ABSTRACT
While whole genome sequencing (WGS) of cell-free DNA (cfDNA) holds enormous promise for detection of molecular residual disease (MRD), its performance is limited by WGS error rate. Here we introduce AccuScan, an efficient cfDNA WGS technology that enables genome-wide error correction at single read-level, achieving an error rate of 4.2 × 10-7, which is about two orders of magnitude lower than a read-centric de-noising method. The application of AccuScan to MRD demonstrated analytical sensitivity down to 10-6 circulating variant allele frequency at 99% sample-level specificity. AccuScan showed 90% landmark sensitivity (within 6 weeks after surgery) and 100% specificity for predicting relapse in colorectal cancer. It also showed 67% sensitivity and 100% specificity in esophageal cancer using samples collected within one week after surgery. When AccuScan was applied to monitor immunotherapy in melanoma patients, the circulating tumor DNA (ctDNA) levels and dynamic profiles were consistent with clinical outcomes. Overall, AccuScan provides a highly accurate WGS solution for MRD detection, empowering ctDNA detection at parts per million range without requiring high sample input or personalized reagents.
ABSTRACT
While whole genome sequencing (WGS) of cell-free DNA (cfDNA) holds enormous promise for molecular residual disease (MRD) detection, its performance is limited by WGS error rate. Here we introduce AccuScan, an efficient cfDNA WGS technology that enables genome-wide error correction at single read level, achieving an error rate of 4.2×10 -7 , which is about two orders of magnitude lower than a read-centric de-noising method. When applied to MRD detection, AccuScan demonstrated analytical sensitivity down to 10 -6 circulating tumor allele fraction at 99% sample level specificity. In colorectal cancer, AccuScan showed 90% landmark sensitivity for predicting relapse. It also showed robust MRD performance with esophageal cancer using samples collected as early as 1 week after surgery, and predictive value for immunotherapy monitoring with melanoma patients. Overall, AccuScan provides a highly accurate WGS solution for MRD, empowering circulating tumor DNA detection at parts per million range without high sample input nor personalized reagents. One Sentence Summary: AccuScan showed remarkable ultra-low limit of detection with a short turnaround time, low sample requirement and a simple workflow for MRD detection.
ABSTRACT
OBJECTIVES: Cancer morbidity and mortality can be reduced if the cancer is detected early. Cell-free DNA (cfDNA) fragmentomics emerged as a novel epigenetic biomarker for early cancer detection, however, it is still at its infancy and requires technical improvement. We sought to apply a single-strand DNA sequencing technology, for measuring genetic and fragmentomic features of cfDNA and evaluate the performance in detecting multiple cancers. METHODS: Blood samples of 364 patients from six cancer types (colorectal, esophageal, gastric, liver, lung, and ovarian cancers) and 675 healthy individuals were included in this study. Circulating tumor DNA mutations, cfDNA fragmentomic features and a set of protein biomarkers were assayed. Sensitivity and specificity were reported by cancer types and stages. RESULTS: Circular Ligation Amplification and sequencing (CLAmp-seq), a single-strand DNA sequencing technology, yielded a population of ultra-short fragments (<100â¯bp) than double-strand DNA preparation protocols and reveals a more significant size difference between cancer and healthy cfDNA fragments (25.84â¯bp vs. 16.05â¯bp). Analysis of the subnucleosomal peaks in ultra-short cfDNA fragments indicates that these peaks are regulatory element "footprints" and correlates with gene expression and cancer stages. At 98â¯% specificity, a prediction model using ctDNA mutations alone showed an overall sensitivity of 46â¯%; sensitivity reaches 60â¯% when protein is added, sensitivity further increases to 66â¯% when fragmentomics is also integrated. More improvements observed for samples representing earlier cancer stages than later ones. CONCLUSIONS: These results suggest synergistic properties of protein, genetic and fragmentomics features in the identification of early-stage cancers.
Subject(s)
Cell-Free Nucleic Acids , Circulating Tumor DNA , Neoplasms , Humans , Early Detection of Cancer , Mutation , Circulating Tumor DNA/genetics , Neoplasms/diagnosis , Neoplasms/genetics , Biomarkers, Tumor/geneticsABSTRACT
Here, we report on the results of a phase I/II trial (NCT00490529) for patients with mantle cell lymphoma who, having achieved remission after immunochemotherapy, were vaccinated with irradiated, CpG-activated tumor cells. Subsequently, vaccine-primed lymphocytes were collected and reinfused after a standard autologous stem cell transplantation (ASCT). The primary endpoint was detection of minimal residual disease (MRD) within 1 yr after ASCT at the previously validated threshold of ≥1 malignant cell per 10,000 leukocyte equivalents. Of 45 evaluable patients, 40 (89%) were found to be MRD negative, and the MRD-positive patients experienced early subsequent relapse. The vaccination induced antitumor CD8 T cell immune responses in 40% of patients, and these were associated with favorable clinical outcomes. Patients with high tumor PD-L1 expression after in vitro exposure to CpG had inferior outcomes. Vaccination with CpG-stimulated autologous tumor cells followed by the adoptive transfer of vaccine-primed lymphocytes after ASCT is feasible and safe.
Subject(s)
Cancer Vaccines/immunology , Immunity , Lymphoma, Mantle-Cell/immunology , T-Lymphocytes/immunology , Adult , Aged , B7-H1 Antigen/metabolism , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/adverse effects , Cell Line, Tumor , Endpoint Determination , Female , Humans , Immunologic Memory , Kaplan-Meier Estimate , Male , Middle Aged , Neoplasm, Residual/immunology , Oligodeoxyribonucleotides , Transplantation, Autologous , Treatment OutcomeABSTRACT
The introduction of novel agents has led to major improvements in clinical outcomes for patients with multiple myeloma. To shorten evaluation times for new treatments, health agencies are currently examining minimal residual disease (MRD) as a surrogate end point in clinical trials. We assessed the prognostic value of MRD, measured during maintenance therapy by next-generation sequencing (NGS). MRD negativity was defined as the absence of tumor plasma cell within 1 000 000 bone marrow cells (<10-6). Data were analyzed from a recent clinical trial that evaluated the role of transplantation in newly diagnosed myeloma patients treated with lenalidomide, bortezomib, and dexamethasone (RVD). MRD negativity was achieved at least once during maintenance in 127 patients (25%). At the start of maintenance therapy, MRD was a strong prognostic factor for both progression-free survival (adjusted hazard ratio, 0.22; 95% confidence interval, 0.15-0.34; P < .001) and overall survival (adjusted hazard ratio, 0.24; 95% confidence interval, 0.11-0.54; P = .001). Patients who were MRD negative had a higher probability of prolonged progression-free survival than patients with detectable residual disease, regardless of treatment group (RVD vs transplant), cytogenetic risk profile, or International Staging System disease stage at diagnosis. These results were similar after completion of maintenance therapy. Our findings confirm the value of MRD status, as determined by NGS, as a prognostic biomarker in multiple myeloma, and suggest that this approach could be used to adapt treatment strategies in future clinical trials.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , High-Throughput Nucleotide Sequencing , Multiple Myeloma/metabolism , Aged , Bone Marrow/metabolism , Bone Marrow/pathology , Bortezomib/administration & dosage , Dexamethasone/administration & dosage , Disease-Free Survival , Female , Follow-Up Studies , Humans , Lenalidomide/administration & dosage , Maintenance Chemotherapy , Male , Middle Aged , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/mortality , Neoplasm, Residual , Plasma Cells/metabolism , Plasma Cells/pathology , Survival RateSubject(s)
Flow Cytometry/methods , Immunoglobulins/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Neoplasm, Residual/genetics , Sequence Analysis, DNA/methods , Adult , Aged , Aged, 80 and over , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Male , Middle Aged , Neoplasm Recurrence, Local , Neoplasm, Residual/diagnosis , Prognosis , Reproducibility of Results , Sensitivity and Specificity , Survival AnalysisSubject(s)
Hematopoietic Stem Cell Transplantation , High-Throughput Nucleotide Sequencing , Multiple Myeloma , Real-Time Polymerase Chain Reaction , Adult , Aged , Autografts , Female , Humans , Male , Middle Aged , Multiple Myeloma/blood , Multiple Myeloma/genetics , Multiple Myeloma/therapy , Neoplasm, Residual , Transplantation, AutologousABSTRACT
In B-cell precursor acute lymphoblastic leukemia, the initial leukemic cells share the same antigen receptor gene rearrangements. However, due to ongoing rearrangement processes, leukemic cells with different gene rearrangement patterns can develop, resulting in subclone formation. We studied leukemic subclones and their distribution in the bone marrow and peripheral blood at diagnosis. Antigen receptor gene rearrangements (IGH, IGK, TRG, TRD, TRB) were analyzed by next-generation sequencing in seven paired bone marrow samples and five paired bone marrow-peripheral blood samples. Background-thresholds were defined, which enabled identification of leukemic gene rearrangements down to very low levels. Paired bone marrow analysis showed oligoclonality in all 7 patients and up to 34 leukemic clones per patient. Additional analysis of evolutionary-related IGH gene rearrangements revealed up to 171 leukemic clones per patient. Interestingly, overall 86% of all leukemic gene rearrangements, including small subclones, were present in both bone marrow samples (range per patient: 72-100%). Paired bone marrow-peripheral blood analysis showed that 83% of all leukemic gene rearrangements in bone marrow were also found in peripheral blood (range per patient: 81-100%). Remarkably, in the paired bone marrow samples and paired bone marrow-peripheral blood samples the vast majority of leukemic gene rearrangements had a similar frequency (<5-fold frequency difference) (96% and 96% of leukemic rearrangements, respectively). Together, these results indicate that B-cell precursor acute lymphoblastic leukemia is generally highly oligoclonal. Nevertheless, the vast majority of leukemic clones, even the minor antigen receptor-defined subclones, are homogeneously distributed throughout the bone marrow and peripheral blood compartment.
Subject(s)
Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Bone Marrow/pathology , Clonal Evolution/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptors, Antigen/genetics , Child , Gene Rearrangement , Humans , Neoplastic Stem Cells/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Receptors, Antigen/metabolismABSTRACT
Patients with HIV are at increased risk for developing B-cell lymphomas likely due in part to chronic antigen stimulation leading to clonal immunoglobulin (Ig) gene rearrangements. Next-generation sequencing (NGS)-based identification of circulating Ig clonotypes has not been well-characterized in HIV-related lymphomas. The AIDS Malignancies Consortium (AMC) enrolled 51 untreated patients with HIV-related B-cell lymphomas and analyzed paired tumor/plasma specimens for Ig clonotypes using an NGS approach (AMC064, NCT00981097). Lymphoma-specific clonotypes (>5% frequency) were identified in 83% (33/40) of tumor specimens. Results from paired tumor/plasma specimens showed identical circulating clonotypes in the plasma from 97% (32/33) of patients. High International Prognostic Index (IPI) scores of 3-4 among patients with B-cell lymphoma correlated with higher lymphoma molecules/million diploid genomes in the plasma compared with lower IPI scores of 0-2, median 77335 vs. 6876, p = .005. Further studies are merited to determine whether plasma clonal Ig DNA is prognostic in HIV-related lymphomas.
Subject(s)
Clonal Evolution/genetics , DNA, Circular , Gene Frequency , Immunoglobulins/genetics , Lymphoma, AIDS-Related/genetics , Adult , Aged , Biomarkers , Female , Gene Rearrangement , Humans , Lymphoma, AIDS-Related/blood , Lymphoma, AIDS-Related/diagnosis , Male , Middle Aged , Neoplasm Staging , Young AdultABSTRACT
Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.
Subject(s)
Antigen Presentation/immunology , Antigens, Neoplasm/immunology , Immunoglobulin Variable Region/immunology , Lymphoma, Mantle-Cell/immunology , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , CD4-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , DNA Mutational Analysis , Epitopes, T-Lymphocyte/immunology , Exome/genetics , Genomics , HLA-D Antigens/immunology , Histocompatibility Antigens Class I/immunology , Humans , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Immunotherapy/trends , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/pathology , Lymphoma, Mantle-Cell/therapy , Mutation , ProteomicsABSTRACT
Recent advances in next-generation sequencing (NGS) have enabled the quantitation of circulating tumour DNA (ctDNA) encoding the clonal rearranged V(D)J immunoglobulin locus. We aimed to evaluate the clonal heterogeneity of follicular lymphoma (FL) in the tumour and the plasma at diagnosis and to assess the prognostic value of the ctDNA level. Plasma samples at diagnosis were available for 34 patients registered in the PRIMA trial (NCT00140582). One tumour clonotype or more could be detected for 29 (85%) and 25 (74%) patients, respectively, in the tumour or plasma samples. In 18 patients, several subclones were detected in the tumour (2 to 71 subclones/cases) and/or in the plasma (2 to 20 subclones/cases). In more than half of the cases, the distribution of subclones differed between the tumour and plasma samples, reflecting high clonal heterogeneity and diversity in lymphoma subclone dissemination. In multivariate analysis, a high level of ctDNA was the only independent factor associated with patients' progression-free survival (HR 4, IC 95 (1.1-37), p=.039). In conclusion, an NGS-based immunosequencing method reveals the marked clonal heterogeneity of follicular lymphoma in patients with FL, and quantification of ctDNA at diagnosis represents a potential powerful prognostic biomarker that needs to be investigated in larger cohorts.
Subject(s)
Biomarkers, Tumor/genetics , Circulating Tumor DNA/genetics , Genes, Immunoglobulin Heavy Chain/genetics , Lymphoma, Follicular/genetics , V(D)J Recombination , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/blood , Chi-Square Distribution , Circulating Tumor DNA/blood , Disease-Free Survival , Female , Genetic Predisposition to Disease , High-Throughput Nucleotide Sequencing , Humans , Lymphoma, Follicular/blood , Lymphoma, Follicular/drug therapy , Lymphoma, Follicular/immunology , Male , Middle Aged , Multivariate Analysis , Phenotype , Predictive Value of Tests , Prospective Studies , Risk Factors , Time Factors , Treatment Outcome , Young AdultABSTRACT
OBJECTIVE: Ankylosing spondylitis (AS), a chronic inflammatory disorder, has a notable association with HLA-B27. One hypothesis suggests that a common antigen that binds to HLA-B27 is important for AS disease pathogenesis. This study was undertaken to determine sequences and motifs that are shared among HLA-B27-positive AS patients, using T cell repertoire next-generation sequencing. METHODS: To identify motifs enriched among B27-positive AS patients, we performed T cell receptor ß (TCRß) repertoire sequencing on samples from 191 B27-positive AS patients, 43 B27-negative AS patients, and 227 controls, and we obtained >77 million TCRß clonotype sequences. First, we assessed whether any of 50 previously published sequences were enriched in B27-positive AS patients. We then used training and test cohorts to identify discovered motifs that were enriched in B27-positive AS patients versus controls. RESULTS: Six previously published and 11 discovered motifs were enriched in the B27-positive AS samples as compared to controls. After combining motifs related by sequence, we identified a total of 15 independent motifs. Both the full set of 15 motifs and a set of 6 published motifs were enriched in the B27-positive AS patients as compared to B27-positive healthy individuals (P = 0.049 and P = 0.001, respectively). Using an independent cohort, we validated that at least some of these motifs were associated with AS, and not simply with B27-positive status. CONCLUSION: We identified TCRß motifs that are enriched in B27-positive AS patients as compared to B27-positive healthy controls. This suggests that a common antigen, presented by HLA-B27 and detected by CD8+ T cells, may be associated with AS disease pathogenesis.
Subject(s)
HLA-B27 Antigen/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Spondylitis, Ankylosing/immunology , Adolescent , Adult , Amino Acid Motifs , Amino Acid Sequence , Female , Humans , Male , Middle Aged , Sequence Analysis , Young AdultABSTRACT
Aggressive induction chemotherapy followed by autologous haematopoietic stem cell transplant (auto-HCT) is effective for younger patients with mantle cell lymphoma (MCL). However, the optimal induction regimen is widely debated. The Southwestern Oncology Group S1106 trial was designed to assess rituximab plus hyperCVAD/MTX/ARAC (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone, alternating with high dose cytarabine and methotrexate) (RH) versus rituximab plus bendamustine (RB) in a randomized phase II trial to select a pre-transplant induction regimen for future development. Patients had previously untreated stage III, IV, or bulky stage II MCL and received either 4 cycles of RH or 6 cycles of RB, followed by auto-HCT. Fifty-three of a planned 160 patients were accrued; an unacceptably high mobilization failure rate (29%) on the RH arm prompted premature study closure. The estimated 2-year progression-free survival (PFS) was 81% vs. 82% and overall survival (OS) was 87% vs. 88% for RB and RH, respectively. RH is not an ideal platform for future multi-centre transplant trials in MCL. RB achieved a 2-year PFS of 81% and a 78% MRD negative rate. Premature closure of the study limited the sample size and the precision of PFS estimates and MRD rates. However, RB can achieve a deep remission and could be a platform for future trials in MCL.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bendamustine Hydrochloride/administration & dosage , Hematopoietic Stem Cell Transplantation/methods , Lymphoma, Mantle-Cell/therapy , Remission Induction/methods , Rituximab/administration & dosage , Adolescent , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Combined Modality Therapy/methods , Combined Modality Therapy/mortality , Cyclophosphamide/administration & dosage , Cytarabine/administration & dosage , Dexamethasone/administration & dosage , Disease-Free Survival , Doxorubicin/administration & dosage , Female , Hematopoietic Stem Cell Transplantation/mortality , Humans , Lymphoma, Mantle-Cell/mortality , Male , Methotrexate/administration & dosage , Middle Aged , Neoplasm, Residual/diagnosis , Transplantation, Autologous , Treatment Outcome , Vincristine/administration & dosage , Young AdultABSTRACT
While immune checkpoint blockade elicits efficacious responses in many patients with cancer, it also produces a diverse and unpredictable number of immune-related adverse events (IRAE). Mechanisms driving IRAEs are generally unknown. Because CTLA-4 blockade leads to proliferation of circulating T cells, we examined in this study whether ipilimumab treatment leads to clonal expansion of tissue-reactive T cells. Rather than narrowing the T-cell repertoire to a limited number of clones, ipilimumab induced greater diversification in the T-cell repertoire in IRAE patients compared with patients without IRAEs. Specifically, ipilimumab triggered increases in the numbers of clonotypes, including newly detected clones and a decline in overall T-cell clonality. Initial broadening in the repertoire occurred within 2 weeks of treatment, preceding IRAE onset. IRAE patients exhibited greater diversity of CD4+ and CD8+ T cells, but showed no differences in regulatory T-cell numbers relative to patients without IRAEs. Prostate-specific antigen responses to ipilimumab were also associated with increased T-cell diversity. Our results show how rapid diversification in the immune repertoire immediately after checkpoint blockade can be both detrimental and beneficial for patients with cancer. Cancer Res; 77(6); 1322-30. ©2016 AACR.
Subject(s)
Antibodies, Monoclonal/pharmacology , CTLA-4 Antigen/antagonists & inhibitors , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Prostatic Neoplasms, Castration-Resistant/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Humans , Ipilimumab , Male , Prognosis , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/pathology , T-Lymphocyte Subsets/drug effects , T-Lymphocytes, Regulatory/drug effectsABSTRACT
Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples. Sixteen of 19 (84%) patients with disease progression after HSCT had detectable ctDNA prior to progression at a median of 3·7 months prior to relapse/progression. Patients with detectable ctDNA 3 months after HSCT had inferior progression-free survival (PFS) (2-year PFS 58% vs. 84% in ctDNA-negative patients, P = 0·033). In multivariate models, detectable ctDNA was associated with increased risk of progression/death (Hazard ratio 3·9, P = 0·003) and increased risk of relapse/progression (Hazard ratio 10·8, P = 0·0006). Detectable ctDNA is associated with an increased risk of relapse/progression, but further validation studies are necessary to confirm these findings and determine the clinical utility of NGS-based minimal residual disease monitoring in lymphoma patients after HSCT.
Subject(s)
DNA, Neoplasm/blood , Hematopoietic Stem Cell Transplantation/standards , Lymphoma/diagnosis , Neoplasm, Residual/diagnosis , Adolescent , Adult , Aged , Death , Disease Progression , Female , Hematopoietic Stem Cell Transplantation/methods , Humans , Immunoglobulins/genetics , Lymphoma/genetics , Lymphoma/therapy , Male , Middle Aged , Receptors, Antigen, T-Cell/genetics , Recurrence , Retrospective Studies , Sequence Analysis, DNA , Transplantation, Homologous , Treatment Outcome , Young AdultABSTRACT
Since the incorporation of tyrosine kinase inhibitors into the treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL), the notion that all patients with "high-risk" ALL uniformly require allogeneic (allo) hematopoietic cell transplantation (HCT) has received increasing scrutiny. Although multiple studies have shown superiority of alloHCT over autologous (auto) hematopoietic cell transplantation for high-risk patients, these findings may be explained, in part, by contamination of the peripheral blood progenitor cell (PBPC) leukapheresis product by residual leukemic cells in patients undergoing autoHCT. We retrospectively evaluated minimal residual disease (MRD) using next-generation sequencing (NGS) in the PBPC leukapheresis product of 32 ALL patients who underwent autoHCT. Twenty-eight patients (88%) had diagnostic samples with quantifiable immunoreceptor rearrangements to follow for MRD. Twelve (38%) patients had Ph+ B-ALL, 12 (38%) had Philadelphia chromosome-negative (Ph-) B-ALL, and 4 (14%) had T cell ALL. With a median follow-up of 41 months (range, 3 to 217), median relapse-free survival (RFS) and overall survival for the entire cohort were 3.2 and 4.2 years, respectively; at 5 years after transplantation, 42% of patients remain alive and relapse free. Using MRD detection at a threshold of ≥ 1 × 10(-6), median RFS for patients with detectable MRD was 6.5 months and was not reached for patients without detectable disease (P = .0005). In multivariate analysis, the only factor significantly associated with relapse was the presence of MRD ≥1 × 10(-6) (odds ratio, 23.8; confidence interval, 1.8 to 312.9; P = .0158). Our findings suggest that NGS for MRD detection can predict long-term RFS in patients undergoing autoHCT for high-risk ALL.
Subject(s)
Hematopoietic Stem Cell Transplantation/standards , High-Throughput Nucleotide Sequencing/methods , Leukapheresis , Neoplasm, Residual/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Adult , Female , Humans , Male , Middle Aged , Philadelphia Chromosome , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Prognosis , Recurrence , Retrospective Studies , Transplantation, Autologous , Young AdultABSTRACT
Chemoimmunotherapy followed by autologous stem cell transplantation (ASCT) is a standard therapy for transplant-eligible patients with newly diagnosed mantle cell lymphoma (MCL). The achievement of complete remission (CR) and minimal residual disease (MRD) negativity are associated with better outcomes. We tested an induction regimen of rituximab/bendamustine followed by rituximab/high-dose cytarabine (RB/RC). This phase 2 study (NCT01661881) enrolled 23 transplant-eligible patients aged 42-69, of whom 70% were MCL international prognostic index low-risk. Patients received three cycles of RB followed by three cycles of RC. The primary endpoint of the trial was the rate of CR after six cycles of therapy, with a rate of 75% considered promising. 96% of patients achieved a CR/unconfirmed CR after treatment, meeting the primary objective. One patient progressed on study, one declined ASCT in CR, and the remaining 21 underwent successful stem cell collection and ASCT. After a median follow-up of 13 months, the progression-free survival rate was 96%. Among 15 MRD-evaluable patients who completed treatment, 93% achieved MRD negativity after RB/RC. In conclusion, RB/RC achieves very high CR and MRD negativity rates in transplant-eligible patients, with a favourable safety profile. RB/RC warrants further comparative studies, and may become a useful alternative to RCHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone)-based induction regimens in this patient population.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/mortality , Adolescent , Adult , Aged , Bendamustine Hydrochloride/administration & dosage , Cytarabine/administration & dosage , Disease-Free Survival , Female , Humans , Lymphoma, Mantle-Cell/blood , Male , Middle Aged , Remission Induction , Rituximab/administration & dosage , Survival RateABSTRACT
Monitoring antigen-specific T cells is critical for the study of immune responses and development of biomarkers and immunotherapeutics. We developed a novel multiplex assay that combines conventional immune monitoring techniques and immune receptor repertoire sequencing to enable identification of T cells specific to large numbers of antigens simultaneously. We multiplexed 30 different antigens and identified 427 antigen-specific clonotypes from 5 individuals with frequencies as low as 1 per million T cells. The clonotypes identified were validated several ways including repeatability, concordance with published clonotypes, and high correlation with ELISPOT. Applying this technology we have shown that the vast majority of shared antigen-specific clonotypes identified in different individuals display the same specificity. We also showed that shared antigen-specific clonotypes are simpler sequences and are present at higher frequencies compared to non-shared clonotypes specific to the same antigen. In conclusion this technology enables sensitive and quantitative monitoring of T cells specific for hundreds or thousands of antigens simultaneously allowing the study of T cell responses with an unprecedented resolution and scale.
Subject(s)
Enzyme-Linked Immunospot Assay , Epitopes, T-Lymphocyte/immunology , High-Throughput Nucleotide Sequencing , Receptors, Antigen, T-Cell/genetics , Receptors, Immunologic/genetics , T-Cell Antigen Receptor Specificity/genetics , T-Cell Antigen Receptor Specificity/immunology , Clonal Evolution/genetics , Clonal Evolution/immunology , Enzyme-Linked Immunospot Assay/methods , Enzyme-Linked Immunospot Assay/standards , Humans , Reproducibility of ResultsABSTRACT
IMPORTANCE: Carfilzomib-lenalidomide-dexamethasone therapy yields deep responses in patients with newly diagnosed multiple myeloma (NDMM). It is important to gain an understanding of this combination's tolerability and impact on minimal residual disease (MRD) negativity because this end point has been associated with improved survival. OBJECTIVE: To assess the safety and efficacy of carfilzomib-lenalidomide-dexamethasone therapy in NDMM and high-risk smoldering multiple myeloma (SMM). DESIGN, SETTING, AND PARTICIPANTS: Clinical and correlative pilot study at the National Institutes of Health Clinical Center. Patients with NDMM or high-risk SMM were enrolled between July 11, 2011, and October 9, 2013. Median follow-up was 17.3 (NDMM) and 15.9 months (SMM). INTERVENTIONS: Eight 28-day cycles were composed of carfilzomib 20/36 mg/m2 on days 1, 2, 8, 9, 15, and 16; lenalidomide 25 mg on days 1 through 21; and dexamethasone 20/10 mg (cycles 1-4/5-8) on days 1, 2, 8, 9, 15, 16, 22, and 23. Patients who achieved at least stable disease subsequently received 24 cycles of lenalidomide extended dosing. MAIN OUTCOMES AND MEASURES: Primary end points were neuropathy of grade 3 or greater (NDMM) and at least very good partial response rates (SMM). Minimal residual disease was also assessed. RESULTS: Of 45 patients with NDMM, none had neuropathy of grade 3 or greater. Of 12 patients with high-risk SMM, the most common of any-grade adverse events were lymphopenia (12 [100%]) and gastrointestinal disorders (11 [92%]). All patients with SMM achieved at least a very good partial response during the study period. Among the 28 patients with NDMM and the 12 with SMM achieving at least a near-complete response, MRD negativity was found in 28 of 28 (100% [95% CI, 88%-100%]), 11 of 12 (92% [95% CI, 62%-100%]) (multiparametric flow cytometry), 14 of 21 (67% [95% CI, 43%-85%]), and 9 of 12 (75% [95% CI, 43%-94%]) (next-generation sequencing), respectively. In patients with NDMM, 12-month progression-free survival for MRD-negative vs MRD-positive status by flow cytometry and next-generation sequencing was 100% vs 79% (95% CI, 47%-94%; P < .001) and 100% vs 95% (95% CI, 75%-99%; P = .02), respectively. CONCLUSIONS AND RELEVANCE: Carfilzomib-lenalidomide-dexamethasone therapy is tolerable and demonstrates high rates of MRD negativity in NDMM, translating into longer progression-free survival in patients achieving MRD negativity. Carfilzomib-lenalidomide-dexamethasone therapy also demonstrates efficacy in high-risk SMM.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Dexamethasone/administration & dosage , Multiple Myeloma/drug therapy , Oligopeptides/administration & dosage , Proteasome Inhibitors/administration & dosage , Thalidomide/analogs & derivatives , Adult , Aged , Aged, 80 and over , Angiogenesis Inhibitors/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Biomarkers, Tumor/genetics , Dexamethasone/adverse effects , Disease-Free Survival , Drug Administration Schedule , Female , Humans , Lenalidomide , Male , Maryland , Middle Aged , Multiple Myeloma/diagnosis , Multiple Myeloma/genetics , Multiple Myeloma/mortality , National Institutes of Health (U.S.) , Neoplasm, Residual , Oligopeptides/adverse effects , Pilot Projects , Proteasome Inhibitors/adverse effects , Risk Factors , Thalidomide/administration & dosage , Thalidomide/adverse effects , Time Factors , Treatment Outcome , United StatesABSTRACT
BACKGROUND: Diffuse large-B-cell lymphoma is curable, but when treatment fails, outcome is poor. Although imaging can help to identify patients at risk of treatment failure, they are often imprecise, and radiation exposure is a potential health risk. We aimed to assess whether circulating tumour DNA encoding the clonal immunoglobulin gene sequence could be detected in the serum of patients with diffuse large-B-cell lymphoma and used to predict clinical disease recurrence after frontline treatment. METHODS: We used next-generation DNA sequencing to retrospectively analyse cell-free circulating tumour DNA in patients assigned to one of three treatment protocols between May 8, 1993, and June 6, 2013. Eligible patients had diffuse large-B-cell lymphoma, no evidence of indolent lymphoma, and were previously untreated. We obtained serial serum samples and concurrent CT scans at specified times during most treatment cycles and up to 5 years of follow-up. VDJ gene segments of the rearranged immunoglobulin receptor genes were amplified and sequenced from pretreatment specimens and serum circulating tumour DNA encoding the VDJ rearrangements was quantitated. FINDINGS: Tumour clonotypes were identified in pretreatment specimens from 126 patients who were followed up for a median of 11 years (IQR 6·8-14·2). Interim monitoring of circulating tumour DNA at the end of two treatment cycles in 108 patients showed a 5-year time to progression of 41·7% (95% CI 22·2-60·1) in patients with detectable circulating tumour DNA and 80·2% (69·6-87·3) in those without detectable circulating tumour DNA (p<0·0001). Detectable interim circulating tumour DNA had a positive predictive value of 62·5% (95% CI 40·6-81·2) and a negative predictive value of 79·8% (69·6-87·8). Surveillance monitoring of circulating tumour DNA was done in 107 patients who achieved complete remission. A Cox proportional hazards model showed that the hazard ratio for clinical disease progression was 228 (95% CI 51-1022) for patients who developed detectable circulating tumour DNA during surveillance compared with patients with undetectable circulating tumour DNA (p<0·0001). Surveillance circulating tumour DNA had a positive predictive value of 88·2% (95% CI 63·6-98·5) and a negative predictive value of 97·8% (92·2-99·7) and identified risk of recurrence at a median of 3·5 months (range 0-200) before evidence of clinical disease. INTERPRETATION: Surveillance circulating tumour DNA identifies patients at risk of recurrence before clinical evidence of disease in most patients and results in a reduced disease burden at relapse. Interim circulating tumour DNA is a promising biomarker to identify patients at high risk of treatment failure. FUNDING: National Cancer Institute and Adaptive Biotechnologies.