Minimal Residual Disease Quantification Research Articles

Abstract 5024: Analytical performance of a genome-wide methylome enrichment platform to detect minimal residual disease from plasma-derived cell-free DNA

Abstract Introduction: Plasma-derived cell-free DNA (cfDNA) can be used to identify cancer signals, including minimal residual disease (MRD), in patients who have undergone curative cancer treatments. The cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) methodology is combined with custom algorithms that leverage differentially methylated regions (DMRs) found in cfDNA to distinguish between cancer and non-cancer signals. This novel non-degradative, tissue-agnostic approach was developed to bypass the limitations of bisulfite-sequencing and tissue-informed methods used in other liquid biopsy technologies. Here we present preliminary analytical performance metrics of an algorithm in development for detecting MRD. Methods: In the genome-wide methylome enrichment platform, plasma-derived cfDNA is subjected to standard library preparation, combined with DNA filler, denatured, and immunoprecipitated using an anti-5-mC antibody. Captured DNA is amplified and sequenced. For this study, a candidate algorithm comprised of differentially methylated regions (DMRs) and 12 cancer types was used to quantify cancer-specific methylation. Control samples from 12 non-cancer donors were used to establish a selected 95% true negative rate threshold. This threshold was used to determine assay sensitivity, which was evaluated using contrived cancer samples intended to mimic low-level circulating tumor DNA (ctDNA) representative of MRD. Samples were contrived by titrating fragmented DNA from three immortalized tumor-derived cell-lines into plasma-derived cfDNA in a titration series targeting <1% ctDNA levels. Cell-lines were of non-small cell lung cancer and head and neck squamous cell carcinoma origin; titration series were replicated for a total of 65 tests. Results: All non-cancer and contrived cancer samples met in-process quality control metrics. This included ≥98.5% methylation specificity and ≥80 million unique molecules. Limit of detection calculations at 95% sensitivity (LoD95) were <0.1%. Conclusions: Tissue-agnostic approaches for detecting cancer signals from plasma have significant benefit, especially in settings where tissue is not accessible for evaluation. However, these tests must have highly sensitive methods of cancer signal detection for clinical applications. Our preliminary analytical data demonstrates the use of a blood-based, tissue- agnostic genome-wide methylome enrichment platform utilizing non-degradative methodology combined with specific algorithms and DMRs for MRD quantification and prognostic prediction, using treatment-naïve plasma samples. Future studies in post-treatment and longitudinal samples are ongoing to evaluate the utility of this genome-wide methylome enrichment platform for cancer management. Citation Format: Shu Yi Shen, Iulia Cirlan, Felicia Vincelli, Ben Brown, Jun Min, Justin Burgener, Junjun Zhang, Yulia Newton, Margaret Gruca, Abel Licon, Jing Zhang, Anne-Renee Hartman, Alan Williams, Hestia Mellert, Daniel D. De Carvalho. Analytical performance of a genome-wide methylome enrichment platform to detect minimal residual disease from plasma-derived cell-free DNA [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 5024.

A Personalized Whole Genome-Informed Assay Targeting Single Mutant in Circulating Tumor DNA Can Identify MRD and Predict Relapse in DLBCL

Introduction: Minimal residual disease (MRD) detected by circulating tumor DNA (ctDNA) has emerged as a promising biomarker in diffuse large B-cell lymphoma (DLBCL). MAESTRO (minor-allele-enriched sequencing through recognition oligonucleotides) was recently developed to aid the detection of low-frequency mutations by enriching for mutant alleles using probes preferentially capturing single-nucleotide variants. We have extended this application - termed MAESTRO-Pool - to analyze personalized MRD variant detection within a cohort-level single assay. We demonstrate high sensitivity to detect MRD using MAESTRO-Pool and the detection of emergent mutations using targeted sequencing of the same samples. Methods: Fifty-nine plasma specimens from 9 patients with relapsed/refractory (R/R) DLBCL treated on a phase II trial (NCT02362997) of post-autologous stem cell transplant (ASCT) pembrolizumab maintenance were tested. Cases were selected based on the availability of genomic DNA from tumor tissue, patient-matched germline DNA, and serial post-ASCT plasma samples (≥ 3 time points). MAESTRO probes were designed to target patient tumor-specific somatic variants using results of baseline tumor-normal whole genome sequencing. Probes were then pooled into an integrated, cohort-level assay (MAESTRO-Pool). Serial samples were compared with an orthogonal, whole-genome, tumor-informed MRD test which does not use mutation enrichment (MRD Tracker; Parsons, HA et al. Clin Cancer Res26, 2556-2564 (2020)) for sensitivity and specificity of variant detection. In addition, sensitivity to detect MRD and predict relapse was compared to that observed with immunoglobulin locus high-throughput sequencing (IgHTS). Additional baits were designed to capture single nucleotide variants (SNVs) previously reported in R/R DLBCL (63 loci in 12 genes), enabling detection of treatment-emergent mutations not identified in baseline tumor specimens. Results: Tumor-normal WGS revealed a median of 433 somatic SNVs per tumor (range 81-1653). The pooled assay comprised 6044 SNV-specific probes. MRD identification was similar for MAESTRO-Pool and MRD Tracker. Among 59 samples, a discrepant MRD call was observed for a single sample where ctDNA was detected at 4 ppm using MRD Tracker, but not detected using MAESTRO-Pool (limit of detection [LoD] 16 ppm). Estimated tumor fractions using MRD Tracker and MAESTRO-Pool were concordant. Even with reduced sequencing requirements of MAESTRO-Pool, we observed a similar median limit of detection (LoD) for MAESTRO-Pool (median 30 ppm, range 1-18,243) and MRD Tracker (median 40 ppm, range 1-4,727 ppm). MAESTRO-Pool identified ctDNA prior to recurrence for all 5 patients who relapsed, including at the earliest available post-ASCT timepoint for 4 of 5 relapsing patients. The time from ctDNA detection to clinical relapse (lead time) was the same or longer for each patient using MAESTRO-Pool (median 178 days, range 69-518) compared to IgHTS (median 44 days, range not detected to 518 days) (p=0.37) (Fig.1a). MAESTRO-POOL was associated with improved sensitivity compared to IgHTS (MAESTRO-Pool sensitivity of 90.5% for samples with matched IgHTS results versus IgHTS sensitivity of 61.9%, p=0.006). Superior sensitivity was primarily driven by MAESTRO-Pool's improved detection of low-frequency (<1000 ppm) mutant alleles. In addition to tracking molecular tumor burden, we identified several de novo mutations in relapsing patients using targeted sequencing of the same samples. Notably, plasma from a patient who progressed at 18.5 months post-ASCT (DL-015) manifested an emergent CREBBP R1446H mutation not detected in the baseline tumor whose allele frequency steadily increased from 0.048% one week after ASCT to 30.533% at relapse (Fig 1b). Conclusion: In this pilot study, MAESTRO-Pool enabled ultrasensitive detection and quantification of MRD with superior sensitivity compared to IgHTS. Complementary targeted sequencing also characterized genetic evolution, including detection of treatment-emergent mutations. Our results support the incorporation of ctDNA testing using MAESTRO-Pool in future prospective trials in DLBCL.

CD33 CAR T-Cells (CD33CART) for Children and Young Adults with Relapsed/Refractory AML: Dose-Escalation Results from a Phase I/II Multicenter Trial

Introduction: Current therapies for patients with acute myeloid leukemia (AML) push the limits of chemotherapy intensity but cure only 30% of adults and 70% of children. Recently developed antibody-based therapies and molecularly-targeted agents only modestly improve outcomes for select patient subsets. Thus, new approaches are needed. Given the success of chimeric antigen receptor (CAR) T-cell therapies for acute lymphoblastic leukemia (ALL), we developed and optimized a novel CD33 CAR T-cell (CD33CART) construct for clinical testing in a multicenter Phase I/II clinical trial in children, adolescents, and young adults (AYA) with relapsed/refractory (r/r) AML. ( Qin H, et al. JITC 2021) We report interim results following completion of the dose-escalation phase. Methods: This multicenter phase I/II clinical trial (NCT03971799) was conducted as a 3+3 dose escalation study through the Pediatric Transplantation and Cell Therapy Consortium with National Marrow Donor Program sponsorship and managed by CIBMTR CRO. Autologous CD33CART product were centrally manufactured on a ClinicMACS Prodigy® by the Biopharmaceutical Development Program at the Frederick National Laboratory for Cancer Research (NCI). Eligibility criteria were r/r AML in subjects < 35 years old with adequate organ/performance status and an identified allogeneic stem cell transplant (SCT) donor. Bone marrow assessment was used for standard morphologic evaluation and central flow cytometric minimal residual disease (MRD) quantification. All subjects received pre-CD33CART lymphodepleting chemotherapy (LD) with fludarabine (75-120 mg/m 2) and cyclophosphamide (900-1000 mg/m 2). CD33CART doses levels (DLs) were: DL1: 3 x 10 5 CD33 CAR+ T-cells/kg; DL2: 1 x 10 6/kg; DL3: 3 x 10 6/kg and DL4: 1 x 10 7/kg. Adverse event grading used CTCAE v5 with incorporation of ASTCT consensus definitions for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). Dose-limiting toxicities (DLTs) included > grade 3 CRS, > grade 3 ICANS, and persistent > grade 3 non-hematologic toxicity that did not resolve < grade 2 in 72 hours. CD33CART persistence was assessed via flow cytometric evaluation of the blood and bone marrow. Data cut-off was June 1, 2023. Results: A total of 24 subjects (median age of 16 years, range 1-34 years), were enrolled, 12 (50%) of whom underwent a prior SCT. CD33CART products were successfully manufactured for 23 subjects and infused into 19. In 4 non-infused subjects with manufactured products, 1 died from progressive disease (PD) prior to LD, 2 transitioned to palliative care and 1 withdrew consent to seek alternative therapy. Manufacturing was not performed in one subject due to death from PD after enrollment. One morbidly obese subject (BMI=49.3) enrolled at DL4 but received treatment at DL3 due to weight-based manufacturing limitations. The median time from enrollment to infusion was 47 days (range, 24-242). (Table) Three subjects each were infused at DL1 and DL2 without DLTs. At DL3, 1 subject experienced DLT (grade 4 CRS) prompting expansion at this dose level, and no subsequent DLTs were observed. At DL4, 1 subject experienced a prolonged grade 3 CRS (> 72 hours) and grade 3 ICANS, both constituting DLT and necessitating expansion of the cohort without additional DLTs seen. Across all dose-levels, CRS was seen in 13 (68%) patients and was > grade 3 in 4 (21%) with onset typically within the first 24 hours post-infusion. Complete remission (CR) was only seen at DL4 and achieved in 2 subjects, both achieving an MRD negative CR alongside myeloid aplasia. One SCT naïve subject developed candidemia during aplasia but was able to proceed to an allogenic SCT, achieved engraftment, and was in remission at Day +100 post SCT. The second patient (post-two prior SCTs) declined third SCT, had spontaneous count recovery and remained in remission until day +119 (MRD+ relapse). Transient CD33CART expansion was detected in 9 (47.4%) subjects overall and in all 6 (100%) subjects at DL4. Conclusions: CD33CART manufacturing is feasible in children and AYAs with r/r AML with acceptable toxicity experienced in treated subjects. CD33CART expansion was best in subjects treated at DL4 (1 x 10 7/kg) with MRD negative CRs and transient myeloid aplasia occurring in 2 of 5 (40%) subjects evaluable for response (Table). Based on early clinical efficacy at DL4, enrollment continues in the phase 2 portion.

Minimal Residual Disease (MRD) Status Predicts Outcomes in Patients with Follicular Lymphoma (FL) Treated with Chemo-Immunotherapy on SWOG S0016

Background. FL is associated with a heterogeneous clinical course, and currently available prognostic indicators are unable to reliably separate patients who exhibit early therapy resistance (POD24, defined as progression within 24 months from diagnosis) from those who achieve “functional cure” following initial therapy. NGS methods developed in recent years enabled quantification of MRD. The ClonoSeq assay (Adaptive Biotechnology, Inc.) has been FDA-cleared to track MRD in several hematologic malignancies. Here we demonstrate that MRD analysis by ClonoSeq assay predicts outcomes in a prospective cohort of patients with advanced stage FL enrolled on SWOG S0016 clinical trial. Methods. SWOG S0016 was a phase III randomized study that compared the safety and efficacy of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) with CHOP-RIT (CHOP + I 133-tositumomab) in patients with FL. Between 2001 and 2008, 531 patients with previously untreated advanced-stage FL (bulky stage II, III-IV) of any pathologic grade were randomly assigned to receive 6 cycles of R-CHOP or CHOP-RIT. We used high-throughput sequencing (HTS) of the IGH and IGK/L loci (ClonoSeq assay) from tumor biopsy specimenstaken prior to treatment to define CDR3 rearrangements that tag the tumor. Subsequently, 1-year bone marrow specimens were screened for residual disease. The baseline dominant clone was defined as ≥10 estimated number of genomes (ENG) and comprised ≥5% of the library. MRD-positive state (MRDp) was defined as a minimum Hamming distance (HD) ≤6 in a patient's post-library (in any clone also detected in a pre-library) and conserved pre-existing SHM (if HD>0). Undetectable MRD (MRDu, at <10 -4) was defined as CRD3 HD>6 in a post-library with a depth >10,000 ENG. Five-year progression-free survival was the primary endpoint (PFS; defined as the time from registration to the first observation of progression or death due to any cause). Landmark analysis was defined at 1 year after registration and used for imputing the 5-year PFS and OS. Overall survival (OS) was defined as the time from registration to date of death due to any cause or last follow-up. PFS and OS estimates and 95% confidence interval were calculated using the Kaplan-Meier method and compared for MRDp and MRDu disease using two-sided log-rank test. HR and 95% CI were calculated using Cox regression model. Results. Tumor biopsy tissue was available for 189 patients. Of these, 24 patients were not evaluable (16 did not consent for banking, 6 were ineligible and 4 had an early event). In 36 patients, pre-libraries did not yield a trackable clone. In total, MRD status could not be ascertained in 47 patients due to absence of a clear trackable clone in either pre- or post-library, contributing to a 28.8% failure rate. Of 116 patients which yielded a trackable clone in both libraries, 83 were MRDu and 33 were MRDp. Baseline characteristics (age, sex, race, β2M, B symptoms, bulky disease, grade 3, stage, and FLIPI risk) were similar between MRDp and MRDu patients; however, MRDp patients were more likely to exhibit morphologic baseline bone marrow involvement by FL (82% vs. 55%, p=0.01). The estimated 5-year PFS was 72% in MRDu patients and 30% in MRD+ patients (Figure); 10-year PFS was 56% and 17%, correspondingly. Meanwhile, 5-year OS was not different (96% for MRDu, 81% for MRD+, p=0.91). Patients with MRDu had a significantly higher best overall response rate than MRDp patients (96% vs 79%, p=0.005), but complete response (CR) rates were similar (41% vs. 30.3%). Patients who received CHOP-RIT had better PFS regardless of MRD status (HR=0.48, p=0.01 for MRDu; HR=0.38, p=0.02 for MRDp), while OS was not statistically different. Five-year PFS was superior in patients who achieved MRDu (75% for CR and 70% for PR) compared with MRDp patients, regardless of response (34%). Patients who had MRDp status had a significantly increased risk of experiencing POD24 relative to MRDu patients (RR=6.5, 95% CI 3.0-14.0; p<0.0001). Conclusion. Here we for the first time demonstrate that MRDu status, as assessed by Clonoseq, predicts improved 5- and 10-year PFS in patients with FL initially treated with chemoimmunotherapy. MRD+ status in the bone marrow at a 1-year timepoint was associated with POD24. Despite a relatively high failure rate to detect trackable sequences using current technology (~29%), MRD assessment by NGS is a promising prognostic tool in FL. *co-senior authors

Inhibition of the PCR by genomic DNA.

qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6. The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer. Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition. Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.

Abstract 1039: Individualized tumor-informed circulating tumor DNA (ctDNA) analysis for postoperative monitoring of non-small cell lung cancer (NSCLC) - the MEDAL study

Abstract Background: Through parallel testing and comparison of personalized and fixed panel minimal residual disease (MRD) assays, to establish the best technique and application strategy of dynamic MRD detection for prognosis prediction and disease assessment among non-small cell lung cancer (NSCLC) patients. Method: We analyzed 760 plasma samples from prospectively enrolled 181 patients with NSCLC recruited to the MEDAL study (NCT03634826), with disease stage I (63%), II (19%) and III (18%). 80% were adenocarcinomas. Plasma samples were collected at baseline (n=157), landmark 3-day and 1-month (n=334), and longitudinal points (n=248) were analyzed. Additional plasma was collected after relapse for 14 patients (n=21). Median follow-up was 1092 days, and 48 patients progressed. We employed a novel personalized tumor-informed technology named PROPHET using deep sequencing of 50 patient-specific variants. The PROPHET was developed to detect MRD with a limit of detection (LoD) of 0.004% and sample-level specificity of greater than 99% in the analytical validation. Detection and quantification of MRD through tumor-informed (TI) and tumor-agnostic (TA) fixed panel assays in the same samples were conducted for a head-to-head comparison. Results: ctDNA was detected by PROPHET prior to treatment in 45% of samples (83%, 75% and 23% for disease stage III, II and I), and showed a higher positive rate than the TI and TA assays (22% and 19%). PROPHET identified 30 more ctDNA positive patients with a median ctDNA fraction of 0.01% at baseline. From the landmark single test, the sensitivity was 45%; integrating longitudinal time points increased the sensitivity to 85%. Landmark PROPHET status was the only risk factor other than clinical features to predict the clinical relapse (p<0.001, multivariate Cox model). MRD positive patients defined by non-canonical variants (n=8) had similar disease-free survival (DFS) as MRD positive patients defined by canonical variants (n=9). Landmark PROPHET-based MRD status combined with clinical TNM stage outperformed TNM stage for prediction of prognosis (p<0.001). Longitudinal MRD achieved negative predictive value (NPV) of 99% with an interval of 150 days, and demonstrated 299 days of longer lead-time than other state-of-the-art fixed-panel assays. Among 16 patients with equivocal radiological diagnosis, all the MRD positive patients relapsed (n=6). Among relapsed patients received next-line treatments, 7 ctDNA negative patients survived or died other disease, 67% (2/3) ctDNA positive patients died from cancer. Sensitivity for bone and brain metastasis was 100% (11/11) and 50% (2/4), respectively. Conclusion: The sensitive tumor-informed personalized MRD approach could provide advantages in prognosis prediction at landmark and disease assessment during surveillance for NSCLC patients. Citation Format: Kezhong Chen, Chenyang Wang, Haifeng Shen, Xi Li, Yichen Jin, Shuailai Wu, Fujun Qiu, Qiang Lu, Di Peng, Shuai Fang, Bing Li, Juan Lv, Jinlei Song, Yang Wang, Shannon Chuai, Zhihong Zhang. Individualized tumor-informed circulating tumor DNA (ctDNA) analysis for postoperative monitoring of non-small cell lung cancer (NSCLC) - the MEDAL study [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 1039.

From Generic RNA-Based MRD Assessment Towards Patient-Specific Genomic (DNA-based) MRD in AML and Other Non-B Cell Acute Leukemia

Pediatric acute myeloid (AML) is considered as a heterogeneous disease affecting 15% of children with acute leukemia. Outcome is still unsatisfactory with an overall survival around 70-80% and a relapse rate of 30-40% after primary treatment. The most important factors determining clinical outcome are genetic aberrations, and early response to treatment. Many studies have demonstrated the prognostic significance of minimal residual disease (MRD) monitoring. Although MRD monitoring is now an integral part in the management (including MRD-guided stratification) of Acute Lymphoblastic Leukemia (ALL) in children (generally done by ASO-qPCR on tumor specific IG/TCR targets), its clinical utility in childhood non-lymphoblastic leukemia (such as AML) is currently unknown/not used. Contemporary approaches to MRD detection in AML rely on PCR amplification of usually fixed genetic abnormalities (MRD-PCR) or on flowcytometric profiling of leukemia-associated immunophenotypes (LAIP). Molecular MRD detection is in general more sensitive than flow-based MRD, but the applicability is lower since recurrent mutational targets (e.g. fusion genes) using conventional technology, are present in only 40-50% of pediatric AML patients. This RNA-based MRD is common practice for CBFb-MYH11, RUNX1-RUNXT1, KMT2A-AFF1, -MLLT1, -MLLT3, and NPM1 exon 12 mutations and standard curves necessary for MRD quantification are available for all mentioned genes. Since the introduction of Whole Transcriptome Sequencing (WTS; RNAseq) as standard diagnostics in our laboratory, we identified various additional fusion genes in AML (increasing available molecular targets up to 75%), but also in other non-B cell malignancies such as SCLL, CEL, HES and BPDCN. By designing patient specific primers/probe sets surrounding the breakpoint sequences, we were able to generate 36 RNA-based MRD assays (including FOP-FGFR1, RANBP2-ALK, DEK-NUP214, ETV6-PDGFRB, KMT2A-AFDN) and monitor MRD during/after therapy. Although RNA-based MRD approach allows additional molecular monitoring in these patients, this type of MRD displays several disadvantages: sensitivity of a certain MRD assay can be low due to low expression levels of the fusion gene or low RNA yield and/or quality. Furthermore, quantification can be hampered by lack of available standard curves. Therefore DNA-based MRD is favorable over RNA-based MRD and Whole Exome Sequencing (WES) or Whole Genomic Sequencing (WGS) can identify the genomic breakpoint sequence in respectively genomic exons or introns. In addition, other genomic aberrations (such as insertions, deletions and point mutations) can be identified by these techniques and used for development of DNA-based MRD. By using this approach we were able to generate 17 additional DNA-based MRD assays (including FOP-FGFR1, RANBP2-ALK, RUNX1-CBFA2T3 NPM1-MLF1 UBTF-TD, p53 deletion), all with very high sensitivity (e.g. around 10-4 to 5x10-5) and patient-derived standard curves to quantify MRD. Figure 1 shows an example for DNA-based MRD monitoring of 3 AML patients harboring the recently described poor-outcome UBTF-TD aberration showing no/slow decrease of MRD in all patients. In conclusion, we show that fusion gene detection by WTS, preferably in combination with mutation analysis by WES/WGS, is providing comprehensive diagnostic results to improve the development of highly sensitive patient specific MRD targets to allow better molecular monitoring (and prediction of relapse), eventually leading to treatment stratification of patients in the future. We will present an update of our results and interesting cases at the upcoming conference. Figure 1: By WES, 3 patients with tandem duplications (TD) in exon 13 of the UBTF gene were identified, and patient specific primers and probes were designed for MRD on follow-up DNA samples. MRD was quantified by using a standard curve derived from serial dilutions from the diagnostic DNA. Quantitative range, sensitivity and background were determined according to the ESG-MRD-ALL guidelines. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Why is it Important to Identify Residual Cells in Leukaemia Patients after Treatment? A Review Article

Introduction: Most children diagnosed with acute lymphoblastic leukemia are cured and are not at risk of relapse. However, 20% of children are at a high risk of experiencing relapse later on in their lives. In order to detect risk and obtain prognostic information, the quantification of minimal residual disease (MRD) can be utilized. Detection of MRD can lead to efficient identification of relapse risk. However, there is limited understanding of the association between MRD and long-term outcomes after treatment in children. Therefore, this systematic review will examine existing literature to determine the strength of association between MRD negativity and relapse risk in children and its importance in the prediction of relapse. Methods: A systematic review of 5 articles centered around ALL in children was analyzed to examine the strength of association between MRD negativity and clinical outcomes of event-free survival (EFS) and overall survival (OS) following the PRISMA guideline. The literature search was done through databases such as NCBI, PubMed, and other childhood oncology databases. The inclusion criteria included peer-reviewed clinical studies that focus on ALL relapse risk and MRD detection. Additionally, reviews, abstracts, and studies with inadequate sample sizes or correlations were excluded. Data were extracted and organized based on criteria of MRD negativity, MRD detection type, and relapse risk level. The data collected from all studies were analyzed through a meta-analysis. The five publications discussed in this article were a total of 11,265 participants. Results: The results portion of your abstract should concisely describe a summary of the main findings. A positive correlation was determined between EFS and OS hazard ratios and MRD detection. Discussion: The analysis of the five publications demonstrated that MRD is an important marker and a strong predictor of relapse in children who are diagnosed with ALL. Conclusion: MRD detection can be proposed as a method of predicting a high risk of relapse in children with ALL. In essence, this literature review has the potential to identify the clinical and therapeutic significance of MRD testing which can be utilized to predict and prevent relapse of ALL in children.

Effective NPM1 plasmid standards selection for minimal/measurable residual disease monitoring in acute myeloid leukemia.

NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming. We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid. With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.

Digital Droplet PCR Is a Reliable Tool to Improve Minimal Residual Disease Stratification in Adult Philadelphia-Negative Acute Lymphoblastic Leukemia

Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P<0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples-2.6% versus 14% (P=0.003)-and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared to RQ-PCR; indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification.

UMAP Based Anomaly Detection for Minimal Residual Disease Quantification within Acute Myeloid Leukemia.

Simple SummaryAcute myeloid leukemia (AML) is the second most frequent leukemia entity in children and adolescents, and definitely the most aggressive variant. Multiparameter flow-cytometry is one of the methodologies most useful to monitor the number of remaining leukemic cells in bone marrow (minimal residual disease, MRD) in AML patients, because it is widely available and applicable to most patients. However, AML flow cytometry data show very complex patterns and identifying leukemic cells in the data is subjective, time-consuming and requires experienced operators who are not available world-wide. In this paper, we approach automatic assessment of AML flow cytometry samples with a novel semi-supervised machine learning model, leveraging implicit expert knowledge stored in a collection of manually assessed samples. Because AML data exhibit a high degree of variability in the patterns of blast cell populations that is difficult to model, the model detects anomalies starting from the appearance of normal cell populations.Leukemia is the most frequent malignancy in children and adolescents, with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) as the most common subtypes. Minimal residual disease (MRD) measured by flow cytometry (FCM) has proven to be a strong prognostic factor in ALL as well as in AML. Machine learning techniques have been emerging in the field of automated MRD quantification with the objective of superseding subjective and time-consuming manual analysis of FCM-MRD data. In contrast to ALL, where supervised multi-class classification methods have been successfully deployed for MRD detection, AML poses new challenges: AML is rarer (with fewer available training data) than ALL and much more heterogeneous in its immunophenotypic appearance, where one-class classification (anomaly detection) methods seem more suitable. In this work, a new semi-supervised approach based on the UMAP algorithm for MRD detection utilizing only labels of blast free FCM samples is presented. The method is tested on a newly gathered set of AML FCM samples and results are compared to state-of-the-art methods. We reach a median -score of 0.794, while providing a transparent classification pipeline with explainable results that facilitates inter-disciplinary work between medical and technical experts. This work shows that despite several issues yet to overcome, the merits of automated MRD quantification can be fully exploited also in AML.

Minimal Residual Disease Prior to and After Haematopoietic Stem Cell Transplantation in Children and Adolescents With Acute Lymphoblastic Leukaemia: What Level of Negativity Is Relevant?

Minimal residual disease (MRD) assessment plays a central role in risk stratification and treatment guidance in paediatric patients with acute lymphoblastic leukaemia (ALL). As such, MRD prior to haematopoietic stem cell transplantation (HSCT) is a major factor that is independently correlated with outcome. High burden of MRD is negatively correlated with post-transplant survival, as both the risk of leukaemia recurrence and non-relapse mortality increase with greater levels of MRD. Despite growing evidence supporting these findings, controversies still exist. In particular, it is still not clear whether multiparameter flow cytometry and real-time quantitative polymerase chain reaction, which is used to recognise immunoglobulin and T-cell receptor gene rearrangements, can be employed interchangeably. Moreover, the higher sensitivity in MRD quantification offered by next-generation sequencing techniques may further refine the ability to stratify transplant-associated risks. While MRD quantification from bone marrow prior to HSCT remains the state of the art, heavily pre-treated patients may benefit from additional staging, such as using 18F-fluorodeoxyglucose positron emission tomography/computed tomography to detect focal residues of disease. Additionally, the timing of MRD detection (i.e., immediately before administration of the conditioning regimen or weeks before) is a matter of debate. Pre-transplant MRD negativity has previously been associated with superior outcomes; however, in the recent For Omitting Radiation Under Majority age (FORUM) study, pre-HSCT MRD positivity was associated with neither relapse risk nor survival. In this review, we discuss the level of MRD that may require pre-transplant therapy intensification, risking time delay and complications (as well as losing the window for HSCT if disease progression occurs), as opposed to an adapted post-transplant strategy to achieve long-term remission. Indeed, MRD monitoring may be a valuable tool to guide individualised treatment decisions, including tapering of immunosuppression, cellular therapies (such as donor lymphocyte infusions) or additional immunotherapy (such as bispecific T-cell engagers or chimeric antigen receptor T-cell therapy).

Frequency and Outcome of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia with BCR-ABL1 Clonal Hematopoiesis after Blast Clearance: Results from the Graaph-2014 Trial

BackgroundIG/TR-based minimal residual disease (MRD) is a faithful marker of response to therapy and the strongest predictor of relapse in acute lymphoblastic leukemia (ALL). In adults with Philadelphia chromosome-positive (Ph+) ALL, MRD is commonly monitored by BCR-ABL1 transcript quantification, although its prognostic significance has not been compared to IG/TR MRD to date. Recently, it has been shown that BCR-ABL1 rearrangement may be found and persist in non-lymphoblastic cells in some patients. In the prospective GRAAPH-2014 trial, using a dual IG/TR and BCR-ABL1 MRD monitoring, we aimed to study the biological and clinical significance of persistent BCR-ABL1 clonal hematopoiesis (CH) in adults with de novo Ph+ ALL.Patients and MethodsThe study comprised 156 adults with de novo Ph+ ALL randomized in the GRAAPH-2014 trial. Bone marrow (BM) and peripheral blood (PB) follow-up (FU) samples were collected after each treatment cycle and before hematopoietic stem cell transplantation (HSCT). MRD monitoring was performed by quantification of both BCR-ABL1 transcripts and IG/TCR clonal rearrangements on all available samples and MRD quantification of genomic BCR-ABL1 breakpoint fusion was also performed for 37 patients. MRD results were considered discordant if more than one log10 difference or positivity/negativity discordance on the same FU sample was evidenced. Cell fractions from 14 peripheral blood mononuclear cells (PBMC) samples (T-cells, B-cells and monocytes) were FACS-sorted and evaluated for BCR-ABL1 expression. Achievement of major molecular response (MMR) defined as <0.1% BCR-ABL1/ABL1 ratio after the 4 treatment cycles (MRD4) was the primary endpoint of the clinical trial.ResultsQuantification of BCR-ABL1 transcripts and IG/TR MRD levels on 876 samples from 156 patients (456 BM and 542 PB) identified 54 out of 142 (38%) evaluable patients with consistently discordant MRD results in at least 3 different timepoints, suggesting the persistence of non-lymphoblastic BCR-ABL1-positive cells in these patients and BCR-ABL1 CH. Possible bias related to variable BCR-ABL1 expression was ruled out by genomic BCR-ABL1 quantification on 263 FU samples (r s=0.89, p<0.0001). Analysis of sorted PBMC fractions from discordant FU samples revealed the expression of BCR-ABL1 in mature B-cells, T-cells and/or monocytes in 11 patients, demonstrating the presence of BCR-ABL1 CH. Baseline characteristics of patients with BCR-ABL1 CH (CH+) showed no difference in terms of age, gender and white blood cell counts, but an enrichment in M-bcr breakpoints as compared to CH- patients (46.3% vs 17.1%, p<0.0001). As expected, the rate of MMR at MRD4 was lower (68% vs 95%, p<0.0001), with a trend for more patients who received alloSCT (38/54 (70%) vs 48/88 (55%), p=0.08).We then evaluated the clinical significance of BCR-ABL1 CH. Unexpectedly, CH+ patients had a significant lower cumulative incidence of relapse (CIR) (panel A, hazard ratio (HR) 0.37, 95% CI [0.15-0.90], p=0.03). However, this lower CIR did not translate into longer disease-free survival (DFS) (panel B, HR=0.85, 95% CI [0.44-1.63], p=0.63). Since an imbalance in alloSCT was observed between CH+ and CH- patients, we tested if the observed difference in CIR may be related to different alloSCT outcomes. When censoring patients at alloSCT, the difference in CIR was indeed no longer significant (subhazard ratio (SHR) 0.48, 95% CI [0.16-1.43], p=0.19). More interestingly, the benefit of alloSCT was restricted to CH- patients. Using alloSCT as a time-dependent variable, DFS was indeed improved by alloSCT in CH- (SHR 0.40, 95% CI [0.17-0.91], p=0.03, panel C) but not in CH+ patients (SHR 1.12, 95%CI [0.32-3.87], p=0.86, panel D) despite a non-significant interaction between CH status and alloSCT.ConclusionMore than one third of adults with de novo Ph+ ALL displays persistent measurable BCR-ABL1 signal during therapy, likely related to BCR-ABL1 CH. Strikingly, this condition is not associated with a higher risk of relapse nor with poorer overall outcomes in the GRAAPH-2014 trial. Moreover, our results suggest that patients with BCR-ABL1 CH may not be good candidates for alloSCT. By contrast, IG/TR MRD may allow to identify patients with the higher risk of relapse. These results highlight the need for implementation of IG/TR MRD in adult Ph+ ALL to guide therapeutic decisions. [Display omitted] DisclosuresRousselot: Incyte, Pfizer: Consultancy, Research Funding. Chalandon: Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Amgen: Other: Advisory Board; Incyte: Speakers Bureau; Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Gilead, Amgen, Jazz, Astra Zenec: Other: Travel EXpenses, Accomodation. Straetmans: Alexion: Membership on an entity's Board of Directors or advisory committees. Huguet: Novartis: Other: Advisor; Jazz Pharmaceuticals: Other: Advisor; Celgene: Other: Advisor; BMS: Other: Advisor; Amgen: Other: Advisor; Pfizer: Other: Advisor. Dombret: Amgen: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Honoraria, Research Funding; Servier: Research Funding; Abbvie: Honoraria; BMS-Celgene: Honoraria; Daiichi Sankyo: Honoraria. Boissel: SANOFI: Honoraria; CELGENE: Honoraria; Servier: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; PFIZER: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; JAZZ Pharma: Honoraria, Research Funding.

Evaluation of Hematopoietic Stem Cell Product As a New Site for Minimal Residual Disease By Next Generation Flow in Multiple Myeloma

Introduction- Next generation flow (NGF) is one of the approaches for testing multiple myeloma (MM) minimal residual disease (MRD) over conventional response assessments. Actually, bone marrow (BM) is the preference site of evaluation because of its sensitivity. Because of its invasively technic, other possible sites for MRD evaluation outside the BM have been studied. In the present study we analyzed the MRD between the BM and the hematopoietic stem cell collected product (HSC product), once the concentration of plasma cell in the HSC product could be higher than peripheric blood sample.Aims- To compare MRD quantification of plasma cell between BM and HSC product after induction from Newly Diagnosed MM(NDMM) Transplant Eligible (TE) patients (pts) exposed to daratumumab, cyclophosphamide, thalidomide and dexamethasone (Dara-CTD) protocol.Methods- The SC product and BM samples were collected after four 28 days cycles of induction therapy from pts treated with Dara-CTd protocol described before by (Crusoe E. et al. Blood 2020; 136 (supplement 1): 17-18). MRD was evaluated by next-generation flow (NGF) based in the EuroFlow® protocol. EuroFlow standards was used to identify clonality and aberrant PC immune phenotype, consisting by EuroFlow 8-color 2-tube method (MM MRD kit, Cytognos, Salamanca), with the acquisition of 5 million events each tube and then merged into a single analysis tube on approximately 10 million events. Plasma cells were identified by CD38 multiepitope and CD138. Other markers were used to detect abnormal phenotypes. For comparison of MRD results, Bland-Altman plot comparing BM-MRD and HSC product-MRD was performed.Results- The first pts was enrolled in November 2018. A total of 24 pts were included, the median age was 60 (range 37- 67 years), 23 (92%) were non-white, 5 (21%) had an R-ISS = 1, 12 (54%) had an R-ISS = 2 and 4 (16%), an R-ISS = 3. Six (25%) pts had high-risk chromosomal abnormalities [del17p, t(4;14) or t(14;16)]. To date, all pts have completed induction and 20 have received transplant. Regarding response rates, after the end of induction (cycle 4), 19 (90%) of the pts obtained > PR and 8 (38%) obtained >VGPR, including three MRD negativity by NGF. 19 pts were analyzed for MRD. Negative MRD in sensitivity <10 -5, >=10 -5 and <10 -4, >=10 -4 evaluated in bone marrow was 4/19(21%), 4/19(21%), 11/19(58%) respectively. Negative MRD in sensitivity <10 -5, >=10 -5 and <10 -4, >=10 -4 evaluated in the HSC product was 13/19(68%), 3/19(16%), 3/19(16%) respectively. Median bone marrow sensitivity 10 -4 lower quartile 10 -5 upper quartile 10 -3. Normal distribution of the differences between BM and SC product MRD was first assessed (Kolmogorov-Smirnov's p < 0.001, n = 19).Discussion-Conclusions- The use of HSC product could enhance the plasma cell concentration and may be an alternative and attractive method for MRD detection that diminished the invasiveness of repetitive bone marrow aspirations and tackling the heterogeneity distribution of MM cells. In this preliminary data the sample size did not allow to show a direct correlation between BM and HCS product. A larger sample would be needed to confirm the hypothesis. [Display omitted] DisclosuresHungria: Amgen, BMS, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings/travel ; Abbvie: Honoraria; Sanofi: Honoraria, Other: Support for attending meetings/travel ; Takeda: Honoraria. De Queiroz Crusoe: Janssen: Research Funding.

Analytical Quality Controls for ddPCR Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia

Abstract Background Droplet digital PCR (ddPCR) is a promising technique for absolute quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but there is no comprehensive quality assurance program to enable its application in clinical laboratories. Current guidelines for real-time quantitative PCR (qPCR) assays targeting immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements needed adaptation for ddPCR to cover droplet generation, intraassay variation, and interassay variation in the absence of standard curves. Methods Six qPCR MRD assays for Ig/TCR gene rearrangements and a standard albumin control gene assay were migrated to a ddPCR platform and used to test 82 remission samples from 6 patients with ALL. Three analytical quality controls (QC) were developed and evaluated for ddPCR MRD detection. Results Analytical QC for droplet number generation (DN-QC), for albumin ddPCR assay performance (Alb-QC) and for patient-specific marker assay performance (PS-QC) were established with pass/fail limits and corresponding QC rules. Compared to established qPCRs, the ddPCR assays had comparable sensitivity and quantitative range. Overall, there was close agreement (91%) of MRD results between qPCR and ddPCR (κ = 0.86, P &amp;lt; 0.0001) and stronger concordance in 32 quantifiable samples (R2 = 0.97, P &amp;lt; 0.0001). Conclusions The use of this newly developed quality control system for ddPCR MRD testing avoids the need to repeat standard curves and provides reliable results comparable to standardized qPCR methods for MRD detection in ALL.

The beginning of a new therapeutic era in acute myeloid leukemia.

In the field of AML, the early 2000s were shaped by the advent of novel molecular biology technologies including high‐throughput sequencing that improved prognostic classification, response evaluation through the quantification of minimal residual disease, and the launch of research on targeted therapies. Our knowledge of leukemogenesis, AML genetic diversity, gene‐gene interactions, clonal evolution, and treatment response assessment has also greatly improved. New classifications based on chromosomal abnormalities and gene mutations are now integrated on a routine basis. These considerable efforts contributed to the discovery and development of promising drugs which specifically target gene mutations, apoptotic pathways and cell surface antigens as well as reformulate classical cytotoxic agents. In less than 2 years, nine novels drugs have been approved for the treatment of AML patients, and many others are being intensively investigated, in particular immune therapies. There are now numerous clinical research opportunities offered to clinicians, thanks to these new treatment options. We are only at the start of a new era which should see major disruptions in the way we understand, treat, and monitor patients with AML.

New Concepts of Flow Cytometry Analysis in Oncohematology: Application to Diagnosis and Follow up (Minimal Residual Disease) in AML, ALL and MDS

IntroductionThe development of mass cytometry (CyTOF), using up to 40 concomitant tags for a given sample, has led to the generation of large databases. In order to properly analyze these extensive files, new software solutions have been devised using either mathematical reduction of multidimensional spaces or clustering methods allowing for two-dimensional representations. Such tools as SPADE (SPanning-tree progression Analysis of Density-Normalized events), t-SNE (t-Stochastic Neighborhood Embedding) and FlowSOM (Flow Self Organizing Map) therefore were published with CyTOF data. They have however seldom been used to analyze classical flow cytometry (FCM) data, although current instruments generate 10-12 parameters analyzes that would be good candidates for such analyzes. Here we report on the new paradigm of using such tools to interpret harmonized classical FCM data in particular for minimal residual disease (MRD).Materials and MethodsMultiparametric FCM was performed using panels and recommendations proposed by the Harmonemia group (Lacombe F et al. Leukemia 2016), on a Navios® instrument (Beckman Coulter, Miami, FL) since 2013, yielding a cohort of diagnosis (D) and follow-up (FU) samples of 460 AML (160 D, 300 FU), 695 ALL (129 D, 566 FU), and 150 MDS (100 D, 50 FU). In parallel, 45 normal bone marrow (NBM) samples (respectively 22 for AML and MDS cases and 23 for ALL cases) were processed in the same conditions as pathological samples with the relevant immunophenotyping panels (three combinations for AML/MDS and 3 others for ALL). All files were first processed using specific R-software scripts and Bioconduct or CRAN packages to normalize fluorescence and light diffusion intensity including shift intensity, compensation and biexponential/logical adjustment. Harmonized flow cytometry files (HF) ([1305 pathological + 45 NBM files] x 3 panels corresponding to a total of 4050 HF files) were then generated and used for further analysis.NBM files were adjusted and merged in a single, events-adjusted, representative file of NBM (R-NBM). For each case, merges of this R-NBM, D and FU files were processed using 3 different analysis ways 1) principal component analysis (PCA), 2) t-SNE and 3) FlowSOM. New resulting parameters were generated for each combination of R-NBM, D and FU merged files, i.e. PCA, t-SNE and FlowSOM. These parameters could then be analyzed using the Kaluza® software (Beckman Coulter). Graphical representations of all analyses were then performed in an automated unsupervised manner with Kaluza®, allowing for a direct comparison of NBM and pathological samples for each case. Interestingly, HF files generated by the R software, being integrated in the system, could also be classically analyzed by Kaluza®, in a supervised manner, by drawing gates around PCA/t-SNE clusters or FlowSOM nodes of interest. Such gates allowed to further analyze data by backgating on classical biparametric representations of antibody combinations as well as on the classical CD45/SSC bone marrow cartography.ResultsBoth t-SNE and FlowSOM provide a clear identification of normal and abnormal subsets, while PCA is less discriminative. Comparative simultaneous representations of the three types of samples - (D), (FU) and (NBM)- allow for both a rapid visualization and accurate quantification of normal and abnormal cell subpopulations. The FlowSOM solution further allows to identify and visually characterize specific subsets both within the diagnostic clone and along normal hematopoietic differentiation. FU samples can then be easily analyzed for the persistence of abnormal immunophenotypic characteristics, both in AML and ALL. Even unsupervised results allow for a rapid appreciation and quantification of MRD. Besides, such comparisons, between NBM and D samples provide a new approach of the complexity of MDS. Moreover, both supervised and unsupervised results were comparable.ConclusionThe combination of automated sophisticated multidimensional representation of numerous subsets and their in-depth analysis by classical manual tools of FCM analysis is a truly revolutionary approach. The strength of FCM, which retains and analyses morphological characteristics of the cells, is thus combined to the precision of mathematical analyzes developed for CyTOF. These versatile new tools open the way for a new era of classical FCM analysis in hematological malignancies. [Display omitted] DisclosuresDupont:Beckman Coulter: Employment.

Challenging the Prognostic Value of MRD: Sequential Tumor-Ablative Conditioning of FA5-Bucy for Ph-Positive Acute Lymphoblastic Leukemia

Background:Minimal residual disease (MRD) quantification prior to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a powerful predictor for post-transplant outcome. Thus, new strategies with modified transplantation procedures are needed to reverse the outcomes of that remain MRD-positive despite multiple induction attempts.OBJECTIVE:To determine the prognostic relevance of MRD status in patients with acute lymphoblastic leukemia (ALL), especially those with Philadelphia chromosome-positive ALL (Ph+ ALL), receiving the conditioning regimen of FA5-Bucy, registered on http:// ClinicalTrials.gov (NCT02328950).METHODS:We retrospectively reviewed our HSCT experience using FA5-Bucy, i.e., 5-day salvage chemotherapy (Fludarabine/Ara-C) and conditioning (Busulfan/ Cyclophosphamide) for patients with ALL. A total of 48 consecutive patients from 2013 to 2016 are analyzed. 11 out of 48 (7 males and 4 females) were diagnosed with Ph+ ALL with the induction treatments including a tyrosine kinase inhibitor (TKI). Median age was 30 years (range, 8-52 years). Among whom four (36.4%) achieved complete remission(CR)at transplantation, Six (54.5%) with detectable residual P190 BCR-ABL positive leukaemic cells, while 1 (9.1%) not in the status of remission with 39.5% blasts in the bone marrow (BM). The other 37 cases were Ph-negative (Ph-) ALL, with the median age of 21 years (range, 2-61 years). Among whom 25 (67.6%) patients were in CR, 6 (16.2%) MRD-positive and 6 (16.2%) with 23.5%-98% BM blasts. The MRD status was monitored by four-color FCM in Ph- group, and by RT-PCR to detect the chimeric bcr-abl mRNA transcript in Ph+ group. Patients were transplanted either from an HLA-identical sibling or haplo-identical related donors. The GVHD prophylaxis consisted of ATG, CsA, MMF and short-term MTX. None of these patients had severe infection or organ failure before HSCT.RESULTS:All patients achieved successful engraftment and full donor chimerism. Patients with Ph+ ALL seems have a better outcome than that with Ph- ALL (3-year OS of 54.5% vs 37.8%, P=0.245). With a median follow-up of 666 (83-1276) days, the 1y-OS and 1y-DFS were 72.7%, 3y-OS and 3y-DFS were 54.5% in Ph+ group, respectively, and non-relapse mortality and relapse incidence were 18.2% and 10%. MRD monitoring is an important tool for relapse prediction in Ph- ALL patients (3-year OS and DFS of MRD-positive vs MRD-negative =16.7% vs 56%, P=0.021).However, the profile of OS and DFS was similar in Ph+ patients with bcr-abl positive to that with bcr-abl negative (3-year OS and DFS of bcr-abl positive vs bcr-abl negative= 57.1% vs 50%, P=0.744). Severe acute-GvHD (aGVHD) occurred in 3/11 patients. Few patients experienced mild-to-moderate toxicity, and main causes of death were aGVHD.CONCLUSION:We concluded that allo-HSCT with the sequential tumor-ablative conditioning of FA5-Bucy offers great benefits for the patients with Ph+ ALL, with the potentially capability of reducing or overcoming the negative impacts of MRD. Thus creates exciting transplant opportunities for patients not in remission to be cured. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Utility of Droplet Digital PCR to Monitor BCR-ABL1 Transcripts of Patients With Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia Post-chimeric Antigen Receptor19/22 T-Cell Cocktail Therapy

Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) accounts for 20–30% of adult patients with ALL, characterized by translocation of t(9, 22). Tyrosine kinase inhibitors (TKIs) have significantly improved the outcome even though there are still some problems including relapse due to drug-resistant mutations and suboptimal molecular remission depth. Previously, we reported the safety and efficacy of sequential infusion of CD19/22 chimeric antigen receptor T-cell (CAR-T) immunotherapy in the treatment of relapsed/refractory (R/R) B-cell neoplasms including cases with Ph+ ALL. Given possible deeper reaction, more patients were expected to reach optimal minimal residual disease (MRD) response. An alternative method, duplex droplet digital PCR (ddPCR) with high sensitivity was established, which could provide absolute quantification of MRD without the need for calibration curves. Here, we retrospectively collected 95 bone marrow samples from 10 patients with R/R Ph+, who received 19/22 CAR-T-cell cocktail therapy. Notably, sequential molecular remission for more than 3 months (SMR3), a significant indicator based on ddPCR after CAR-T infusion was established, which was defined as a sequential molecular remission for not <3 months with negative MRD. In this cohort, no recurrence was observed in six patients achieving SMR3, where four of whom accepted allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CAR-T cell regimen. Unfortunately, the other four patients who did not reach SMR3 relapsed, and did not receive extra specific treatment except CAR-T regimen. To sum up, ddPCR may be an alternative, especially when nucleic acid was insufficient in clinical practice. No achievement of SMR3 may be an early warning of potential relapse after CAR-T and indicating the initiation of other therapies including allo-HSCT.

Validation of a PCR-Based Next-Generation Sequencing Approach for the Detection and Quantification of Minimal Residual Disease in Acute Lymphoblastic Leukemia and Multiple Myeloma Using gBlocks as Calibrators

Detection of minimal residual disease (MRD) to guide therapy has been a standard practice in treatment of childhood acute lymphoblastic leukemia (ALL) for decades. In multiple myeloma (MM), a clear correlation is found between absence of MRD and longer survival. Quantitative allele-specific oligonucleotide (qASO)-PCR is the standard molecular method for MRD detection in these hematologic malignant tumors. However, this technique has some drawbacks that can be overcome by next-generation sequencing (NGS). In this study, NGS is validated as an alternative method for qASO-PCR for MRD detection in both ALL and MM. MRD results obtained by NGS and qASO-PCR were compared in 59 and 39 bone marrow samples of 33 and 14 patients with ALL and MM, respectively. Our results indicate that the use of gBlocks as calibrators makes the NGS approach a powerful tool to quantify MRD. With an input of 400 ng of DNA (corresponding to approximately 7×104 cells), a limit of detection of 0.01% can be achieved. The specificity of the NGS-MRD technique was 100%, and a correlation with qASO-PCR for quantifiable MRD results of 0.93 and 0.91 was found in ALL and MM, respectively. Especially for MM, the higher applicability (100%) of the NGS-MRD protocol, compared with qASO-PCR (57%), was clearly demonstrated. These results demonstrate that NGS is an even better alternative to qASO-PCR.