Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.

Genetic alterations and MRD refine risk assessment for KMT2A-rearranged B-cell precursor ALL in adults: a GRAALL study.

KMT2A-rearranged (KMT2A-r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is widely recognized as a high-risk leukemia in both children and adults. However, there is a paucity of data on adults treated in recent protocols, and the optimal treatment strategy for these patients is still a matter of debate. In this study, we set out to refine the prognosis of adult KMT2A-r BCP-ALL treated with modern chemotherapy regimen and investigate the prognostic impact of comutations and minimal residual disease (MRD). Of 1091 adult patients with Philadelphia-negative BCP-ALL enrolled in 3 consecutive trials from the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL), 141 (12.9%) had KMT2A-r, with 5-year cumulative incidence of relapse (CIR) and overall survival (OS) rates of 40.7% and 53.3%, respectively. Molecular profiling highlighted a low mutational burden in this subtype, reminiscent of infant BCP-ALL. However, the presence of TP53 and/or IKZF1 alterations defined a subset of patients with significantly poorer CIR (69.3% vs 36.2%; P = .001) and OS (28.1% vs 60.7%; P = .006) rates. Next, we analyzed the prognostic implication of MRD measured after induction and first consolidation, using both immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements and KMT2A genomic fusion as markers. In approximately one-third of patients, IG/TR rearrangements were absent or displayed clonal evolution during the disease course, compromising MRD monitoring. In contrast, KMT2A-based MRD was highly reliable and strongly associated with outcome, with early good responders having an excellent outcome (3-year CIR, 7.1%; OS, 92.9%). Altogether, our study reveals striking heterogeneity in outcomes within adults with KMT2A-r BCP-ALL and provides new biomarkers to guide risk-based therapeutic stratification.

Prognostic value of combined WT1 and multiparameter flow cytometry assessment for measurable residual disease after induction in non-APL acute myeloid leukemia.

The objective of this study was to investigate the expression pattern of Wilms tumor 1 (WT1) gene at diagnosis, complete remission (CR) and relapse status in non-acute promyelocytic leukemia (non-APL) acute myeloid leukemia (AML) patients, and further explore the prognostic value of measurable residual disease (MRD) assessment by WT1 gene and multiparameter flow cytometry (MFC). Our results showed that the average expression level of WT1 was 4026 ± 616.1 copies/104 ABL at diagnosis, 155.3 ± 36.03 copies/104 ABL at CR, and 1766 ± 238.8 copies/104 ABL at relapse, with statistically significant differences (p = .000). ROC analysis showed that WT1 expression levels were 118.1 copies/104 ABL and MFC-MRD was 0.155%, which had good predictive efficacy for relapse of patients during consolidation therapy. Both WT1-MRD and MFC-MRD had a significant impact on relapse-free survival (RFS) and overall survival (OS). Patients with WT1-MRD positive or MFC-MRD positive were associated with worse RFS (HR 3.840, 95% CI 1.582-9.320, p = .003), (HR 4.464, 95% CI 1.841-10.984, p = .001) and worse OS (HR 2.963, 95% CI 1.058-8.295, p = .039), (HR 3.590, 95% CI 1.254-10.280, p = .017). Besides, compared with patients who were negative for both WT1-MRD and MFC-MRD, patients who were positive both WT1-MRD and MFC-MRD were associated with worse RFS (HR 6.200, 95% CI 2.206-17.430, p = .001) and worse OS (HR 4.886, 95% CI 1.388-17.197, p = .013). This study demonstrates that combined assessment of MRD by WT1 and MFC improves relapse and prognosis prediction in non-APL AML patients, and may help guide interventions for disease relapse.

Utility of Measurable Residual Disease (MRD) Assessment in Mantle Cell Lymphoma.

OPINION STATEMENT: Mantle cell lymphoma (MCL) treatment advances have significantly improved disease-free remission, with greater focus in clinical trials being placed on measurable residual disease (MRD) as a marker of subclinical disease assessment. While this concept is used extensively in other haematological neoplasms, there is yet to be a consensus on the threshold for MRD in MCL that demonstrates prognostic and therapeutic significance, and in this context has yet to reach routine clinical practice. The historical long-term method for MCL MRD assessment has been real-time quantitative polymerase chain reaction (PCR), targeting the clonal immunoglobulin heavy locus (IGH) rearrangement or the IGH::CCND1 translocation rearrangement. A significant problem at present relates to identifying alternative assays for patients who do not have a suitable molecular target by this method. This article reviews existing techniques used in MRD assessment for MCL and describes novel methods which may overcome existing limitations, including next-generation sequencing modalities. The use of circulating tumour DNA is explored, with techniques such as CAPP-Seq and PhasED-Seq demonstrating promise in B-lymphoproliferative disorders, though application in MCL requires further study. The other aspect of practice using MRD is identifying therapeutic options which can address a subclinical molecular relapse. Developing suitable interventions that can alter the disease trajectory based on longitudinal MRD kinetics are needed to justify its incorporation into standard care.

Ultrasensitive NGS MRD assessment in Ph+ ALL: Prognostic impact and correlation with RT-PCR for BCR::ABL1.

Reverse transcription polymerase chain reaction (RT-PCR) for BCR::ABL1 is the most common and widely accepted method of measurable residual disease (MRD) assessment in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL); however, RT-PCR may not be an optimal measure of MRD in many cases of Ph+ ALL. We evaluated the clinical impact of a highly sensitive next-generation sequencing (NGS) MRD assay (sensitivity of 10-6 ) and its correlation with RT-PCR for BCR::ABL1 in patients with Ph+ ALL. Overall, 32% of patients had a discordance between MRD assessment by RT-PCR and NGS, and 31% of patients who achieved NGS MRD negativity were PCR+ at the same timepoint. Among eight patients with long-term detectable BCR::ABL1 by PCR, six were PCR+/NGS-. These patients generally had stable PCR levels that persisted despite therapeutic interventions, and none subsequently relapsed; in contrast, patients who were PCR+/NGS+ had more variable PCR values that responded to therapeutic intervention. In a separate cohort of prospectively collected clinical samples, 11 of 65 patients (17%) with Ph+ ALL who achieved NGS MRD negativity had detectable BCR::ABL1 by PCR, and none of these patients relapsed. Relapse-free survival and overall survival were similar in patients who were PCR+/NGS- and PCR-/NGS-, suggesting that PCR for BCR::ABL1 did not provide additional prognostic information in patients who achieved NGS MRD negativity. NGS-based assessment of MRD is prognostic in Ph+ ALL and identifies patients with low-level detectable BCR::ABL1 who are unlikely to relapse nor to benefit from therapeutic interventions.

Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia.

Undetectable measurable residual disease (MRD) is associated with favourable clinical outcomes in chronic lymphocytic leukaemia (CLL). While assessment is commonly performed using multiparameter flow cytometry (MFC), this approach is associated with limitations including user bias and expertise that may not be widely available. Implementation of unsupervised clustering algorithms in the laboratory can address these limitations and have not been previously reported in a systematic quantitative manner. We developed a computational pipeline to assess CLL MRD using FlowSOM. In the training step, a self-organising map was generated with nodes representing the full breadth of normal immature and mature B cells along with disease immunophenotypes. This map was used to detect MRD in multiple validation cohorts containing a total of 456 samples. This included an evaluation of atypical CLL cases and samples collected from two different laboratories. Computational MRD showed high correlation with expert analysis (Pearson's r > 0.99 for typical CLL). Binary classification of typical CLL samples as either MRD positive or negative demonstrated high concordance (>98%). Interestingly, computational MRD detected disease in a small number of atypical CLL cases in which MRD was not detected by expert analysis. These results demonstrate the feasibility and value of automated MFC analysis in a diagnostic laboratory.

ctDNA as promising tool for the assessment of minimal residual disease (MRD) and the need of an adjuvant treatment in gastroesophageal adenocarcinoma.

Gastroesophageal adenocarcinoma is a challenging disease due to its poor prognosis and the presence of few therapeutic options. For these reasons, it is mandatory to identify the subgroup of patients who are at high risk for relapse after curative-intention surgery. In the last years, liquid biopsy has aroused great interest in cancer treatment for its feasibility and the possibility to capture tumor heterogeneity in a real-time way. In postoperative setting, the interest is directed to the identification of Minimal Residual Disease (MRD), defined as isolated or small cluster of cancer cells that residues after curative-intention surgery, and are undetectable by conventional radiological and clinical exams. This review wants to summarize current evidence on the use of liquid biopsy in gastroesophageal cancer, focusing on the detection of ctDNA in the postoperative setting and its potential role as a guide for treatment decision.

Circulating Tumor DNA-Based MRD Assessment in Patients with CLL Treated with Obinutuzumab, Acalabrutinib, and Venetoclax.

PURPOSE: With the advent of highly efficacious time-limited combination treatments of targeted agents in chronic lymphocytic leukemia (CLL), minimal residual disease (MRD) assessment has gained importance as a measure for therapeutic success and as a surrogate for progression-free survival. The currently most widely used method is multicolor flow cytometry, which detects circulating CLL cells in the peripheral blood. However, it seems to be less sensitive for the detection of MRD in the lymph node compartment. PATIENTS AND METHODS: To evaluate whether a cell-free approach can overcome this limitation, we performed serial assessments of circulating tumor DNA (ctDNA) in patients with CLL treated with obinutuzumab, acalabrutinib, and venetoclax in the phase II CLL2-BAAG trial. Patient-specific variability, diversity, joining (VDJ) rearrangements as well as somatic driver mutations were tracked before, during and after treatment by digital droplet PCR in blood plasma. Furthermore, these were systematically compared to matched flow cytometry data. RESULTS: In the 381 sample pairs, ctDNA and flow cytometry yielded highly concordant results. However, clone-specific ctDNA was detected in 44 of 152 samples (29%) that were assessed as undetectable MRD (uMRD) by flow cytometry (defined as less than one CLL cell in 10,000 normal leukocytes). 29 ctDNA-negative samples showed detectable MRD >10-4 by flow cytometry. Also, somatic driver mutations were detected with a similar sensitivity compared with patient-specific VDJ rearrangements in plasma. In patients with predominantly nodal residual disease, ctDNA compared favorably with 4-color flow cytometry and seemed to more accurately reflect the entire disease burden across compartments. CONCLUSIONS: On the basis of these findings, ctDNA-based MRD assessment appears to be a promising method to complement cell-based MRD approaches like flow cytometry that focus on circulating CLL cells in the peripheral blood.

NPM1-mutation-based measurable residual disease assessment after completion of two courses of post-remission therapy is a valuable clinical predictor of the prognosis of acute myeloid leukemia.

Recent studies have reported that measurable residual disease (MRD) analysis using NPM1 mutations helps determine whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) is indicated in acute myeloid leukemia (AML) patients. However, the optimal timing and cutoff value for measuring MRD using genomic DNA remain undetermined. This study aimed to investigate the optimal timing and cutoff value to ascertain the value of NPM1 mutation in MRD assessment. NPM1-mutated MRD was quantified by real-time polymerase chain reaction of bone marrow samples from 56 patients with NPM1-positive AML who achieved hematological remission. The area under the receiver-operating characteristic curve was greatest when MRD was assessed after two courses of post-remission therapy with a cutoff value of 0.010% (specificity, 68.4%; sensitivity, 87.0%). Patients whose MRD was below the cutoff value throughout the course of treatment had significantly better overall survival and relapse-free survival rates. Of the 33 patients who did not undergo transplantation during the first remission, all of the 11 who were never MRD-negative at any point experienced a relapse. Evaluating MRD with a cutoff value of 0.010% after two courses of post-remission therapy helps predict prognosis and determine the indication for allo-HSCT.

Assessment of Clonotypic Rearrangements and Minimal Residual Disease in Lymphoid Malignancies.

CONTEXT.­: Measurable (minimal) residual disease (MRD) is an independent prognostic factor for survival outcomes in patients with lymphoid and plasma cell malignancies and has been incorporated into consensus criteria regarding treatment response, strategy, and clinical trial endpoints. clonoSEQ (a next-generation sequencing [NGS]-MRD assay) uses multiplex polymerase chain reaction and NGS to identify clonotypic rearrangements at the immunoglobulin (Ig) H, IgK, IgL, T-cell receptor (TCR)-ß, and TCR-γ loci, as well as translocated B-cell lymphoma 1/IgH and 2/IgH sequences for MRD assessment. Additionally, it can be used to confirm diagnoses of cutaneous T-cell lymphoma (CTCL). OBJECTIVE.­: To review the technical aspects of our experience using the clonoSEQ Assay in routine clinical practice. DESIGN.­: In this single-center experience, 390 patients with lymphoid and plasma cell malignancies were assessed with the NGS-MRD Assay at a central laboratory. RESULTS.­: Median time from arrival of the shipment to initiation of the assay (defined as captured in Adaptive's secure tracking system) was 2.1 hours. Overall, 317 patients had 1 or more samples submitted for sequence identification. Of these, 290 (91.5%) had trackable sequences identified. The median calibration rate of samples by malignancy (where n ≥ 10 samples, excluding CTCL samples) was 88.1%, across a variety of fresh and archived sample sources (177 of 201 samples). TCR-ß and/or TCR-γ clonotypes were identified in 40 of 95 samples (42.1%) from 66 patients with suspected CTCL. CONCLUSIONS.­: This NGS-MRD Assay is a valuable and sensitive tool for monitoring MRD in patients with plasma cell and lymphoid malignancies and assisting in the diagnosis of CTCL.

Interlaboratory Analytical Validation of a Next-Generation Sequencing Strategy for Clonotypic Assessment and Minimal Residual Disease Monitoring in Multiple Myeloma.

CONTEXT.­: Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. OBJECTIVE.­: To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. DESIGN.­: The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. RESULTS.­: Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. CONCLUSIONS.­: Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.

Molecular Genomic Assessment Using a Blood-based mRNA Signature (NETest) is Cost-effective and Predicts Neuroendocrine Tumor Recurrence With 94% Accuracy.

INTRODUCTION: Identification of residual disease after neuroendocrine tumor (NET) resection is critical for management. Post-surgery imaging is insensitive, expensive, and current biomarkers ineffective. We evaluated whether the NETest, a multigene liquid biopsy blood biomarker, correlated with surgical resection and could predict recurrence. METHODS: Multicenter evaluation of NET resections over 24 months (n = 103): 47 pancreas, 26 small bowel, 26 lung, 2 appendix, 1 duodenum, 1 stomach. Surgery: R0 (83), R1/R2 (20). One millilitre of blood was collected at D0 and posroperative day (POD) 30. Transcript quantification by polymerase chain reaction (normal: ≤20), CgA by NEOLISA (normal ≤108 ng/mL). Standard-of-care (SoC) follow-up costs were calculated and compared to POD30 NETest-stratification approach. Analyses: Wilcoxon-paired test, Chi-square test. D BIOMARKERS: NETest: 103 of 103 (100%)-positive, whereas 23 of 103 (22%) were CgA-positive (Chi-square = 78, P < 0.0001).In the R0 group, the NETest decreased 59 ± 28 to 26 ± 23 (P < 0.0001); 36% (30/83) remained elevated. No significant decrease was evident for CgA. In the R1/R2 group the NETest decreased but 100% remained elevated. CgA levels did not decrease.An elevated POD30 NETest was present in R0 and 25 (83%) developed radiological recurrences. Normal score R0 s (n = 53) did not develop recurrence (Chi-square = 56, P < 0.0001). Recurrence prediction was 94% accurate with the NETest. COST EVALUATION: Using the NETest to stratify postoperative imaging resulted in a cost-savings of 42%. CONCLUSION: NETest diagnosis is more accurate than CgA (100% vs 22%). Surgery significantly decreased NETest. An elevated POD30 NETest predicted recurrence with 94% accuracy and post-surgical POD30 NETest follow-up stratification decreased costs by 42%. CgA had no surgical utility. Further studies would define the accuracy and cost-effectiveness of the NETest in the detection of postoperative recurrent disease.

Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

CD304/neuropilin-1 is a very useful and dependable marker for the measurable residual disease assessment of B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Measurable residual disease (MRD) assessment using multicolor flow cytometry (MFC) has become the center point of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC-based MRD assay further. Herein, we evaluated the utility of a new marker, CD304/neuropilin-1, in the assessment of MFC-based MRD. METHODS: Expression patterns of CD304 were studied in leukemic blasts from BCP-ALL patients and in normal precursor B cells (NPBC) from uninvolved non-BCP-ALL bone marrow samples using 10-color MFC. MRD was monitored at end-of-induction (EOI; Days 35-40) and end-of-consolidation (Day 78-80) time points. RESULTS: We studied CD304 expression in 300 pediatric BCP-ALL patients and found it positive in BCP-ALL blasts in 41.7% of diagnostic samples. It was significantly associated with ETV6-RUNX1 (p < .001) as well as BCR-ABL1 (p = .019) and inversely associated with TCF3-PBX1 fusion gene (p = .0012). It was found clearly negative in NPBC. EOI-MRD was detectable in 152/300 (50.7%; ≥0.01% in 35.33% and <0.01% in 15.33%) samples, in which CD304 was positive in 72/152 (47.4%) diagnostic and 63/152 (41.4%) MRD samples. It was positive in 45.7% (21/46) of low-level (<0.01%) MRD samples. In comparison with diagnostic samples, its expression was retained in 68.06% (49/72), lost in 31.94% (23/72), and gained in 14/80 (17.5%) of EOI-MRD samples. CONCLUSIONS: CD304 is commonly expressed in leukemic blasts of BCP-ALL. It is very useful in distinguishing residual disease from hematogones and is a fairly dependable marker. Hence, it is a valuable addition for enhancing the sensitivity and applicability of MFC-based MRD assay in BCP-ALL.

Best practices for the assessment of measurable residual disease (MRD) in multiple myeloma.

Therapeutic advances in multiple myeloma have led to durable, deep remissions in a subset of patients. However, outcomes of patients achieving a complete response are not homogeneous. In recent years, measurable residual disease (MRD) has emerged as a prognostic biomarker. While several technologies have been evaluated to detect MRD, two assessment technologies are most frequently utilized in patients with multiple myeloma. Next-generation flow (NGF) uses flow cytometry to identify malignant plasma cells through the presence of immunologic markers located on the cell surface. Next-generation sequencing (NGS) analyzes for the presence of sequences in immunoglobulin genes that were previously identified as markers of that specific patient's plasma cell malignant clone. Both methods are included in criteria for MRD by the International Myeloma Working Group, which defines MRD negativity as less than 10-5. Recently, the NGS-based clonoSEQ® Assay obtained clearance from the US Food and Drug Administration, with a limit of detection of less than 10-6 given proper sample input. Based on available evidence correlating attainment of MRD negativity with outcomes, MRD assessment has been incorporated into ongoing clinical trials. Analyses will provide additional insight into the correlation between MRD and outcome. This monograph examines the available trial data and provides recommendations on how to incorporate MRD assessment into clinical management.