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Featured Publications
Next-Generation Sequencing in Adult B Cell Acute Lymphoblastic Leukemia Patients
Biology of Blood and Marrow Transplantation | January, 2017We used next generation sequencing (NGS) of the immunoglobulin genes to evaluate residual disease in 153 specimens from 32 patients with adult B cell ALL enrolled in a single, multi-center study. The sequencing results were compared to multi-parameter flow cytometry (MFC) data in 66 specimens (25 patients) analyzed by both methods. There was a strong concordance (82%) between the methods in the qualitative determination of the presence of disease. However, in 17% of cases leukemia was detected by sequencing, but not by MFC. In 54 bone marrow (BM) and peripheral blood (PB) paired specimens, the burden of leukemia detected by NGS was lower in PB than BM, although still detectable in 68% of the 28 paired specimens with positive BM.
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Next-generation sequencing-based detection of circulating tumour DNA after allogeneic stem cell transplantation for lymphoma
British Journal of Haematology | December, 2016Next-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.
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CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients
The Journal of Clinical Investigation | April, 2016T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR–T cell products were prepared from unselected T cells.
Immunotherapy with a CAR–T cell product of defined composition enabled identification of factors that correlated with CAR–T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR–T cell dosing strategies that mitigated toxicity and improved disease-free survival.
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TCR Sequencing Can Identify and Track Glioma-Infiltrating T Cells after DC Vaccination
Cancer Immunology Research | March, 2016Although immunotherapeutic strategies are emerging as adjunctive treatments for cancer, sensitive methods of monitoring the immune response after treatment remain to be established. We used a novel next-generation sequencing approach to determine whether quantitative assessments of tumor-infiltrating lymphocyte (TIL) content and the degree of overlap of T-cell receptor (TCR) sequences in brain tumors and peripheral blood were predictors of immune response and overall survival in glioblastoma patients treated with autologous tumor lysate–pulsed dendritic cell immunotherapy. A statistically significant correlation was found between a higher estimated TIL content and increased time to progression and overall survival.
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T-cell receptor profiling in cancer
Molecular Oncology | September, 2015Immunosequencing is a platform technology that allows the enumeration, specification and quantification of each and every B- and/or T-cell in any biologic sample of interest. Thus, it provides an assessment of the level and distribution of all the clonal lymphocytes in any sample, and allows “tracking” of a single clone or multiple clones of interest over time or from tissue to tissue within a given patient. It is based on bias-controlled multiplex PCR and high-throughput sequencing, and it is highly accurate, standardized, and sensitive.
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Multiplex Identification of Antigen-Specific T Cell Receptors Using a Combination of Immune Assays and Immune Receptor Sequencing
PLOS ONE | October, 2015Monitoring 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.
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Gut and liver T-cells of common clonal origin in primary sclerosing cholangitis-inflammatory bowel disease
Background & Aims: Recruitment of gut-derived memory T-cells to the liver is believed to drive hepatic inflammation in primary sclerosing cholangitis (PSC). However, whether gut-infiltrating and liver-infiltrating T-cells share T cell receptors (TCRs) and antigenic specificities is unknown. We used paired gut and liver samples from PSC patients with concurrent inflammatory bowel disease (PSC-IBD), and normal tissue samples from colon cancer controls, to assess potential T cell clonotype overlap between the two compartments.
Methods: High-throughput sequencing of TCRb repertoires was applied on matched colon, liver and blood samples from patients with PSC-IBD (n = 10), and on paired tumor-adjacent normal gut and liver tissue samples from colon cancer patients (n = 10). Results:An average of 9.7% (range: 4.7–19.9%) memory T cell clonotypes overlapped in paired PSC-IBD affected gut and liver samples, after excluding clonotypes present at similar frequencies in blood. Shared clonotypes constituted on average 16.0% (range: 8.7–32.6%) and 15.0% (range: 5.9–26.3%) of the liver and gut memory T-cells, respectively. A significantly higher overlap was observed between paired PSC-IBD affected samples (8.7%, p = 0.0007) compared to paired normal gut and liver samples (3.6%), after downsampling to equal number of reads.
Conclusion: Memory T-cells of common clonal origin were detected in paired gut and liver samples of patients with PSC- IBD. Our data indicate that this is related to PSC-IBD pathogenesis, suggesting that memory T-cells driven by shared antigens are present in the gut and liver of PSC-IBD patients. Our findings support efforts to therapeutically target memory T cell recruitment in PSC-IBD.
Lay summary: Primary sclerosing cholangitis (PSC) is a devastating liver disease strongly associated with inflammatory bowel disease (IBD). The cause of PSC is unknown, but it has been suggested that the immune reactions in the gut and the liver are con- nected. Our data demonstrate for the first time that a proportion of the T-cells in the gut and the liver react to similar triggers, and that this proportion is particularly high in patients with PSC and IBD.
Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment
Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3hi, CD127lo Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro–expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2–sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.
The T-cell receptor repertoire influences the tumor microenvironment and is associated with survival in aggressive B-cell lymphoma
Several studies have high-lighted the importance of various aspects of the tumor microenvironment upon the clinical outcome in lymphoma. However, the role of the intra-tumoral T-cell receptor (TCR) repertoire has not been established. This is the first intra-tumoral TCR repertoire study of a large Diffuse Large B-cell Lymphoma (DLBCL) cohort. There have been several studies in solid cancers examining the dynamics of the TCR-repertoire after treatment with immune checkpoint inhibitors. However here, particular attention has been paid to the association with progression free and overall survival and TCR repertoire after conventional front-line (i.e. non checkpoint blockade) therapy. Observations in the setting of a non-defined tumor antigen (EBV-ve DLBCL) were contrasted with those with a well-defined tumour antigen (EBV+ DLBCL) and with a nonlymphomatous cancer (melanoma) that is typically associated with a high mutational load. The composition of the TCR repertoire should be factored into the rational design of immune-based therapies.
ABC transporters and NR4A1 identify a quiescent subset of tissue-resident memory T cells
Immune surveillance in tissues is mediated by a long-lived subset of tissue-resident memory T cells (Trm cells). A putative subset of tissue-resident long-lived stem cells is characterized by the ability to efflux Hoechst dyes and is referred to as side
population (SP) cells. Here, we have characterized a subset of SP T cells (Tsp cells) that exhibit a quiescent (G0) phenotype in humans and mice. Human Trm cells in the gut and BM were enriched in Tsp cells that were predominantly in the G0 stage of the cell cycle. Moreover, in histone 2B-GFP mice, the 2B-GFP label was retained in Tsp cells, indicative of a slow-cycling phenotype. Human Tsp cells displayed a distinct gene-expression profile that was enriched for genes overexpressed in Trm cells. In mice, proteins encoded by Tsp signature genes, including nuclear receptor subfamily 4 group A member 1 (NR4A1) and ATP-binding cassette (ABC) transporters, influenced the function and differentiation of Trm cells. Responses to adoptive transfer of human Tsp cells into immune-deficient mice and plerixafor therapy suggested that human Tsp cell mobilization could be manipulated as a potential cellular therapy. These data identify a distinct subset of human T cells with a quiescent slow-cycling phenotype, propensity for tissue enrichment, and potential to mobilize into circulation, which may be harnessed for adoptive cellular therapy.
Deep Sequencing of T-Cell Receptor DNA as a biomarker of clonally expanded TILs in breast cancer after immunotherapy
In early stage breast cancer, the degree of tumor-infiltrating lymphocytes (TILs) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryo-immunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 x 106 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared to monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring, and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T cell responses to therapy and for thestudy of cryo-immunotherapy in early-stage breast cancer.
Atezolizumab in combination with bevacizumab enhances antigen-specific T-cell migration in metastatic renal cell carcinoma
Anti-tumour immune activation by checkpoint inhibitors leads to durable responses in a variety of cancers, but combination approaches are required to extend this benefit beyond a subset of patients. In preclinical models tumour-derived VEGF limits immune cell activity while anti-VEGF augments intra-tumoral T-cell infiltration, potentially through vascular normalization and endothelial cell activation. This study investigates how VEGF blockade with bevacizumab could potentiate PD-L1 checkpoint inhibition with atezolizumab in mRCC. Tissue collections are before treatment, after bevacizumab and after the addition of atezolizumab. We discover that intra-tumoral CD8þ T cells increase following combination treatment. A related increase is found in intra-tumoral MHC-I, Th1 and T-effector markers, and chemokines, most notably CX3CL1 (fractalkine). We also discover that the fractalkine receptor increases on peripheral CD8þ T cells with treatment. Furthermore, trafficking lymphocyte increases are observed in tumors following bevacizumab and combination treatment. These data suggest that the anti-VEGF and anti-PD-L1 combination improves antigen-specific T-cell migration.
Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens
Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRa. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRa, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2þ MAITcells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAITantigens are derived from riboflavin metabolites, this suggests that TRAV12-2þ clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites.
A Public Database of Memory and Naive B- Cell Receptor Sequences
The vast diversity of B-cell receptors (BCR) and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.
Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency
Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRb sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21 lo B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease. The Journal of Immunology, 2016, 197: 000–000.
Chemotherapy and radiation therapy elicits tumor specific T cell responses in a breast cancer patient
Background: Experimental evidence and clinical studies in breast cancer suggest that some anti-tumor therapy regimens generate stimulation of the immune system that accounts for tumor clinical responses, however, demonstration of the immunostimulatory power of these therapies on cancer patients continues to be a formidable challenge. Here we present experimental evidence from a breast cancer patient with complete clinical response after 7 years, associated with responsiveness of tumor specific T cells.
Methods: T cells were obtained before and after anti-tumor therapy from peripheral blood of a 63-years old woman diagnosed with ductal breast cancer (HER2/neu+++, ER-, PR-, HLA-A*02:01) treated with surgery, followed by paclitaxel, trastuzumab (suspended due to cardiac toxicity), and radiotherapy. We obtained a leukapheresis before surgery and after 8 months of treatment. Using in vitro cell cultures stimulated with autologous monocyte- derived dendritic cells (DCs) that produce high levels of IL-12, we characterize by flow cytometry the phenotype of tumor associated antigens (TAAs) HER2/neu and NY-ESO 1 specific T cells. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and Tumor Infiltrating Lymphocytes (TILs) were performed in order to correlate both repertoires prior and after therapy.
Results: We evidence a functional recovery of T cell responsiveness to polyclonal stimuli and expansion of TAAs specific CD8+ T cells using peptide pulsed DCs, with an increase of CTLA-4 and memory effector phenotype after anti-tumor therapy. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and TILs showed that whereas the TCR-Vβ04-02 clonotype is highly expressed in TILs the HER2/neu specific T cells are expressed mainly in blood after therapy, suggesting that this particular TCR was selectively enriched in blood after anti-tumor therapy.
Conclusions: Our results show the benefits of anti-tumor therapy in a breast cancer patient with clinical complete response in two ways, by restoring the responsiveness of T cells by increasing the frequency and activation in peripheral blood of tumor specific T cells present in the tumor before therapy.
T cell receptor diversity in the human thymus
Analyzing the CDR3 Repertoire with respect to TCR—Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS
V-D-J rearrangement of the TCR—beta chain follows the 12/23 rule and the beyond 12/23 restriction. Currently, the proportion and characteristics of TCR—beta chain V—J rearrangement is unclear. We used high-throughput sequencing to compare and analyze TCR—beta chain V-J rearrangement and V-D-J rearrangement in the CDR3 repertoires of T cells from the PBMCs of six volunteers and six BALB/c mice. The results showed that the percentage of V-J rearrangement of the volunteers was approximately 0.7%, whereas that of the mice was 2.2%. The clonality of mice V-J rearrangement was significantly reduced compared with the V-D-J rearrangement, whereas the clonality of human V-J rearrangement was slightly reduced compared with the V-D-J rearrangement. V-J rearrangement in CDR3 involved the significant usage of N, S, F and L, whereas V-D-J rearrangement in CDR3 involved the significant usage of R and G. The levels of V deletion and J deletion in V-J rearrangement were significantly reduced compared with V-D-J rearrangement. TRBD and TRBJ usage in V-J rearrangement differedfrom that of V-D-J rearrangement, including dominant usage of TRBV and TRBJ and their pairing. Taken together, these results provide new ideas and technology for studies of V-D-J rearrangement and V-J rearrangement in the CDR3 repertoire.
Definitive chemoradiation alters the immunologic landscape and immune checkpoints in head and neck cancer
Background: Preclinical and clinical studies suggest potential synergy between high dose per fraction focal radiation and immunotherapy. However, conventionally fractionated radiation regimens in combination with concurrent chemotherapy are more commonly administered to patients as definitive treatment and may have both immune-stimulating and -suppressive effects.
Methods: We prospectively collected longitudinal samples from head and neck squamous cell carcinoma patients receiving definitive radiation therapy. We quantified changes in populations of circulating immune cells and chemokines CXCL9, 10, and 16. Analyses of humoral and cellular immune responses were conducted in select patients via proteomic analysis and T-cell receptor sequencing.
Results: Treatment not only increased circulating CD-8þ T-effector cells, but also myeloid-derived suppressor cells, regulatory T cells, and checkpoint receptor-expressing T cells, particularly PD-1þ T cells. Significant decreases in CXCL10 and increases in CXLC16 were noted. Treatment also increased the percentage of unique and dominant TCR clones, and increased humoral responses as measured by proteomic array.
Conclusions: Our results suggest that fractionated chemoradiation leads to quantifiable effects in circulating immune mediators, including a balance of stimulatory and suppressive mechanisms. These results suggest future combinations with immune checkpoint blockade.
Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor
Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this, we developed a multi-step strategy that involved TCRB deep sequencing of the CD8 + PD-1 + T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid, personalized, TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity.
Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor
Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8+ and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR+ T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR+ T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.
Antigen-specificity of T-cell Infiltrates in Biopsies with T-cell Mediated Rejection and BK Polyomavirus Viremia: Analysis by Next Generation Sequencing
This study interrogates the antigen-specificity of inflammatory infiltrates in renal biopsies with BK polyomavirus (BKPyV) viremia (BKPyVM) with or without allograft nephropathy (BKPyVN). PBMC from 5 healthy HLA-A0101 subjects were stimulated by peptides derived from the BKPYV proteome or polymorphic regions of HLA. Next generation sequencing (NGS) of the T-cell receptor (TCR) cDNA was performed on peptide stimulated PBMC and 23 biopsies with T-cell mediated rejection (TCMR) or BKPyVN. Biopsies from patients with BKPyVM or BKVPyVN contained 7.7732 times more alloreactive than virus reactive clones. Biopsies with TCMR also contained BKPyV-specific clones, presumably a manifestation of heterologous immunity. The mean cumulative T-cell clonal frequency was 0.1378 for alloreactive clones and 0.0375 for BKPyV reactive clones. Samples with BKPyVN and TCMR clustered separately in dendrograms of V-family and J-gene utilization patterns. Dendrograms also revealed that V-gene, J-gene, and D-gene usage patterns were a function of HLA type. In conclusion, biopsies with BKPyVN contain abundant allospecific clones that exceed the number of virus reactive clones. The T-cell component of tissue injury in viral nephropathy appears to be mediated primarily by an 'innocent bystander' mechanism in which the principal element is secondary T-cell influx triggered by both anti-viral and anti-HLA immunity.
IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection
In HIV-1–infected patients, increased numbers of circulating CD8+ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B+, CD8+ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B+ phenotype in CD8+ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8+ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8+ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8+ T cell expansion that is linked to increased morbidity and mortality in treated patients.
TCR repertoire sequencing identifies synovial Treg cell clonotypes in the bloodstream during active inflammation in human arthritis
Objectives: The imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation.
Methods: We compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions.
Results: We found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells.
Conclusions: Using an innovative antigen-agnostic approach, we uncovered a population ofbona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity.
High-throughput sequencing reveals restricted TCR VB usage and public TCRB clonotypes among pancreatic lymph node memory CD4+ T cells and their involvement in autoimmune diabetes
Islet-reactive memory CD4+ T cells are an essential feature of type 1 diabetes (T1D) as they are involved in both spontaneous disease and in its recurrence after islet transplantation. Expansion and enrichment of memory T cells have also been shown in the peripheral blood of diabetic patients. Here, using high-throughput sequencing, we investigated the clonal diversity of the TCRβ repertoire of memory CD4+ T cells in the pancreatic lymph nodes (PaLN) of non-obese diabetic (NOD) mice and examined their clonal overlap with islet-infiltrating memory CD4 T cells. Both prediabetic and diabetic NOD mice exhibited a restricted TCRβ repertoire dominated by clones expressing TRBV13-2, TRBV13-1 or TRBV5 gene segments. There is a limited degree of TCRβ overlap between the memory CD4 repertoire of PaLN and pancreas as well as between the prediabetic and diabetic group. However, public TCRβ clonotypes were identified across several individual animals, some of them with sequences similar to the TCRs from the islet-reactive T cells suggesting their antigen-driven expansion. Moreover, the majority of the public clonotypes expressed TRBV13-2 (Vβ8.2) gene segment. Nasal vaccination with an immunodominat peptide derived from the TCR Vβ8.2 chain led to protection from diabetes, suggesting a critical role for Vβ8.2+ CD4+ memory T cells in T1D. These results suggest that memory CD4+ T cells bearing limited dominant TRBV genes contribute to the autoimmune diabetes and can be potentially targeted for intervention in diabetes. Furthermore, our results have important implications for the identification of public T cell clonotypes as potential novel targets for immune manipulation in human T1D.
Identification of a CD4 T-cell epitope in the hemagglutinin stalk domain of pandemic H1N1 influenza virus and its antigen-driven TCR usage signature in BALB/c mice
The stalk region of the influenza virus hemagglutinin is relatively well conserved compared with the globular head domain, which makes it a potential target for use as a universal vaccine against influenza. However, the role of CD4 T cells in the hemagglutinin stalk-specific immune response is not clear. Here we identified a mouse CD4 T-cell epitope that encompasses residues HA2113-131 from the hemagglutinin stalk domain after a sub-lethal infection of influenza. In response to stimulation with the identified epitope, splenocytes derived from the infected mice showed significant polyfunctionality as shown by IL-2, TNF-α and IFN-γ production as well as degranulation. Moreover, mice immunized with the peptide corresponding to this CD4 T-cell epitope exhibited interindividual sharing of the CD4 T-cell receptor β sequences, and they had a higher survival rate following a challenge with a lethal dose of pandemic H1N1 influenza virus. Thus, our data demonstrated a crucial role of hemagglutinin stalk-specific CD4 T cells in the host immune response against influenza virus infection.
Featured Publications
IgH-V(D)J NGS-MRD Measurement Pre- and Early Post- Allo-Transplant Defines Very Low and Very High Risk ALL Patients
Blood | May, 2015Positive detection of minimal residual disease (MRD) by multichannel flow cytometry (MFC) prior to hematopoietic cell transplantation (HCT) of patients with ALL identifies patients at high risk for relapse, but many pre-HCT MFC-MRD negative patients also relapse, and the predictive power MFC-MRD early post-HCT is poor. To test whether the increased sensitivity of next-generation sequencing (NGS-MRD) better identifies pre- and post-HCT relapse risk, we performed IgH V(D)J NGS-MRD on 56 patients with B-cell ALL enrolled in Children's Oncology Group (COG) trial ASCT0431. NGS-MRD predicted relapse and survival more accurately than MFC-MRD (p<0.0001), especially in the MRD negative cohort (relapse 0% vs. 16%; p=0.02, 2yr OS 96% vs. 77%; p=0.003).
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Next-generation sequencing-based detection of circulating tumour DNA after allogeneic stem cell transplantation for lymphoma
British Journal of Haematology | December, 2016Next-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.
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Immunoglobulin and T-cell Receptor Gene High-Throughput Sequencing Quantifies Minimal Residual Disease in Acute Lymphoblastic Leukemia and Predicts Post-Transplant Relapse and Survival
Biology of Blood and Marrow Transplantation |Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10−4 after induction predicts subsequent relapse. Likewise, MRD ≥ 10−4 in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL.
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Prognostic Value of Deep Sequencing Method for Minimal Residual Disease Detection in Multiple Myeloma
Blood | May, 2014We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively.
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Treatment With Carfilzomib-Lenalidomide-Dexamethasone With Lenalidomide Extension in Patients With Smoldering or Newly Diagnosed Multiple Myeloma
JAMA Oncology | September, 2015Importance: 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).
VIEW
Next-Generation Sequencing in Adult B Cell Acute Lymphoblastic Leukemia Patients
Biology of Blood and Marrow Transplantation | January, 2017We used next generation sequencing (NGS) of the immunoglobulin genes to evaluate residual disease in 153 specimens from 32 patients with adult B cell ALL enrolled in a single, multi-center study. The sequencing results were compared to multi-parameter flow cytometry (MFC) data in 66 specimens (25 patients) analyzed by both methods. There was a strong concordance (82%) between the methods in the qualitative determination of the presence of disease. However, in 17% of cases leukemia was detected by sequencing, but not by MFC. In 54 bone marrow (BM) and peripheral blood (PB) paired specimens, the burden of leukemia detected by NGS was lower in PB than BM, although still detectable in 68% of the 28 paired specimens with positive BM.
VIEWPoster #2943 Quantitative Baseline Circulating Tumor DNA Levels Correlate with GM-CSF Response to Idiotype Vaccine in Untreated Mantle Cell Lymphoma
Background: Mantle cell lymphoma (MCL) is an incurable lymphoma that responds to various forms of immunotherapy. Previously, we reported that specific anti-idiotype tumor responses as measured by GM-CSF levels after vaccine were associated with overall survival (OS) and time to next treatment (TTNT) following DA-EPOCH-R in untreated MCL at 11 years of follow-up (Dunleavy ASCO 2012). Levels of GM-CSF prior to idiotype vaccine also were correlated with post-vaccine GM-CSF levels suggesting that pre-existing anti-tumor immune response predict idiotype vaccine responsiveness. High-throughput DNA sequencing methods can detect and quantify circulating tumor-specific DNA (ctDNA) in peripheral blood of MCL patients prior to therapy and can be monitored for clearance after therapy. We hypothesized that pre-treatment ctDNA levels may be a surrogate marker for ongoing host anti-tumor immunity. Here, we analyzed the relationship of pre- and post-treatment levels of ctDNA to predict GM-CSF response and clinical outcomes in untreated MCL.
Methods: DA-EPOCH-R was administered x 6, followed by 5 cycles of Id-vaccine beginning at least 12 weeks later in untreated MCL. Id protein was produced by hybridoma technology, conjugated to keyhole limpet hemocyanin (KLH), and administered together with GM-CSF x 5 over 6 months. Pre- and post-vaccine samples were tested in parallel to assess humoral and cellular immune responses. Pre-treatment formalin-fixed paraffin embedded tissue specimens were de-identified and sent for identification of tumor-specific clonotypes. Tumor DNA was amplified using locus-specific primer sets for the immunoglobulin heavy-chain locus (IGH) complete (IGH-VDJH), IGH incomplete (IGH-DJH), and immunoglobulin k locus (IGK). Amplified products were sequenced and tumor-specific clonotypes were quantitated in peripheral blood mononuclear cells before treatment as ctDNA and after treatment as MRD.
Results: Characteristics of all 26 pts: median age 57 (r 22-73), male sex 73%, PS 1 (0-2), MIPI (low-65%; interm-16%; high-19%), and blastoid 15%. Responses to DA-EPOCH-R: CR-92%, PR-8%. Immune analyses were performed in 24 pts; vaccine not produced in 1 pt and 1 pt progressed before immune analyses. Baseline tumor was available for clonotype calibration in 20 patients. With a potential follow-up of 14 years, the median OS of the entire cohort is 9.0 years and 10/26 (38%) patients are still alive. Pre-treatment levels of ctDNA were inversely correlated with normalized antitumor GM-CSF response (r=0.49, p=0.035) but not with white blood cell count (r=0.18, p=0.46), LDH (r=0.21, p=0.39) or MIPI scores (r=22, p=0.37). Patients with low pre-treatment ctDNA levels had longer OS than patients with high levels of ctDNA, but this did not reach statistical significance (11.01 y vs. 5.71 y, p=0.67). Eight of 20 of patients (40%) were MRD positive after DA-EPOCH-R despite achieving CR/CRu. Patients who achieved MRD-zero had a trend towards improved OS (NR vs. 8.2 years, p=0.15).
Conclusions: Lower pre-treatment ctDNA levels correlate with normalized GM-CSF response to idiotype vaccine and may be a surrogate marker of ongoing host anti-tumor immunity. In this small study, ctDNA levels did not correlate with traditional markers of tumor proliferation. There is a trend towards improvement in OS in patients who achieve MRD-zero. Future studies are underway to explore the biologic significance of pre- and post-treatment ctDNAlevels in MCL.
Poster #3286 Next-Generation Sequencing Based Minimal Residual Disease Assessment in Peripheral Blood RNA from Multiple Myeloma Patients
Oral #377: Comparison of MRD Detection by MFC, NGS, and PET-CT in Patients at Different Treatment Stages for Multiple Myeloma
Introduction: The achievement of Minimal Residual Disease (MRD) negativity has become a new standard of care for patients with multiple myeloma (MM). However the methods used for MRD detection, including multi-color flow-cytometry (MFC) and next generation sequencing (NGS), although both highly sensitive have different levels of sensitivity and are prone to a different range of errors. In addition MM is not distributed evenly through the marrow and biopsies are taken only from a single site. In contrast imaging studies using PET-CT can review an extended area and will detect focal lesions and extra-medullary disease. Studies incorporating all of these modalities are rare despite the importance of understanding how they perform together.
We report on the comparison and clinical relevance of MRD testing by MFC, NGS and PET-CT in patients at different stages of their disease, including newly diagnosed MM (NDMM) as well as relapsed/refractory MM (RRMM) and in patients in long standing remission.
Methods: We investigated 100 patients, who all had at least achieved a VGPR and underwent MRD detection by 8 color MFC, NGS using the ClonoSEQ assay (Adaptive Biotechnologies Corp.) and imaging with PET-CT. Patients were at different disease stages and were categorized as either 1) undergoing upfront therapy for NDMM (n=53 including induction, post Stem Cell Transplant [SCT] and maintenance), 2) remission after completed therapy (n=32) or 3) RRMM (n=5). Detection thresholds were 1 MM cell in 105 using 8 parameter MFC and 1MM cell in 106 by NGS.
Results: Of the 100 patients analyzed, a myeloma clonal rearrangement was detected in 90 patients (90%). For patients with long-term MM history, clonal rearrangements could be identified in BM samples up to 13 years ago. Fifty-three of 90 patients (59%) were undergoing upfront therapy for newly diagnosed MM and the proportion of MRD negativity by MFC and NGS increased with treatment stage and was highest during maintenance. Of these 53 patients, six were undergoing induction therapy, none of them had achieved MRD negativity by MFC or by NGS. Eight patients were post-transplant and MFC was negative in 6 (75%) and 2 were negative by NGS (25%). Thirty-nine of the 53 patients were undergoing maintenance therapy. Of these 26 were negative by MFC (50%) and 17 (44%) by NGS.
32 of the 90 patients (35.5%) were off any therapy after achieving complete remission during treatment and completion of maintenance. MFC was negative in 19 of these 32 patients (60%), yet only 15 patients had an undetectable tumor burden by NGS (47%). For 14 patients with very long term remission (>5years) and who had remained off treatment for at least 2 years, MRD by MFC was negative in all of them and 11 showed no residual tumor burden by NGS (78.5%).
Five patients were status post treatment for relapsed and refractory disease (5.5%). Of interest is that MRD burden by molecular analysis was overall very low, with 4 patients having <0.01% detectable tumor load by MFC and NGS, yet PET-CT showed active and newly involving lesions in 4 of these 5 patients suggesting a typical pattern of macro-focal disease. Apart from these 4 relapsed patients, PET-CT was positive in 5 patients that were undergoing upfront therapy. In these cases however, activity and number of focal lesions was improving to prior imaging, indicating that response to treatment was ongoing.
Conclusions:
The study shows:
1) Almost half of patients who have achieved remission and completed treatment for NDMM are still MRD positive by NGS. Further evaluation will be necessary to determine whether MRD positivity in this patient population inevitably leads to clinical relapse and 6 months follow up of this patient population will be presented at ASH 2016.
2) The proportion of patients that achieve MRD negativity increases with treatment and is highest during and after maintenance. Detection of MRD should hence be measured sequentially throughout treatment to identify the time point of best response, which could further have an impact of overall prognosis.
3) Relapsed/refractory patients can present with non-secretory focal lesions without any significant MM burden on random aspirate. In these patients imaging studies, such as PET-CT, are crucial for the detection and monitoring of their disease.
Poster #2064: Next Generation Sequencing (NGS) Based Minimal Residual Disease (MRD) Testing Is Highly Predictive of Overall and Progression Free Survival in the Total Therapy Trials and Shows Different Prognostic Implications in High Vs Standard Risk Multiple Myeloma
Introduction: Achieving complete remission (CR) improves outcomes in multiple myeloma (MM). We have shown that 50% of patients enrolled in Total Therapy (TT) trials achieve CR within one year of enrollment irrespective of risk as defined by the GEP70 risk signature. Nevertheless, the majority of patients with high risk (HR) MM show an early relapse with grim prognosis, while most standard risk (SR) patients tend to relapse late, most often years after completion of maintenance therapy. Evaluation of minimal residual disease (MRD) has been shown to be of prognostic relevance in patients in CR with MRD negativity being associated with better outcomes. Here we report the impact of next generation sequencing (NGS)-based MRD assessment in TT patients who have achieved at least a very good partial response (VGPR) at two different time points during their treatment protocol: first after auto stem cell transplant (ASCT at 4-8 months of enrollment) and secondly during maintenance therapy (12-24 months).
Materials and Methods: In brief, our TT protocols incorporate induction therapy consisting of Velcade and Thalidomide in conjunction with chemotherapy (Cisplatin, Doxorubicin, Cytoxan, Etoposide) followed by ASCT, consolidation and three years of maintenance with Velcade, Revlimid and Dexamethasone. HR and SR were assigned using the GEP70 risk signature. For MRD testing, we included 119 patients who were treated on our TT3b -TT6 protocols and who achieved at least VGPR and had bone marrow samples available at 4-8 months post ASCT and 12-24 months into maintenance. Thirty-eight patients had HR MM (32%), 75 patients had SR MM (63%) and no GEP70 data was available for 6 patients (5%).
For NGS-based MRD assessment (Adaptive Biotechnologies Corp), genomic DNA was amplified using locus-specific primer sets for immunoglobulin heavy-chain complete (IGH-VDJH) and incomplete (IGH-DJ) as well as for immunoglobulin κ locus (IGκ). The amplified products underwent sequencing and clonal gene rearrangements were analyzed. MRD levels were calculated at a sensitivity level of 1 x 10-5. PET-CT and/or MRI were obtained at same time point as MRD assessment to evaluate presence of focal lesions.
Results: Using MRD status post-ASCT and during maintenance as a predictor, there were significant differences for PFS and OS in HR and SR MM. In HR disease, MRD positivity post ASCT was associated with significantly worse clinical outcomes with 2 year PFS/OS at 33% and 52% for MRD positive patients compared to 82%and 83% in MRD negative patients. Further 5 year PFS and OS for the same patient group were 11% and 24% for MRD positivity compared to 45% and 75% for HR patients that had achieved MRD negativity.
In contrast to the HR group, there was no significant difference between MRD positivity and negativity in SR patients at the time point post ASCT we have investigated. In this latter patient group a significant difference only became obvious later during maintenance with 5 year PFS and OS at 55% and 63% for MRD positive patients compared to 83% and 93% for MRD negative patients.
Of the 54 SR patients that were MRD positive post ASCT, 25 (46%) became negative later during maintenance, while in HR disease only 4 of 21 MRD positive patients post ASCT became negative during maintenance (19%).
Importantly, a stratified analysis of our MRD data showed that even during maintenance the majority of cases in the favorable CD-2 subgroup were MRD positive, indicating that MRD results should further be interpreted in the context of patients’ molecular subgroup.
Conclusions: Our data suggest that HR patients that do not achieve MRD negativity after their first ASCT have a very high likelihood of disease progression and death within 24 months. In contrast, SR patients that are still MRD positive post ASCT tend to have continuing response to treatment and MRD testing only becomes prognostic during maintenance. Of interest is that a high proportion of HR patients that achieve MRD negativity post ASCT and a smaller proportion of MRD negative SR patients still relapse within 5 years of enrollment suggesting that remaining residual MM cells are not detected by molecular MRD testing. Imaging studies with PET-CT and MRI could further stratify these patients as they detect any residual focal lesions that are not assessed by molecular MRD testing. We have combined PET-CT/MRI data for all of the 119 patients that were included in this study and analyzed data will be presented at ASH 2016.
Oral #246 Evaluation of Minimal Residual Disease (MRD) in Relapsed/Refractory Multiple Myeloma (RRMM) Patients Treated with Daratumumab in Combination with Lenalidomide Plus Dexamethasone or Bortezomib Plus Dexamethasone
Introduction: Daratumumab (DARA) is a human monoclonal IgG1κ CD38 antibody that functions through multifaceted mechanisms of action, including CDC, ADCC, ADCP, induction of apoptosis and immunomodulatory activity (Krejcik et al, Blood 2016;128:384-394). DARA was added to standard of care (SOC) regimens in two randomized, controlled, phase 3 trials in RRMM: POLLUX (DARA+lenalidomide/dexamethasone [DRd] vs lenalidomide/dexamethasone [Rd]) and CASTOR (DARA+bortezomib/dexamethasone [DVd] vs bortezomib/dexamethasone [Vd]). The addition of DARA to these regimens resulted in significant improvements in PFS (hazard ratio for PFS: 0.37 and 0.39 for POLLUX and CASTOR, respectively) and ORR (POLLUX: 93% DRd vs 76% Rd; CASTOR: 83% DVd vs 63% Vd) (Dimopoulos MA et al, N Engl J Med 2016; in press; Palumbo A et al, N Engl J Med 2016; in press). To determine the ability of DARA to drive deep clinical responses beyond complete response (CR), MRD was assessed in both POLLUX and CASTOR using next-generation sequencing (NGS) of B cell receptor (Ig). This is the first comprehensive and prospective study of MRD to date in randomized phase 3 clinical trials of RRMM patients and investigates the ability of DARA-containing regimens to drive deep responses in this challenging population.
Methods: MRD was assessed in POLLUX (blinded to treatment group) at the time of suspected CR, and at 3 and 6 months post-suspected CR for patients who maintained this response. Similarly, in CASTOR, MRD was assessed for patients at the time of suspected CR (blinded to treatment group) and at 6 months and 12 months after first dose (at the end and 6 months after end of Vd background therapy, respectively). MRD was assessed on bone marrow aspirate samples prepared using Ficoll and evaluated by the ClonoSEQTM assay (Adaptive Biotechnologies, Seattle, WA, USA) at sensitivities of 0.01% (1 cancer cell per 10,000 nucleated cells or 10-4), 0.001% (10-5), and 0.0001% (10-6). The MRD negative rate per treatment arm was determined as the proportion of patients with negative MRD at any time point after the first dose and compared using the likelihood-ratio test. To allow for a stringent, unbiased evaluation of MRD in these studies, the entire intent-to-treat population was evaluated where patients were considered MRD positive if they had only MRD positive test result or had no MRD assessment. In POLLUX, 63% in DRd and 87% in Rd did not receive a MRD assessment, and for CASTOR, 76% in DVd and 87% in Vd were not assessed for MRD.
Results: Median duration of follow-up was 13.5 months and 7.4 months in POLLUX and CASTOR, respectively. The addition of DARA to SOC regimens (Rd in POLLUX or Vd in CASTOR) resulted in significantly higher MRD negative rates at all three thresholds examined (10-4, 10-5, and 10-6; Table). Additionally, patients who achieved MRD negative status experienced fewer PFS events compared with MRD positive patients at a threshold of 10-5(Figure). Among pts who achieved CR or better, the rate of MRD negativity was at least 3-fold higher across all sensitivity thresholds in the DARA + SOC treatment groups compared with SOC alone. In MRD positive patients, PFS was significantly longer in the DARA + SOC treatment groups compared with SOC alone treatment arms. Updated data from both studies will be presented at the annual meeting.
Conclusion: These two studies represent the first randomized, controlled, prospective evaluation of MRD in the RRMM phase 3 clinical trial setting and demonstrate that DARA-containing therapies are able to drive patients to remarkably deep levels of clinical response. Regardless of the SOC component, DARA-containing regimens consistently demonstrated ≥3-fold increase in MRD negative rate compared with the control groups at all evaluated thresholds. Importantly, since patients who achieved MRD negative status demonstrated low PFS event rates, the deep clinical responses induced by DARA may lead to improved survival.
Oral #238 A Novel Evolutionary Pattern Revealed Using Deep Sequencing of Immunoglobulin Loci at Diagnosis and over the Course of Treatment in Multiple Myeloma Patients
Introduction: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. As multiple myeloma is considered a clonal disease originating from the transformation of a single plasma cell, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. We previously observed that multiple Ig sequences related by somatic hypermutation (SHM) may be present in some MM patients at diagnosis. Here we provide an expanded observation in a very large cohort of the patients, and perform mutational analysis of the oligoclonal myeloma clonotypes observed at diagnosis and post-treatment, revealing changes in the relative frequency of the MM clonotypes and emergence of new Ig clones.
Methods: 620 MM patients enrolled in IFM/DFCI and Hospital 12 de Octubre trials were included in this analysis. The next-generation sequencing (NGS)-based immunosequencing platform was used to detect evidence of oligoclonality at the Ig heavy chain loci. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from DNA and/or RNA isolated from purified CD138+ MM cells collected at the time of diagnosis. MM-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic (dx) sample. The highest frequency MM clonotype in a dx sample is termed the "index clonotype." DNA and/or RNA isolated from dx AND post-treatment bone marrow samples were assessed for evidence of evolved MM clonotypes.
Results: We identified Ig clones in 367 RNA samples and 430 DNA samples from the cohort. We first looked for cases with evidence that myeloma cells have two unrelated origins. We found 11/620 (1.8%) cases at diagnosis, which had evidence of unrelated clones as evident by having three IgH or two functional sequences. In 8 of the 11 cases (72.6%), we had multiple samples to analyze, including two samples at diagnosis or diagnosis/post-treatment pairs. In 4 of the 8 samples, we saw dramatically different relative frequencies of unrelated clones in these samples suggesting that these unrelated clones are likely to be present in two distinct cells. We then considered cases where we found two IgH sequences that are related to each other by SHM at diagnosis. Overall 79 (12.7%) of 620 samples had more than one evolved clone; of these 63/367 (17.2%) of RNA dx samples showed evidence of evolved clones via SHM, while 22/430 patients (5.1%) showed evidence of evolved clones related to the index clone via SHM in DNA samples from diagnosis. Mutant clonotypes had an average of 3.9 to 4.5 mutations in the CDR3 region. We also noted mixed isotypes in 13 clones from 13 patients at diagnosis. The majority of related clones observed in the RNA samples are present at very low frequencies (<10-4), as the greater sequencing depth in RNA allows for identification of low frequency clones. 304 post-treatment samples from 206 patients were MRD positive and were assessed for the presence of clonal evolution. In 27/304 follow-up samples (8.8%) and 7/206 patients (3.4%), an evolved clone related to the index clone was observed even though the period between diagnosis and post-treatment samples was only 6 months. In 6 patients, a substantial change in the relative index and unrelated clone frequencies was observed from the dx to post-treatment time points suggesting a differential sensitivity to treatment.
Conclusions: We confirm presence of multiple evolved clonotypes in a substantial percentage of diagnostic MM samples in a large cohort of patients. The evolution of multiple clones related by SHM indicates that SHM remains active after myeloma development and may also impact other non-Ig sites. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. The very high depth of our sequencing also indicates that the emergence of new IgH clones may be newly acquired mutations in the Ig gene, driven by some ongoing genomic mutation process. Thus, these evolved myeloma clonotypes may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy.
Improved Efficacy After Incorporating Autologous Stem Cell Transplant (ASCT) Into KRD Treatment with Carfilmozib (CFZ), Lenalinomide (LEN), and Dexamethasone (DEX) in Newly Diagnosed Multiple Myeloma
Background
In a phase 1/2 trial (N=53), extended treatment with KRd without (w/o) ASCT was highly active in newly diagnosed myeloma (NDMM) with stringent complete response (sCR) 55% and 3-year progression-free survival (PFS) 79%.
Aims
In a subsequent phase 2 trial, we are evaluating whether extended KRd can be further improved by incorporating ASCT (KRd+ASCT). We report results from both trials after completion of enrollment into KRd+ASCT and after completion of KRd w/o ASCT (median follow-up [f/u] 4 years) as a historical control.
Methods
Both studies enrolled patients (pts) with NDMM based on similar eligibility criteria except KRd+ASCT excluded transplant ineligible pts. The treatment schemas were generally similar between studies. In KRd+ASCT, pts received: four 28-day cycles of induction with CFZ IV 36 mg/m2 on Days (D) 1-2, 8-9, 15-16 (CFZ 20 mg/m2 for D1-2, C1 only), LEN PO D121 at 25 mg, DEX PO 40 mg/wk; followed by stem cell collection (SCC), melphalan 200 mg/m2 and ASCT; then KRd consolidation (C5-8) using the same doses and schedule except LEN 15 mg in C5 with the option to escalate to prior dose, and DEX reduced to 20 mg/wk; then KRd maintenance (C9-18) using the same doses as in C8 except CFZ on Days 1-2, 15-16 only. In KRd w/o ASCT, transplant-eligible pts underwent SCC after C4 then resumed KRd, and KRd maintenance was longer (C9-24). Both studies recommended single-agent LEN off study. The primary endpoint in KRd+ASCT is sCR at the end of C8. We hypothesized that an improvement of sCR from 30% at the end of C8 (historical KRd w/o ASCT) to >50% (KRd+ASCT) represents added benefit of ASCT with 5% type I error (2 sided) and supports further evaluation. Minimal residual disease (MRD) is evaluated by 10-color multiparameter flow cytometry (MFC, threshold 10-4-10‑5) and by next-generation sequencing (NGS, LymphoSIGHTTM, threshold at 10-6 for MRD negativity).
Results
The current KRd+ASCT study enrolled 76 pts with 72 evaluable. Baseline characteristics were comparable between the KRd+ASCT and KRd w/o ASCT study populations, including median age (59 and 59y) and high-risk IMWG cytogenetics (36% and 33%). In the ongoing KRd+ASCT, 69 pts proceeded to ASCT at data cut-off (Jan 1, 2016), 50 completed KRd consolidation, and 26 KRd maintenance, with remaining pts on treatment, except 1 patient who progressed prior to transplant. At the end of C8, sCR was 72% for KRd+ASCT (n=50) vs 30% for KRd w/o ASCT (n=44) and 88% (n=26) vs 51% (n=41) at the end of C18. At median f/u of 17.8 months, 2-year PFS was 99% for KRd+ASCT vs 92% for KRd w/o ASCT at median f/u of 47.5 months. In KRd+ASCT, MRD by MFC was negative in 94% of pts tested (n=31) at the end of C8 and 95% of pts tested (n=19) at the end of C18. In KRd w/o ASCT, 4-year PFS was 69% overall, 78% in MRD-negative pts vs 60% in positive/unknown pts by MFC, and 100% in pts with MRD negative status by NGS. Updated MRD analyses and outcomes based on MRD status by both MFC and NGS will be presented at the meeting. The types and rates of adverse events (AEs) pre- and post-ASCT were comparable to AEs in KRd w/o ASCT.
Conclusion
Recognizing limitations of cross-study comparisons, KRd+ASCT shows superior outcomes vs historical KRd w/o ASCT, supporting further evaluation in the randomized setting. Both KRd studies compare favorably to other NDMM studies.
High Throughput Sequencing as a Measure of Early Response to Therapy in Childhood ALL
Background
Early response to induction chemotherapy has been demonstrated to be a highly significant prognostic factor in the outcome of children with acute lymphoblastic leukemia. Multiparametric flow cytometry (mpFC) has been the routinely used methodology in the US for determination of this response. New high throughput sequencing (HTS) technologies of rearranged immune receptor (TCR and Ig) genes have raised the possibility of a more accurate, sensitive, and standardizable approach to determination of early response to therapy in ALL patients.
Aims
In this study, we investigated whether the Adaptive Biotechnologies assay of IgH and TCRG would be able to quantify residual disease at the end of induction therapy for children with ALL and be of prognostic value with regard to outcome (event free survival) in these patients.
Methods
The first study involved a total of 480 patients enrolled on Children’s Oncology Group (COG) clinical trials AALL0331 and AALL0232 for whom mpFC measurement of residual disease and outcome data are available. A second study involved samples from 73 patients enrolled in a former Pediatric Oncology Group trial, POG 9905, who were mpFC negative at d29. For increased statistical power the patients in POG 9905 were selected for analysis so that ~50% had relapsed disease determined in follow-up monitoring.MpFC was performed at COG reference laboratories the University of Washington or Johns Hopkins Hospital as part of the evaluation for MRD. Genomic DNA was extracted from frozen bone marrow specimens collected at diagnosis and at day 29 post the start of induction therapy. High throughput sequencing of CDR3 regions of IGH and TCRG was performed on all samples, with the testing laboratory blinded to patient outcome. Diagnostic and d29 matched samples from a given patient were sequenced and dominant clonal CDR3 sequences from diagnosis were searched for in the corresponding d29 sample. Both the presence and the frequency of the MRD clone relative to the total IGH repertoire and total nucleated cell population were determined.
Results
The assays defined the dominant clonal sequences in 93% of the patients. 70% of this subgroup was found to have residual disease present at d29. Clones from some of the patients demonstrated a single “trackable” sequence while clones from other patients demonstrated multiple trackable sequences either within or between the two immune receptor loci being assessed. 60 % of the residual disease detected by HTS was previously reported as MRD negative by mpFC. For “standard risk” patients, 53% were positive for MRD by HTS and negative by mpFC. With the combined COG data, using a MRD cutoff of 10-4, HTS was able to define a correlation with event-free survival (p=0.0003). Furthermore, for the “standard risk” patients, being MRD positive or negative as determined by the more sensitive HTS assay was also correlated with outcome (p=0.02). In addition, a correlation was noted between poorer outcome and a “germline” or TCRG only (i.e. IgH loci retained in the germline configuration) genotype (p=0.04). In the second study of patients enrolled in the POG 9905 trial, the more sensitive HTS cut-off of 10-5 was correlated with outcome (p=0.0228).
Conclusion
This is the largest patient cohort studied to date for which mpFC, HTS, and outcome data are available. This work suggests that HTS is an accurate and standardized assay whose increased sensitivity compared to mpFC is relevant to determination of patient outcome.
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