approach. Limitations of MFC-based MRD analyses include the lack of standardization, the reliance on a high-quality marrow aspirate, and variable sensitivity. Emerging techniques that look to improve the detection of leukemic cells use dimensional reduction analysis, incorporating more leukemia specific markers and identifying leukemic stem cells. This review will discuss current methods together with new and emerging techniques to determine the role of MFC MRD analysis.

scholarly journals A Phase 2 Open-Label, Multicenter, Dose Optimization Clinical Study of the Safety, Tolerability, and Pharmacokinetic (PK) and Pharmacodynamic (PD) Profiles of Cfi-400945 As a Single Agent or in Combination with Azacitidine or Decitabine in Patients with Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-25
Author(s):  
Brian A Jonas ◽  
Dale L. Bixby ◽  
Joseph M Brandwein ◽  
Karen W.L. Yee ◽  
Tracy Murphy ◽  
...  
Keyword(s):  
Health Care ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Food Effect ◽  
Phase 1 ◽  
Single Agent ◽  
High Fat ◽  
For Profit ◽  
Health Care Company ◽  
Care Company

Background: CFI-400945 is a potent, selective, orally administered, first-in-class inhibitor of the serine/threonine kinase, Polo-like kinase 4 (PLK4). PLK4 is a highly conserved master regulator of centriole duplication, and is critical for maintenance of genomic integrity. Aberrant expression of PLK4 results in a number of effects including the centrosome amplification often seen in aneuploid cancers, pointing to a potentially causative role for PLK4 in genome instability and cancer progression. A Phase 1 study has been completed evaluating CFI-400945 as a monotherapy in solid tumors, showing a tolerable safety profile and promising signs of activity. As acute myeloid leukemia (AML) is a disease characterized by genomic instability, it is of significant interest as a potential indication for the clinical evaluation of CFI-400945. pre-clinical studies, CFI-400945 showed potent activity towards leukemia cell lines and primary human samples in vitro, as well as marked efficacy in two subcutaneous models of leukemia, specifically the MV4-11 FLT3-ITD AML. A Phase 1 trial in AML was initiated at the Princess Margaret Cancer Center in 2018, and of six patients evaluable for response, two (33%) achieved complete remission (CR), and 3 patients (50%) had stable disease (with one patient having a 78% reduction in marrow blast count) [re: Murphy et al, ASH 2020]. These promising results have led to a plan for an expanded trial examining CFI-400945 in AML, particularly focused on complex karyotype (CK). Study Design and Methods: The study will have 4 parts, Part 1A (1A): a single agent dose escalation portion, Part 1B (1B): a food effect portion once the MTD of 1A is determined, and combinations with azacitidine (2A), and decitabine (2B). For parts 1A and 1B, patients with relapsed and/or refractory AML, MDS, or CMML after >1 prior therapy will be included. Patients with MDS or CMML must have progressed or had a lack of response after at least 4 cycles of hypomethylating agents. For parts 2A and 2B, patients should have relapsed and/or refractory AML or untreated MDS or CMML. Untreated patients who decline or are ineligible for intensive therapy may be included. The study will use a standard 3 + 3 design. The maximum tolerated dose (MTD) will be defined as the dose level where the number of dose limiting toxicities (DLTs) is <1 out of 6 at highest dose level below the maximally administered dose. Biomarker Selection and companion Diagnostics: No biomarker based pre-selection of patients. PD evaluations will include blast reduction and markers of mitosis. Study Treatment and Endpoints: Part 1: Each cycle will be 28 days (21 days on/7 days off). Starting does of CFI-400945 will be 32mg po. Once the MTD of 1A is determined, 1B will explore the food effect of a high fat meal on the PK of CFI-400945 at the MTD. 2A and 2B will explore the combination of CFI-400945 with standard dose of either azacitidine (2A) or decitabine (2B). The starting dose of CFI-400945 will be 32mg po for 21 days on/7 off. The DLTs will be defined as NCI CTCAE V5.0 Grade >3 related non-hematologic event(s) occurring during cycle 1 or prolonged pancytopenia in the presence of a hypocellular bone marrow > day 42 without evidence of disease. The efficacy endpoints for AML, MDS, and CMML include the overall response rate, and the CR rate per standard criteria. The aim of the food effect part of the study is the asses the effect of high fat food on the PK of CFI-400945. The safety endpoint is the incidence of treatment emergent adverse events. PK endpoints include evaluations of parameters such as half-life, AUC, etc. Exploratory endpoints include eval of minimal residual disease, genomic alterations and other molecular features associated with response and biological effects of PLK4 inhibition. Disclosures Jonas: Amgen: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; AbbVie: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; GlycoMimetics: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Sigma Tau: Research Funding; Jazz: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Tolero: Consultancy; Treadwell: Consultancy; Forty Seven: Research Funding; Accelerated Medical Diagnostics: Research Funding; AROG: Research Funding; Daiichi Sankyo: Research Funding; F. Hoffmann-La Roche: Research Funding; Forma: Research Funding; Genentech/Roche: Research Funding; Hanmi: Research Funding; Incyte: Research Funding; LP Therapeutics: Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding. Bixby:GlycoMimetics: Research Funding. Brandwein:Pfizer: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Astellas: Honoraria; Taiho: Honoraria; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria. Michelson:Treadwell Therapeutics: Consultancy. Bray:TIO Discovery: Current Employment; Treadwell Therapeutics: Current Employment. Roberts-Thomson:Treadwell Therapeutics: Current Employment. Borthakur:BioTherix: Consultancy; FTC Therapeutics: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; Oncoceutics: Research Funding; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; BioLine Rx: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; BioLine Rx: Consultancy; PTC Therapeutics: Research Funding; Curio Science LLC: Consultancy; Cyclacel: Research Funding; GSK: Research Funding.

scholarly journals Investigation of measurable residual disease in acute myeloid leukemia by DNA methylation patterns

Leukemia ◽  
2021 ◽  
Author(s):  
Tanja Božić ◽  
Chao-Chung Kuo ◽  
Jan Hapala ◽  
Julia Franzen ◽  
Monika Eipel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Amplicon Sequencing ◽  
Multiparametric Flow Cytometry ◽  
Methylation Patterns ◽  
Cg Dinucleotides ◽  
Measurable Residual Disease ◽  
Acute Myeloid

AbstractAssessment of measurable residual disease (MRD) upon treatment of acute myeloid leukemia (AML) remains challenging. It is usually addressed by highly sensitive PCR- or sequencing-based screening of specific mutations, or by multiparametric flow cytometry. However, not all patients have suitable mutations and heterogeneity of surface markers hampers standardization in clinical routine. In this study, we propose an alternative approach to estimate MRD based on AML-associated DNA methylation (DNAm) patterns. We identified four CG dinucleotides (CpGs) that commonly reveal aberrant DNAm in AML and their combination could reliably discern healthy and AML samples. Interestingly, bisulfite amplicon sequencing demonstrated that aberrant DNAm patterns were symmetric on both alleles, indicating that there is epigenetic crosstalk between homologous chromosomes. We trained shallow-learning and deep-learning algorithms to identify anomalous DNAm patterns. The method was then tested on follow-up samples with and without MRD. Notably, even samples that were classified as MRD negative often revealed higher anomaly ratios than healthy controls, which may reflect clonal hematopoiesis. Our results demonstrate that targeted DNAm analysis facilitates reliable discrimination of malignant and healthy samples. However, since healthy samples also comprise few abnormal-classified DNAm reads the approach does not yet reliably discriminate MRD positive and negative samples.

scholarly journals Duplex Sequencing with Patient-Specific Hybrid Capture Panels Reveals Ultra-Low Frequency Measurable Residual Disease in Pediatric Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Jacob Higgins ◽  
Fang Yin Lo ◽  
Michael J. Hipp ◽  
Charles C. Valentine ◽  
Lindsey N. Williams ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Residual Disease ◽  
Limit Of Detection ◽  
Low Frequency ◽  
Patient Specific ◽  
Pediatric Acute Myeloid Leukemia ◽  
Hybrid Capture ◽  
Pediatric Aml ◽  
Duplex Sequencing ◽  
Measurable Residual Disease

Sensitive and specific detection of measurable residual disease (MRD) after treatment in pediatric acute myeloid leukemia (AML) is prognostic of relapse and is important for clinical decision making. Mutation-based methods are increasingly being used, but are hampered by the limited number of common driver gene mutations to target as clone markers. Additional targets would greatly increase MRD detection power. However, even in cases with many AML-defining mutations, it is the limited accuracy of current molecular methods which establishes the lower bounds of sensitivity. Here we describe an ultrasensitive approach for disease monitoring with personalized hybrid capture panels targeting hundreds of somatic mutations identified by whole genome sequencing (WGS), and using extremely accurate Duplex Sequencing (DS) in longitudinal samples. In a pilot cohort of 13 patients we demonstrate detection sensitivities several orders of magnitude beyond currently available single locus testing or less accurate sequencing. With multi-target panels, overall power for MRD detection is cumulative across sites. For example, if a patient has MRD at a true frequency of 1/30,000, sequencing a single mutant site to 10,000x molecular depth would be unlikely to detect MRD. However, sequencing 10 sites each to 10,000x would effectively total 100,000x informative site depth, increasing power to >95%. However, standard sequencing assays are insufficiently accurate to achieve this theoretical limit of detection (LOD). DS enables accurate detection of individual variants to <10-5 with an error rate <10-7 and, thus, can achieve MRD sensitivities below one-in-one-million. Marrow aspirates were collected from 13 uniformly treated pediatric AML patients at time of diagnosis (TOD), during treatment (end of induction, EOI), in remission (end of therapy, EOT), and at relapse. 9/13 patients relapsed. DNA from TOD was analyzed by WGS. Germline variants were excluded and somatic single nucleotide variants (SNVs) were targeted by a custom probe panel designed for each patient. An average of 170 SNVs were targeted per patient (range 53-200). More than 90% of the SNVs were noncoding. Longitudinal samples were then analyzed with DS, which compares sequences from both strands of each DNA molecule to eliminate technical noise and reveal biological mutation signal with extreme accuracy and sensitivity. A median of 82% of WGS SNVs were validated by DS in the TOD DNA, and the vast majority of those were also present at relapse. Relapsers had more SNVs at diagnosis than non-relapsers. EOT samples were sequenced to an average Duplex molecular depth of 29,400x, with a maximum of 61,283x. The figure below shows time course plots tracking SNVs at diagnosis, EOT and relapse for 2 patients. Among mutations validated in TOD samples, a median of only 8 (5%) were detected per EOT sample (range 0-66 mutations). MRD was detected in 8/9 relapsers. Targeting 1 or even 10 SNVs would therefore have missed MRD in the majority of these patients. Among relapsers, median EOT SNV VAF was 0.069%. The lowest single VAF detected per EOT sample ranged from 0.036% to 0.002%. The presence of an SNV at diagnosis and relapse implies that it must truly be present at EOT, whether or not it is detected. Therefore, if a small minority of leukemic mutations are detected at EOT, the true overall MRD frequency is much lower than the LOD at any single site. In the only relapser where MRD was not detected, targeted SNVs were present at diagnosis and relapse, so additional sequencing depth at EOT would eventually reveal ultra-low frequency mutations. Among patients that did not relapse by the end of the study, median VAF at EOI (the latest time point DNA available) was 0.0258%. Therefore, non-relapsers have a lower median VAF at EOI than relapsers do even later at EOT, potentially indicating very early on that treatment is more successful. This study shows excellent performance of DS-based assays for detecting MRD with patient-specific panels. We have demonstrated that among large panels of validated somatic SNVs present at time of diagnosis, a median of 5% are identified at EOT in eventual relapsers. DS detected MRD in 8/9 patients, and at a median VAF well below the limit of detection of any other sequencing technology. Comprehensive personalized hybrid selection panels coupled with DS represents a powerful option for MRD monitoring in pediatric AML and potentially other cancers. Figure Disclosures Higgins: TwinStrand Biosciences: Current Employment. Lo:TwinStrand Biosciences: Current Employment. Hipp:TwinStrand Biosciences: Current Employment. Valentine:TwinStrand Biosciences: Current Employment. Williams:TwinStrand Biosciences: Current Employment. Radich:TwinStrand Biosciences: Research Funding. Salk:TwinStrand Biosciences: Current Employment.

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