High-Throughput Sequencing of T-Cell Receptor Gene Loci for Minimal Residual Disease Monitoring in T Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2545-2545
Author(s):  
David Wu ◽  
Anna Sherwood ◽  
Stuart S. Winter ◽  
Kimberly Dunsmore ◽  
Mignon L Loh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Post Treatment ◽  
Pre Treatment ◽  
Higher Sensitivity

Abstract Abstract 2545 There is increasing evidence for the utility of minimal residual disease (MRD) assessment in predicting clinical outcomes of patients with T cell lymphoblastic leukemia (T-ALL). Evaluation of MRD by PCR-based analysis of T-cell receptor (TCR) genes has a sensitivity of 10−5, but requires the use of individualized patient-specific primers, which is laborious, expensive and difficult to implement for real-time, clinical decision-making. Multi-parametric flow cytometry is currently limited to a sensitivity of 10−4, requires viable cells, and is poorly standardized. High-throughput DNA sequencing offers the potential to equal or surpass the higher sensitivity of PCR-based MRD testing with reduced cost, improved turn-around time, and better standardization. Paired samples of pediatric T-ALL from 14 patients enrolled on Children's Oncology Group AALL0434 were obtained at diagnosis and at day 29 post-induction therapy. The complementarity determining regions (CDR3) regions of TCRB and TCRG were sequenced for all 28 specimens using an Illumina GA2 platform as previously described (see Blood, 114(19):4099–4107, 2009 and Sci Transl Med. 3(90):90ra61, 2011). Pre-treatment samples were used to obtain unique TCR sequences for the leukemic clone, and post-treatment samples were assessed for the frequency of each TCR sequence as a percentage of the total. The frequency of each sequence was also enumerated in post-treatment samples from all other patients to evaluate specificity. These results were compared to MRD results obtained by 9-color flow cytometry per trial protocol. Eleven of 14 pre-treatment samples (78.6%) had a detectable clonal population based on TCRG sequence analysis, and 10 of these also had a clonal TCRB sequence. Five samples exhibited an additional unique TCRG sequence, consistent either with rearrangement of both TCRG loci or the presence of two clonal subpopulations. Two of 3 cases without a detectable clonal TCR gene sequence had the immunophenotype of early thymic precursor (ETP) T-ALL and would be expected to have germline TCRB and TCRG genes. No other cases were ETP. Clones were found in all 5 informative post-treatment samples positive for MRD by flow cytometry, as well as at a low level in 3 additional patients without MRD by flow cytometry, suggesting superior sensitivity for sequencing. The background sequence frequencies were very low (0–10−5) in other patient post-treatment samples, being slightly higher for TCRG than for TCRB, consistent with germline sequence diversity. We demonstrate the potential of high-throughput sequencing for analysis of MRD in pediatric T-ALL. The number of cases in which the assay is informative (78.6%) is similar to that seen with standard PCR MRD methods, but evaluation of more cases is needed. MRD by sequencing appears to have a higher sensitivity than current flow cytometric methods, although direct comparison of MRD frequencies from the two techniques is problematic and will require normalization. The strong association of ETP status and lack of clonal TCR sequence identification at diagnosis suggests utility in identifying this poor outcome subset of T-ALL. Disclosures: Sherwood: Adaptive TCR, Seattle, WA: Employment, Equity Ownership. Wood:Becton, Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive TCR, Seattle, WA: Consultancy, Equity Ownership, Patents & Royalties.

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 Intra-Tumoral Blast Heterogeneity and Implications for Minimal Residual Disease Detection in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1076-1076
Author(s):  
Nina Friesgaard Öbro ◽  
Lars Peter Ryder ◽  
Hans Ole Madsen ◽  
Mette Klarskov Andersen ◽  
Birgitte Klug Albertsen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Flow Sorting ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Minimal Residual

Abstract Introduction:The early treatment response, measured as minimal residual disease (MRD), is the most important tool for treatment stratification in T-cell acute lymphoblastic leukemia (T-ALL). Flow cytometry-based MRD (Flow-MRD) monitoring, in addition to the PCR-MRD method, is often important to ensure a sensitive MRD marker. Additionally, Flow-MRD investigation may add biological information to the MRD result itself, and allow cell sorting for biological and functional analyses. Flow-MRD in T-ALL consists of identification of cells with immature T-cell phenotype in bone marrow. However, important pitfalls in Flow-MRD, e.g. treatment-related marker modulation and intra-tumoral immunophenotypic heterogeneity, are poorly described. The aim of this study was to explore the implications of these pitfalls on T-ALL MRD detection and on the concordance between the two MRD methods. Potentially both PCR- and Flow-MRD methods might miss blast subpopulations, which is important if subpopulations have divergent chemosensitivity. Methods:The patient cohort included 49 Danish T-ALL patients (1-45 years of age) treated according to the NOPHO ALL2008 protocol. Standard PCR- and flow cytometry-based MRD data were obtained as part of routine MRD monitoring. We investigated intra-tumoral heterogeneity of the leukemia-associated immunophenotype by flow cytometry (diagnostic BM samples), including clonal T-cell receptor gene-rearrangements in flow-sorted blast subpopulations (22 patients). Immunophenotypic MRD markers (including assessment of modulation) were re-evaluated at follow-up in MRD-positive patients. Flow-MRD was validated by PCR-MRD analysis in flow-sorted cell populations (61 follow-up BM samples, 32 patients). Results:At diagnosis, more than 80% of the T-ALL patients had a heterogeneous immunophenotype, most often involving CD1a, CD4, and TdT. The degree of overall heterogeneity, as defined by the number of markers with heterogeneous expression showing distinct blast subpopulations, did not show association to day29 PCR-MRD. Except for one patient, the dominant T-cell receptor clonal gene rearrangements were conserved across phenotypically diverse blasts. Immunophenotypic changes in MRD-positive patients at early follow-up often included subpopulation-loss and/or marker down-modulation of CD1a, TdT and/or CD4. The marker modulations were frequently independent of each other in different subpopulations. Overall, flow cytometry-based identification of blasts and normal cells at Flow-MRD time points was verified by PCR in the flow-sorted cells: In patients where at least 90% of the blasts showed aberrant marker expression at diagnosis, the flow-sorted MRD cells were concordantly PCR-positive, and flow-sorted phenotypically normal cells were similarly PCR-negative in all but three samples that had very high MRD levels (>20%). However, many patients had only partly-informative immunophenotypes (less than 90% of blasts having aberrant marker). Three discrepant cases with Flow-MRD underestimation showed loss of CD1a- and TdT and down-modulation of CD99, verified in flow-sorting experiments. Conclusions and Discussion: We show that intra-tumoral immunophenotypic heterogeneity—a possible result of genetic instability—is common in T-ALL patients and involves several immaturity and T-linage markers commonly used in Flow-MRD. The dominant PCR-MRD targets are in most cases conserved across the diverse blast subpopulations at diagnosis, but in rare cases PCR-MRD might miss a subpopulation. The observed immunophenotypic changes in T-ALL blasts and blast subpopulations at early follow-up, including reduction of immaturity markers, represent important pitfalls in Flow-MRD. Flow-sorting experiments verified that, when all blasts of heterogeneous immunophenotypes were informative, MRD identified by flow cytometry at follow-up was highly concordant with PCR-MRD markers in sorted cells. The T-ALL blast heterogeneity and marker modulations, which are possibly treatment protocol-specific, are important to take into account to obtain reliable Flow-MRD and thus correct treatment stratification of T-ALL patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies

Blood ◽  
2015 ◽  
Vol 125 (26) ◽  
pp. 3996-4009 ◽  
Author(s):  
Jacques J. M. van Dongen ◽  
Vincent H. J. van der Velden ◽  
Monika Brüggemann ◽  
Alberto Orfao
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Minimal Residual Disease ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Pcr Analysis ◽  
Advantages And Disadvantages ◽  
Minimal Residual

Abstract Monitoring of minimal residual disease (MRD) has become routine clinical practice in frontline treatment of virtually all childhood acute lymphoblastic leukemia (ALL) and in many adult ALL patients. MRD diagnostics has proven to be the strongest prognostic factor, allowing for risk group assignment into different treatment arms, ranging from significant treatment reduction to mild or strong intensification. Also in relapsed ALL patients and patients undergoing stem cell transplantation, MRD diagnostics is guiding treatment decisions. This is also why the efficacy of innovative drugs, such as antibodies and small molecules, are currently being evaluated with MRD diagnostics within clinical trials. In fact, MRD measurements might well be used as a surrogate end point, thereby significantly shortening the follow-up. The MRD techniques need to be sensitive (≤10−4), broadly applicable, accurate, reliable, fast, and affordable. Thus far, flow cytometry and polymerase chain reaction (PCR) analysis of rearranged immunoglobulin and T-cell receptor genes (allele-specific oligonucleotide [ASO]-PCR) are claimed to meet these criteria, but classical flow cytometry does not reach a solid 10−4, whereas classical ASO-PCR is time-consuming and labor intensive. Therefore, 2 high-throughput technologies are being explored, ie, high-throughput sequencing and next-generation (multidimensional) flow cytometry, both evaluating millions of sequences or cells, respectively. Each of them has specific advantages and disadvantages.

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.

scholarly journals Nonmyeloablative allogeneic transplantation achieves clinical and molecular remission in cutaneous T-cell lymphoma

2020 ◽  
Vol 4 (18) ◽  
pp. 4474-4482 ◽  
Author(s):  
Wen-Kai Weng ◽  
Sally Arai ◽  
Andrew Rezvani ◽  
Laura Johnston ◽  
Robert Lowsky ◽  
...  
Keyword(s):  
T Cell ◽  
High Throughput Sequencing ◽  
Residual Disease ◽  
Chronic Gvhd ◽  
Survival Rates ◽  
Stage Iv ◽  
Allogeneic Transplantation ◽  
Cell Receptor ◽  
Advanced Stage ◽  
Molecular Remission

Abstract The majority of patients with refractory, advanced-stage mycosis fungoides (MF) or Sézary syndrome (SS) have a life expectancy of <5 years. Here, we report a phase 2 study of a novel nonmyeloablative allogeneic transplantation strategy tailored for this patient population. This study has completed the enrollment, and 35 patients (13 MF, 22 SS) have undergone transplant as planned. The majority (80%) of the patients had stage IV disease and received multiple previous systemic therapies. All patients had active disease at the time of conditioning using total skin electron beam therapy, total lymphoid irradiation, and antithymocyte globulin, and received allograft infusion as outpatients. Cyclosporine or tacrolimus and mycophenolate mofetil were used for graft-versus-host disease (GVHD) prophylaxis. Patients tolerated the transplant well, with 1- and 2-year nonrelapse mortality of 3% and 14%, respectively. The day +180 cumulative incidence of grade 2 to 4 acute GVHD was 16%, and the 2-year incidence of moderate/severe chronic GVHD was 32%. With a median posttransplant follow-up of 5.4 years, the 2-, 3-, and 5-year overall survival rates were 68%, 62%, and 56%. Using high-throughput sequencing of the T-cell receptor for minimal residual disease monitoring, we observed that 43% achieved molecular remission, which was associated with a lower incidence of disease progression or relapse (9% vs 87%; P = .02). Our study also showed that patients who were aged ≥65 years at the time of allotransplant had similar clinical outcomes compared with younger patients. Thus, we have developed an alternative and potentially curative nonmyeloablative allogeneic transplant regimen for patients with advanced stage MF/SS. This trial was registered at www.clinicaltrials.gov as #NCT00896493.

scholarly journals Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor

2017 ◽  
Vol 137 (6) ◽  
pp. e131-e138 ◽  
Author(s):  
Tiago R. Matos ◽  
Menno A. de Rie ◽  
Marcel B.M. Teunissen
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
Research Techniques
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