Sensitive Detection Of Minimal Residual Disease Research Articles

Abstract 965: Urine cell-free DNA multi-omics combining copy number analysis with fragmentomics to detect minimal residual disease in bladder cancer patients

Abstract Background: Standard-of-care for patients with localized muscle-invasive bladder cancer (BC) is neoadjuvant chemotherapy followed by radical cystectomy (RC). Bladder-sparing treatments are limited by our current inability to sensitively detect minimal residual disease (MRD). Here, we analyzed urine, a noninvasive and proximal biofluid, from patients with bladder cancer and utilized an approach that incorporates copy number-derived tumor fraction as well as fragmentomics to sensitively detect MRD and predict pathologic complete response (pCR). Methods: We acquired urine preoperatively from 51 BC patients (80% muscle-invasive) on the day of standard-of-care RC, and after neoadjuvant chemotherapy in 57% of patients. We performed whole genome sequencing (WGS) of urine cfDNA (median depth: 10X) from all 51 BC patients and 13 healthy adults. Tumor fraction level based on genome-wide copy number alterations was estimated using ichorCNA. We inferred fragment size and genomic coverage of urine cfDNA using Picard Tools. Then, we computed the short-to-long (S/L) fragment ratio for genomic bins in each sample. The S/L ratio per bin was derived by partitioning the genome into 100 kb bins and evaluating the ratio of GC-corrected short fragments (50-150 bp) to long fragments (151-250 bp) within each bin. A normalized genome-wide S/L score was calculated by averaging across all bins. Results: In our cohort of 51 BC patients, 45% of patients achieved pCR (n = 23) while 55% had residual disease detected in their surgical sample (no pCR; n = 28). Patients with no pCR had significantly higher copy number-derived tumor fractions in urine compared to patients with pCR (median 10.6% vs 1.9%, p = 0.0002) and healthy adults (n = 12) (median 10.6% vs 1.8%, p = 0.003). Tumor fraction achieved an AUC of 0.89 for classification of No pCR from pCR with sensitivity of 82% and specificity of 92%. Presumed cancer-free patients (healthy and pCR) had significantly longer urine cfDNA median fragment sizes than patients with no pCR (median 177 bp vs 156 bp, p = 0.002). Presumed cancer-free patients (healthy and pCR) also had significantly lower S/L ratio scores than no pCR patients (mean .98 vs 1.5, p = 0.007). The top 100 bins with maximally differential S/L ratio between these two patient groups were enriched for genes involved in KEGG pathways implicated in cancer and immune signaling (autophagy, transcriptional misregulation in cancer, Th1 differentiation) indicating biological relevance. Conclusion: Copy number alteration-derived tumor fraction inference combined with fragmentomic analysis of urine cell-free DNA can enable sensitive detection of MRD to predict pCR and reveal potentially onco-relevant pathways. Citation Format: Arpit Panda, Irfan Alahi, Pradeep S. Chauhan, John Sheng, Nathan Colon, Cayce Nawaf, Alexander Shiang, Peter K. Harris, Faridi Qaium, Eric H. Kim, Melissa A. Reimers, Woodson Smelser, Zachary L. Smith, Aadel A. Chaudhuri. Urine cell-free DNA multi-omics combining copy number analysis with fragmentomics to detect minimal residual disease in bladder cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 965.

Sensitive MRD Detection from Lymphatic Fluid after Surgery in HPV-Associated Oropharyngeal Cancer.

Our goal was to demonstrate that lymphatic drainage fluid (lymph) has improved sensitivity in quantifying postoperative minimal residual disease (MRD) in locally advanced human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) compared with plasma, and leverage this novel biofluid for patient risk stratification. We prospectively collected lymph samples from neck drains of 106 patients with HPV (+) OPSCC, along with 67 matched plasma samples, 24 hours after surgery. PCR and next-generation sequencing were used to quantify cancer-associated cell-free HPV (cf-HPV) and tumor-informed variants in lymph and plasma. Next, lymph cf-HPV and variants were compared with TNM stage, extranodal extension (ENE), and composite definitions of high-risk pathology. We then created a machine learning model, informed by lymph MRD and clinicopathologic features, to compare with progression-free survival (PFS). Postoperative lymph was enriched with cf-HPV compared with plasma (P < 0.0001) and correlated with pN2 stage (P = 0.003), ENE (P < 0.0001), and trial-defined pathologic risk criteria (mean AUC = 0.78). In addition, the lymph mutation number and variant allele frequency were higher in pN2 ENE (+) necks than in pN1 ENE (+) (P = 0.03, P = 0.02) or pN0-N1 ENE (-) (P = 0.04, P = 0.03, respectively). The lymph MRD-informed risk model demonstrated inferior PFS in high-risk patients (AUC = 0.96, P < 0.0001). Variant and cf-HPV quantification, performed in 24-hour postoperative lymph samples, reflects single- and multifeature high-risk pathologic criteria. Incorporating lymphatic MRD and clinicopathologic feature analysis can stratify PFS early after surgery in patients with HPV (+) head and neck cancer. See related commentary by Shannon and Iyer, p. 1223.

Personalised circulating tumour DNA assay with large-scale mutation coverage for sensitive minimal residual disease detection in colorectal cancer

BackgroundPostoperative minimal residual disease (MRD) detection using circulating-tumour DNA (ctDNA) requires a highly sensitive analysis platform. We have developed a tumour-informed, hybrid-capture ctDNA sequencing MRD assay.MethodsPersonalised target-capture panels for ctDNA detection were designed using individual variants identified in tumour whole-exome sequencing of each patient. MRD status was determined using ultra-high-depth sequencing data of plasma cell-free DNA. The MRD positivity and its association with clinical outcome were analysed in Stage II or III colorectal cancer (CRC).ResultsIn 98 CRC patients, personalised panels for ctDNA sequencing were built from tumour data, including a median of 185 variants per patient. In silico simulation showed that increasing the number of target variants increases MRD detection sensitivity in low fractions (<0.01%). At postoperative 3-week, 21.4% of patients were positive for MRD by ctDNA. Postoperative positive MRD was strongly associated with poor disease-free survival (DFS) (adjusted hazard ratio 8.40, 95% confidence interval 3.49–20.2). Patients with a negative conversion of MRD after adjuvant therapy showed significantly better DFS (P < 0.001).ConclusionTumour-informed, hybrid-capture-based ctDNA assay monitoring a large number of patient-specific mutations is a sensitive strategy for MRD detection to predict recurrence in CRC.

Dynamic ctDNA to inform the precise management of resected NSCLC: LUNGCA-2 study.

8528 Background: Circulating tumor DNA (ctDNA) has been reported to be valuable for minimal residual disease (MRD) detection, but there is a lack of studies comprehensively assessing whether dynamic ctDNA monitoring can inform whole-course precise postoperative cancer management. In the present study (LUNGCA-2), we analyzed data obtained from longitudinal ctDNA monitoring of stage I-III NSCLC patients in the LUNGCA cohort, and sought to assess the use of dynamic ctDNA monitoring for precise whole-course postoperative management of NSCLC patients. Methods: All eligible NSCLC patients underwent curative-intent surgery and subsequently received adjuvant therapies when indicated by clinical practice guidelines for NSCLC. The clinical follow-up was scheduled to be every 3-6 months mainly involving CT and/or MRI. Fresh frozen or paraffin-embedded tissues of primary tumors were collected intraoperatively for baseline somatic mutation profiling. Blood samples were collected before surgery, at 3 days and 1 month after surgery, and then every 3-6 months at clinical visits up to 3 years postoperatively. 233 patients with a total of 2123 plasma samples were ultimately included in the longitudinal ctDNA analysis in the current study. NGS assays were performed with a custom-designed panel that covered 769 cancer-related genes via the MinerVA platform (Genecast Biotechnology Co., Ltd). Results: Among the cohort, 166 patients harboring targetable mutations in the primary tumor tissue, 15 were preadjuvant ctDNA positive, and among them, only 25% patients treated with adjuvant TKI therapy experienced relapse during follow-up, and patients received ACT or no adjuvant therapy had a relapse rate of 100%. TKI treatment tended to improve RFS compared with no treatment (HR 0.1, P = 0.025), but ACT failed to improve RFS (HR 1.1, P = 0.834). Among all 82 patients receiving adjuvant therapy, the pre- and post-adjuvant therapy postoperative ctDNA status with a negative-negative or positive-negative pattern had similar and favorable RFS ( P = 0.545) but that patients with a positive-positive or negative-positive pattern had significantly inferior RFS than those with a negative-negative pattern (both P &lt; 0.001). For recurrence monitoring, posttreatment ctDNA MRD status had a positive predictive value of 100% and a negative predictive value of 91.8%, and the sensitivity of MRD detection was affected by the organ of relapse. Among positive ctDNA MRD patients, faster increase of ctDNA concentration correlated with more imminent clinical relapse. The median lead time from ctDNA detection to radiological relapse was 273 days. Moreover, ctDNA negativity after relapse was associated with favorable survival. Conclusions: These findings highlight the clinical significance of ctDNA dynamics for the precise whole-course postsurgical management of NSCLC patients.

Abstract 3384: Evaluation of a tumor informed MRD assay with contrived breast cancer samples

Abstract The non-invasive detection of circulating tumor DNA (ctDNA) from plasma has been shown to have clinical value for detection of minimal residual disease (MRD), emergence of resistance, and predicting treatment response. The sensitive detection of MRD following curative treatment allows for the identification of patients destined to recur. Higher assay sensitivities enable the longest lead times to clinical recurrence. Evaluations using several contrived pan-cancer samples have shown that the Inivata RaDaR tumor-informed personalized assay has a high sensitivity to detect ctDNA. The tumor-informed analysis utilizes sequencing of the tumor tissue to identify up to 48 clonal somatic variants, which are selected to generate a personalized panel to track the variants in patient plasma. To evaluate this technology in a disease specific manner, we generated a set of contrived plasma samples from commercial breast cancer patients and healthy individuals. The biological materials allowed us to generate a plasma sample with defined range of tumor content between 0.2 and 0.001% variant allele fraction (VAF). The sample dilutions were intended to recapitulate challenging cell-free DNA inputs that can be encountered in a clinical setting (median input: 5.76 ng [range 0.22 to 103.73]. Through our pan-cancer and breast cancer evaluations, we were able generate both sensitivity metrics along with a small limit of detection (LOD) study. The assay successfully detected tumor fractions in samples tested at 1X and 1.5X of the established LOD95% of 0.0011% VAF. RaDaR was able to detect the majority of MRD+ samples at 0.5X LOD. We will present data showing the level of sensitivity based on using both 2000 and 20000 genomic equivalents. The RaDaR assay showed high sensitivity for pan-cancer as well as ER+ breast cancer, which is known to have a low TMB compared to other subtypes. In this albeit small LoD study the Inivata assay was highly sensitive for the detection of ctDNA which is imperative to confidently detect ctDNA in advance of overt clinical recurrence. Citation Format: Hugh Russell, Paul LaBrousse, Daniel Stetson, Greg Jones, Giovanni Marsico, Tim Forshew, Robert McEwen, Elza De Bruin, Christopher Abbosh, Darren Hodgson, Brian Dougherty, James Hadfield, Carl Barrett. Evaluation of a tumor informed MRD assay with contrived breast cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3384.

The Value of PET/CT Combined with Next-Generation Flowcytometry in Minimal Residual Disease Detection and Prognostic Evaluation of Multiple Myeloma

Background: Minimal residual disease (MRD) plays an increasingly vital role in efficacy evaluation of multiple myeloma (MM), especially in the era of novel drugs. Currently, the mainstream MRD evaluation methods include next-generation sequencing (NGS), next-generation flowcytometry (NGF) and positron emission tomography/computed tomography (PET/CT). However, it remains to be solved which method holds the best sensitivity and specificity. Our research intends to compare the sensitivity between 11C-acatate, a novel PET tracer with the traditional one 18F-FDG, and meanwhile, explore the prognostic value of PET/CT and NGF. Methods: Our research included MM patients treated in our center between 2015.11 to 2021.11, who received standard medication and reached at least partial response. The patients were examined by 11C-acatate PET/CT (AC-PET), 18F-FDG PET/CT (FDG-PET) as well as Euroflow MRD assessment. Baseline characteristics including ISS stage, cytogenetic abnormalities, treatment and response were collected. Statistical analysis was conducted using SPSS 24.0, with Χ2 test or McNemar test for Categorical variables, and Kaplan-Meier analysis or COX regression model for survival analysis. Results: A total of 54 patients were included, with 42 patients received dual-tracer PET/CT, 10 patients received FDG-PET only and 2 patients received AC-PET only. 37 patients also underwent NGF MRD evaluation. The average age of all patients were 58.5 years, with ISS III patients accounting for 53.7% and cytogenetically high-risk patients accounting for 60.4%. 46.3% of patients completed autologous stem cell transplantation, and 90.7% of patients reached at least very good partial response. A total of 11 patients found minimal residual disease by PET/CT, with a sensitivity of 18.2% by AC-PET and 7.7% by FDG-PET. Although it appeared that AC-PET had superior sensitivity compared with FDG-PET, the difference was statistically insignificant (p=0.14). The paired test of dual-tracer PET/CT scan revealed a same result (p=0.13). In patients conducted NGF, the MRD positive rate was 40.5%. Paired test between NGF and PET/CT in these patients demonstrated that sensitivity of NGF was significantly higher than PET/CT (p=0.01). In terms of progression-free survival (PFS), single-factor analysis showed that ISS stage II or III, response not reaching stringent complete response and positive MRD by NGF were all adverse prognostic factor, with p value of 0.019, ＜0.001 and 0.008, respectively. Cox regression analysis further determined that positive MRD by PET/CT was also an independent prognostic factor. Conclusion: The novel tracer AC-PET is advantageous over traditional FDG-PET in sensitivity for MRD detection, although not statistically significant. However, the sensitivity NGF is still superior than either method of PET/CT. In terms of PFS, NGF and PET/CT are both independent prognostic factor.

Genotyping and Minimal Residual Disease (MRD) Assessment in cfDNA By the Euroclonality-NGS DNA Capture (EC-NDC) Panel in Mantle Cell Lymphoma (MCL)

Background Modern concepts for the treatment of mantle cell lymphomas (MCL) increasingly integrate clinical and biological variables for risk-adapted treatment strategies. Novel next-generation sequencing (NGS) technologies are highly suited for tumor genotyping as well as disease monitoring by minimal residual disease (MRD) assessment, identifying patients (pts) with a high risk of relapse. Here, we investigated whether the EuroClonality (EC)-NGS DNA Capture (EC-NDC) assay is a) applicable for genotyping of MCL in peripheral blood (PB) and plasma cell-free (cf)DNA and b) suitable for MRD assessment in plasma as compared to the amplicon-based EuroClonality Immunoglobulin-based (IG-)NGS assay. Patients and methods A total of 109 PB samples (55 baseline and 54 interim staging) from a cohort of MCL treated within the TRIANGLE trial by the European MCL network (NCT02858258) were selected according to a PB infiltration ≥10% at diagnosis. From prospectively collected PB samples in national reference labs, DNA from the cellular blood compartment (PBC) and plasma cfDNA was extracted. For targeted capture, the EC-NDC assay (Univ8® Genomics, UK) was used to simultaneously detect immunoglobulin and T cell receptor (IG/TR) rearrangements, structural variants (SV) and single nucleotide variants (SNVs) in 72 genes (Stewart, Blood Adv, 2021). For diagnostic PBC and cfDNA samples, IG clonotypes were called with ≥6 unique reads and ≥5% of annotated IG reads, SV were called with ≥4 unique reads and SNV were called with ≥6 unique reads at ≥4% variant allele frequency (VAF). For amplicon-based MRD analysis the EuroClonality IG-NGS (IG-NGS) assay was used with IG clonotypes called with ≥5% of annotated IG reads and at ≥1 log higher abundance to the polyclonal background. MRD was analyzed by IG-NGS in PBC using 8 µg PBC DNA to reach a sensitivity of 10-6. For MRD analysis in cfDNA, we aimed to use ≥5000 genome equivalents (GE) for both IG-NGS and EC-NDC. Data were analyzed by ARResT/Interrogate (Bystry, Bioinformatics, 2017) with an adapted pipeline for targeted capture in cfDNA. MRD status was defined as positive by detecting a lymphoma-specific IG clonotype and/or SV at ≥1 read, or alternatively by retrieval of ≥1 disease-specific mutation at ≥3 reads. Results Total cfDNA amounts at baseline were high (mean 92.6 ng/mL plasma) and even increasing at interim staging (mean 123.8 ng/mL) while decreasing by 10-fold in 38 samples at end of treatment (EoT)(mean 11.45 ng/mL). At baseline, a tumor-specific IGH-VJ clonotype was detected by IG-NGS in all 55 PBC and cfDNA samples. Genotyping by EC-NDC showed fully concordant results in 55 PBC and cfDNA samples with respect to IG clonotypes, SV and SNV, except for 3 PBC samples with low usable reads covering IGH-VJ regions. The presence of a t(11;14)(q13;q32) translocation was confirmed in 54/55 pts in PBC and cfDNA, while one patient with unknown t(11;14) translocation status by FISH was negative by EC-NDC. EC-NDC identified 106 putative somatic SNVs (VAF 2.7-75%, median 30%) in PBC and cfDNA, and among those were genes described as drivers in MCL (ATM (25/55), TP53 (11/55), KMT2D (9/55), SAMHD1 (5/55), CCND1 (5/55) and 18 other genes (Fig. 1). Six pts with detectable t(11;14) and IG rearrangement did not show additional SNVs. On average, 6 MRD markers/pt were identified (range: 3-10). At interim staging, MRD was analyzed in 54 PBC and 44 cfDNA samples. Twenty-five (47%) pts were MRD+ in PBC by IG-NGS. Twenty (46%) pts were MRD+ in cfDNA by EC-NDC (8 cases with ≥2 markers, 4 with 2 markers, and 8 with 1 marker). From 10/44 (23%) cfDNA samples MRD+ by IG-NGS, 9 were also MRD+ by ED-NDC. From 44 pts with parallel analysis of PBC and cfDNA, 14 (32%) were MRD+ and 18 (41%) were MRD-, while 12 (27%) showed discordant results. Conclusion To the best of our knowledge, this is the first analysis comparing targeted capture and amplicon assays for application in PBC and cfDNA in MCL. The capture-based EC-NDC assay allows a comprehensive liquid biopsy-based genotyping of MCL. In addition, both IG-NGS and EC-NDC assays are suitable for MRD assessment in cfDNA with evidence of more sensitive MRD detection EC-NDC due to multiple MRD targets detected and traced by this assay. Clinical data are still needed to explore the potential prognostic impact of MRD assessment in plasma vs PBC. Updated MRD analysis at EoT and correlation with clinical data will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PB1800: DEEPDECON: A DEEP-LEARNING METHOD FOR DETECTING MINIMAL RESIDUAL DISEASE OF ACUTE MYELOID LEUKEMIA

Background: Acute myeloid leukemia (AML) is a heterogeneous disease that the myeloid lineages of hematopoietic progenitor cells (blasts) lose the ability of normal differentiation and build up in the bone marrow. The ability to identify minimal residual disease (MRD) below the morphology-based 5% blast threshold has significant impact in clinical outcome. Flow cytometry and real-time quantitative polymerase chain reaction [qPCR]) are the most common methods for MRD detection while next-generation sequencing (NGS) is emerging as a more sensitive method. Aims: Here, we report DeepDecon, a deep neural network model that utilizes NGS RNAseq data to detect MRD of AML. Methods: DeepDecon makes use of the non-linear transformation among the latent layers and learns to extract high-order representations of the input gene expression. Meanwhile, DeepDecon doesn’t need to select a subset of genes. The neural network will optimize and assign different weights to all genes to minimize the prediction error. A challenging issue with deep learning models is the need for large data sets of diverse examples for model training to obtain robust and good performance. With single-cell RNAseq data, we digitally create a large number of bulk RNAseq samples with known AML fraction for model training. Results: When applied to clinical data TARGET-AML from GDC Data Portal (https://portal.gdc. cancer.gov/), DeepDecon demonstrated better performance than other machine learning algorithms, including MuSic, NNLS, Bisque, RNA-Sieve, and Scaden, for detecting MRD of AML. Summary/Conclusion: Deepdecon can be applied to clinical NGS data for accurate and sensitive detection of MRD in AML treatment. It will significantly improve the clinical outcomes of current AML therapy.

Abstract 5163: A high sensitivity, tumor-informed liquid biopsy platform, designed to detect minimal residual disease at part per million resolution

Abstract Tumor-informed liquid biopsy approaches have proven promising for detecting minimal residual disease (MRD) and recurrence of cancer following surgical resection or other therapy. However, current liquid biopsy MRD assays typically detect ctDNA in a range above 30 to 300 parts per million (PPM), leaving a significant fraction of MRD cases undetected, particularly soon after surgery and in early stage cancers where ctDNA can be at very low levels. To address this, we have developed NeXT Personal™, a tumor-informed liquid biopsy assay that achieves sensitivity down to 1 PPM, therefore enabling earlier detection of MRD and recurrence. NeXT Personal leverages tumor/normal whole genome sequencing to design personalized MRD liquid biopsy panels for each patient. The panel is composed of &amp;gt;1,200 somatic tumor variants enabling higher sensitivity MRD detection in plasma through tracking of larger numbers of high quality and lower noise variants. This allows the platform to achieve high sensitivity across cancer types and stages, including early stage cancers and low mutational burden tumors, utilizing ~4 mL of plasma. Two independent methods were used to establish utility and performance: a proprietary cell-line media system, and well-characterized matched tumor-normal-plasma patient samples. Samples were serially diluted to &amp;lt;1 PPM, with replicates used to confirm performance. Digital droplet polymerase chain reaction (ddPCR) was used to orthogonally validate platform performance to the limit of detection (LOD) of ddPCR. Characterization of MRD LOD in three cell-line media systems, HCC1143, HCC38, and HCC1937, yielded accurate and reproducible detection of signal across a broad range of concentrations, to a lower limit of 1-2 PPM. We then used our platform to characterize MRD LOD in a set of serially diluted patient samples, demonstrating sensitivity down to as low as 1 PPM, with high specificity in normal control samples. Finally, we demonstrated the performance of NeXT Personal with matched tumor-normal-plasma patient samples (8 different cancer types, stages II-IV). In this series, NeXT Personal detected cancers down to 0.8 PPM with high specificity demonstrated across a set of healthy normal donor samples. We estimate that ~50% of the cases in this set of patients would not have been detected by other commercially available liquid biopsy MRD platforms. NeXT Personal achieved highly sensitive and specific MRD detection, reproducibly demonstrating a LOD down to 1 PPM in different cancer types and cell line dilutions, representing approximately 10 to 100 times higher sensitivity than other liquid biopsy MRD approaches. The high sensitivity of NeXT Personal potentially enables MRD detection across a broad variety of cancers and stages, including typically challenging early stage, low mutational burden, and low-shedding cancers. Citation Format: Sean Michael Boyle, Gabor Bartha, John Lyle, Jason Harris, Josette Northcott, Dan Norton, Rachel Marty Pyke, Fabio C. P. Navarro, Alexander Stram, Christian Haudenschild, Rose Santiago, Robin Li, Chris Nelson, Yelia Huo, Manju Chinnappa, Qi Zhang, Lloyd Hsu, John West, Richard O. Chen. A high sensitivity, tumor-informed liquid biopsy platform, designed to detect minimal residual disease at part per million resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5163.

Igh-V(D)J NGS-MRD Measurement in Pediatric B-Acute Lymphoblastic Leukemia

IntroductionB-acute lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. Early response to induction chemotherapy is one of the important prognostic factors in B-ALL. However, the analytic sensitivity for flow cytometry (FC) is only 10 -4. The feasibility of using next-generation sequencing (NGS) of immunoglobulin for the determination of minimal residual disease (MRD) in B-ALL has been demonstrated. This study aimed to investigate the performance of NGS techniques measuring immunoglobulin heavy chain (IgH)-variable, diversity, and joining (V[D]J) clonal rearrangements compared with FC in detecting MRD for children with B-ALL and to predict the clinical outcome of B-ALL patients.MethodsNewly diagnosed younger than 18 years old B-ALL patients who received the treatment strategy of South China children's leukemia Group (SCCLG)-ALL 2016 were recruited. DNA extracted from bone marrow cells at all available time points for each patient was submitted to Simcere diagnostics for sequencing using Illumina NovaSeq platform. We performed IgH V(D)J NGS and FCM on the bone marrow serially obtained at diagnosis (D0), 15 days at induction therapy (D15), 33 days at induction therapy (D33) and then at the end of induction therapy (EOI). We defined MRD positive (MRD +) by IgH V(D)J NGS and FCM as more than 1 blast cell among 10 4 and 10 6 bone marrow cells, respectively. The sensitivity of MRD detection by IgH V(D)J NGS and FCM, and the association of MRD status with clinicopathological characteristics were investigated. Statistical analysis was performed through SPSS Statistics 22. Enumeration data and correlation between MRD data and clinicopathological characteristics were compared by Chi-square test or Fisher's exact test. This trial was registered at www.clinicaltrials.gov as # NCT04977895.ResultsAs of July 27, 2021, 22 patients (median age, 4.5 years; range, 3.0-7.3) were enrolled in the study. Three patients (13.6%) had a t (9;22) translocation consistent with Philadelphia chromosome positive disease. According to risk stratifications, 8 (36.4%), 8 (36.4%), and 2 (9.1%) patients were classified as low risk (LR), intermediate risk (IR), and high risk (HR) groups, respectively. The remaining 4 patients are still under treatment and have not been classified. We identified leukemic IgH clones in 100% of the diagnostic samples and 68.2% (15/22) of the patients were polyclonal. In 11 patients whose samples of all the four timepoints (D0, D15, D33, EOI) have been tested in parallel by FCM and IgH V(D)J NGS, the frequencies of patients with MRD + were 30.4% vs. 90.9% at D15 (P＜0.05) by FCM and IgH V(D)J NGS. IgH V(D)J NGS MRD monitoring could identify MRD + patients with frequency of 45.5% and 18.2% among patients achieved MRD negativity by FCM at D33 (P＜0.05) and EOI (P = 0.46). With an MRD detection limit of 10 -6, 90.9% (10/11), 36.4% (4/11) and 18.2% (2/11) patients were MRD negative by FCM but positive by the NGS test at D15, D33 and EOI, respectively. This suggested that the sensitivity of IgH V(D)J NGS was significantly higher than that of FCM. Correlation of the measured MRD between the two methods in the entire cohort (r = 0.7934, P < 0.0001) as well as in the concordant cases (r = 0.5558, P = 0.0032) was very high. There was a high discordant rate with NGS identifying more patients MRD + at this threshold. Furthermore, NGS MRD was positive but the FCM MRD was negative in 13 samples (P < 0.0001). In addition, positive MRD status of D33 by NGS was significantly associated with the age of B-ALL patients, patients under 6 years more frequently harbored detectable MRD compared with those ≥ 6 years old (87.5% vs. 11.1%, P < 0.01). There was no patient relapsed after a medium follow-up of 10.5 months.ConclusionsWe demonstrated the higher sensitivity of IgH-V(D)J NGS in MRD detection of B-ALL, which implies that NGS MRD monitoring could be helpful for more accurate risk stratifications and more precise treatment according to risk stratifications. Further study with a larger sample size and a longer follow-up period is need. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Calibration-free NGS quantitation of mutations below 0.01% VAF

Quantitation of rare somatic mutations is essential for basic research and translational clinical applications including minimal residual disease (MRD) detection. Though unique molecular identifier (UMI) has suppressed errors for rare mutation detection, the sequencing depth requirement is high. Here, we present Quantitative Blocker Displacement Amplification (QBDA) which integrates sequence-selective variant enrichment into UMI quantitation for accurate quantitation of mutations below 0.01% VAF at only 23,000X depth. Using a panel of 20 genes recurrently altered in acute myeloid leukemia, we demonstrate quantitation of various mutations including single base substitutions and indels down to 0.001% VAF at a single locus with less than 4 million sequencing reads, allowing sensitive MRD detection in patients during complete remission. In a pan-cancer panel and a melanoma hotspot panel, we detect mutations down to 0.1% VAF using only 1 million reads. QBDA provides a convenient and versatile method for sensitive mutation quantitation using low-depth sequencing.

Identification of Chemo-Resistant Residual Cell Population in Pediatric AML of Complete Remission By Single Cell RNA Sequencing

Although majority of pediatric acute myeloid leukemia (pAML) undergo complete remission (CR) after chemotherapy, more than 30% of which eventually relapse, leading to a dismal outcome. Chemo-resistant cells at CR, measured as minimal residual disease (MRD), are thought to be origin for relapse. Although more sensitive detection of MRD has been achieved through genetic mutations, the pathological and clinical significance for most of the detected mutations remain unknown, largely due to a lack of understanding for the biological context where the mutation resides. The idea of retention of leukemia stem cells (LSCs) as a mechanism of chemo-resistance has not been demonstrated post-therapy in patients. Here we applied single cell RNA sequencing (scRNA-seq) on 14 pAML who met the criteria for clinical CR (n=11) and partial remission (PR, n=3) to determine the cellular heterogeneity and cancerous feature of the residual cells that can survive chemotherapy at remission. These patients carried common genetic mutations such as AML1-ETO, CBFB-MYH11 and FLT3-ITD, which represents genetically diverse WHO subtypes. To maximize the power to detect tumor cells that can survive chemotherapy, we used 54 unsorted total bone marrow (BM) and/or peripheral blood (PB) nucleated cells collected at diagnosis and Day 26 (D26) of the first cycle of chemotherapy for 10X genomics' scRNA-seq. Cells from each pAML were compiled into one UMAP together with normal reference using unsupervised clustering to distinguish tumor clusters from normal. Clusters were defined to be tumor if patient's diagnosis contributed at least 80% of the cells, and were confirmed by the presence of somatic mutations and/or known AML mRNA expression signature associated with chromosome translocations. By projecting onto the closest normal hematopoietic cells based on transcription features, the tumor cells were classified as one of the 12 cell types (HSC/MPP, LMPP, GMP, MEP, E/B/M, CLP, classical/nonclassical Monocyte, cDC, pDC, and pre/inmature Neutrophil like cells). Consistently with findings in adult AML, cell populations were heterogeneous at diagnosis with 5-9 distinct clusters. Interestingly, majority (9/11) of the patients had 1-6 tumor clusters detected (mean 52 cells per cluster) at D26 post-chemotherapy. These D26 residual tumor cells possessed mutations originally detected by genomic sequencing and/or known AML signatures, and consistently clustered with tumor cells from diagnosis. These residual tumor cells accounted for average 1.4% of total BM cells at D26, while the morphological examination and flow cytometry analysis of MRD showed average 4.0% and 0.71% of tumor cells. To further evaluate the chemo-resistant potential of identified residual tumor cells, we focused on three distinct features known to be associated with chemo-resistance in mouse models, including LSC activity, active oxidative phosphorylation (OXPHOS) or leukemic-regenerating cell (LRC) state. Among the total 18 residual tumor clusters detected at D26, 33.3% (6/18) exhibited expression signatures associated with at least one chemo-resistant features. The remaining clusters consisted more differentiated progenitor/monocyte-like cells. Specifically, three patients (1 PR and 2 CR) had the HSC/MPP or LMPP-like clusters possessing strong LSC and OXPHOS-associated signatures. The remaining one patient had cDC-like cluster expressing reported LRC signature. Importantly, all these four patients had either unfavorable cytogenetics or persistence of driver mutations detected by PCR. Taken together, these data showed that pediatric AMLs represented heterogeneous populations at both diagnosis and remission. Among the residual tumor clusters that survived chemotherapy, a small fraction (6/18) were HSC/MPP-like, LMPP-like and cDC-like cells with known chemo-resistant expression features. These findings provide the first in vivo characterization of cellular heterogeneity in chemo-treated pAML with complete remission. Further studies are needed to determine the molecular characteristics of these residual cells that may convey chemo-resistance and to determine whether the presence of these cells are associated with increased risk of relapse. Disclosures No relevant conflicts of interest to declare.

Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph+ B cells but not bone marrow stem/progenitors

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Highly Sensitive Minimal Residual Disease Detection by Biomimetic Multivalent Aptamer Nanoclimber Functionalized Microfluidic Chip.

Minimal residual disease (MRD) offers a highly independent prognostic factor for leukemia patients. However, challenges confronting conventional MRD assays are high invasiveness, as well as limited detection sensitivity and clinical applicability. Inspired by the self-adaptive skeleton and multiple suckers or tendrils of climbing plants, a biomimetic Multivalent Aptamer Nanoclimber (MANC)-functionalized microfluidic chip (MANC-Chip) is reported for minimally invasive, highly sensitive and clinically applicable MRD detection in the peripheral blood of T-cell acute lymphoblastic leukemia patients. The MANCs are synthesized by a simple co-polymerization reaction. Due to their flexible structure and cooperative multivalent effect, MANCs dramatically enhance the binding affinity of aptamers targeting leukemia cells. A deterministic lateral displacement-patterned microfluidic chip is designed to further increase the collision probability between MANCs and leukemia cells. Benefiting from the synergistic effect of multivalent binding and enhanced collision, a high capture efficiency of 92.2% for leukemia cells is achieved. Moreover, the captured leukemia cells can be released with high efficiency of 88.9% and high viability of 93.8% via nuclease treatment prior to downstream analysis. Overall, the excellent features of MANC-Chip make it very useful for precise detection of MRD and better understanding of leukemia.

Cluster of differentiation 97 as a biomarker for the detection of minimal residual disease in common acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disorder. Clinical parameters, immunophenotype, cytogenetic factors, and minimal residual disease (MRD) are among currently used factors in risk stratification and therapy determination of ALL patients. MRD is gaining importance nowadays for therapy efficacy, follow-up, and relapse risk estimation. Recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. Cluster of differentiation (CD) 97 is one of the markers that show overexpression in pediatric ALL. In this study, we aimed to assess the value of CD97 as a biomarker for MRD detection in pediatric ALL. Patients and methods This cohort study was conducted on 30 newly diagnosed patients with B-ALL. There were 16 male and 14 female patients with a mean age of 8.38±4.21. Twenty patients were in the low-risk group and 10 patients were in the high-risk group and were treated according to modified CCG 1991. A panel of monoclonal antibodies was used, with special emphasis on CD10, CD19, CD34, and CD97 at diagnosis and at day 14 postinduction of chemotherapy for MRD detection. Results The percentage of CD19/CD97, CD34/CD97, and CD10/CD97 at day 0 was 57.15±21.74, 57.73±21.20, and 57.87±20.77, whereas at day 14 it was 6.09±2.50, 10.67±8.89, and 5.97±2.44, respectively (P<0.001). CD97 was expressed in 81.5% of patients at diagnosis and was not detected at day 14 (P<0.001). One patient had blast counts more than 5% by light microscopy, whereas 29 patients had MRD more than 0.1 by flow cytometry at day 14 (P<0.001). Conclusion CD97 can be used for MRD tracing in pediatric ALL.

Time Points, Dynamics and Clinical Impact of Minimal Residual Disease (MRD) Monitoring in AML with NPM1 mutation: A Study on 428 Patients

Abstract Introduction: Investigation of minimal residual disease (MRD) using NPM1 as a target has been proven to be of importance in AML. Guidelines for best schedules and implication on clinical use need to be defined. Aims: To better define the clinical impact and to suggest strategies for MRD monitoring in AML with NPM1 mutation. Patients and Methods: Between 2005 and 2015 we investigated 428 AML patients (pts) with NPM1 mutation at diagnosis and at a minimum of 2 follow-up time points. All pts had to achieve at least once a complete molecular remission (CMR) to be considered for this study. Sensitivity for MRD detection was at least 1:10,000. The median age of the cohort was 57 years (range: 18-85 yrs) and comprised of 198 males and 230 females. 3,039 samples (median number of samples per pts: 7, range: 2-35) were studied during course of disease. Molecular techniques applied included gene scan, sequencing and quantitative real-time PCR at diagnosis and quantitative real-time PCR during follow-up. Median time between 2 investigations was 2.8 months (mo; range: 0.3-71.0 mo). All pts were treated with standard protocols according to genotype and age. Allogeneic bone marrow or stem cell transplantation was performed in 136 pts (31.8%). Results: NPM1 type A mutation was the most frequent mutation type (317/428, 74.1%), followed by type B and D (36/428, 8.4% and 23/428, 5.4%), respectively. 25 other NPM1 types occurred at frequencies between 0.2 and 3.7%, in total demonstrating the expected distribution of NPM1 mutation types in an adult AML cohort. Subgroups of these pts were analyzed for FLT3-ITD (n=421) and mutations in DNMT3A (n=236). 122/421 (29%) pts showed a FLT3-ITD. In 96/236 (41%) DNMT3A was mutated. Further in 33/235 (14%) both genes were mutated. 103/235 (44%) screened for all three genes had a sole NPM1 mutation. All sole NPM1 mutated study pts achieved the CMR after a median of 4.1 mo (range: 1.0-8.6 mo). The presence of an additional DNMT3A mutation (CMR after a median of 4.4 mo, range 1.0-8.7) or a FLT3-ITD (CMR after a median of 2.7 mo, range 1.0-8.7) or both mutations (CMR after a median of 4.1 mo, range 1.1-7.9 mo) had no influence on time to achieve CMR. After achievement of CMR an increase of NPM1 ratio was detected in 185/428 (43%) pts. The median time to loss of CMR was 5.1 mo (range: 0.4-88 mo). In more detail, 42/185 of these patients also had FLT3-ITD, 53/109 had DNMT3A mutations and 13/109 had mutations in both genes. Patients with a DNMT3A mutation showed more often loss of CMR (40/60, 67%), while FLT3-ITD and FLT3-ITD/DNMT3A mutated patients showed no significant influence on loss of CMR ratio (46% and 48%, respectively) maybe due to number of cases. In 152/185 molecular relapses further follow up samples after loss of CMR were available. The median time between detected loss of CMR and the next follow-up sample was 2.0 mo. Due to treatment intervention 46/152 patients achieved a second CMR and 27/152 a decrease in NPM1 ratio. However, in 79/152 a further increase leading to clinical relapse was observed. The increase after loss of CMR was in median 13-fold between first and second sample after CMR was lost. Importantly, keeping periods between two MRD samplings at an interval of 3 mo allowed the detection of nearly all cases of first relapse at the molecular level. Addressing the sensitivity levels of the assays applied to bone marrow (BM) versus peripheral blood (pB) samples showed a 1.6 fold higher sensitivity for BM samples (median copies of reference gene, 14,628 vs 9,363). Due to the comparable sensitivities pB can be investigated until a first increase on the molecular level is detectable, followed by BM sampling for confirmation 4 weeks later. Conclusions: 1) NPM1 has proven to be a good marker for MRD monitoring in AML. 2) Time to CMR is short with a median of 4.1 mo. 3) An increase of NPM1 in all cases is followed by relapse after a median of 5.1 mo, if no treatment intervention has been initiated before. 4) Time intervals for MRD should be no longer than 3 mo, pB can be used. 5) Transplantation should already be planned after first molecular increase is detected. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Worseg:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

A Comparison of Minimal Residual Disease Detection Among ASO-PCR, Dd-PCR and Deep-Sequencing in Patients with Multiple Myeloma Who Underwent Autologous Stem Cell Transplantation

▪Background: Most patients with multiple myeloma (MM) are considered to be incurable, and relapse, due to minimal residual disease (MRD), is the main cause of death among these patients. Even though allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR) of immunoglobulin heavy chain gene rearrangement has been used to assess the MRD in MM due to its excellent sensitivity and specificity, ASO-qPCR has a major limitation in its relative quantification method because it needs a reference standard curve, which is usually made with dilutions of diagnostic myeloma DNA or from plasmids containing the target IgH rearrangement gene. Recently, droplet digital PCR (ddPCR) was developed to perform reliable absolute quantification of target genes. Based on the principle of single target gene detection, the sensitivity can be increased when the larger amount of DNA is analyzed. Here we assessed the prognostic value of MRD assessment in autografts from MM patients in the autologous stem cell transplantation (ASCT) setting using ASO-qPCR, ddPCR and next-generation sequencing (NGS) approaches.Methods: Twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT without any post-ASCT therapy were retrospectively analyzed. Median age 57 (range 39-67); males 11, females 12; ISS 1 (n=7), 2 (n=12), 3 (n=3), not assessed (n=1). 11 patients were analyzed by G-banding and FISH (t(4;14), del17p, t(14;16)) and 4 patients showed high-risk chromosomal abnormalities (t(4;14) (n=2), t(14;16) (n=1), -13 by G-banding (n=1)). All patients had achieved a very good partial response (VGPR) or better after ASCT. Analyzed samples included: (1) BM slides from 20 MM patients at diagnosis, (2) fresh/frozen BM cells from 3 MM patients at diagnosis, and (3) obtained autografts. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). ddPCR was performed by the QX200 Droplet Digital PCR system (Bio-RAD Inc.) with a total 6000 ng of genomic DNA combined with the same ASO-primers and TaqMan-probes used in the ASO-qPCR. Droplets were generated by the QX200 droplet generator. End-point PCR (40 cycles) was performed on a C1000 Touch Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX200 droplet reader and analyzed by QuantaSoft 1.7.4 (Bio-Rad Inc). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014).Results: Nineteen patients could be analyzed by ASO-qPCR and ddPCR, while all 23 patients could be analyzed by NGS. We compared MRD results in autografts between ddPCR and NGS. We observed a high correlation between ddPCR and NGS results of MRD (r=0.82, P<0.0001). Although 19 samples were MRD negative by ASO-qPCR (MRDASO (-)), 12 samples were MRD positive by NGS (0.6-46 x 10-6, median 3.5 x 10-6) (MRDNGS (+)) and 7 samples were MRD positive by ddPCR (1.2-102 x 10-6, median 7.5 x 10-6) (MRDddPCR (+)), demonstrating the higher sensitivity of NGS (10-6 or higher) and ddPCR compared to ASO-qPCR (10-4 -10-5). Two high-risk chromosomal abnormality cases (t(4;14) (n=1), -13 by G-banding (n=1)) could achieve MRDNGS (-) in autograft. We evaluated the association of clinical outcome with MRD in autografts using ddPCR and NGS. To investigate the value of sensitive MRD detection by ddPCR and NGS, we compared PFS in 7 MRDddPCR (+) cases (Group 1) with 12 MRDddPCR (-) cases (Group 2) and 12 MRDNGS (+) cases (Group 3) with 11 MRDNGS (-) (Group 4). Group 2 and Group 4 showed significantly better PFS than Group 1 (P = 0.028) and Group 3 (P=0.033), respectively (Figure 1A and 1B). We also compared 5 MRDddPCR (-) & MRDNGS (+) cases (Group 5) with 11 MRDNGS (-) cases (Group 4). Group 4 tended to show better PFS than Group 5 (P=0.063, Figure 1C).Conclusions: In this study, we showed the prognostic value of ddPCR and NGS-based MRD assessment in autografts of patients with MM. Although the ddPCR had improved sensitivity in detecting MRD in autografts and demonstrated higher prognostic value compared with ASO-qPCR, the NGS platform showed the highest sensitivity and prognostic value among these methods. [Display omitted] DisclosuresZheng:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

Prognostic significance of sequencing-based MRD detection in multiple myeloma

Stem cell transplantation in conjunction with therapeutic agents such as proteasome inhibitors and immunomodulatory drugs can dramatically improve response rates and the prognoses of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. We utilized a deep sequencing method, which employs consensus primers and next-generation sequencing (NGS), to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone. This technique has been shown to have 1-2 log greater sensitivity than both allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) (sensitivity 10(-5)) and multiparameter flow cytometry (MFC) (sensitivity at least 10(-4)). To investigate the value of sensitive detection of MRD in autograft by NGS, we compared progression-free survival (PFS) in 11 MRDNGS(-) cases (Group 1) with that in 12 MRDNGS(+) cases in which MRD was not detected by ASO-PCR (MRDASO(-)) (Group 2). Neither group received any post-ASCT therapy. Group 1 showed a better PFS than Group 2 (P=0.027). MRD-negativity in autografts, as revealed by NGS, is more closely associated with durable remission of MM than that revealed by ASO-PCR.

A Comparison Between Next-Generation Sequencing and ASO-qPCR For Minimal Residual Disease Detection In Multiple Myeloma: The Clinical Value In ASCT Setting

BackgroundAlthough molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We utilized a sequencing method, termed the LymphoSIGHT™ platform, which employs consensus primers and high-throughput sequencing to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone. The sequencing method is quantitative at frequencies above 10-5 and the lower limit of detection is below 10-6. Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. MethodsWe compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting. Because myeloma cells exist patchily in bone marrow (BM), myeloma cells in PBSC autografts may reflect the whole amount of tumor in vivo. Thirty-six Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or complete response (CR) after ASCT. BM slides from 28 MM patients and fresh BM cells from 8 MM patients at diagnosis as well as autografts were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using next-generation sequencing (NGS). Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in follow-up samples (Faham et al, Blood 2012). ResultsMRD in autografts could be assessed 36 of 36 (100%) by NGS and 30 of 36 (83%) by ASO-qPCR. MRD in autografts was detected in 27 of 36 (75%) by NGS and 11 of 30 (37%) by ASO-qPCR (Figure 1A). Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, ASO-qPCR could not detect myeloma cells at MRD levels of 10-5 or lower. Two cases where MRD was not detected by NGS (MRDNGS(-)) and 14 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide while 7 MRDNGS(-) cases and 13 MRDNGS(+) cases were followed without post-ASCT therapy. The best post-ASCT responses were as follows: 6 (67%) mCR, 1 (11%) sCR and 2 (22%) VGPR in 9 MRDNGS(-) cases; 2 (14%) mCR, 2 (14%) sCR, 2 (14%) CR, 8 (58%) VGPR in 14 MRDNGS(+) cases with post-ASCT therapy; 2 (15%) sCR, 10 (78%) VGPR and 1 (7%) PR in 13 MRDNGS(+) cases without post-ASCT therapy. The MRDNGS(-) cases tended to show a better PFS than the MRDNGS(+) cases with post-ASCT therapy (P = 0.400) and showed a significantly better PFS than those without post-ASCT therapy (P = 0.032) (Figure 1B) although overall survival rates were comparable among the three groups. To investigate the value of sensitive detection by NGS, we compared PFS in 7 MRDNGS(-) cases (Group 1) with the 6 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 tended to show a better PFS than Group 2 (P = 0.091) (Figure 1C). This underscores the value of sensitive detection of MRD in MM. [Display omitted] ConclusionsA high correlation between NGS and PCR MRD results was observed, and MRD-negativity in PBSC autografts revealed by NGS may be more closely associated with durable remission of MM than that revealed by ASO-qPCR. Disclosures:Zheng:Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment.

Significance Of Minimal Residual Disease Detected By Multi-Color Flow Cytometry In Childhood Acute Myeloid Leukemia With The Intermideate-Risk Cytogenetics and Negative FMS-Like Tyrosine Kinase 3 Internal Tandem Duplication: A Report Of The Tokyo Children's Cancer Study Group

Abstract Background Response to induction chemotherapy is one of the most important predictors of outcome in acute myeloid leukemia (AML) as well as cytogenetics and molecular genetics. Measurement of minimal residual disease (MRD) by flow cytometry is an informative method for assessment of initial treatment response, but the heterogeneity of leukemia-associated antigens and antigen shifts during treatment limit its sensitivity and specificity. We prospectively evaluated the prognostic prevalence of MRD monitoring using multi-color flow cytometry in children with AML treated on the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. Patients and methods From January 2007 to October 2010, 34 children with newly diagnosed de novo AML were enrolled on the AML-MRD study conducted by the Tokyo Children's Cancer Study Group. The median age at the diagnosis was 8 years (1 month- 15 years), and 17 patients were boys and 17 were girls. They were all treated with the JPLSG AML-05 trial. In AML-05, enrolled patients received two induction courses and those achieving a complete remission (CR) were treated according to risk classification at which all the core binding factor-AML cases and good initial response based on morphology after induction 1 were assigned to low risk (LR) group, cases presented with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), unfavorable cytogenetics including monosomy7, 5q-,t(16;21), and with poor initial response were assigned to high risk (HR) group, the others were assigned to intermediate risk (IR) group. Allogeneic hematopoietic stem cell transplantation (HSCT) was indicated for the HR patients after the third or later treatment courses. Among the 34 patients enrolled on the AML-MRD study, 8 presented with LR, 22 with IR, and 4 with HR regarding the cytogenetics and FLT3-ITD status. MRD of 63 bone marrow samples were analyzed after induction 1 (BMA-2) and induction 2 (BMA-3) by four-color flow cytometry using 9 AML-associated antigens. A threshold level for MRD-positivity was set at the point of 0.1%. Results Sixty-two (98.4%) of 63 bone marrow samples were evaluable for MRD. Thirteen (39.4%) of 33 samples and 8 (27.6%) of 29 showed MRD-positivity at BMA-2 and BMA-3 respectively. Among the patients with MRD-positivity, 12 at BMA-2 and 7 at BMA-3 were diagnosed as achieving CR by morphology. MRD was associated neither specific FAB subtype nor white blood cell count at diagnosis, but all 3 patients with FLT3-ITD showed the MRD-positivity at BMA-2. Although 3-year probability of event free survival (3-yr EFS) at BMA-2 or BMA-3 was similar between patients with and without MRD; 53.8% (n= 13) vs 70.0% (n= 20) (p=0.30) and 50.0% (n= 8)vs 62.0%(n= 21) (p=0.36), respectively, 3-yr EFS of those with MRD at BMA-2 or BMA-3 was significantly worse compared to those without MRD; 33.3% (n= 9) vs 83.3% (n= 12) (p=0.02) and 20.0%(n= 5) vs 76.9% (n= 13) (p=0.04), respectively, in the group of the IR cytogenetics and negative FLT3-ITD. Multivariate analysis indicated that the MRD detected by multi-color flow cytometry was solely associated with worse outcome in this group. Conclusion Highly sensitive detection of MRD by multi-color flow cytometry was possible in children with AML. The present study suggests that MRD monitoring may have a prognostic relevance in childhood AML with the IR cytogenetics and negative FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.