Comparison of High-Throughput Sequencing and Flow Cytometry for Measuring Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia: A Children's Oncology Group Cohort

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1440-1440
Author(s):  
Charles Gawad ◽  
Michael J. Borowitz ◽  
Gary V. Dahl ◽  
Meenakshi Devidas ◽  
Malek Faham ◽  
...  
Keyword(s):  
High Throughput ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Repertoire ◽  
Employment Equity ◽  
Clonal Rearrangement ◽  
Post Induction ◽  
Equity Ownership

Abstract Abstract 1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we compared the ability of the sequencing assay to measure MRD to that of FC in diagnostic and post-induction samples from 6 ALL patients. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity (D), and joining (J) gene segments from genomic DNA in diagnostic and follow-up bone marrow samples from 6 ALL patients. Amplified products were sequenced to obtain >1 million reads per sample and were analyzed using algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the diagnostic sample. The presence of the tumor-specific clonotype was then monitored in post-induction samples. Absolute quantification was performed by normalizing the patient's reads to internal reference DNA. We then analyzed concordance between MRD results obtained by sequencing and FC. Results: We detected a high-frequency IgH clonal rearrangement in 5/6 diagnostic ALL samples. MRD was assessed in the 5 post-induction samples from these patients (Table 1). Deep coverage of all MRD samples was obtained, with each original IgH molecule generating ∼20 sequencing reads, ensuring the detection of a single leukemic cell if present in the sample. Leukemic clones were detected in 4/5 follow-up samples (Table 1). In the positive samples, the number of detected leukemic molecules ranged from 12 to over 6,000 and the MRD level ranged from 0.008% to 0.3%. MRD results were concordant with FC in 3 of 5 patients and were consistent with the patient's clinical courses. In one patient we detected MRD at 0.008%, a level below the sensitivity of FC, which was negative. In another sample, FC detected MRD of 0.01–0.1%, but no leukemic clones were detected by the sequencing assay despite the fact that the sample contained sufficient cell input (almost 2 million cells). The patient remained in continuous remission. Evaluation of additional paired diagnostic and post-induction samples and their association with clinical outcomes is ongoing. Conclusions: We show the application of a high-throughput sequencing method for MRD detection in childhood ALL. IgH clonal rearrangements were detected in 5/6 (83%) of samples using the sequencing assay. The absence of a clonal rearrangement in 1/6 of patients was anticipated and is likely to be mitigated by the presence of a clonal rearrangement in another immunoglobulin or T cell receptor gene. Experiments are ongoing to assess the presence of clonal rearrangements in these receptors (i.e., IgH D-J, IgK, TRB@, TRD@ or TRG@) in the diagnostic samples. In 3/5 patients there was concordance between FC and sequencing-based MRD detection. In one patient, sequencing detected MRD at a level below the threshold of FC. The last patient was negative by sequencing but positive by FC and has not relapsed. Further analysis of the sensitivity and specificity of the sequencing platform compared to FC using additional paired diagnostic and post-induction samples is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Detection of Multiple Myeloma Cells in Peripheral Blood Using High-Throughput Sequencing Assay

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 321-321
Author(s):  
Amitabha Mazumder ◽  
Malek Faham ◽  
Mark Fiala ◽  
Mark Klinger ◽  
Thomas Martin ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
High Frequency ◽  
Genomic Dna ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Plasma Dna ◽  
Employment Equity ◽  
Clonal Rearrangement ◽  
Dna And Rna ◽  
Equity Ownership

Abstract Abstract 321 Background: 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 (BM), and related organ or tissue impairment. MM patients are monitored during therapy and posttherapy using immunoglobulin, M-protein and free light chain assays. Assessing pathological myeloma cells using flow cytometry and RT-PCR has been shown to have superior prognostic value. However, the sensitivity of these techniques has generally limited their use to assessment of BM. In order to determine whether myeloma minimal residual disease could be detected in peripheral blood (PB), we assessed a cohort of MM patients using a sequencing based platform, LymphoSIGHT, with a sensitivity of 1 cancer cell per million leukocytes. Methods: We obtained from 4 sources (UCSF, NYU, Washington Univ, commercial source) pairs of BM and PB samples from 60 MM patients at different points of disease. BM samples were used to identify the clonal MM sequence and detection of that sequence in the PB was assessed. For some patients BM/PB sample pairs were obtained from >1 time point resulting in a total of 78 pairs. In addition blood and bone marrow plasma samples were available for 44 and 6 patients, respectively, to assess presence of the myeloma clonotype in cell free DNA. Altogether there were 206 samples. BM samples were either available as BM mononuclear cells (BMMC) or bead enriched CD138+ cells. Using universal primer sets, we amplified IgH@ variable (V), diversity (D), and joining (J) gene segments from genomic DNA and/or RNA samples, the incomplete IgH rearrangement (DJ), and IgK from genomic DNA. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype determination. Myeloma-specific IgH, IgK, and DJ clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in all PBMC (DNA and RNA), BM plasma (DNA), and PB plasma (DNA) samples using the same IgH and in some samples using the IgK sequencing assays. A quantitative and standardized measure of clone level among all leukocytes in each PB or BM sample was determined using internal reference DNA. Here we describe data on 46/60 patients; data from all 60 patients will be presented. Results: In BM samples, we detected the myeloma clonal rearrangement of at least one receptor (“calibrating receptor”) in 34/46 (74%) of MM patients (Table 1). The calibration rate varied by receptor, with 30/46 (65%) patients calibrating with IgH, 14/43 (33%) with IgH DJ, and 22/43 (51%) with IgK (Table 1). Identification of myeloma-specific clonal rearrangement is based on presence at high frequency and may not occur in samples from patients with low disease load (e.g., post-treatment). Of the 12 non-calibrating patients, only 3 had high disease load. The myeloma clonotype that was identified in the BM was also detected in PBMC in 22/30 (73%) and 28/30 (93%) patients with the DNA and RNA IgH analysis, respectively (Table 2). IgK DNA analysis showed the presence of the myeloma clonotype in 9/10 PBMC samples, all of which were concordant with IgH results. The myeloma clonotype that was identified in the BM was also detected in the cell-free BM and PB samples in 5/5 and 7/11 patients, respectively, using the IgH DNA assay. The evaluation of blood plasma and PBMC were at times complementary in detecting the myeloma. Conclusions: Results from the application of a high-throughput sequencing method for detection of myeloma-specific clonotypes in 46 MM patients are shown. A clonal rearrangement was detected in 74% of MM BM samples. Importantly, 93% of peripheral blood samples from 30 patients showed evidence of circulating myeloma in PBMC. Analysis of BM and PB samples from 14 additional MM patients as well as association of the level of myeloma in PBMC and BM with clinical measures is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Klinger:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Comparison of Minimal Residual Disease Detection Using High-Throughput Sequencing and Allele-Specific Oligonucleotide PCR Methods in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2532-2532 ◽  
Author(s):  
Marian Harris ◽  
Malek Faham ◽  
Lei Fang ◽  
Martin Moorhead ◽  
Jianbiao Zheng ◽  
...  
Keyword(s):  
High Frequency ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Lymphoblastic Leukemia ◽  
Patient Specific ◽  
Employment Equity ◽  
Allele Specific ◽  
Equity Ownership ◽  
B Lineage ◽  
Minimal Residual

Abstract Abstract 2532 Background: The clinical management of patients with pediatric B-lineage acute lymphoblastic leukemia (B-ALL) relies on combinations of multiagent anticancer drugs and risk-stratified treatment. The prognostic significance of minimal residual detection (MRD) in pediatric B-ALL has been demonstrated in multiple cohorts. Allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin or T-cell receptor rearrangements, a method for MRD detection, requires the development of patient-specific reagents and cannot detect clonal evolution. ASO-PCR also has limited coverage, with clonal rearrangements being detected in only 90% of patients. We developed the sequencing-based LymphoSIGHT platform to address these limitations. Here we report the results of a pilot study of MRD detection using both the sequencing assay and ASO-PCR in paired diagnostic and end-of-induction samples from 7 B-ALL patients. Analysis of 82 additional patients is ongoing. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity, and joining gene segments from genomic DNA in diagnostic and follow-up bone marrow samples. Amplified products were sequenced to obtain >1 million reads (20× coverage per B-cell), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then assessed in the end-of-induction sample. A quantitative and standardized measure of MRD level among all leukocytes in the sample was determined using internal reference DNA. Following identification of IgH clonal rearrangements and MRD assessment using the sequencing assay, we examined the MRD results obtained at Boston Children's Hospital using ASO-PCR. Among the 7 patients analyzed to date, 6 patients were in complete remission at the time of the second sample; 1 patient had persistent evidence of disease. Sequencing was performed blinded to all clinical and ASO-PCR information on these patients. Results: With the sequencing platform, we detected a high-frequency IgH clonal rearrangement in all 7 diagnostic ALL samples. The leukemic clonotype that was identified at diagnosis was detected in the end-of-induction sample in each of the 7 patients. The quantitative range of the leukemic sequence in MRD samples ranged over 5 orders of magnitude. MRD results were concordant between sequencing and ASO-PCR in 5 of 7 patients. The detected MRD level differed by > 10 fold in 2 patients. In patient 1, sequencing detected high MRD, while low MRD was detected by ASO-PCR; this patient relapsed 1 year later while still on therapy. In patient 6, sequencing detected low MRD, while ASO-PCR detected high MRD. This patient remains in complete remission after 7 years. In patient 7, sequencing and ASO-PCR concordantly detected low MRD; this patient relapsed after completion of therapy. Conclusions: Results from the application of a high-throughput sequencing method for MRD detection in childhood B-ALL are shown. The sequencing assay does not require development of patient-specific reagents, which will reduce cost and laboratory turnaround time. This data, along with the laboratory workflow improvements, support the use of the sequencing assay as a next-generation MRD test for B-ALL. Analysis of samples from 82 patients is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Fang:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Robust Detection Of Minimal Residual Disease In Unselected Patients With B-Cell Precursor Acute Lymphoblastic Leukemia By High-Throughput Sequencing Of IGH

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2550-2550
Author(s):  
David Wu ◽  
Ryan O Emerson ◽  
Anna Sherwood ◽  
Mignon L. Loh ◽  
Anne Angiolillo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Lymphoblastic Leukemia ◽  
Post Treatment ◽  
Employment Equity ◽  
Equity Ownership ◽  
Pre Treatment

Abstract High-throughput sequencing (HTS) of immunoglobulin heavy chain genes (IGH) may be useful for detecting minimal residual disease (MRD) in acute lymphoblastic leukemia. We previously demonstrated the first application of high-throughput sequencing for the detection of minimal residual disease in T-cell precursor acute lymphoblastic leukemia (TPC-ALL) (Sci. Transl. Med. 4(134):134ra63. 2012). Recently, Faham and colleagues considered deep sequencing for MRD detection in B-cell precursor acute lymphoblastic leukemia (BPC-ALL) (Blood 120(26):5173-80, 2012). As this prior analysis in BPC-ALL apparently focused only on samples known to have a clonal rearrangement in IGH, the potential applicability and wide-spread utility of sequencing of IGH in unselected clinical samples for MRD has not been tested. Here, we consider an unselected cohort of patients enrolled in Children Oncology Group AALL0932 trial and use residual material from 99 patient samples submitted for routine multi-parametric flow cytometry (mpFC) at U. of Washington. One sample failed in the initial DNA extraction step and was not further considered. We show using high-throughput sequencing that clonal IGH rearrangements can be identified in 92 of the remaining 98 pre-treatment samples, using a definition of a V-D-J or D-J rearrangement comprising at least 10% of total nucleated cells (Fig. 1A). Similar to our prior findings in TPC-ALL, we find three subsets of patients—1) those for whom MRD is not detected by either flow cytometry or HTS; 2) those for whom MRD is detected both by flow cytometry and HTS; and 3) those for whom MRD is detected only by HTS, but not flow cytometry (Fig. 1B). There were no false negative results by HTS as compared to flow cytometry.Figure 1Measurement of clonal IGH rearrangement by high-throughput sequencing (HTS) or immunphenotypically abnormal B lymphoblast population by multi-parametric flow cytometry in pre-treatment (A) or day 29 post-treatment (B) residual samples. Results are reported for both HTS (red) and mpFC (blue) as clone frequency per total nucleated cells.Figure 1. Measurement of clonal IGH rearrangement by high-throughput sequencing (HTS) or immunphenotypically abnormal B lymphoblast population by multi-parametric flow cytometry in pre-treatment (A) or day 29 post-treatment (B) residual samples. Results are reported for both HTS (red) and mpFC (blue) as clone frequency per total nucleated cells. In the third group (HTS+positive, flow cytometry-negative), a subset of these patients, (5 of 28) had MRD detectable by HTS at a level within the expected sensitivity of flow cytometry. We hypothesized that in these cases that post-treatment MRD sequences may be present within the maturing B cell compartment that is not immunophenotypically aberrant by flow cytometry. To test this hypothesis, we analyzed eight additional post-treatment samples that were negative for MRD by flow cytometry. The mature B-cell fraction was collected by triple, flow cytometry-sorting and then sequenced by HTS for IGH rearrangements to search for the index clone defined in the corresponding, paired pre-treatment samples. Although a limited finding, diagnostic index IGH sequence was indeed identified in one of eight samples, in only the mature B-cell fraction, which is consistent with the proportion of cases with high-level MRD detected by HTS but which was missed by flow cytometry. Taken together, our results provide additional support for assessment of MRD in acute lymphoblastic leukemia by high-throughput sequencing. Our findings argue that precise quantification of the level of MRD by HTS will be important, and suggest that clonal IGH rearrangement sequences may be detected in an immunophenotypically normal population of mature B cells that may not be detected by flow cytometry. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Kirsch:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Wood:Becton Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

scholarly journals Deep Sequencing Approach for Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1388-1388
Author(s):  
Malek Faham ◽  
Jianbiao Zheng ◽  
Martin Moorhead ◽  
Victoria Carlton ◽  
Patricia Lee Stow ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Employment Equity ◽  
Equity Ownership ◽  
Minimal Residual

Abstract Abstract 1388 Background: The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and avoid over- or under-treatment.1 The measurement of minimal residual disease (MRD) during therapy has now emerged as the most important predictor of outcome in ALL.2 We developed the LymphoSIGHT platform, a high-throughput sequencing method, which universally amplifies antigen-receptor gene segments and can identify all leukemia-specific sequences at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In this study, we determined the sensitivity and specificity of this method, delineated the extent of clonal evolution present at diagnosis, and compared its capacity to measure MRD to that of flow cytometry and allele-specific oligonucleotide PCR (ASO-PCR) in follow-up samples from >100 patients with ALL. Methods: Using the sequencing assay, we analyzed diagnostic bone marrow samples from 100 ALL patients for clonal rearrangements of immunoglobulin (IgH@) and T cell receptor (TRB@, TRD@, TRG@) genes, as well as the extent of clonal evolution present at diagnosis. We assessed the capacity of the sequencing assay to detect MRD using diagnostic samples from 12 ALL patients carrying 13 leukemic IgH clonal rearrangements. Serial dilutions were prepared in normal peripheral blood mononucleated cells, at a range between <1 in 1 million to >1 in 1,000 cells. We also assessed MRD in follow-up samples from 106 ALL patients and analyzed concordance between MRD results obtained by the sequencing assay, flow cytometry and ASO-PCR. Results: In diagnostic bone marrow samples, we detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 100 ALL samples; 94 samples had at least 2 calibrating receptors at diagnosis, with 51 having 3 or more. We also detected a variable degree of clonal evolution: the number of evolved clones in each sample ranged from 0 to 6933, with 39 (37%) samples having 1–50 evolved clones and 17 (16%) >50 (Figure 1). In experiments with mixtures of normal and leukemic cells, the sequencing assay unequivocally and accurately detected leukemic signatures in all dilutions up to a concentration of at least one leukemic cell in 1 million leukocytes. In direct comparisons with established MRD assays performed on follow-up samples from patients with B-ALL, sequencing detected MRD in all 28 samples positive by flow cytometry, and in 35 of the 36 positive by ASO-PCR; it also revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively (Figure 2). Conclusions: The sequencing assay is precise, quantitative, and can detect MRD at levels below 1 in 1 million leukocytes (0.0001%), i.e., represents sensitivity 1–2 orders of magnitude higher than standard flow cytometric and ASO-PCR methods. Our assay also allows monitoring of all leukemic rearrangements regardless of their prevalence at diagnosis, which abrogates the risk of false-negative MRD results due to clonal evolution. Finally, the sequencing assay utilizes a set of universal primers and does not require development of patient-specific reagents. These data, together with the results of our comparison with standard MRD assays in clinical samples, strongly support the use of the sequencing assay as a next-generation MRD test for ALL. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Research Funding. Zheng:Sequenta: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta: Employment, Equity Ownership, Research Funding. Carlton:Sequenta: Employment, Equity Ownership, Research Funding.

A Complementary Role of High Throughput Sequencing and Multiparameter Cytometry for Minimal Residual Disease (MRD) Detection in Chronic Lymphocytic Leukemia (CLL):an European Research Initiative (ERIC) Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1976-1976
Author(s):  
Andy Rawstron ◽  
Claudia Fazi ◽  
Neus Villamor ◽  
Julio Delgado ◽  
Remi Letestu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Limit Of Detection ◽  
Pearson Correlation ◽  
Post Treatment ◽  
Employment Equity ◽  
Equity Ownership ◽  
Minimal Residual

Abstract BACKGROUND. The detection of minimal residual disease (MRD) at the level of 0.01%/10-4 or above is a strong independent predictor of reduced progression-free (PFS) and overall survival (OS) in patients with CLL treated with chemoimmunotherapy. Although newer agents such as B-cell receptor pathway inhibitors can result in prolonged survival without achieving complete response, there remains a important role for MRD analysis in assessing therapeutic strategies aimed at disease eradication and cure. This is particularly important in front-line trials for fit patients which now require at least five years of follow-up if PFS is used as an endpoint. The feasibility of using MRD as a surrogate or intermediate endpoint for accelerated approval of new treatments is under review by regulatory agencies but further prospective validation is required. At the same time technology is rapidly evolving and high-throughput sequencing (HTS) technologies now detect MRD at the 0.0001%/10-6 level. It is therefore important to determine the most effective approaches for quantifying MRD that are compatible with previous studies but sufficiently sensitive for current treatments. AIMS. This collaborative project had two objectives. First, to identify the simplest and most flexible flow cytometry panel capable of detecting MRD at the 0.01%/10-4 or lower, that is compatible with published data and independent of instrument/reagent manufacturer. Second, to compare the flow cytometry approach with HTS analysis using the ClonoSEQ assay (Adaptive Biotechnologies, Seattle, WA). METHODS AND RESULTS. A core panel of antibodies for MRD detection was identified by testing an 8-marker combination in 52 samples (27 post-treatment and 25 dilution study) and re-analysing data with serial exclusion of single markers to determine redundancy. A 1-tube core panel of CD19, CD20, CD5, CD43, CD79b, and CD81 was identified and validated against the previously published 2-tube 6-marker and 4-tube 4-marker ERIC panels in 76 samples (19 post-treatment and 57 dilution study). The results showed good concordance (for log-transformed data above the LoQ, linearity=0.977, Pearson correlation co-efficient=0.983, average difference=0.026 log, 95% limit of agreement 0.20log) and a limit of detection of 0.001%/10-5 for the 1-tube core panel. Inter-operator variation was similar to CML MRD monitoring with both experienced operators, or inexperienced cytometrists after ~1 hour of specific education, achieving a 95% limit of agreement less than 0.3log in samples with MRD levels ranging from 0.0001 – 100%. The flow cytometry approach was compared with the ClonoSEQ HTS assay in 109 samples (21 dilution study and 88 post-treatment samples, complete data currently available on 13/88). The assay was applicable to the vast majority CLL patients, often with two clonal markers. There was 94% concordance at the 0.01% (10-4) threshold (15 samples with ≥0.01% CLL by both methods, 14 samples with <0.01% by both methods, 1 sample with 0.03% CLL by HTS and <0.003% CLL by flow cytometry, and 1 sample with 0.005% CLL by HTS and 0.012% by flow cytometry. HTS detected CLL IGH sequences in 22% (7/31) samples with no detectable CLL cells by flow cytometry (i.e. CLL level 0.0001-0.001%, 3/13 patient samples and 4/18 dilution samples). HTS demonstrated a relatively high variability in quantification, as seen in previous studies, but with a clear superiority in the limit of detection and good linearity (linearity=0.905, Pearson correlation co-efficient=0.870, average difference=0.078 log, 95% limit of agreement 1.5 log). CONCLUSIONS. The 1-tube 6-marker flow cytometry core panel is compatible with published studies, manufacturer-independent and flexible, providing directly quantitative results to 0.001%/10-5 without requiring a pre-treatment sample. HTS requires further work to standardise the quantitative analysis and prospective validation but the ClonoSEQ assay is applicable to >95% of CLL patients, does not require viable cells and is extremely sensitive, detecting residual disease in a significant proportion of cases with <0.01% CLL. The results indicate that flow cytometry and HTS are complementary technologies with a combined approach offering the most reliable way of quantifying CLL at the 0.01%/10-4 threshold while allowing higher sensitivity in clinical trials aimed at disease eradication. Disclosures Rawstron: Roche: Honoraria; Biogen Idec: Consultancy; Gilead: Consultancy, Honoraria; Abbvie: Honoraria; BD Biosciences: Intrasure reagent Patents & Royalties; Celgene: Honoraria; GSK: Honoraria. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Sanders:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Hallek:Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; GSK: Honoraria; Gilead: Honoraria. Hillmen:Roche: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

scholarly journals Molecular Detection of Minimal Residual Disease Precedes Morphological Relapse and Could be Used to Identify Relapse in Pediatric and Young Adult B-Cell Acute Lymphoblastic Leukemia Patients Treated with Tisagenlecleucel

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1551-1551 ◽  
Author(s):  
Michael A. Pulsipher ◽  
Xia Han ◽  
Máire Quigley ◽  
Gabor Kari ◽  
Susana Rives ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Post Treatment ◽  
Employment Equity ◽  
Advisory Boards ◽  
Sensitivity Level ◽  
Equity Ownership

Abstract Introduction Detection of minimal residual disease (MRD) is an important predictor of patient outcome following treatment of B-cell acute lymphoblastic leukemia (B-ALL). We assessed concordance between two MRD assays, with different assay sensitivities, to determine which MRD detection method could support early relapse detection. Immunoglobulin next generation sequencing (Ig NGS) and flow cytometry (FC) were tested in samples from two clinical trials ELIANA (NCT02435849) and ENSIGN (NCT02228096) for pediatric relapsed and refractory B-ALL patients treated with tisagenlecleucel. We also assessed whether using blood with Ig NGS would be comparable to BM testing with FC. Finally we analyzed whether clonal evolution, as detected by Ig NGS, occurred during of the course of therapy for both CD19+ and CD19- relapse patients. Methods In this analysis, bone marrow and peripheral blood specimens at screening (pre-tisagenlecleucel infusion), post-infusion and relapse were tested. Ig NGS was performed in 300 samples from 88 patients. 237 samples from 83 patients also had FC MRD results available. MRD was measured on fresh blood and bone marrow using a 3-tube FC assay (CD10, CD19, CD13, CD20, CD22, CD33, CD34, CD38, CD45, CD58, CD123). The FC MRD assay has a lower limit of sensitivity of 0.01% of white blood cells. Ig NGS detection of MRD was performed using the Adaptive Biotechnology's NGS MRD assay. MRD quantitative values, along with the qualitative MRD calls at each assay sensitivity level (10-4, 10-5 and 10-6) were reported. At baseline, 85 out of 88 samples had informative clones. Results and Conclusions To examine the comparability of flow cytometry and Ig NGS methods in assessing MRD, baseline and post-treatment samples were tested. Baseline samples, which had a high disease burden, showed 100% MRD concordance between both assays. However, post-treatment, where the leukemic burden was dramatically reduced, Ig NGS detected a greater number of MRD positive samples compared to FC, at each sensitivity level tested (10-4, 10-5 and 10-6). At the highest sensitivity level of 10-6, Ig NGS was able to detect 18% more MRD positive post-treatment samples. Importantly, Ig NGS was able to detect MRD positivity 1-4 months ahead of clinical relapse in a small number of relapsed patients, whether relapse was CD19+ or CD19-. This may provide an important window of opportunity for pre-emptive treatment while a patients' tumor burden is still low. In B-ALL, it has previously been described that MRD levels can be one to three logs lower in blood compared to bone marrow (VanDongen JJ et al. Blood 2015). Our results support these findings whereby MRD burden in bone marrow was higher than in blood using both FC and Ig NGS. We next set out to determine if the increased sensitivity afforded by the Ig NGS assay could provide a level of MRD detection in the blood comparable to FC in the bone marrow. In patients with matching data available, Ig NGS was able to detect more MRD positive blood samples than FC MRD positive bone marrow samples. This suggests that monitoring of MRD using Ig NGS in the blood holds the potential to be used as a surrogate for FC MRD in bone marrow. The relationship between MRD and prognosis was examined. Patients who were MRD negative by both Ig NGS and FC at the end of first month post-infusion had better progression-free survival and overall survival compared to those with detectable MRD. Tumor clonality will be further analyzed to understand sub-clone composition at baseline and clonal evolution following tisagenlecleucel treatment. Taken together, these results highlight the importance of using a highly sensitive assay, such as Ig NGS, when monitoring for MRD. MRD detection by Ig NGS holds the potential to identify early response/relapse in patients, which could provide a window of opportunity for additional intervention before morphological relapse. Ongoing studies with larger patient groups will provide further information on the applicability of Ig NGS MRD detection and its association with long-term outcome in tisagenlecleucel-treated pediatric r/r B-ALL patients. Disclosures Pulsipher: Novartis: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Consultancy; Amgen: Honoraria; Adaptive Biotech: Consultancy, Research Funding. Han:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Quigley:Novartis Pharmaceuticals Corporation: Employment. Kari:Adaptimmune LLC: Other: previous employment within 2 years; Novartis Pharmaceuticals Corporation: Employment. Rives:Shire: Consultancy, Other: Symposia, advisory boards ; Amgen: Consultancy, Other: advisory board ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards . Laetsch:Bayer: Consultancy; Eli Lilly: Consultancy; Pfizer: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy; Loxo Oncology: Consultancy. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Qayed:Novartis: Consultancy. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Baruchel:Shire: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Servier: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Celgene: Consultancy. Bader:Cellgene: Consultancy; Riemser: Research Funding; Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Novartis: Consultancy, Speakers Bureau. Yi:Novartis Pharmaceuticals Corporation: Employment. Kalfoglou:Novartis Pharmaceuticals Corporation: Employment. Robins:Adaptive Biotechnologies: Consultancy, Employment, Equity Ownership, Patents & Royalties. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership. Görgün:Novartis Pharmaceuticals Corporation: Employment. Bleickardt:Novartis Pharmaceuticals Corporation: Employment. Wong:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties.

On-Going Evolution Of IGH In B-Cell Precursor Acute Lymphoblastic Leukemia Does Not Substantially Affect Day 29, Post-Treatment MRD Quantification By High-Throughput Sequencing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1341-1341
Author(s):  
David Wu ◽  
Ryan O Emerson ◽  
Anna Sherwood ◽  
Mignon L. Loh ◽  
Anne Angiolillo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Lymphoblastic Leukemia ◽  
Post Treatment ◽  
Cell Precursor ◽  
Employment Equity ◽  
Vh Replacement ◽  
Igh Locus ◽  
Equity Ownership ◽  
Pre Treatment

Abstract High-throughput sequencing (HTS) of immunoglobulin heavy chain genes (IGH) may be useful for detecting minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukemia (BPC-ALL), particularly in the context of massive clonal evolution at the IGH locus, as previously identified by others (Gawad et al., Blood 120(22):4407-17, 2012; and Faham et al., Blood 120(26):5173-80, 2012). This on-going rearrangement of IGH may limit detection of MRD in post-treatment samples by traditional molecular-based methods, typically real-time PCR using patient-specific primers or probes. Here, we examine the extent to which evolution of IGH in unselected pre-treatment samples from patients with BPC-ALL affects detection of MRD in day 29 post-treatment samples by high-throughput sequencing of IGH. Of 99 samples from an unselected series from the Children’s Oncology Group trial AALL0932, we find that 92 of 98 samples have a clonal IGH gene rearrangement in pre-treatment samples. One sample failed at the outset during the DNA extraction step. Of the remaining 92 cases with pre-treatment VDJ or D-J rearrangements, 82 had evidence of on-going recombination in which VH replacement was identified in clones, each having conserved D-J rearrangements. The average number of clones was 192, but ranged from 1 to over 2000 unique sequences. In cases with VH replacement, an average of 4.12% of IGH sequences was made up of VH-replaced sequences. In post-treatment samples that were MRD positive, the predominant clone in pre-treatment samples was typically the most frequent clone. Clones consistent with VH replacement were found in 19 patients; in one patient, the only MRD detected was a single clone consistent with VH replacement at a level of ∼1 in 1,000,000. In the other 18 post-treatment MRD positive cases, the dominant clone identified pre-treatment was also dominant post-treatment: on average, 3.2% of total IGH rearrangements matched the dominant clone post-treatment, while only 0.027% of IGH rearrangements were consistent with VH replacement of the major clone. Among pre-treatment samples in which VH replaced clones were detected, all VH replaced clones together were 12% as large as the dominant clone on average. Among post-treatment samples, VH replaced clones were on average 14% as large as the dominant clone, indicating little change in the relative proportions of the dominant clone and VH replaced sub-clones. These findings together suggest that on-going rearrangement of the IGH locus is not likely to be important for clonal tumor evolution within the time frame of initial chemotherapy, as no substantial change in clonal diversity as assessed by IGH sequencing is evident. In other words, on-going rearrangement of IGH appears to be neutral with respect to therapy-induced selection of tumor clones that may represent early (day 29) relapse. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Kirsch:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Wood:Becton Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

Detection Of Recurrent/Persistent Disease By T-Cell Receptor Repertoire Profiling In Patients With Mature T-Cell Neoplasm

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2614-2614
Author(s):  
Anna Sherwood ◽  
Harlan Robins ◽  
Jonathan R. Fromm ◽  
Harvey A. Greisman ◽  
Daniel E. Sabath ◽  
...  
Keyword(s):  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Recurrent Disease ◽  
Cell Lineage ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Employment Equity ◽  
Equity Ownership ◽  
Clinical Measures

Abstract Identification of recurrent or persistent disease in T-cell neoplasms is important for individualized patient care. While patients with T-cell lineage lymphomas and leukemias are a small subset of all lymphoma and leukemia patients, the incidence of refractory disease in these patients can be higher than patients with B-cell lineage neoplasms. We recently developed a method to sequence the diversity of the TCR CDR3 rearrangements (Blood. 2009; 114(19): 4099-107) that exploits the capacity of high-throughput sequencing (HTS) to document the diverse repertoire of TCRB CDR3 chains simultaneously. These assays can describe both the breadth of T-cell repertoire and quantify individual clones. For example, we have shown in that T-cell clones as rare as 1:100,000 total T cells can be accurately quantified (Journal of Immunological Methods 2012;375:14-9). This technology thus provides a potential opportunity to track the presence and frequency of clones in the context of an evolving, adaptive immune system, during the course of ongoing therapy. While our recent study demonstrated the first application of high-throughput sequencing to the evaluation of minimal residual disease in precursor acute T lymphoblastic leukemias (Sci. Transl. Med. 4(134):134ra63. 2012), we focus here on mature T-cell neoplasms to demonstrate the broad potential of this technology to contribute to the post-therapeutic monitoring of T cell neoplasia. We amplified the TCRB repertoire of 35 index samples to identify high-frequency TCRB rearrangements. Clones were classified as neoplastic if occurring at a proportion greater than 7 standard deviations above the mean frequency of the most abundant rearranged TCRB in control samples of either blood, bone marrow, or lymphoid tissues. Samples that lacked a detectable TCRB clone were excluded. For each patient, at least one subsequent follow-up sample was available. For these 59 samples, we sequenced the TCRB repertoire to screen for the corresponding index clone. We find that for most samples, high-throughput sequencing concurs with currently available, routine clinical measures of disease, such as clinical flow cytometry or PCR-based evaluation of TCRG rearrangement. High-throughput sequencing of TCRB was concordant in 46 samples with identification of the index clones and in 7 additional samples without the identification of the index clones. However, 5 of 59 samples were only positive for recurrent disease based on HTS only, and 1 of the 59 samples was only positive for recurrent disease based on current diagnostic technology but not HTS. We find that for most samples, high-throughput sequencing concurs with currently available, routine clinical measures of disease, such as clinical flow cytometry or PCR-based evaluation of TCRG rearrangement and T-cell repertoire analysis may be useful for clinical laboratory evaluation of patients with T-cell neoplasms.Table 1High-throughput sequencingPositiveNegativeRoutine clinical testingPositive461Negative57 Disclosures: Sherwood: Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Rieder:Adaptive Biotechnologies: Employment, Equity Ownership. Wood:Becton Dickinson and Company, NJ, USA: Research Funding.

scholarly journals Impact of Modified Thalidomide Dosing in Bortezomib/Thalidomide/Dexamethasone for Patients with Newly Diagnosed Multiple Myeloma Who Are Transplant-Eligible: A Matching-Adjusted Indirect Comparison

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4739-4739
Author(s):  
Pieter Sonneveld ◽  
Maria-Victoria Mateos ◽  
Adrián Alegre ◽  
Thierry Facon ◽  
Cyrille Hulin ◽  
...  
Keyword(s):  
Peripheral Neuropathy ◽  
Board Of Directors ◽  
Research Funding ◽  
Indirect Comparison ◽  
Employment Equity ◽  
Advisory Committees ◽  
Post Transplant ◽  
Post Induction ◽  
Adjusted Indirect Comparison ◽  
Equity Ownership

Introduction: For patients with newly diagnosed multiple myeloma (NDMM) who are transplant-eligible, bortezomib/thalidomide/dexamethasone (VTd) is a standard of care (SoC) for induction and consolidation therapy. Clinical practice has evolved to use a modified VTd dose (VTd-mod; 100 mg thalidomide daily), which is reflected in recent treatment guidelines. As VTd-mod has become a real-world SoC, a matching-adjusted indirect comparison (MAIC) of the VTd-mod dose from recent clinical trials versus the dose included in the label (VTd-label; ramp up to 200 mg thalidomide daily) was performed to understand the effect on efficacy of modified VTd dosing for patients with NDMM who are transplant-eligible. Methods: For each outcome (overall survival [OS], progression-free survival [PFS], overall response rates [ORR] post-induction and post-transplant, and rate of peripheral neuropathy), a naïve comparison and a MAIC were performed. Data for VTd-label were obtained from the phase 3 PETHEMA/GEM study (Rosiñol L, et al. Blood. 2012;120[8]:1589-1596). Data for VTd-mod were pooled from the phase 3 CASSIOPEIA study (Moreau P, et al. Lancet. 2019;394[10192]:29-38) and the phase 2 NCT00531453 study (Ludwig H, et al. J Clin Oncol. 2013;31[2]:247-255). Patient-level data for PETHEMA/GEM and CASSIOPEIA were used to generate outcomes of interest and were validated against their respective clinical study reports; aggregate data for NCT00531453 were extracted from the primary publication. Matched baseline characteristics were age, sex, ECOG performance status, myeloma type, International Staging System (ISS) stage, baseline creatinine clearance, hemoglobin level, and platelet count. Results: Patients received VTd-mod (n = 591) or VTd-label (n = 130). After matching, baseline characteristics were similar across groups. For OS, the naïve comparison and the MAIC showed that VTd-mod was non-inferior to VTd-label (MAIC HR, 0.640 [95% CI: 0.363-1.129], P = 0.121; Figure 1A). VTd-mod significantly improved PFS versus VTd-label in the naïve comparison and MAIC (MAIC HR, 0.672 [95% CI: 0.467-0.966], P = 0.031; Figure 1B). Post-induction ORR was non-inferior for VTd-mod versus VTd-label (MAIC odds ratio, 1.781 [95% CI: 1.004-3.16], P = 0.065). Post-transplant, VTd-mod demonstrated superior ORR in both the naïve comparison and MAIC (MAIC odds ratio, 2.661 [95% CI: 1.579-4.484], P = 0.001). For rates of grade 3 or 4 peripheral neuropathy, the naïve comparison and MAIC both demonstrated that VTd-mod was non-inferior to VTd-label (MAIC rate difference, 2.4 [⁻1.7-6.49], P = 0.409). Conclusions: As naïve, indirect comparisons are prone to bias due to patient heterogeneity between studies, a MAIC can provide useful insights for clinicians and reimbursement decision-makers regarding the relative efficacy and safety of different treatments. In this MAIC, non-inferiority of VTd-mod versus VTd-label was demonstrated for OS, post-induction ORR, and peripheral neuropathy. This analysis also showed that VTd-mod significantly improved PFS and ORR post-transplant compared with VTd-label for patients with NDMM who are transplant-eligible. A limitation of this analysis is that unreported or unobserved confounding factors could not be adjusted for. Disclosures Sonneveld: Takeda: Honoraria, Research Funding; SkylineDx: Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding. Mateos:Janssen, Celgene, Takeda, Amgen, Adaptive: Honoraria; AbbVie Inc, Amgen Inc, Celgene Corporation, Genentech, GlaxoSmithKline, Janssen Biotech Inc, Mundipharma EDO, PharmaMar, Roche Laboratories Inc, Takeda Oncology: Other: Advisory Committee; Janssen, Celgene, Takeda, Amgen, GSK, Abbvie, EDO, Pharmar: Membership on an entity's Board of Directors or advisory committees; Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Takeda Oncology.: Speakers Bureau; Amgen Inc, Janssen Biotech Inc: Other: Data and Monitoring Committee. Alegre:Celgene, Amgen, Janssen, Takeda: Membership on an entity's Board of Directors or advisory committees. Facon:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hulin:celgene: Consultancy, Honoraria; Janssen, AbbVie, Celgene, Amgen: Honoraria. Hashim:Ingress-Health: Employment. Vincken:Janssen: Employment, Equity Ownership. Kampfenkel:Janssen: Employment, Equity Ownership. Cote:Janssen: Employment, Equity Ownership. Moreau:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

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