scholarly journals Validation of Next Generation Sequencing-Based Clonality Assays for Diagnosis and Minimal Residual Disease Tracking in Lymphoid Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2959-2959
Author(s):  
Haiyan Wu ◽  
Feng Li ◽  
Jiacheng Cai ◽  
Ke Zhang ◽  
Xin Zheng ◽  
...  
Keyword(s):  
Cell Lines ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Clinical Samples ◽  
Blast Phase ◽  
Lymphoid Malignancies ◽  
Mixed Reaction ◽  
Generation Sequencing ◽  
Clonality Assessment ◽  
Minimal Residual

Abstract Introduction: B or T cell receptor (BCR/TCR) clonal rearrangements have served as important diagnostic markers and minimal residual disease (MRD) tracking markers to guide treatment decisions in lymphoid malignancies. Next generation sequencing (NGS)-based BCR/TCR clonality assessment allows easier sample preparation, higher sensitivity and simpler standardization than flow cytometry and polymerase chain reaction (PCR)-based assays. Here, we developed NGS-based BCR/TCR clonality assays to identify and track disease-associated clonotypes of IGH, IGK, IGL, TCRB and TCRG rearrangements and BCL1/2-IGH translocations in lymphoid malignant cells. Our studies validated the analytical performance of the assays using genomic DNA (gDNA) from cell lines and patient samples diagnosed with acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), lymphoma and lymphoid blast phase chronic myeloid leukemia (BP-CML), as well as peripheral blood gDNA and circulating tumor DNA (ctDNA) from lymphoma patients. Methods: Our BCR/TCR clonality assays were based on two rounds of multiplex PCR followed by NGS. In the first-round PCR, the sequences of complementary determining region 3 (CDR3) in rearranged immune receptor genes were amplified by multiplex primers, and sample-specific index and NGS adapters were then added in the second-round PCR. Sequencing was performed on NovaSeq 6000 System and processed with customized bioinformatics pipelines. BCR clonality assays could identify IGH (V H-D H-J H or D H-J H), IGK (V κ-J κ, V κ-Kde and intronRSS-Kde) and IGL (V λ-J λ) rearrangements, as well as BCL1-IGH and BCL2-IGH translocations. TCR clonality assays could detect TCRB (V β-D β-J β) and TCRG (V γ-J γ) rearrangements. To evaluate the performance of our assays, we detected BCR or TCR clonality in gDNA from 4 cell lines and 40 clinical samples with ALL, MM, CLL and lymphoma, as well as paired chronic- and blast-phase samples of CML. Limit-of-detection (LOD) was estimated by clinical samples and cell lines with a background of peripheral blood gDNA from healthy donors. Linearity of detection was established with gDNA of cell lines spiked into normal gDNA to generate across orders of magnitude of clonal frequencies. To ensure consistent performance of the assays, we tested separate reactions and a single mixed reaction for IGH VDJ, IGH DJ, IGK and IGL. In addition, peripheral blood gDNA matching aforementioned lymphoma cases and ctDNA samples were also tested by our assays. Results: Both BCR clonality of B-cell lymphoid malignancies (B-ALL, MM, CLL and lymphoma) and TCR clonality of T-cell lymphoid malignancies (T-ALL and lymphoma) showed above 90% positive detection rate and mostly positive in more than one receptor gene. Chronic and blast phase samples from the same CML patient showed an identical dominant clonotype. The assays had high sensitivity, with LoD defined between 1 to 2 malignant cells in both BCR and TCR clonality assessment. Linearity was observed with clonal frequencies from 1 to 10 -6, which indicated consistence between observed and expected frequencies. The sequencing results with a single adjusted mixed reaction for IGH VDJ, IGH DJ, IGK and IGL were comparable to that with separate reactions, suitable for both diagnosed samples and MRD samples, suggesting the robustness of the assays. Our testing results also showed that peripheral blood gDNA of lymphoma patients carried identical clonotypes found in malignant tissues and ctDNA. Conclusion: We characterized and validated the performance of our NGS -based BCR/TCR clonality assays. We also demonstrated its potential application as a highly sensitive tool for diagnosis and MRD tracking for lymphoid malignancies, including ALL, MM, CLL, lymphoma and even CML at risk of lymphoid blast transformation. Disclosures No relevant conflicts of interest to declare.

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

2020 ◽  
Vol 9 (12) ◽  
pp. 3818
Author(s):  
Paula Aguirre-Ruiz ◽  
Beñat Ariceta ◽  
María Cruz Viguria ◽  
María Teresa Zudaire ◽  
Zuriñe Blasco-Iturri ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Mixed Chimerism ◽  
Clinical Relapse ◽  
Next Generation ◽  
Myeloid Neoplasms ◽  
Generation Sequencing ◽  
Minimal Residual

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.

Next-Generation Sequencing Based Minimal Residual Disease Assessment in Peripheral Blood RNA from Multiple Myeloma Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3286-3286
Author(s):  
Jeffrey L. Wolf ◽  
Katherine A. Kong ◽  
Jenai Wilmoth ◽  
Joshua Bayes ◽  
Amy Marsala ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
High Frequency ◽  
Dna Analysis ◽  
Residual Disease ◽  
Employment Equity ◽  
Dna And Rna ◽  
Equity Ownership ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract Introduction: Multiple myeloma (MM) is characterized by the presence of monoclonal protein (M-protein) in serum and/or urine, clonal plasma cell accumulation in bone marrow, and related organ or tissue impairment. MM patients are monitored during and after therapy using immunoglobulin, M-protein and free light chain assays. Minimal residual disease (MRD) assessment of bone marrow samples from MM patients in complete remission (CR) using next-generation sequencing (NGS)-based technology has been shown to have prognostic value, and previous studies have demonstrated evidence of circulating myeloma cells in peripheral blood. In this prospective study, we compared the sensitivity of RNA- and DNA-based MRD assays in peripheral blood (PB) and bone marrow (BM) samples collected from MM patients. Methods: Matched BM and PB samples were obtained from 50 MM patients in various stages of their disease. Diagnostic BM samples were used to identify clonal immunoglobulin sequences (i.e. clonotypes) unique to each MM patient. Matched BM and PB samples were then assessed for the presence of the MM clonotype. Using universal primer sets, we amplified immunoglobulin (IgH) variable (V), diversity (D), and joining (J) gene segments, the incomplete IgH-DJ rearrangement, and IgK receptors. Genomic DNA samples were assessed for all 3 receptors, while RNA samples were only assessed for the functional IgH-VDJ and IgK receptors. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype quantification. Myeloma-specific IgH, IgK, and IgH-DJ clonotypes were identified for each patient based on their high frequency at diagnosis. The presence of the myeloma clonotype was then assessed in BM cells (DNA), PB cells (DNA and RNA), and PB plasma (DNA) samples. Myeloma clonotype levels were calculated as previously described (Martinez-Lopez et al. 2014). Results: High-frequency myeloma clonotypes were identified in the BM of all 50 patients. We performed a sequence-level assessment of each clonotype to determine whether it was also evaluable in RNA. Non-functional clonotypes (e.g. IgH-DJ and kappa deleting element clones, frameshift/nonsense mutations, nonfunctional V or J segments, loss of cysteine in CDR3) were excluded from the analysis. 37 evaluable RNA clonotypes were identified in 28 of the 50 patients (56%). We compared the sensitivity of MRD assessment using DNA and RNA extracted from PB mononuclear cells. 20 clonotypes were qualitatively concordant in both DNA and RNA. 17 clonotypes were discordant, with all 17 clonotypes being detectable in RNA but not in DNA (Figure 1A). Only 4 of 21 (19%) clonotypes detectable in RNA were also detectable in DNA in the PB. Thus, RNA provides a clear sensitivity advantage over DNA analysis in PB cellular samples. We then investigated whether the sensitivity advantage conferred by RNA in PB cells was equivalent or superior to the sensitivity of DNA analysis in BM cells. On a patient level, 23 patients were qualitatively concordant in the PB and BM assays. 5 patients were discordant, with all patients being positive in BM and negative in PB. 4 of the discordant patients were positive at low levels in the BM (1-10 MM clonotype molecules per 1 million cells). On a clonotype level, 28 clonotypes were qualitatively concordant in BM DNA and PB RNA. 9 clonotypes were discordant, with all 9 clonotypes being positive in BM DNA and negative in PB RNA (Figure 1B). Nevertheless, PB RNA detected MRD in 21 of 30 with measurable disease in the BM DNA (ie, 70% sensitivity). These results demonstrate that MRD assessment using PB RNA is more informative than PB DNA in MM; however, despite the sensitivity improvement provided by RNA, MRD assessment in the BM DNA remains the superior sample source for MRD assessment, particularly when achievement of MRD negativity is the goal of therapy. Conclusions: NGS-based MRD assessment of BM in myeloma patients has been shown to have prognostic value. PB-based MRD assessment would improve clinical MRD assessment and monitoring paradigms. Our results demonstrate that RNA provides increased sensitivity for blood-based MRD assessment. Additional assay optimization to improve the fraction of clonotypes evaluable in RNA and to enhance sensitivity compared with BM is necessary before PB MRD monitoring in MM patients can be incorporated into routine clinical practice. Figure Figure. Disclosures Wolf: Telomere Diagnostics: Consultancy; Pharmacyclics: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Bayes:Adaptive Biotechnologies Corp: Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Martin:Sanofi: Research Funding; Amgen: Research Funding.

scholarly journals Minimal Residual Disease Detection By Next Generation Sequencing in Adult B-Cell Acute Lymphoblastic Leukemia (ALL) Patients Treated on SWOG Trial S0333

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2399-2399 ◽  
Author(s):  
Olga Sala Torra ◽  
Megan Othus ◽  
David W Williamson ◽  
Brent L. Wood ◽  
Ilan Kirsch ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
B Cell ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Next Generation ◽  
Employment Equity ◽  
Equity Ownership ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract We used next generation sequencing (NGS) of the immunoglobulin genes to evaluate minimal residual disease (MRD) in 153 specimens from 32 patients with newly diagnosed adult B cell ALL enrolled in the phase II SWOG S0333 multi-center study. We used the clonoSEQTM assay developed by Adaptive Biotechnologies that detects 1 leukemic cell in a background of 1 million nucleated cells and focuses in the B cell receptor (Ig). Initially, a set of pre-study specimens was sequenced in order to identify the precise sequence of the VDJ or DJ fragments. Clones representing more than 5% of the total repertoire of IgH molecules profiled were considered potentially leukemic. The follow-up specimen IgH repertoire sequences were compared to the diagnostic clonal ones and the leukemic marker sequence(s) previously identified were searched for explicitly. At least one Ig clonotype was detected in 29/32 (91%) cases analyzed. The 3 remaining cases were reviewed, and in 2 cases the specimens available for NGS had been reported as having no blasts by morphology. The leukemic clonal sequence was a complete VDJ rearrangement in 17/32 patients (53%), an incomplete DJ rearrangement in 8/32 patients (25%), and in 3/32 cases both VDJ and DJ rearrangements coexisted. One patient had a kappa light chain rearrangement. 17/32 (53%) cases contained more than one IgH rearrangement at diagnosis (median=2, range: 1 - 4). One of our patients is a potential case of therapy driven clonal selection. He presented at diagnosis with two related clones, one representing 75% of VDJ sequences and the second one 18%. At relapse, the second clone was responsible for most of the VDJ sequences (95%). The NGS results were compared to the MRD results detected by multiparameter flow cytometry (MFC) in 66 specimens analyzed by both methods. The concordance between the methods in the qualitative determination of the presence or absence of leukemia was 82% (54/66). In 12 specimens (18%) MRD was detected by sequencing but not by MFC. One specimen had MRD detected at very low levels by MFC and was negative by NGS. Our study includes 54 paired bone marrow (BM) and peripheral blood (PB) specimens. The median values of leukemia detected by NGS were 6-fold higher in BM than PB (range: 0.38 - 821-fold). Twenty-five pairs show no detectable MRD in either specimen. MRD was still detectable in 20 of the remaining 29 PB cases (for one of the pairs the BM specimen was negative). In 6/9 (67%) pairs of samples with disease detectable in BM but not in PB by NGS, no MRD was detected by MFC in the BM specimen. Lastly, outcome analysis was conducted in 21/32 patients with specimen available for MRD studies at the time of registration to second induction. Patients without MRD by NGS had a 5-year relapse free survival (RFS) of approximately 80%, while patients with MRD positive by both NGS and flow have the poorest outcome (p = 0.003) (see Figure). Patients with MRD detectable only by NGS have and intermediate RFS (p = 0.078, and p = 0.04 when compared to patients with MRD negative by both techniques, and patients with leukemia detected both by NGS and flow respectively). These results suggest that MRD detection by immunoglobulin gene sequencing is a very sensitive technique, and may identify patients with an excellent survival. Moreover, the increased sensitivity of the method may allow peripheral blood testing to supplement routine marrow sampling for MRD determination. Figure 1 Figure 1. Disclosures Williamson: Adaptive Biotechnologies: Employment, Equity Ownership. Kirsch:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership.

Analysis of minimal residual disease in bone marrow by NGF and in peripheral blood by mass spectrometry in newly diagnosed multiple myeloma patients enrolled in the GEM2012MENOS65 clinical trial.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 8010-8010
Author(s):  
Noemi Puig ◽  
Bruno Paiva ◽  
Teresa Contreras ◽  
M. Teresa Cedena ◽  
Laura Rosiñol ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Prognostic Value ◽  
Residual Disease ◽  
Newly Diagnosed ◽  
Time Points ◽  
Minimal Residual

8010 Background: Analysis of minimal residual disease (MRD) in the bone marrow (BM) of patients with multiple myeloma (MM) is accepted by the IMWG to evaluate treatment efficacy and is a well-established prognostic factor. However, there is an unmet need to explore the clinical value of MRD in peripheral blood (PB). Methods: Newly diagnosed MM patients enrolled in the GEM2012MENOS65 trial received six induction (Ind) cycles of bortezomib, lenalidomide, and dexamethasone (VRD) followed by autologous stem cell transplantation (ASCT) and 2 further cycles of consolidation (Cons) with VRD. MRD was analyzed in BM using Next Generation Flow (NGF) and in serum by Mass Spectrometry (MS) using IgG/A/M, κ, λ, free κ and free λ specific beads, both after Ind, at day 100 after ASCT, and after Cons. Sequential samples from the first 184 patients were analyzed. Results: Results of both methods were in agreement (NGF+/MS+ and NGF-/MS-) in 83% of cases post-Ind (152/184), 80% post-ASCT (139/174) and 76% post-Cons (128/169). Stratifying by the log range of MRD by NGF, discordances (NGF+/MS- and NGF-/MS+) seemed to increase at the lower MRD ranges, being 22%, 21% and 19% from ≥10−5 to <10−4 and 21%, 21%, 23% at ≥x10−6(post-Ind, ASCT and Cons, respectively). Analysis of discordances showed that they could be partly explained by the higher percentages of cases found to be positive by MS as compared by NGF at part of the time-points analyzed and at each log range of MRD. From ≥10−5 to <10−4, MRD was detected by NGF in 36%, 28%, 20% of cases post-Ind, ASCT and Cons, respectively vs MS in 37%, 29%, 21% of them; at ≥x10−6, NGF was positive in 11%, 14%, 19% of cases vs MS in 23%, 19% and 16% of them. Considering NGF as a reference, the negative predictive value (NPV) of MS per MRD range (≥10−5 to <10−4 and ≥x10−6, respectively) was: post-Ind: 83% (p<0,0001), 94% (p=0,034); post-ASCT 86% (p<0,0001), 90% (p=0,022); post-Cons 89% (p<0,0001), 85% (p=0,0469). Despite these discordances, the prognostic value of each technique in terms of undetectable MRD and progression-free survival (PFS) was consistent at all time-points (Table) and further, discordant cases (NGF+/MS- and NGF-/MS+) did not display a significantly different PFS as compared to NGF-/MS- cases. Conclusions: The results of MRD assessed by NGF in BM and by MS in PB show a significant concordance and are associated with a similar prognostic value analyzed in terms of PFS. Given its high NPV, MRD in peripheral blood by MS provides a gateway for BM aspiration/biopsy and MRD assessment by NGF.[Table: see text]

Droplet digital PCR for the simultaneous analysis of minimal residual disease and hematopoietic chimerism after allogeneic cell transplantation

2019 ◽  
Vol 57 (5) ◽  
pp. 641-647 ◽  
Author(s):  
Miguel Waterhouse ◽  
Dietmar Pfeifer ◽  
Jesus Duque-Afonso ◽  
Marie Follo ◽  
Justus Duyster ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Digital Pcr ◽  
Mixed Chimerism ◽  
Clinical Samples ◽  
Molecular Remission ◽  
Remission Status ◽  
Hematopoietic Chimerism ◽  
Minimal Residual

Abstract Background Minimal residual disease (MRD) and hematopoietic chimerism testing influences clinical decision and therapeutic intervention in patients after allogeneic stem cell transplantation (HSCT). However, treatment approaches to induce complete donor chimerism and MRD negativity can lead to complications such as graft-versus-host disease (GvHD) and marrow aplasia. Therefore, there is a need for comprehensive characterization of the molecular remission status after transplantation. Methods We analyzed 764 samples from 70 patients after HSCT for the simultaneous measurement of chimerism and molecular targets used for MRD testing with a digital PCR (dPCR) platform. Results Mixed chimerism (MC) was detected in 219 samples from 37 patients. The mean percentage of host derived DNA in these clinical samples was 4.3%. Molecular relapse with a positive MRD marker and/or increased WT1 expression was observed in 15 patients. In addition to WT1 overexpression, other MRD positive markers were: NPM1 (Type A, B, K), DNMT3A (R882H), MLL-PTD, IDH1 (R132H) and KRAS (G12S). Increasing MC was observed in 15 patients. This group of patients showed either a positive MRD marker, increased WT1 expression or both. Next, we analyzed whether MC or the molecular target for MRD was first detected. MC and MRD marker positivity in this group was first detected in six and two patients, respectively. In the remaining seven patients MC and MRD positivity was detected simultaneously. Conclusions The combination of MRD and chimerism markers in a dPCR platform represents a practical, sensitive and accurate diagnostic tool for the comprehensive assessment of the molecular remission status of patients undergoing HSCT.

Flow cytometric minimal residual disease assessment of peripheral blood in acute lymphoblastic leukaemia patients has potential for early detection of relapsed extramedullary disease

2018 ◽  
Vol 71 (7) ◽  
pp. 653-658 ◽  
Author(s):  
Alissa Keegan ◽  
Karry Charest ◽  
Ryan Schmidt ◽  
Debra Briggs ◽  
Daniel J Deangelo ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Minimal Residual Disease ◽  
Lymphoblastic Leukaemia ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Diagnostic Value ◽  
Disease Assessment ◽  
Extramedullary Disease ◽  
Systemic Relapse ◽  
Minimal Residual

ObjectivesTo evaluate peripheral blood (PB) for minimal residual disease (MRD) assessment in adults with acute lymphoblastic leukaemia (ALL).MethodsWe analysed 76 matched bone marrow (BM) aspirate and PB specimens independently for the presence of ALL MRD by six-colour flow cytometry (FC).ResultsThe overall rate of BM MRD-positivity was 24% (18/76) and PB was also MRD-positive in 22% (4/18) of BM-positive cases. We identified two cases with evidence of leukaemic cells in PB at the time of the extramedullary relapse that were interpreted as MRD-negative in BM.ConclusionsThe use of PB MRD as a non-invasive method for monitoring of systemic relapse may have added clinical and diagnostic value in patients with high risk of extramedullary disease.

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