NGS-based Minimal Residual Disease Research Articles

Clonotypic VDJ Rearrangements in Mixed Phenotype Acute Leukemia can be Successfully Utilized to Track Minimal Residual Disease.

Multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) can both be used to identify a neoplastic clonotype by targeting CDR3 and assessing rearrangements in IgH, IgK, IgL, TCR-β, and TCR-gamma loci. The clonotypic sequence can be robustly used to track minimal residual disease (MRD). The ability to track MRD by NGS in mixed phenotype acute leukemia (MPAL) is unknown and warrants investigation. Institutional Review Board (IRB) approval was obtained. Central Moffitt Cancer Center (MCC) database was searched to locate any patients with MPAL from over 600,000 entries. Patient charts were manually curated to identify those with clonoSEQ data, and clinical data was procured from the electronic medical record (EMR). Twenty-nine patients with MPAL were identified. Only 2 patients with clonoSEQ testing were found. Both demonstrated a B/myeloid phenotype, and both were bilineal. NGS (clonoSEQ) identified 4 dominant (IGH) (patient A; 8/2019) and 2 dominant sequences (patient B; 10/2019), respectively. In both patients, clonoSEQ testing successfully tracked minimal residual disease and mirrored clinical disease burden. This report is the first to confirm the utility of NGS-based MRD tracking in patients with MPAL and shows increased sensitivity of NGS over MRD flow cytometry.

NSABP C-14/Exact Sciences 16-002: CORRECT-MRD II—Second colorectal cancer clinical validation study to predict recurrence using a circulating tumor DNA assay to detect minimal residual disease.

TPS241 Background: Detectable circulating tumor DNA (ctDNA) after resection of early-stage solid tumors has been associated with very high risk of recurrence, suggesting ctDNA is evidence of minimal residual disease (MRD). Several studies are ongoing to investigate the role of ctDNA in the optimal management of patients (pts) with colorectal cancer using different assay technologies. Methods: This is a prospective, observational, multicenter study in the United States and Canada of 750 pts who have undergone complete surgical resection for stage II or III colorectal cancer, who have FFPE tissue available from the primary resection sufficient for a novel bespoke MRD assay and are willing to provide serial whole blood specimens for ctDNA analysis. Participants are asked to provide study specimens after definitive surgical resection, pre-recurrence follow-up, and clinical recurrence (if applicable). Recently amended eligibility criteria include inclusion of rectal cancer pts who have completed neo-adjuvant therapy and surgical resection, as well as enrollment of all stage II and III pts regardless of microsatellite stability status. The Oncotype Colon Recurrence Score® will be assessed on all pts from their surgical specimen, if criteria are met for this testing. ctDNA will be analyzed with an NGS-based tumor-informed MRD assay that identifies somatic genomic alterations from DNA derived from the patient’s tumor tissue, subtracts germline variants, and detects a selected subset of tumor-specific (bespoke) ctDNA in their blood. All primary tumor specimens will undergo full exome and transcriptome sequencing using the Onco ExTra assay. If there is evidence of disease recurrence, the metastatic tissue will also undergo Onco ExTra testing and results will be shared with participants. The primary objective is to validate the association of post-definitive therapy and pre-recurrence follow-up ctDNA positivity with recurrence-free interval (RFI). Further objectives are to assess the: sensitivity and specificity of ctDNA positivity for subsequent clinical recurrence; contribution of post-surgery baseline, post-adjuvant therapy, and pre-recurrence follow-up ctDNA results on RFI; time from positive ctDNA to clinical recurrence in participants who had a positive ctDNA result; and to compare the Oncotype Colon Recurrence Score estimate of 3-year recurrence risk with the observed 3-year recurrence rate. The primary analysis will use a Cox proportional hazards regression applied to the RFI with ctDNA result (positive or negative) measured after surgery, or end of adjuvant therapy if received, and serially after that as a single, time-dependent covariate. Enrollment has currently surpassed the 50% target. Protocol#: NSABP C-14 / Exact Sciences 16-002 NCT#: 05210283. Support: NSABP Foundation, ExactSciences. Clinical trial information: NCT05210283.

NSABP C-14: CORRECT-MRD II—Second colorectal cancer clinical validation study to predict recurrence using a circulating tumor DNA assay to detect minimal residual disease.

TPS284 Background: Detectable ctDNA after resection of early-stage solid tumors has been associated with very high risk of recurrence, suggesting ctDNA is evidence of minimal residual disease (MRD). Several studies are ongoing to investigate the role of ctDNA in the optimal management of pts with colorectal cancer using different assay technologies. Methods: This is a prospective, observational, multicenter study in the United States and Canada of 750 patients who have undergone complete surgical resection for stage II or III colorectal cancer, who have FFPE tissue available from the primary resection sufficient for a novel bespoke MRD assay and are willing to provide serial whole blood specimens for ctDNA analysis. Participants are asked to provide study specimens after definitive surgical resection, pre-recurrence follow-up, and clinical recurrence (if applicable). Recently amended eligibility criteria include inclusion of rectal cancer patients who have completed neo-adjuvant therapy and surgical resection, as well as enrollment of all stage II and III patients regardless of microsatellite stability status. The Oncotype Colon Recurrence Score will be assessed on all patients from their surgical specimen if criteria are met for this testing. ctDNA will be analyzed with an NGS-based tumor-informed MRD assay that identifies somatic genomic alterations from DNA derived from the patient’s tumor tissue, subtracts germline variants, and detects a selected subset of tumor-specific (bespoke) ctDNA in their blood. All primary tumor specimens will undergo full exome and transcriptome sequencing using the Oncomap ExTra assay. If there is evidence of disease recurrence, the metastatic tissue will also undergo Oncomap ExTra testing, which will be shared with participants. The primary objective is to validate the association of post-definitive therapy and pre-recurrence follow-up ctDNA positivity with recurrence-free interval (RFI). Further objectives are to assess the: sensitivity and specificity of ctDNA positivity for subsequent clinical recurrence; contribution of post-surgery baseline, post-adjuvant therapy, and pre-recurrence follow-up ctDNA results on RFI; time from positive ctDNA to clinical recurrence in participants who had a positive ctDNA result; and compare the Oncotype Colon Recurrence Score estimate of 3-year recurrence risk with the observed 3-year recurrence rate. The primary analysis will use a Cox proportional hazards regression applied to the RFI with ctDNA result (positive or negative) measured at post-surgical baseline (or end of adjuvant therapy if used) and serially after that as a single, time-dependent covariate. Protocol#: NSABP C-14 / ES 16-002. Support: NSABP Foundation, ExactSciences Clinical trial information: 05210283 .

Comparison of the Analytical Performance of EXENT®, a Mass Spectrometry-Based Assessment of M-Protein, to SPEP and NGS-Based MRD in Multiple Myeloma Patient Samples

Background: serum protein electrophoresis (SPEP) and B-cell receptor next generation gene sequencing (NGS) are methodologies used to monitor the amount of a monoclonal protein in multiple myeloma (MM) patients and to evaluate minimal residual disease (MRD), respectively. Mass spectrometry (MS) is a methodology currently being explored to provide a serum/plasma-based quantitative assessment of M-protein, which could also serve as a surrogate of MRD. Here we present the analytical performance of SPEP and MRD compared to the MALDI-TOF MS-based assay EXENT. Methods: SPEP, NGS (Clonoseq v 2.0, Adaptive Biotechnologies), and MS (EXENT®, The Binding Site) assays were performed to assess M-protein in 65 serum samples from 22 MM patients who participated in a phase 3 clinical trial (CASTOR, NCT02136134), in which patients received Daratumumab, Bortezomib and Dexamethasone (DVd) or Bortezomib and Dexamethasone (Vd). Irrespective of treatment, serum samples collected at three time points were selected from each subject, including 1) baseline prior to treatment, 2) available sample at or most approximately prior to the first confirmed complete response (CR) according to IMWG criteria with SPEP equaled 0, and 3) last available sample prior to disease progression (PD) with SPEP equaled 0. M-protein concentration measured by EXENT was compared to that measured by SPEP. Data was analyzed by using either all obtained values (n=62) or only non-zero values (n=18) by EXENT or SPEP. NGS-based MRD results (n=26) used for the comparison were those sampled within 60 days of the samples used for the EXENT analysis. Results: M-protein concentration by EXENT significantly correlated with SPEP measurements by using all obtained values (r=0.986, p<0.0001). The correlation was also significant when non-zero values were used in the analysis (r=0.969, P<0.0001). The lower limit of quantification (LLOQ) of EXENT was 0.015 g/L, while that of SPEP was approximately 1 g/L. With the better sensitivity, EXENT was able to detect M-protein up to 167 days prior to PD when SPEP detected none (figure). MS-based assessments were concordant with NGS-based MRD results. A 76.9% (20 of 26) agreement was observed when comparing EXENT with MRD at a sensitivity of 10-5. EXENT purified samples were reflexed to analysis by the more sensitive MS-based methodology LC-MS. The agreement between LC-MS and MRD at a sensitivity of 10-6 was 92.3% (24 of 26). In addition, we observed several useful features unique to MS-based assays. These include distinguishing daratumumab from the endogenous M-protein, detecting isotype switching, and the ability of detecting light chain glycosylation. Conclusion: EXENT significantly correlated with SPEP while EXENT demonstrated a higher sensitivity in quantification of M-protein in serum than SPEP. A high degree of agreement was observed between EXENT/LC-MS and NGS-based MRD. With its non-invasive nature and early detection of relapse, EXENT can be used as a complementary assay in MM patient management to guide bone marrow based MRD assessment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AML-234 Prognostic Value of FLT3-ITD Residual Disease in Acute Myeloid Leukemia

FLT3-internal tandem duplications (FLT3-ITD) are among the most common genetic molecular abnormalities in acute myeloid leukemia (AML) patients. Here, the prognostic impact of the presence of minimal residual disease (MRD) of FLT3-ITD in AML patients is assessed by next-generation sequencing (NGS). The prognostic significance of FLT3-ITD in AML in relation to other concurrent gene mutations and allelic mutational burden has remained a subject of scientific controversy. Detection of FLT3-ITD MRD by RQ-PCR is restricted by patient-specific variables, such as sequence, position, and length. However, systematic studies analyzing the applicability of FLT3-ITD MRD detection with NGS techniques are currently lacking. Here, we evaluate the impact of the presence of FLT3-ITD MRD detected by NGS on treatment outcome in the context of current prognostic factors at diagnosis and MRD status measured by multiparameter flow cytometry (MFC) and mutant NPM1. In 161 de novo AML patients with an FLT3-ITD enrolled in the HOVON-SAKK clinical trials, NGS was performed at diagnosis and ultra-deep in CR after induction chemotherapy. Presence of FLT3-ITD MRD was correlated to incidence of relapse and overall survival (OS). NGS-based FLT3-ITD MRD was present in 47 of 161 (29%) AML patients. Presence of FLT3-ITD MRD was associated with increased risk of relapse (4-year CIR, 75% FLT3-ITD MRD vs. 33% no FLT3-ITD MRD; P<0.001) and inferior OS (4-year OS, 31% FLT3-ITD MRD vs. 57% no FLT3-ITD MRD; P<0.001). In multivariate analysis, detection of FLT3-ITD MRD in CR confers independent prognostic significance for relapse (hazard ratio, 3.55; P<0.001) and OS (hazard ratio 2.51; P<0.001). Presence of FLT3-ITD MRD exceeds the prognostic value of most of the generally accepted clinical and molecular prognostic factors, including the FLT3-ITD allelic ratio at diagnosis and MRD status of mutant NPM1 and MFC. Presence of FLT3-ITD MRD in CR detected by NGS after induction chemotherapy identifies AML patients with an increased risk of relapse and inferior OS that outweighs the significance of currently accepted prognostic factors. This finding offers support for the use of FLT3-ITD MRD as a clinically relevant biomarker for dynamic disease risk assessment in AML.

381P A tumor-informed, hybrid-capture based ctDNA assay for minimal residual disease (MRD) detection in colorectal cancer (CRC) patients after curative surgery

MRD detection using ctDNA analysis after curative treatment may help identify patients at higher risk of relapse and improve treatment outcomes by enabling personalized treatment. We have analyzed the clinical performance of a recently developed tissue-informed, hybrid-capture NGS-based ctDNA MRD assay in CRC.

Minimal residual disease detection by next-generation sequencing in multiple myeloma: Promise and challenges for response-adapted therapy.

Assessment of minimal residual disease (MRD) is becoming a standard diagnostic tool for curable hematological malignancies such as chronic and acute myeloid leukemia. Multiple myeloma (MM) remains an incurable disease, as a major portion of patients even in complete response eventually relapse, suggesting that residual disease remains. Over the past decade, the treatment landscape of MM has radically changed with the introduction of new effective drugs and the availability of immunotherapy, including targeted antibodies and adoptive cell therapy. Therefore, conventional serological and morphological techniques have become suboptimal for the evaluation of depth of response. Recently, the International Myeloma Working Group (IMWG) introduced the definition of MRD negativity as the absence of clonal Plasma cells (PC) with a minimum sensitivity of <10−5 either by next-generation sequencing (NGS) using the LymphoSIGHT platform (Sequenta/Adaptative) or by next-generation flow cytometry (NGF) using EuroFlow approaches as the reference methods. While the definition of the LymphoSIGHT platform (Sequenta/Adaptive) as the standard method derives from its large use and validation in clinical studies on the prognostic value of NGS-based MRD, other commercially available options exist. Recently, the LymphoTrack assay has been evaluated in MM, demonstrating a sensitivity level of 10−5, hence qualifying as an alternative effective tool for MRD monitoring in MM. Here, we will review state-of-the-art methods for MRD assessment by NGS. We will summarize how MRD testing supports clinical trials as a useful tool in dynamic risk-adapted therapy. Finally, we will also discuss future promise and challenges of NGS-based MRD determination for clinical decision-making. In addition, we will present our real-life single-center experience with the commercially available NGS strategy LymphoTrack-MiSeq. Even with the limitation of a limited number of patients, our results confirm the LymphoTrack-MiSeq platform as a cost-effective, readily available, and standardized workflow with a sensitivity of 10−5. Our real-life data also confirm that achieving MRD negativity is an important prognostic factor in MM.

NSAB C-14: CORRECT-MRD II—Second colorectal cancer clinical validation study to predict recurrence using a circulating tumor DNA assay to detect minimal residual disease.

TPS3632 Background: Patients (pts) with stage II and III colon cancer (CC) have unique post-operative decisions regarding adjuvant chemotherapy (ACT). There is a subset of stage II pts with defined clinicopathologic features associated with poor prognosis who may benefit from ACT, although more discriminating and objective predictors of benefit are needed. In addition, there may be a subset of Stage III CC pts who could tolerate a de-escalation of ACT or who may require intensification of ACT to improve clinical outcome. Detectable ctDNA after resection of early-stage solid tumors has been associated with very high risk of recurrence, suggesting ctDNA is evidence of minimal residual disease (MRD). Several studies are ongoing to investigate the role of ctDNA in the optimal management of pts with CC using different assay technologies. Methods: This is a prospective, observational, multicenter study in the United States and Canada of 750 pts who have undergone complete surgical resection for stage II or III CC, have FFPE tissue available from the primary resection sufficient for a novel bespoke MRD assay, and are willing to provide serial whole blood specimens for ctDNA analysis. Subjects are asked to provide study specimens at baseline, pre-recurrence follow-up, and clinical recurrence (if applicable) study visits. ctDNA will be analyzed with an NGS-based MRD assay that identifies somatic genetic alterations from DNA derived from the pt’s tumor tissue, subtracts germline variants, and detects a subset of these tumor-specific (bespoke) ctDNA in the pt’s blood. The primary objective is to validate the association of post-definitive therapy and pre-recurrence follow-up ctDNA positivity with recurrence-free interval (RFI). Further objectives are to assess the: sensitivity and specificity of ctDNA positivity for subsequent clinical recurrence; contribution of post-surgery baseline, post-adjuvant therapy, and pre-recurrence follow-up ctDNA results on RFI; time from positive ctDNA to clinical recurrence in subjects who had a positive ctDNA result; and compare the Oncotype Colon Recurrence Score estimate of 3 yr recurrence risk with the observed 3 yr recurrence rate. The primary analysis will use a Cox proportional hazards regression applied to the RFI with ctDNA result (positive or negative) measured at post-surgical baseline (or end of ACT if ACT was used) and serially after that as a single, time-dependent covariate. Protocol: 16-002/NSABP C-14. Support: NSABP Foundation. Clinical trial information: 05210283.

Comparison of MRD Detection in Autografts in Multiple Myeloma between Novel High-Sensitivity Euroflow-NGF and NGS

Background: Autologous stem cell transplantation (ASCT) remains the gold-standard treatment for multiple myeloma (MM). To date, we have reported the prognostic value of minimal residual disease (MRD) detection in autografts in an ASCT setting using EuroFlow next-generation flow (NGF) and next-generation sequencing (NGS) (Takamatsu et al., ASH 2018, 2020). The main problem with NGF is its lower sensitivity (2 × 10 -6) compared with that of NGS (<1 × 10 -6).Methods: We reanalyzed 11 autografts in which the MRD were negative on NGF but positive (n = 7) or negative (n = 4) on NGS (Takamatsu et al., ASH 2018, abstract #258) using 5-20 mL of autografts with NGF to increase the sensitivity of MRD detection. Additionally, we enrolled 9 patients with newly diagnosed MM, from whom 5-20 mL of apheresed autografts were cryopreserved. We included 20 patients with newly diagnosed MM. The median age at ASCT was 60 (range, 45-67) years, and the patients included 12 men and 8 women at International Staging System I (n = 3), II (n = 13), and III (n = 4), 6 of whom harbored high-risk chromosomal abnormalities, including t(4;14) (n = 3), t(14;16) (n = 1), del17p (n = 1), and t(4;14) and del 17p (n = 1). All patients received bortezomib-based chemotherapy for induction followed by melphalan at a dose of 200 mg/m 2 for conditioning before ASCT. Three patients received consolidation therapy with carfilzomib-lenalidomide-dexamethasone (n = 2) or bortezomib-lenalidomide-dexamethasone (n = 1), and 18 patients received lenalidomide (n = 16), thalidomide (n = 1), or thalidomide and lenalidomide (n = 1) maintenance. Frozen autografts (n = 20) were thawed for MRD assessment using NGF and NGS. The NGF method was based on a previous report (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD assay (Seattle, WA) (Ching et al., BMC Cancer 2020). The NGF method was modified to increase the sensitivity of MRD detection by capturing up to 6 × 10 7 cells.Results: Frozen autografts were used in this study; therefore, we performed a sensitivity test using a dilution of frozen/thawed primary MM cells in an autograft with NGF. The sensitivity test revealed a strong correlation between 5 × 10 -7 and 1 × 10 -4 MRD levels (Figure 1A; r = 0.999, P <0.0001). Next, MRD in autografts (n = 20) was evaluated using NGF and NGS. The sensitivity of NGS was 1.7 × 10 -7-8.7 × 10 -5 (median, 7 × 10 -7) using 2-4 mL of autografts; the sensitivity of NGF was 1.6 × 10 -7-3.7 × 10 -6 (median, 3 × 10 -7) using up to 20 mL of autografts based on the detection of ≥10 abnormal cells. MRD levels in autografts using NGF and NGS were correlated (Figure 1B; r = 0.979, P <0.0001). There was no discrepancy in the MRD negativity between both methods except for two cases (MRD-negative on NGF/positive on NGS [n = 2]). All high-risk chromosomal abnormality cases (n = 6) revealed MRD levels <10 -5 butonly three patients achieved MRD levels <10 -6. The best responses included 16 cases of a stringent complete response and 4 of a very good partial response. Three patients with positive MRD in the autograft exhibited progression. With a median follow-up period of 41 months after ASCT, the 3-year progression-free survival (PFS) was 90%, and the 3-year overall survival was 95%. There was no significant difference in the PFS based on the MRD levels between NGF and NGS. MRD negative cases tended to show better PFS than MRD positive ones (Figure 1C; P = 0.077 by NGF; P = 0.111 by NGS).Conclusion: The modified EuroFlow-NGF method may be used to assess MRD in frozen/thawed autografts, and its sensitivity may increase up to 5 × 10 -7, which is comparable to that of NGS. [Display omitted] DisclosuresYamashita: Janssen: Honoraria; Bristol-Myers Squibb: Honoraria; celgene: Honoraria; Takeda: Honoraria. Nakao: Novartis Pharma: Honoraria; Symbio: Consultancy; Kyowa Kirin: Honoraria; Alexion Pharma: Research Funding. Takamatsu: Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

P-043: Comparison of MRD detection of autografts in multiple myeloma between novel high-sensitivity EuroFlow-NGF and NGS

<h3>Background</h3> Autologous stem cell transplantation (ASCT) is still a gold standard treatment in multiple myeloma (MM). So far, we have reported the prognostic value of minimal residual disease (MRD) detection in autografts at ASCT setting using EuroFlow next-generation flow (NGF) and next-generation sequencing (NGS) (Takamatsu et al, ASH 2018). The main problem of EuroFlow-NGF is its lower sensitivity (2×10^-6) compared with that of NGS (<1×10^-6). <h3>Methods</h3> The study enrolled 9 newly-diagnosed MM patients whose frozen autografts' cells were preserved. The median age at ASCT was 52 (range 47-61) years and included 4 males and 5 females at ISS I (n=2), II (n=6) and III (n=1). Of there, 4 patients harbored high-risk chromosomal abnormalities including t(4;14) (n=1), t(14;16) (n=1), del17p (n=1), and t(4;14) and del 17p (n=1). All patients received bortezomib-based chemotherapy for induction together with melphalan at 200 mg/m² for conditioning before ASCT. Two patients received consolidation therapy with carfilzomib-lenalidomide-dexamethasone (KRD) and all patients received lenalidomide maintenance until progressive disease. Frozen autografts (n=9) and primary myeloma cells (n=1) were thawed for MRD assessment by EuroFlow-NGF and NGS. The EuroFlow-NGF method was based on the previous report (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Ching et al., BMC Cancer 2020). The EuroFlow-NGF method was modified to increase the sensitivity of MRD by capturing cells up to 5×10⁷. <h3>Results</h3> Because frozen autografts were used in this study, we performed the sensitivity test using the dilution of frozen/thawed primary MM cells in an autograft by EuroFlow-NGF. The sensitivity test revealed a strong correlation between 1×10^-7 and 1×10^-4 of MRD level (r=0.9996, p<0.0001). Next, MRD in autografts (n=9) was evaluated using EuroFlow-NGF and NGS. The sensitivity of NGS was 6×10^-7–8.7×10^-5 (median, 8×10^-7) using 2–4 mL of autografts; the sensitivity of EuroFlow-NGF was 4×10^- ⁷–8×10^-7 (median, 5×10^-7) using up to 20 mL of autografts. MRD levels in autografts using EuroFlow-NGF and NGS correlated with one another (r = 0.9964, P < 0.0001). There was no discrepancy of MRD levels between both methods: negative (n=2), 10^-6-10^-5 (n=4), 10^-5-10^-4 (n=2), 10-4 < (n=1). All high-risk chromosomal abnormality cases (n=4) achieved MRD level <10^-5. The response on day 100-300 post-ASCT included 6 sCR, 2 CR and 1 VGPR before consolidation/maintenance therapy and all patients achieved sCR at two years post-ASCT and maintained it at three years post-ASCT during lenalidomide maintenance. No progression was observed. <h3>Conclusions</h3> This modified EuroFlow-NGF method can assess MRD of frozen/thawed autografts and its sensitivity can be increased up to 4×10^-7 that is comparable to NGS.

Detection of Circulating Clonal Plasma Cells Using Next Generation Sequencing in Patients with AL Amyloidosis and Low Plasma Cell Burden: A Novel Discovery

Background: AL amyloidosis is a bone marrow disorder in which clonal plasma cells produce light chains that misfold and deposit in vital organs, such as the kidneys and heart, leading to organ failure and eventual death. Treatment is directed towards the clonal plasma cell population in an effort to halt the production of toxic light chains and recuperate organ function. Pallidini et al. demonstrated that almost 50% of patients with AL amyloidosis who achieved a complete hematologic response to prior therapy had minimal residual disease (MRD) detectable in their bone marrow by multiparametric flow cytometry (MPF)(1). Next generation gene sequencing (NGS) has been a successful tool in measuring MRD among patients with multiple myeloma(2) though it has yet to be evaluated in AL amyloidosis, a disease with a much smaller plasma cell burden at baseline (typically 5-10%), making the task of isolating an initial clonal sequence even more challenging. We sought to evaluate NGS as a method of isolating a clonal population of plasma cells among 10 treatment-naïve patients with systemic AL amyloidosis in a first-ever feasibility study.Methods: Patients were eligible if they had systemic AL amyloidosis with no prior treatment and without evidence of concurrent active multiple myeloma. In this study, feasibility was deemed successful if discovery of a clone could be achieved in 3 out of 10 of patients. Five cc's of peripheral blood and bone marrow aspirate were collected from each patient and processed for CD138 selection and DNA isolation/purification. De-identified samples were sent to Adaptive Biotech Inc. (Seattle, WA) for initial clonal identification using Adaptives NGS MRD assay. Genomic DNA was amplified by implementing consensus primers targeting the IGH complete (IGH-VDJH) locus, IGH incomplete (IGH-DJH) locus, immunoglobulin κ locus (IGK) and immunoglobulin l locus (IGL). The amplified product was sequenced and a clone identified based on frequency.Results: The average patient age was 63 years old (range: 45 to 74), 50% of which were female. All 10 patients with treatment naïve, biopsy-proven systemic AL amyloidosis had measurable disease based on serum electrophoresis and immunofixation and/or serum free light chain assay (Table 1). Three patients had only a free lambda monoclonal protein and the remaining 7 patients had a clonal heavy chain with an associated light chain. Bone marrow biopsies demonstrated clonal plasmacytosis of 20% or lower. The NGS MRD assay from Adaptive identified trackable clones in 8 patients (80%) (see Table 1). Three patients had at least one trackable sequence (range: 1 to 5 sequences) in the peripheral blood and 7 patients had at least one trackable sequence in the bone marrow aspirate (range: 1 to 7 sequences). Of the 3 patients with detectable clones in the peripheral blood, one had a clone that was not found in the bone marrow. No correlation was seen between the detection of a clone and standard measures of plasma cell tumor burden (SIFE, SPEP, UIFE, UPEP, and sFLCs).Conclusion: Due to the low clonal burden in patients with AL amyloidosis, it is often difficult to assess disease status, especially post-treatment. Here we report identification of an initial clone in 8 out of 10 treatment-naïve patients with systemic AL amyloidosis using NGS-based method. Importantly, we detected circulating clonal plasma cells in the peripheral blood in 3 patients. These encouraging results using NGS-based MRD detection method may enhance disease monitoring and improve patient care in this rare disease. These preliminary results have led us to expand our study to 30 patients who will be monitored for NGS-based MRD assessment following therapy.REFERENCES1. Palladini G, Massa M, Basset M, Russo F, Milani P, Foli A, et al. Persistence of Minimal Residual Disease By Multiparameter Flow Cytometry Can Hinder Recovery of Organ Damage in Patients with AL Amyloidosis Otherwise in Complete Response. Abstr 3261. 2016;2. Ladetto M, Brüggemann M, Monitillo L, Ferrero S, Pepin F, Drandi D, et al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia. 2014;28:1299-307. [Display omitted] DisclosuresJacob:Adaptive Biotechnologies: Employment, Equity Ownership.

Assessment of Minimal Residual Disease by Next Generation Sequencing in Peripheral Blood as a Complementary Tool for Personalized Transplant Monitoring in Myeloid Neoplasms.

Patients with myeloid neoplasms who relapsed after allogenic hematopoietic stem cell transplant (HSCT) have poor prognosis. Monitoring of chimerism and specific molecular markers as a surrogate measure of relapse is not always helpful; therefore, improved systems to detect early relapse are needed. We hypothesized that the use of next generation sequencing (NGS) could be a suitable approach for personalized follow-up post-HSCT. To validate our hypothesis, we analyzed by NGS, a retrospective set of peripheral blood (PB) DNA samples previously evaluated by high-sensitive quantitative PCR analysis using insertion/deletion polymorphisms (indel-qPCR) chimerism engraftment. Post-HCST allelic burdens assessed by NGS and chimerism status showed a similar time-course pattern. At time of clinical relapse in 8/12 patients, we detected positive NGS-based minimal residual disease (NGS-MRD). Importantly, in 6/8 patients, we were able to detect NGS-MRD at time points collected prior to clinical relapse. We also confirmed the disappearance of post-HCST allelic burden in non-relapsed patients, indicating true clinical specificity. This study highlights the clinical utility of NGS-based post-HCST monitoring in myeloid neoplasia as a complementary specific analysis to high-sensitive engraftment testing. Overall, NGS-MRD testing in PB is widely applicable for the evaluation of patients following HSCT and highly valuable to personalized early treatment intervention when mixed chimerism is detected.

Minimal Residual Disease in Autografts and Bone Marrow of Patients with Multiple Myeloma: 8-Color Multiparameter Flow Cytometry (EuroFlow-NGF) Vs. Next-Generation Sequencing

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognoses of patients with multiple myeloma (MM). However, most patients with MM ultimately relapse due to minimal residual disease (MRD). Next-generation multiparameter flow cytometry (MFC) (EuroFlow-NGF) and next-generation sequencing (NGS) are currently the standard methods to assess MRD. Aims: To compare the prognostic value of MRD detection in autografts and bone marrow (BM) cells using 8-color MFC (EuroFlow-NGF) and NGS (Adaptive Biotechnologies), and also MRD levels between fresh and cryopreserved autografts using NGF. Methods: The study enrolled 52 newly-diagnosed MM patients who underwent ASCT. The median age ASCT was 61 (range 41-69) years and included 29 males and 23 females at ISS I (n = 17), II (n = 23), and III (n = 12). Of these, 18 patients harbored high-risk chromosomal abnormalities including t(4;14) (n = 15), del17p and t(4;14) (n = 2), and complex (n = 1). Bortezomib-based chemotherapy was used for induction together with melphalan at 140 mg/m2 (n = 1) and 200 mg/m2 (n = 51) for conditioning before ASCT. 39 of 52 (75%) patients received maintenance therapy until progressive disease. The best responses achieved post-ASCT included 30 sCR, 4 CR, 15 VGPR, and 3 PR. Forty autografts, one from each MM patient, were analyzed using NGF and NGS protocols, and BM cells at pre/post-ASCT and autografts derived from 16 patients were analyzed using NGS. The EuroFlow-NGF method uses standard sample preparation; large numbers of cells are evaluated using an optimized 8-color antibody panel that facilitates accurate identification of discrimination between phenotypically aberrant plasma cells (aPCs) and their normal counterparts (Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). Eight additional autografts were used to assess MRD in both fresh and cryopreserved samples by NGF. Results: MRD was evaluated in 48 of 52 autografts (92%) using NGF and in 44 of 52 autografts (85%) using NGS. We identified aPCs in autografts based on multivariate analysis of individual cell populations (e.g., CD56+, CD19−, CyIgκ+, and CD117+). As the results of NGF revealed a strong correlation with respect to MRD in fresh vs. thawed autografts (r = 0.999, P &amp;lt; 0.0001), MRD was subsequently evaluated in thawed autografts. The sensitivity of NGF was 1 × 10−5-2 × 10−6; the sensitivity of NGS was 1 × 10−6. 28 of 48 (58%) of the autografts were MRD-positive by NGF; 30 of 44 (68%) of the autografts were MRD-positive by NGS. MRD levels in autografts using NGF and NGS correlated with one another (r = 0.69, P &amp;lt; 0.0001; Fig. 1A). MRD negative in autografts by NGF cases (MRDNGF (-)) and MRDNGS (-) tended to show better progression-free survival (PFS) than MRDNGF (+) (P = 0.195) and MRDNGS (+) (P = 0.156), respectively. Furthermore, MRDNGS (-) showed significantly better overall survival (OS) than MRDNGS (+) (P = 0.03) (Fig. 1C) while MRDNGF (-) showed better OS than MRDNGF (+) (P = 0.09) (Fig. 1B). Our data revealed only a minimal correlation between MRD in the autografts (median 1.1 × 10−5,range 0-7.29 × 10−4) and in the BM cells at pre-ASCT (median 5.05 × 10−3,range 6 × 10−6-2.64 × 10−1; r = 0.09, P = 0.7) or at post-ASCT (median 2.11 × 10−4,range 0-9.09 × 10−3; r = 0.14, P = 0.6); MRD detected in the autografts was &amp;gt; 27 times lower than that detected in pre-ASCT BM cells, and MRD detected in the post-ASCT BM cells was &amp;gt; 3 times lower than that detected in pre-ASCT BM cells except for one case in which the ratio was increased by two times. Interestingly, while MRD was detected in all BM cells at pre-ASCT (n = 16), 4 of 16 (25%) of these autografts were MRDNGS-negative. The median of MRD levels of the 4 cases in pre-ASCT and post-ASCT BM cells were 4.14 × 10−4 (range 6-583 × 10−6)and 1.8 × 10−5 (range 0-27 × 10−6), respectively. Conclusion: Although EuroFlow-NGF is a rapid and accurate method for detecting MRD, NGS was more sensitive and provided greater prognostic value than EuroFlow-NGF. Disclosures Takamatsu: Adaptive Biotechnologies: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; SRL: Consultancy, Research Funding. Takezako:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Research Funding; Abbvie: Research Funding. Nakao:Symbio: Consultancy; Kyowa Kirin: Honoraria; Alexion: Research Funding; Novartis: Honoraria.

Upgraded Standardized Minimal Residual Disease Detection by Next-Generation Sequencing in Multiple Myeloma

Minimal residual disease (MRD) is one of the most powerful prognostic factors in multiple myeloma. Therefore, standardization and easy operation of MRD testing are crucial. Previously, we validated the sensitivity of 10-5 with spike in of plasmid controls for a standardized next-generation sequencing (NGS) approach based on triplicate measurements of bone marrow by LymphoTrack-MiSeq platform. To improve the technique, we replaced spike-in plasmid controls by genomic DNA from myeloma cells. A spike-in control of 0.001% was consistently detected in all 19 samples tested, confirming a uniform sensitivity of 10-5 of this upgraded protocol. MRD was detected in 14 of 19 patients (78%), with a significant (P=0.04) impact on progression-free survival based on high versus low MRD levels. Reproducibility of detection was confirmed by the extremely small interrun variation tested in three patients. In nine patients, MRD was tested in parallel by allele-specific oligonucleotide real-time quantitative PCR. NGS showed an improved sensitivity and provided quantification of MRD for cases assigned positive but not quantifiable by real-time quantitative PCR, obviating the need of patient-specific probes/primers. In summary, the use ofgenomic DNA as spike-in control simplifies NGS detection of MRD while preserving the sensitivity of 10-5. Validity and reproducibility of the standardized procedure were verified, and the prognostic impact of NGS-based MRD in myeloma was confirmed.

Archived Bone Marrow Smears Are an Excellent Source for NGS-Based Mutation Detection in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease with a great variety of somatic driver mutations. Each AML carries specific (combinations of) acquired mutations, which enables minimal residual disease (MRD) detection in virtually every patient with next-generation sequencing (NGS). In recent years there has been an increasing interest to conduct longitudinal molecular MRD detection to better estimate prognosis, monitor treatment efficacy, as well as prediction of an impending relapse. The development of molecular monitoring on archived samples has been slow because only limited numbers of samples collected during the course of the disease were biobanked. We have systematically investigated whether archived May-Giemsa Grünwald (MGG)-stained bone marrow slides could be an alternative source of material to conduct NGS-based molecular MRD detection in AML. Since MGG-stained bone marrow evaluations are performed at high frequency to evaluate the response on therapy and repopulation of the bone marrow, we focused on these bone marrow slides taken at regular intervals during treatment.We initial tested DNA isolation protocol on MGG-stained and unstained morphology slides, which were archived over the years. The DNA could be isolated from the MGG-stained (n=16) and unstained bone marrow slides (n=16). However, we were unable to isolate genomic DNA from all of the unstained slides (11 out of 16). Furthermore, the DNA concentration from the unstained slides was much lower than the MGG-stained slides. We next isolated DNA from MGG-stained bone marrow slides of 43 AML samples at diagnosis and relapse of which the mutation status of genes frequently mutated in myeloid diseases was known. DNA isolated from Ficoll-purified mononuclear cells of the same pairs was previously sequenced by NGS using the Illumina Trusight Myeloid panel. NGS-based mutation screening with the same panel on DNA obtained from morphology slides revealed that detection of the driver mutations was feasible. In fact, all known mutations were detectable at diagnosis and relapse. Interestingly, in 91% (39 out of 43) of cases, the variant allele frequency (VAF) of the mutations were virtually identical to those detected using the DNA from MNCs. Representative examples are shown in Figure 1 (A and B). In a minority of samples, the VAFs differed between MNCs and bone marrow slides (3 cases VAF higher in MNCs and 1 case VAF lower in MNCs). These findings indicate that DNA isolated from bone marrow slides are a rich source for NGS-based mutation detection in AML at diagnosis and relapse.We next sought to evaluate the use of MGG-stained bone marrow slides to study the mutations kinetics in time from AML diagnosis to relapse. To perform this analysis 30 paired-AML (diagnosis - relapse) with variable time to relapse (range: 200 to 4004 days) were selected. We collected the archived bone marrow slides at different time points between diagnosis and relapse, isolated DNA and performed NGS-based mutation detection. The NGS analysis of the 30 paired-AML cases showed different patterns of mutation dynamics and clonal evolution. Representative examples are shown in Figure 1 (C and D). Interestingly, by this systematic analyses of somatic mutations at various time points clear distinction could be made between leukemia-specific mutations and mutations associated with clonal hematopoiesis at the time of complete remission in the individual AML cases.Our study uncovers an unutilized source of material for mutation detection at diagnosis and relapse in AML. These materials are widely available and well archived. Furthermore, our study reveals the potential use of MGG stained bone marrow morphology slides to track mutations and (sub)clones in AML, which is applicable to virtually every AML patient and possibly valuable for other hematological malignancies as well. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Residual Allelic Burden Measured By Next-Generation Sequencing (NGS) at Remission Provides Limited Prognostic Value to Predict Relapse and Long-Term Outcome in Core Binding Factor Acute Myeloid Leukemia (CBF-AML)

IntroductionNGS-based detection of minimal residual disease (MRD) has been successfully demonstrated for its correlation with relapse risk in AML. However, in a subtype of AML, CBF-AML, its clinical relevance of residual allelic burden at complete remission (CR) has not been fully explored. The standard MRD detection method in CBF-AML is quantitative PCR (qPCR). In this study, we aimed to explore applicability and feasibility of NGS-based MRD detection in CBF-AML taking various approaches, rather than simply using residual allele burden at CR.Patients and MethodsFifty-three patients (pts) diagnosed with CBF-AML were enrolled in this study (31 pts with RUNX1-RUNX1T1 and 22 pts with CBFB-MYH11). All 53 patients achieved complete remission (CR). We performed targeted deep sequencing on 84 genes in 106 samples collected at diagnosis and at CR as well as T-cell (n = 53, CD3+) fraction as a control using Illumina Hiseq 2500. Mean on-target coverage for 159 sequenced was 1,572x. The level of RUNX1-RUNX1T1 was measured at diagnosis and at CR for 29 patients using qPCR.ResultsAt diagnosis, 99 mutations from 49 pts (n = 49/53, 92%) were detected, where median number of mutations for 49 pts was 2 (range 1-6). Consistent with previous studies, KIT (36%), NRAS (32%), KRAS (17%), ASXL2 (15%) were commonly mutated. Among mutations detected at diagnosis, cKIT-D816 mutation and mutations in genes in DNA methylation pathway (DNMT3A and TET2) were associated with higher risk of relapse (5.29, [1.89 - 14.87], p = 0.002 and 3.15 [1.07 - 9.26], p= 0.037, respectively). In CR samples, 46 mutations from 32 pts were still detectable (46/99, 46%, mean VAF: 0.60%, range 0.04%-6.28%, Fig A). Only 4 mutations from 2 pts were over 2.5% (2 in TET2, 1 in ASXL1, and 1 in U2AF1). When tracing back at diagnosis, allelic burden of 46 mutations detected at remission were higher than 53 cleared mutations (p < 0.002), indicating clonal mutations are more likely to be detected at CR (Fig A). They were mostly in genes associated with activated signaling (32/46, 70%). When considering complete clearance rate, mutations in genes associated with activated signaling, DNA methylation, and spliceosome tended to be persistent at CR (32/64, 4/5, and 1/1), whereas mutations in cohesin complex and chromatin modifiers were mostly completely cleared (0/8 and 4/13).We then assessed clinical relevance of mutation clearance from various perspectives. We did not find association of mutation clearance at 0.3% (MC03) with OS (p = 0.43) or with relapse risk (p = 0.8, Fig B). Complete mutation clearance also did not show significant association with OS and relapse risk. Among 29 pts with RUNX1-RUNX1T1 with available qPCR data, 20 pts were MRD-positive by qPCR at CR. Nine pts who achieved MRD-negative also achieved MC03 as well. When considering only MRD-positive pts, achievement of MC03 did not affect OS (p = 0.69) and relapse incidence (p = 0.86, Fig C). Lastly, we assessed whether persistence of high risk mutations at CR (cKIT-D816, DNMT3A, and TET2) is associated with higher risk of relapse, but complete clearance of KIT-D816 mutation also did not affect OS and relapse incidence (p = 0.94 and p = 0.40, respectively, Fig D). We were not able to analyze DNMT3A and TET2 mutations as only 1/5 mutation was cleared.ConclusionCurrent study demonstrates that low residual allelic burden measured by NGS at CR does not provide additional clinically relevant information in addition to baseline mutation profile nor qPCR-based MRD in CBF-AML [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of Minimal Residual Disease Detection in Autografts of Patients with Multiple Myeloma between 8-Color Multiparameter Flow Cytometry (EuroFlow) and Next-Generation Sequencing

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis 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. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM.Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts.Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used.Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively.Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. [Display omitted] DisclosuresTakamatsu:Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Simple deep sequencing-based post-remission MRD surveillance predicts clinical relapse in B-ALL

BackgroundNext-generation sequencing (NGS) of the rearranged immunoglobulin heavy-chain gene has emerged as a highly sensitive method to detect minimal residual disease (MRD) in B acute lymphoblastic leukemia/lymphoma (B-ALL). However, a sensitive and easily implemented NGS methodology for routine clinical laboratories is lacking and clinical utility of NGS-MRD surveillance in a post-remission setting to predict clinical relapse has not been determined.MethodsHere we described a simple and quantitative NGS platform and assessed its performance characteristics, quantified NGS-MRD levels in 122 B-ALL samples from 30 B-ALL patients, and explored the clinical merit of NGS-based MRD surveillance.ResultsThe current NGS platform has an analytic sensitivity of 0.0001% with excellent specificity and reproducibility. Overall, it performs better than routine multi-color flow cytometry (MCF) in detecting MRD. Utilizing this assay in MRD surveillance in a post-remission setting showed that it detected conversion to positive MRD (CPMRD) in patients with NGS-based molecular remission much earlier than MCF, and that positive MRD conversion could be detected as early as 25.6 weeks prior to clinical relapse in closely surveilled patients. Post-remission CPMRD, but not NGS-based MRD positivity at end of induction, can accurately predict clinical relapse in our limited cohort of B-ALL patients.ConclusionsThis pilot proof-of-concept study illustrates the clinical utility of a simple, sensitive, and clinically feasible MRD detection platform in post-remission NGS-based MRD surveillance and early relapse detection in B-ALL patients.

An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants

Monitoring of minimal residual disease (MRD) has become an important clinical aspect for early relapse detection during follow-up care after cancer treatment. Still, the sensitive detection of single base pair point mutations via Next-Generation Sequencing (NGS) is hampered mainly due to high substitution error rates. We evaluated the use of NGS for the detection of low-level variants on an Ion Torrent PGM system. As a model case we used the c.1849G > T (p.Val617Phe) mutation of the JAK2-gene. Several reaction parameters (e.g. choice of DNA-polymerase) were evaluated and a comprehensive analysis of substitution errors was performed. Using optimized conditions, we reliably detected JAK2 c.1849G > T VAFs in the range of 0.01–0.0015% which, in combination with results obtained from clinical data, validated the feasibility of NGS-based MRD detection. Particularly, PCR-induced transitions (mainly G > A and C > T) were the major source of error, which could be significantly reduced by the application of proofreading enzymes. The integration of NGS results for several common point mutations in various oncogenes (i.e. IDH1 and 2, c-KIT, DNMT3A, NRAS, KRAS, BRAF) revealed that the prevalent transition vs. transversion bias (3.57:1) has an impact on site-specific detection limits of low-level mutations. These results may help to select suitable markers for MRD detection and to identify individual cut-offs for detection and quantification.

Prognostic value of sequencing-based minimal residual disease detection in patients with multiple myeloma who underwent autologous stem-cell transplantation.

BackgroundMost patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper response are required.Patients and methodsWe retrospectively analyzed 125 patients with MM who underwent high-dose melphalan plus autologous stem-cell transplantation (ASCT) to detect MRD in autograft/bone marrow (BM) cells using a next-generation sequencing (NGS)-based method and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR).ResultsNGS-based method was applicable to 90% and this method had at least one to two logs greater sensitivity compared to ASO-PCR. MRD negative by NGS [MRDNGS(−)] (defined as <10−6) in post-ASCT BM cases (n = 26) showed a significantly better progression-free survival (PFS) (96% at 4 years, P < 0.001) and overall survival (OS) (100% at 4 years, P =0.04) than MRDNGS(+) in post-ASCT BM cases (n = 25). When restricting the analysis to the 39 complete response cases, patients who were MRDNGS(−) (n = 24) showed a significantly better PFS than those that were MRDNGS(+) (n = 15) (P =0.02). Moreover, MRDNGS(−) in post-ASCT BM cases (n = 12) showed significantly a better PFS than MRDNGS(+) cases (n = 7) where MRD was not detected by ASO-PCR (P = 0.001). Patients whose autografts were negative by NGS-based MRD assessment (<10−7) (n = 19) had 92% PFS and 100% OS at 4 years post-ASCT. Conversely, the NGS-based MRD positive patients who received post-ASCT treatment using novel agents (n = 49) had a significantly better PFS (P = 0.001) and tended to have a better OS (P= 0.214) than those that were untreated (n = 33).ConclusionsLow level MRD detected by NGS-based platform but not ASO-PCR has significant prognostic value when assessing either the autograft product or BM cells post-ASCT.