TCRβ Sequencing Research Articles

Suppressed immune microenvironment and repertoire in brain metastases from patients with resected non-small-cell lung cancer

BackgroundThe tumor immune microenvironment (TIME) of lung cancer brain metastasis is largely unexplored. We carried out immune profiling and sequencing analysis of paired resected primary tumors and brain metastases of non-small-cell lung carcinoma (NSCLC). Patients and methodsTIME profiling of archival formalin-fixed and paraffin-embedded specimens of paired primary tumors and brain metastases from 39 patients with surgically resected NSCLCs was carried out using a 770 immune gene expression panel and by T-cell receptor beta repertoire (TCRβ) sequencing. Immunohistochemistry was carried out for validation. Targeted sequencing was carried out to catalog hot spot mutations in cancer genes. ResultsSomatic hot spot mutations were mostly shared between both tumor sites (28/39 patients; 71%). We identified 161 differentially expressed genes, indicating inhibition of dendritic cell maturation, Th1, and leukocyte extravasation signaling pathways, in brain metastases compared with primary tumors (P < 0.01). The proinflammatory cell adhesion molecule vascular cell adhesion protein 1 was significantly suppressed in brain metastases compared with primary tumors. Brain metastases exhibited lower T cell and elevated macrophage infiltration compared with primary tumors (P < 0.001). T-cell clones were expanded in 64% of brain metastases compared with their corresponding primary tumors. Furthermore, while TCR repertoires were largely shared between paired brain metastases and primary tumors, T-cell densities were sparse in the metastases. ConclusionWe present findings that suggest that the TIME in brain metastases from NSCLC is immunosuppressed and comprises immune phenotypes (e.g. immunosuppressive tumor-associated macrophages) that may help guide immunotherapeutic strategies for NSCLC brain metastases.

Abstract A085: High infiltration of NK cells expressing elevated LAG-3 in a subgroup of renal cell carcinoma patients

Abstract Background: Renal cell carcinoma (RCC) is considered one of the most immunogenic cancers with the highest number of indel mutations, frequent infiltration of T-cells, presence of many antigen-specific T-cell clones, and high immuno-oncologic (IO) sensitivity. Since less is known about natural killer (NK) cells in RCC, our primary aim was to investigate the intratumoral phenotype of NK cells as well as further assess the overall immune landscape of the tumors, peripheral blood (PB) and adjacent healthy kidney tissue, which may be critical for patient prognosis and predictions to targeted immunotherapies. Methods: We used multi-parameter flow cytometry together with a comprehensive immunostaining panel containing a total of 56 fundamental markers to cancer immunology to immunophenotype the tumor, adjacent healthy kidney tissue and presurgical peripheral blood (PB) samples from 31 RCC patients who underwent partial or radical nephrectomies. To study the intratumoral T-cell clonalities, T-cell receptor beta (TCRβ) deep sequencing was carried out with eight tumors. Results: Using hierarchical clustering (Spearman correlation distance and Ward linkage) and correlation analyses (Spearman correlation), we discovered that our patient cohort clustered into two distinct subgroups defined by a high (NKhigh, n=11; mean 29.7%) and low (NKlow, n=20; mean 9.4%) percentage of NK cells among the intratumoral lymphocyte population. Accordingly, the NKhigh subgroup had a lower percentage of T-cells (mean 36.9%) than the NKlow group (mean 65.7%), and overall, a significant negative correlation between T and NK cells was discovered. Our TCRβ sequencing results revealed a positive correlation between T-cell clonality and the intratumoral T-cell percentage, whereas the higher proportion of tumor NK cells associated with low T-cell clonality, possibly due to a polyclonal T-cell population. When we compared the expressions of the most clinically relevant IO markers (LAG-3 and PD-1) on the NK cells, LAG-3 was more expressed in the NKhigh group than in the NKlow group (21.3% vs 9.8%; p=0.08). In contrast, no differences were observed with PD-1. Clinical parameters such as tumor grade (Fuhrman), weight, size (diameter), the presence of necrosis, gender, or age of the patients did not differ between the two subgroups. To examine the overall immune landscape of RCC, we compared the cells from the tumor, PB, and healthy kidney tissue of seven patients. Our results showed that tumors have more NK cells compared to their corresponding T-cell-rich PB and healthy tissue counterparts, supporting our findings that some tumors accumulate NK cells. Compared to the adjacent healthy tissue, PD-1 and LAG-3 expressions were higher in the intratumoral CD8 cells. The expressions of PD-1 and LAG-3 on PB CD8+ T-cells or NK cells did not correlate with their intratumoral counterparts, whereas a positive correlation was found between the PB and tumor CD4+ T-cells for both LAG-3 and PD-1. Conclusions: Our study has led to the discovery of two distinct RCC tumor subgroups with differential expressions in the clinically leading molecules. These results suggest that immunophenotyping RCC patients may effectively aid in selecting those who will benefit the most from immune checkpoint inhibition therapies such as anti-PD1 and -LAG3. Prospective analyses on spatial immunoprofiling by multiplexed immunohistochemistry and mutational load by exome sequencing will be assessed next in order to find answers to why some tumors are NK-dominant, with expectations of understanding further the biologic differences between the two tumor types. Citation Format: Moon Hee Lee, Petrus Järvinen, Harry Nisén, Oscar Brück, Mette Ilander, Satu Mustjoki, Kreutzman Anna. High infiltration of NK cells expressing elevated LAG-3 in a subgroup of renal cell carcinoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A085.

T Cell Repertoire Evolution after Allogeneic Bone Marrow Transplantation: An Organizational Perspective.

High-throughput sequencing (HTS) of human T cell receptors has revealed a high level of complexity in the T cell repertoire, which makes it difficult to correlate T cell reconstitution with clinical outcomes. The associations identified thus far are of a broadly statistical nature, precluding precise modeling of outcomes based on T cell repertoire development following bone marrow transplantation (BMT). Previous work has demonstrated an inherent, mathematically definable order observed in the T cells from a diverse group of donors, which is perturbed in recipients following BMT. In this study, T cell receptor (TCR)-β sequences from HLA-matched related donor and recipient pairs are analyzed to further develop this methodology. TCR-β sequencing from unsorted and sorted T cell subsets isolated from the peripheral blood samples of BMT donors and recipients show conservation and symmetry of VJ segment usage in the clonal frequencies, linked to the organization of the gene segments along the TCR locus. This TCR-βVJ segment translational symmetry is preserved post-transplantation and even in cases of acute graft-versus-host disease (aGVHD), suggesting that GVHD occurrence represents a polyclonal donor T cell response to recipient antigens. The complexity of the repertoire is significantly diminished after BMT, and the T cell clonal hierarchy is altered post-transplantation. Low-frequency donor clones tended to take on a higher rank in the recipients following BMT, especially in patients with aGVHD. Over time, the repertoire evolves to a more donor-like state in the recipients who did not develop GVHD as opposed to those who did. The results presented here support new methods of quantifying and characterizing post-transplantation T cell repertoire reconstitution.

Oligoclonal Expansion of Cd8+ T Cells during Idiopathic Multicentric Castleman Disease Flares Suggests an Antigen Driven Process

Castleman disease (CD) describes a group of heterogeneous diseases defined by shared characteristic lymph node histopathology and is classified based on the number of regions of enlarged lymph nodes. Multicentric CD (MCD) involves multiple regions of lymphadenopathy as well as systemic inflammation, cytopenias, and vital organ dysfunction due to a cytokine storm that often includes interleukin-6. In ~50% of patients, the pathogenic driver is Kaposi sarcoma-associated/human herpesvirus-8 (HHV-8) in the context of immunosuppression. In contrast, the etiologic driver in HHV8-negative MCD (idiopathic or iMCD) is unknown. To date, most research has focused on descriptive characterization of the enlarged lymph nodes, and the pathological cell types driving iMCD pathogenesis remain unidentified. Given that lymphoid cells circulate through the blood and lymph nodes, are able to produce high levels of cytokines upon activation, and are the primary cell types responsible for the enlarged lymph nodes in iMCD and other related diseases, we first performed a detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) obtained from iMCD patients in remission (n=16), iMCD patients during disease flare (n=6) and healthy donors (HD) (n=15). PBMCs were isolated by density gradient and either stained immediately or cryopreserved for future analyses. A HD sample was drawn at the same time as each experimental sample and processed and analyzed in parallel. Our initial hypothesis was that analysis of iMCD flare PBMCs would reveal an abnormal myeloid or lymphocyte subset. Thus, we stained and analyzed PBMCs for standard lineage markers: CD11b, CD15, CD19, CD3, CD56 and CD14. However, we observed no gross differences in population frequencies during either remission or flare compared to HD. Additionally, no differences in the proportions of natural killer T cells (CD3+CD56+), or CD4+ or CD8+ lymphocytes were observed. However, more refined examinations of the lymphocyte sub-sets based upon activation status revealed an increased proportion of activated memory (CD62LlowHLA-DR+) CD8+ cells during iMCD flare compared to HD and iMCD patients in remission and a decreased proportion of naïve (CD62L+CD45RA+) CD8+ cells compared to HD (p<0.05 for each comparison, Bonferroni corrected 2-tailed t-test). Interestingly, for one patient followed over a 7-month period, during which time he experienced two disease flares, the proportion of activated memory CD8+ cells mirrored the re-emergence of clinical disease symptoms, rising to 80% of all CD8+ lymphocytes during disease flare and waning as flares subsided. We next questioned whether the expansion of memory CD8+ T cells represented a diverse population of T cells with unique TCRs or alternatively, reflected the expansion of one or a few dominate TCR clones. Thus, we performed bulk TCRβ sequencing on genomic DNA from peripheral CD8+ cells obtained during the patient's partial remission between his two flares and at the start of his second disease flare (flare 2). Interestingly, we found that TCRs from CD8+ cells showed an overrepresentation of only a few clonotypes, indicating increased oligoclonality; the top 20 clones accounted for 45% (partial remission) and 52% (flare 2) of bulk CD8+ reads. Furthermore, the top copy number rearrangement comprised 7.5% (partial remission) and 10% (flare 2) of the CD8+ cell repertoires (healthy subjects' CD8+ T cell top copy number rearrangement is typically below 5%). Sequencing of activated (HLA-DR+) and memory (CD45RO+) CD8+ populations also revealed increased clonality. In these populations, the top 20 clones accounted for 45% (CD45RO+) and 33% (HLA-DR+) of sequencing reads during the patient's partial remission and increased to 61% (CD45RO+) and 54% (HLA-DR+) of sequencing reads during flare 2. Further, the top TCRβ sequences persisted over time, being identified in both the partial remission and flare 2 samples. This study identifies quantitative and phenotypic differences in CD8+ cell populations during iMCD disease flare. Furthermore, we present evidence suggesting a role for oligoclonal T cells in iMCD, as our TCRβ sequencing findings reveal a substantial accumulation of only a few TCR clonotypes in CD8+ populations during disease flare. Cumulatively, these results suggest that TCR signaling, due to antigen stimulation or T cell dysregulation may be involved in iMCD pathogenesis. DisclosuresFajgenbaum:Janssen Pharmaceuticals, Inc.: Research Funding.

Profiling T Cell Receptor Repertoires in Phase I/II Clinical Trials of Donor Treg Infusion for the Treatment of Chronic Graft-Versus-Host Disease

IntroductionUnder the auspices of the EC-Horizon 2020 project “TREGeneration”, we are conducting five clinical phase I/II trials investigating the safety and efficacy of donor regulatory T cell (Treg) infusion in patients with chronic Graft-versus-Host Disease (cGvHD), a serious complication following hematopoietic stem cell transplantation (HSCT). Workpackage 4 evaluates T cell receptor (TCR) repertoire changes by high-throughput sequencing following the infusion of donor Tregs.MethodsFor repertoire analysis, four different T cell subsets (Treg, conventional CD4 T cells - Tcon, total CD4 and CD8 T cells) are FACS sorted from the infused product and from the cGvHD patients' peripheral blood before the infusion and at 10 time-points up to 12 months post infusion (Fig. 1A). To monitor TCR repertoire changes over time, we developed a novel quantitative “DeepImmune” technique for TCRβ amplification and sequencing. We amplify all TCRβ chain family transcripts from total RNA using a normalized primer set including molecular identifiers for unbiased and quantitative determination of TCRβ copy numbers. We have normalized the concentrations of our TCRβ primers using synthetic TCRβ gene sequences, thus minimizing amplification biases during RT-PCR to ≤3-fold differences among gene families. The inclusion of molecular identifiers during the RT-PCR step allows us to computationally remove additional biases introduced during the secondary PCR step for Illumina library preparation. Thus, our method results in ultra-deep characterization of immune receptor repertoires for sensitive detection of differences among samples and sample cohorts. Depending on T cell numbers in the samples, we routinely identify 1,000 - 150,000 clonotypes per sample.ResultsAs first quality control for repertoire analysis, we cluster the top 200 TCRβ clonotypes from the different sequenced T cell subsets for each patient, resulting in heatmaps showing prominent repertoire differences among the analyzed T cell subsets (Fig. 1B; infused patient example shown). Next, we track the fate of infused donor Treg cells in the time course samples of the patients after Treg infusion, allowing us to quantify the number and frequency of TCRβ clonotypes that persist or expand in the patients after the infusion. We report that TCRβ clonotypes specific to the infused Treg cells (which were not present in the patients before Treg infusion) can be detected in the patients' blood after the infusion. Infusion-specific Treg clonotypes can reach up to 10-15% of the Treg repertoire of the patients and usually decline over time (Fig. 1C; boxplot shows clonotype overlap between infusion-specific Treg clonotypes and patient Treg clonotypes from five patients of the Lisbon cohort over 12 months). In the Tcon compartment of the patients, infusion-specific Treg clonotypes typically reach only 1-2% of the Tcon repertoire of the patients and also decline over time (Fig. 1D), similarly to what is observed in the CD8 T cell compartment (data not shown). This suggests that infused Tregs are not reverting to Tcons, an important control and promising result. We apply the same type of analysis to track clonotypes that were shared between the infused product and the patient prior to infusion, in addition to those clonotypes that were unique to the patient before infusion and not present in the infused product.ConclusionBy applying our robust and sensitive TCRβ sequencing platform to samples from cGvHD patients infused with donor Treg, we are able to detect unique clonotypes of the infused product up to 1 year post-infusion. It remains to be be determined if there is any correlation between the level of detection of the infused clonotypes and the clinical responses of cGVHD to donor Treg therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase 1/2 Study of Brentuximab Vedotin in Combination with Nivolumab in Patients with Relapsed or Refractory Classic Hodgkin Lymphoma: Part 3 (Concurrent Dosing) Results and Updated Progression-Free Survival Results from Parts 1 and 2 (Staggered Dosing)

Phase 1/2 Study of Brentuximab Vedotin in Combination with Nivolumab in Patients with Relapsed or Refractory Classic Hodgkin Lymphoma: Part 3 (Concurrent Dosing) Results and Updated Progression-Free Survival Results from Parts 1 and 2 (Staggered Dosing)

Next-Generation Sequencing Reveals a T Cell Receptor Signature Characteristic of Patients with Aplastic Anemia

Background. Acquired aplastic anemia (AA) is a bone marrow failure syndrome, in which patients' hematopoietic stem cells are destroyed, resulting in pancytopenia. The exact mechanism and biological process leading to AA remain largely unknown. Bone marrow destruction is perceived as an immune-mediated process, which is supported by elevated cytotoxic T lymphocyte (CTL) counts, responsiveness to immunosuppressive therapy and skewed CTL T cell receptor (TCR) repertoire. Although there is a well-established role of T cells in the pathology of AA, the putative antigen behind the autoimmune response, and thus, the TCRs recognising the antigen are still unrevealed.Methods. Our cohort comprised of 130 samples, consisting of bone marrow and/or peripheral blood samples from AA patients (n=52) from diagnosis and/or follow-up phases and from healthy controls (n=27). We performed TCRβ sequencing (immunoSEQ, Adaptive Biotechnologies) on sorted AA and healthy CTLs (n=25 and n=27) or AA mononuclear cells (MNCs, n=27). To gain in-depth understanding of the TCR-repertoire, we built two novel analysis methods: 1) an unsupervised clustering method to characterize epitope-specific T cells based on the amino acid -level similarities of TCRs and 2) a probability based classifier to identify AA based on TCR data in the CTL (discovery) and MNC (validation) cohorts.Results. To fully appreciate the complex nature of AA pathology, we divided the cytotoxic T cell response in two distinct categories, private and public response. Private response comprises T cell clones found only in individual patients, and this compartment could explain the variation in treatment responses and disease severity across patients. In the CTL TCR repertoire, we identified patient exclusive expanded T cell clones (>1% frequency in TCR repertoire, n = 317) and treatment responding clones (n = 364). Clustering analysis revealed that there is significant amino acid -level similarity between the TCRs of the private clones. We found several epitope-specific TCR clusters that were associated with different treatment responses, disease severities and HLA-DR15 risk-allele positivity.Interestingly, we discovered that the public response (CTL clones that are statistically enriched in AA patients compared to healthy controls) could discriminate AA from healthy TCR repertoire. Based on these publicly enriched TCRs, we built a classifier which could identify AA CTL TCR repertoire from healthy controls with 97% accuracy (F-score, revieved from leave-one-out cross-validation). We tested our classifier with the validation cohort. The accuracy to diagnose AA based on the TCR repertoire data in the MNC cohort was 0.72. Furthermore, the public clones that differentiated best the AA cases from healthy controls showed statistically significant peptide similarity. These public TCRs occurred also in healthy controls, but with smaller frequencies.When combining the private and public response TCRs, we discovered that some of AA patients' most expanded and treatment responding clones clustered in the same putative epitope-specific clusters with the public TCRs, showing an interesting intersection between public and private signatures. In addition, the comparison of the interesting private and public TCRs against databases of TCR sequences of known antigen specificities hinted that some of these clones may originally target known viral species (CMV, EBV and Influenza A), suggesting a role of these common pathogens in the development of AA.Discussion. CTLTCR repertoire analysis of AA patients revealed a TCR signature that was typical of AA patients, but varied between different patients, and it was validated with an independent dataset. Thus, we could design a TCR-based framework which could identify AA patients based on their TCR repertoire, independently of sample type. A future application for our classifier could be distinguishing AA from other AA-like diseases, like hypoplastic myelodysplastic syndrome. Furthermore, we found groups of TCRs that look similar on amino acid level, and hence these clones may target the same epitope. These TCR clusters were associated with clinical features. Amino acid similarity -based TCR signatures or TCR classifiers have not previously been published for AA or any other autoimmune diseases, and thus our pioneering tools could be utilized to study pathogenesis of other T cell mediated diseases as well. DisclosuresEbeling:Boehringer Ingelheim: Consultancy; Celgene: Speakers Bureau; Otsuka Pharma Scandinavia AB: Consultancy. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy. Mustjoki:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria; Ariad: Research Funding; Pfizer: Honoraria, Research Funding.

Quantitative Assessment of T Cell Clonotypes in Human Acute Graft-versus-Host Disease Tissues

Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-β deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated.

PO-394 Performance of a targeted T cell receptor beta immune repertoire sequencing panel in several FFPE tissue types – a tool for interrogation of the tumour microenvironment

IntroductionT cell receptor beta (TCRβ) immune repertoire analysis by next-generation sequencing is a valuable tool for research studies of the tumour microenvironment and potential immune responses to cancer immunotherapy. Here we describe a TCRβ sequencing assay that leverages the low sample input requirements of AmpliSeq library preparation technology to extend the capability of targeted immune repertoire sequencing to include FFPE samples which can often be degraded and in short supply.Material and methodsThis assay targets the highly diverse CDR3 region which allows for T cell clone identification and frequency measurement which, when combined, can provide a broad view of the immune landscape within tissue samples. The assay allows for functionality with either RNA or DNA input, with little modification of the library construction workflow, as well as flexibility in sequencing throughput and sample multiplexing capability.Results and discussionsTo evaluate assay accuracy, we sequenced libraries including known amounts of 30 well-studied T cell lymphoma rearrangements, as well as evaluation of samples comprised of known numbers of sorted T cells to ensure the absence of false rearrangement reporting. T cell repertoires were evaluated from as low as 5 ng to as large as 1 ug of input from samples of varying repertoire diversity, such as sorted T cells, peripheral blood leukocytes, fresh-frozen tissue, and FFPE tissue from a variety of normal and cancerous tissues such as lung, colon, brain, spleen, lymph node, and thymus. In parallel, we have developed a low input qPCR assay, based on known T cell markers, which allows for a quantitative measure of the T cell content across all sample types applicable to the TCRβ sequencing assay. This sample quantification test acts to guide optimal sample input ranges for library construction and required sequencing depth. Finally, we present the CDR3 TCRβ sequencing assay including a dual barcoding approach to extend sensitivity for detection of rare clones.ConclusionThese data present a T cell immune repertoire sequencing solution for application in a wide range of sample types, in particular, challenging FFPE samples. We find that the assay is capable of profiling repertoire metrics from FFPE samples over a large range of input amounts from several normal and tumour tissue types. In addition, we demonstrate use of a qPCR assay for quantification of sample T cell content to guide sample input for TCRβ immune repertoire sequencing experiments.

A comparison of IL-5 and IL-10-producing antigen-specific clonal T cell repertoires

Abstract Allergic rhinitis affects up to 40% of North Americans, and is associated with potentially debilitating symptoms. Allergen-specific immunotherapy (AIT) is a disease-modifying treatment associated with Treg induction and elevated IL-10 production. To date, it is unknown if IL-10-producing T cells are induced de novo or if AIT modulates allergen-specific Th2 cells to become tolerogenic. To investigate whether IL-5 and IL-10-producing antigen-specific T cells share a common T cell repertoire, we performed a longitudinal TCRβ sequence analysis of Timothy grass (TG)-specific T cells from donors who received AIT for 15 months. As a control, TCRβ sequencing was also performed on TG-specific T cells from an allergic donor. TG-specific cells were expanded by 14-day in vitro culture with a pool of immuno-dominant TG-derived T cells epitopes (TG pool). A pertussis epitope pool was used in parallel as a control. Subsequently, cells were restimulated with the same pool and allergen-specific cells were isolated by cytokine capture with column enrichment, sorting IL-5 and IL-10 -producing cells. The TCRβ repertoire was sequence from DNA isolated from sorted cells. Preliminary results revealed that in the TG pool-specific cells, some clonal overlap was observed between IL-10+ and IL-5+ cells, suggesting that a fraction of IL-10+ cells may originate from IL-5+ cells, but some are generated de novo. Longitudinal analysis of the allergen-specific TCR repertoire is a reliable method to determine if tolerogenic T cells are induced de novo or if they are modulated T cells that originate from the same repertoire as allergen-specific Th2 cells.

Combined immunotherapy encompassing intratumoral poly-ICLC, dendritic-cell vaccination and radiotherapy in advanced cancer patients

BackgroundCombination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methodsIn this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24h with poly-ICLC (Hiltonol), TNF-α and IFN-α. On days+8 and+10 of each cycle, patients received intratumoral image-guided 0.25mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600mg/m2 was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre- and post-treatment PBMC from patients achieving durable stable disease (SD) were studied by IFNγ ELISPOT-assays responding to tumor-lysate loaded DC and by TCRβ sequencing. ResultsCombined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABR+ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-β and IFN-α mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1β concentrations, especially in patients presenting SD. IFNγ-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. ConclusionsThis radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy.

A preclinical study of tumor-infiltrating lymphocytes in NSCLC.

161 Background: Multiple clinical studies have shown that adoptive cell transfer (ACT) of autologous tumor-infiltrating lymphocytes (TIL) is remarkably effective in melanoma patients. Non-small cell lung cancer (NSCLC) shares similarities with melanoma in terms of mutational burden and sensitivity to immune checkpoint inhibitors. We therefore sought to test whether TIL ACT may represent a viable option for the treatment of NSCLC patients. We utilized tissue collected from patients enrolled on the prospective ImmunogenomiC prOfiling of Non-small cell lung cancer (ICON) study. Methods: TIL and tissue-infiltrating lymphocytes were expanded ex vivo from 97 freshly resected early-stage localized NSCLC tumors and 39 matched uninvolved lung tissues. Growth and functional characteristics of TIL were assessed via flow cytometry, TIL-tumor reactivity assays, and analysis of TCRβ sequencing data. Results: NSCLC showed an increased proportion of CD3+ lymphocytes within the tumor-infiltrating leukocyte component as compared to matched normal lung tissue. The TIL compartment included a suppressed CD8+ T cell subset expressing significantly higher levels of PD-1 and lacking cytolytic potential compared to T cells expanded from normal tissue. TIL contained a higher proportion of proliferating (Ki67+) CD8+CD103+ tissue-resident memory (TRM) cells expressing activation markers such as CTLA4, LAG3, PD1 and ICOS, and increased CD4+ Tregs. Despite a highly immunosuppressive environment, TIL expansion was achieved with a success rate of 68% (n = 97) but appeared hindered in patients undergoing neoadjuvant chemotherapy treatment prior to surgery (56.2%, n = 16 vs 72.5% success rate in therapy-naïve patients). In addition, expansion efficiency (number expanded and time of culture) of TIL and matched lung residing lymphocytes were significantly associated (r = 0.379, p = 0.017, n = 39). Importantly, expanded CD8+ TIL products were oligoclonal and showed reactivity toward autologous tumors. Conclusions: Although NSCLC TIL are functionally inhibited in vivo they can be successfully expanded ex vivo and demonstrate recognition of autologous tumor cells. These data suggest that TIL can potentially be used for adoptive T cell-based immunotherapy in NSCLC.

The T-Cell Receptor Repertoire Predicts Interim-PET in Patients with DLBCL Treated with R-CHOP: An Observational Study from a Prospective Clinical Trial

▪T-cell infiltration of the tumor microenvironment (TME) in DLBCL is a key determinant of response to chemo-immunotherapy (Keane, Lancet Haem 2015). We have previously shown that greater diversity of the T-cell receptor (TCR) repertoire within the TME is correlated with improved survival following R-CHOP in DLBCL (Keane, CCR 2017). There are limited data on the impact of the intratumoral TCR repertoire on interim-PET (iPET), the relationship between intratumoral and circulating TCRs, and on dynamic changes of the TCR during therapy. In this study, we interrogated the TCR repertoire in a subset of DLBCL patients treated on the prospective Australasian Leukaemia Lymphoma Group NHL21 study (Hertzberg, Haematologica 2017), in which all patients had 4x RCHOP prior to iPET risk stratification.The CDR3 region of TCRβ chain underwent high-throughput unbiased TCRβ sequencing (Adaptive Biotechnologies). Metrics included: productive templates (total functional T-cells), productive rearrangements (functional T-cells with distinct specificity), productive clonality (repertoire unevenness due to clonal expansions), and maximal frequency clones (% most dominant single clone). Matched intratumoral diagnostic samples, blood at pre-therapy and post-cycle 4 (at the time of iPET) were tested. 42 patients (enriched for iPET+ cases) had sufficient material for testing.Median age was 55 (range 22-69) years and 72% were males. IPI was low/intermediate/high in 13/63/25% respectively. Cell of origin (COO) by Lymph 2CX method (nanoString) was ABC in 30%, and GCB in 44%. 40% were iPET+. In tissue, there was a median of 4652 productive templates, translating into 2998 productive rearrangements identified.Notably, the clonal repertoire of intratumoral TCRs in iPET+ patients was larger than iPET-ve patients (productive clonality 8.1 vs 5.1 x10-2, p=0.04), whereas the numbers of functional T-cells did not vary between groups. Comparing the tumor with the blood samples showed a high, but variable, degree of overlap between peripheral blood and the TME - TCR repertoire. Median number of top 100 tumor tissue clones shared in peripheral blood was 53.5 (range, 1-97) in pre-therapy and 39.5 (range, 0-93) in post-therapy blood, indicating that the both the circulation and the tumor likely contribute to immune-surveillance.In pre-therapy blood, the median productive templates and productive rearrangements were 44,950 (range, 6,003-273,765) and 29,090 (range, 5,190-152,706), and the median clonality was 8.5 (1.46-45.3) x 10-2. There were no differences between iPET+ and iPET-ve patients in these parameters. However, there was a marked change in T-cell composition between time points. Interestingly, in iPET-ve patients clonality measures were increased, with productive clonality 9.4 vs 14.4 x10-2, p=0.03; and % maximum productive frequency 3.39 vs 5.89, p=0.04.These findings demonstrate that the intratumoral TCR repertoire, and sequential blood sampling provide important information on outcome in DLBCL treated with RCHOP. A highly clonal T-cell repertoire in the TME was associated with iPET positivity after 4 cycles of R-CHOP. In line with findings in solid cancers treated with checkpoint blockade, development of clonal responses in peripheral blood was associated with iPET negativity. These findings indicate that clones expanded during therapy may be important in tumor clearance but that highly clonal T-cell responses in the tumor at diagnosis may hinder expansion of other T-cell responses to neoantigens. The circulating TCR composition is representative of the TME. These findings will assist the rationale design and therapeutic monitoring of novel immuno-therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3567: Sequencing the human TCRβ repertoire on the Ion S5™ System

Abstract Next-generation Sequencing (NGS) is proving an important tool in increasing understanding of the human immune system, and thereby cancer immunology. αβ-T cells are the primary constituents of human cell-mediated adaptive immunity. The antigen specificity of each αβ-T cell is encoded in the 500-600 bp transcript encompassing the variable portion of the rearranged TCRα and TCRβ subunits, which can be read via NGS in a process termed repertoire sequencing. Until now, the main challenge the field faces is the lack of a technology that can provide a contiguous read of 600 bp to minimize the complexity of designing bias-prone primers and informatics challenges of stitching short reads. Here we leverage the long read capability of Ion 530™ chip to comprehensively sequence all three CDR domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire profiling studies. Initial testing with Leukocyte total RNA demonstrates that this multiplex PCR assay produced repertoires that were much more similar to data derived from 5’RACE protocol than the commonly used BIOMED2 primer set. This result suggested that the use of long reads minimizes bias by allowing targeting of less variable regions. To further assess the performance of the assay, we designed a model system of 30 plasmid controls containing common human T-cell CDR3 sequences. Each plasmid was amplified individually and sequenced to confirm the detection of a single clonal population. Analytical sensitivity of the assay and accuracy of the accompanied analysis solution were further evaluated by spiking in plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results showed the assay offers linearity over 5 orders of magnitude of decreasing input concentration. In summary, we have demonstrated a NGS workflow for TCRβ sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less than 48 hours. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Denise S. Topacio-Hall, Tim Looney, Yongming Sun, Lauren Miller, Elizabeth Linch, Geoffrey Lowman, Lifeng Lin, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the human TCRβ repertoire on the Ion S5™ System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2017-3567

Molecular characterization of immune-related severe adverse events (irSAE).

3076 Background: Immune checkpoint inhibitors (ICIs; anti-CTLA-4 and anti-PD-1) have shown clinical success in many cancers, but may cause rare irSAE. The molecular features of irSAE have not been extensively explored. Therefore, we characterized the immune composition of tissue affected by ICI-mediated inflammation with a focus on colitis and neurologic toxicity. Methods: We performed retrospective T-cell receptor (TCRβ) sequencing, RNA-sequencing (HTG EdgeSeq; &gt; 2500 immune-related genes), and digital spatial profiling (NanoString) for 20 protein markers in 10 regions across tissues representing ICI-induced colitis, autoimmune inflammation (e.g. Crohn’s) and normal colon. Matched tumors were also included in a subset. We also analyzed the encephalitic and healthy brain of a unique presentation of anti-PD-1-induced encephalitis. Results: Patient-matched melanoma and colitis biopsies (n = 3) demonstrated shared T cell clones in all samples ranging from several shared clones to several hundred (0.4%, 2.7%, and 3% of rearrangements, respectively), including high-frequency clones. Shared TCRβ sequences were also identified among and between colon-irSAE and Crohn’s specimens. Gene expression patterns of inflammation in colon-irSAE resembled that of Crohn’s disease in principle components and clustering analysis, highlighting likeness between these diseases/SAEs. NanoString digital spatial profiling of regions of inflammation across samples showed higher CD68 and PD-L1 positivity in colon-irSAE specimens versus normal colon or Crohn’s specimens and reduced beta-catenin levels in both Crohn’s and colon-irSAE specimens relative to normal controls. Finally, we detected a high degree of TCR clonality in the encephalitic brain, including a single sequence present in ~20% of &gt; 12,000 T cells, suggesting a distinct antigen-specific response. Conclusions: We report the molecular characteristics of irSAE in colon and brain specimens from patients receiving ICIs. Highly clonal TCRβ sequences were frequently detected, particularly in a unique case of encephalitis-irSAE. Furthermore, we identify molecular distinctions and similarities between autoimmune and colon-irSAEs at the gene expression and proteomic levels.

Tracking the interplay between circulating and tumor-infiltrating T cells using AmpliSeq-based Ion Torrent TCRβ immune repertoire sequencing.

e14518 Background: TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to future cancer immunotherapy. Here we describe a multiplex PCR-based TCRβ sequencing assay that takes advantage of the exceptionally low base-call error rate and long read capability of the Ion S5 530 chip. The S530 chip provides &gt; 15M reads, allowing for sample sequencing in multiplex. The research assay utilizes AmpliSeq technology and Framework 1 and Constant gene targeting primers to provide comprehensive coverage of TCRβ CDR 1, 2 and 3 from RNA input with a hands-on time of less than 30 minutes. CDR region sequences reveal the antigen specificity of the receptor and may be genetically modified to increase TCR affinity for tumor antigen. Methods: We evaluated assay performance by sequencing TCRβ rearrangements from normal donor peripheral blood leukocyte (PBL) cDNA that had been spiked with 30 reference rearrangements taken from the literature. We then used our assay to evaluate the extent of clonal overlap between matched tumor infiltrating lymphocyte (TIL) and peripheral blood leukocyte repertoires in an individual with squamous cell carcinoma of lung. Results: Results from sequencing of spike-in reference rearrangements indicate that the assay is accurate and sensitive over 5 logs of input template amount while showing minimal amplification bias. Sequencing of matched PBL and TIL repertoires revealed that a subset of the PBL repertoire (8%) consisted of clones also found in TIL. Technical replicates showed high concordance (r &gt; .96) in the frequency of detected clones, indicating that the results were reproducible and samples were sequenced to an appropriate depth. Conclusions: In summary, these data suggest that AmpliSeq-based multiplex PCR and Ion Torrent sequencing provide unbiased, reproducible, scalable, complete, and accurate information for immune repertoire research sequencing applications.

The T-cell Receptor Repertoire Influences the Tumor Microenvironment and Is Associated with Survival in Aggressive B-cell Lymphoma.

Purpose: To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in de novo diffuse large B-cell lymphoma (DLBCL) and the impact of TCR on survival.Experimental Design: We performed high-throughput unbiased TCRβ sequencing on a population-based cohort of 92 patients with DLBCL treated with conventional (i.e., non-checkpoint blockade) frontline "R-CHOP" therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString. The primary endpoints were 4-year overall survival (OS) and progression-free survival (PFS).Results: The TCR repertoire within DLBCL nodes was abnormally narrow relative to non-diseased nodal tissues (P < 0.0001). In DLBCL, a highly dominant single T-cell clone was associated with inferior 4-year OS rate of 60.0% [95% confidence interval (CI), 31.7%-79.6%], compared with 79.8% in patients with a low dominant clone (95% CI, 66.7%-88.5%; P = 0.005). A highly dominant clone also predicted inferior 4-year PFS rate of 46.6% (95% CI, 22.5%-76.6%) versus 72.6% (95% CI, 58.8%-82.4%, P = 0.008) for a low dominant clone. In keeping, clonal expansions were most pronounced in the EBV+ DLBCL subtype that is known to express immunogenic viral antigens and is associated with particularly poor outcome. Increased T-cell diversity was associated with significantly elevated PD-1, PD-L1, and PD-L2 immune checkpoint molecules.Conclusions: Put together, these findings suggest that the TCR repertoire is a key determinant of the TME. Highly dominant T-cell clonal expansions within the TME are associated with poor outcome in DLBCL treated with conventional frontline therapy. Clin Cancer Res; 23(7); 1820-8. ©2016 AACR.

Clinical significance of T cell clonality and expression levels of immune-related genes in endometrial cancer.

Immune microenvironment characterized by T cell clonality as well as expression signatures of immune-related genes in endometrial cancer tissues may play significant roles in clinical outcome of patients. We aimed to investigate the clinical significance of immune-related gene expression and TCR repertoire in endometrial cancer. Using total RNAs extracted from 32 endometrioid endometrial cancer cases, we performed quantitative real-time PCR to measure mRNA expression levels of immune-related genes including TRB, CD8, GZMA, HLA-A, CD11c and PD-L1. Higher mRNA expression levels of CD8 (P=0.039) and CD11c (P=0.046) in the 32 tissue samples were significantly associated with longer progression-free survival (PFS). Expression levels of CD8 (P<0.001) and CD11c (P=0.048) were also significantly associated with longer PFS in 540 cases in TCGA database. We also performed T cell receptor β (TCRβ) sequencing of tumor-infiltrating lymphocytes (TILs) on an Illumina MiSeq platform. To evaluate clonal expansion of TCRβ clonotypes, we adjusted the number of abundant TCRβ clonotypes by TRB mRNA expression levels and examined TCR clonality with the expression levels of immune-related genes and clinicopathological factors. The cases with high clonal T cell expansion along with high PD-L1 expression in cancer tissues was related to higher mRNA expression levels of CD8 (P<0.001), GZMA (P<0.001) and HLA-A (P=0.027), showed a significantly longer PFS (P=0.015), indicating a possibility that these parameters may serve as faborable prognostic factors. Considering clinical stage, mRNA expression of CD8 (P=0.037), GZMA (P=0.027) and HLA-A (P=0.022) was significantly higher in tumors at an early stage. Thus, we identified clinical and prognostic significance of immune microenvironment including the T cell clonality of TILs as well as PD-L1 and CD11c mRNA expression levels in endometrial cancer tissues.

Tumor-Infiltrating Merkel Cell Polyomavirus-Specific T Cells Are Diverse and Associated with Improved Patient Survival.

Tumor-infiltrating CD8+ T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8+ T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, "KLL") and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer+ CD8+ T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry. TCRβ (TRB) sequencing was performed on tetramer+ cells from PBMCs or TILs (n = 14) and matched tumors (n = 12). Functional avidity of T-cell clones was determined by IFNγ production. We identified KLL tetramer+ T cells in 14% of PBMC and 21% of TIL from MCC patients. TRB repertoires were strikingly diverse (397 unique TRBs were identified from 12 patients) and mostly private (only one TCRb clonotype shared between two patients). An increased fraction of KLL-specific TIL (>1.9%) was associated with significantly increased MCC-specific survival P = 0.0009). T-cell cloning from four patients identified 42 distinct KLL-specific TCRa/b pairs. T-cell clones from patients with improved MCC-specific outcomes were more avid (P < 0.05) and recognized an HLA-appropriate MCC cell line. T cells specific for a single MCPyV epitope display marked TCR diversity within and between patients. Intratumoral infiltration by MCPyV-specific T cells was associated with significantly improved MCC-specific survival, suggesting that augmenting the number or avidity of virus-specific T cells may have therapeutic benefit. Cancer Immunol Res; 5(2); 137-47. ©2017 AACR.

Daratumumab in Combination with Lenalidomide Plus Dexamethasone Induces Clonality Increase and T-Cell Expansion: Results from a Phase 3 Randomized Study (POLLUX)

Daratumumab in Combination with Lenalidomide Plus Dexamethasone Induces Clonality Increase and T-Cell Expansion: Results from a Phase 3 Randomized Study (POLLUX)