T-cell Clones Research Articles

Treatment of indolent cutaneous T-cell lymphoma with hypofractionated radiation therapy in three cats.

Feline indolent cutaneous T-cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL. To report the clinical outcome of three cats with ICL treated with hypofractionated electron-beam radiotherapy (RT). Three privately owned cats with ICL. Medical records and client surveys were reviewed. A diagnosis of probable ICL was based on history, clinical presentation and histopathological findings, and confirmed using CD3 immunohistochemical analysis and PCR for antigen receptor gene rearrangement (PARR). All cats were treated with hypofractionated RT (four fractions of 8Gy). All cats presented with skin lesions characterised by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Before hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T-cell receptor gamma gene rearrangement. After RT, two cats showed histological improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histological resolution of lesions with only minimal residual lymphocytes. One cat was determined to have a complete clinical response while the other showed partial responses. No acute adverse effects of radiation were observed; chronic effects included leukotrichia, partial alopecia and mild fibrosis. All clients reported improvement in quality of life for their cats. Clinical and histological improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.

Abstract 2737: Clinical and epidemiologic predictors of clonal immune responses in colorectal cancer

Abstract The quantity and quality of immune responses in colorectal cancers (CRC) are widely variable and have important clinical, therapeutic, and prognostic implications. We studied clinical and epidemiologic factors that might influence T-cell quantity and clonality within colorectal adenocarcinomas to better understand the drivers of diverse immune responses. Incident cases of CRC from the Molecular Epidemiology of Colorectal Cancer Study (MECC) were interviewed, and 6,006 cases had complete epidemiologic data. Archived tumor blocks were retrieved from 3,865 (64.4%) cases, and all were reviewed by a single expert pathologist who quantified TILS/hpf. Sufficient tissue for macrodissection and measurement of TCR abundance and clonality using the immunoSEQ assay (Adaptive Biotechnologies) was available and completed for 2,750 cases. Logistic regression, negative binomial regression and linear regression models were used to evaluate potential associations between clinical and epidemiologic variables for: TILS/hpf, TCR abundance, and T-cell clonality. The stage distribution was representative of cancer incidence in the population, and the MSI-H phenotype was observed in 14.2% of cases. Clinical, pathologic, and epidemiologic variables including aspirin, alcohol, diet, hormone use, physical activity, smoking, and statins were assessed in relation to immune measures. Among other findings, >5 years of statins (p<0.001) and daily aspirin (p=0.037) were each strongly associated with T-cell clonality. Our study suggests that important parameters of the adaptive immune response may be mediated by modifiable factors. Mechanisms regulating immune responses in CRC may have implications for chemoprevention as well as immunotherapy. Citation Format: Stephen B. Gruber, Joseph D. Bonner, Sidney S. Lindsey, Ya-Yu Tsai, Rebeca Sanz-Pamplona, M. Henar Alonso, Marilena Melas, Hedy S. Rennert, Kevin J. McDonnell, Gregory E. Idos, Christopher P. Walker, W. Martin Kast, Diane Da Silva, Harlan S. Robins, Joel K. Greenson, Victor Moreno, Stephanie L. Schmit, Gad Rennert. Clinical and epidemiologic predictors of clonal immune responses in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2737.

Abstract 835: T-cell abundance, clonality and disease specific survival in colorectal cancer

Abstract Immune responses to colorectal cancer (CRC) are important prognostic factors as measured by either tumor infiltrating lymphocytes per high powered field or the presence of Crohn's like lymphoid reaction. New technologies to quantify specific features of immune responses include immunoSeq (Adaptive Biotechnologies), which accurately quantifies T-cell receptor (TCR) abundance and T-cell receptor clonality. Here we measured the independent contributions of TCR abundance and TCR clonality to 5-year CRC-specific survival in the Molecular Epidemiology of Colorectal Cancer (MECC) study. The MECC study consented, interviewed, and followed 6,006 incident cases of CRC diagnosed between March 31, 1998 and July 1, 2017 (median follow-up time = 6.9 years). Archived paraffin blocks were retrieved, and 2,750 cases had sufficient tissue and adequate DNA extraction to perform the immunoSeq assay. Analyses were restricted to 1,625 samples with more than 100 T-cells to permit accurate quantification of TCR abundance and TCR clonality as measured by Simpson Clonality Index. TCR abundance was log2 transformed for analyses, and Simpson Clonality Index was log2-transformed and z-scale normalized separately by assay version. In unadjusted models, dichotomized log2 transformed TCR abundance (Hazard Ratio (HR) = 0.57, 95% confidence interval (CI) 0.47-0.71, p=9.77E-08) and dichotomized TCR clonality (HR = 0.77, 95% CI 0.63-0.94, p=0.01) were each statistically significantly associated with 5-year CRC-specific survival. In a model with adjustment for age, sex, microsatellite instability, stage and assay version, both dichotomized log2 transformed TCR abundance (HR = 0.61, 95% CI, 0.49-0.75, p=3.03E-06), and dichotomized TCR clonality (HR = 0.81, 95% CI, 0.66-0.99, p=0.048) were associated with improved 5-year disease-specific survival. Conclusion: TCR abundance and TCR clonality are independent prognostic indicators in CRC. Citation Format: Joseph Bonner, Sephanie L. Schmit, Sidney S. Lindsey, Ya-Yu Tsai, Rebeca Sanz-Pamplona, M. Henar Alonso, Marilena Melas, Hedy S. Rennert, Kevin J. McDonnell, Gregory Idos, Christopher P. Walker, W. Martin Kast, Diane Da Silva, Harlan S. Robins, Joel K. Greenson, Victor Moreno, Gad Rennert, Stephen B. Gruber. T-cell abundance, clonality and disease specific survival in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 835.

Abstract 1488: Viral molecular mimicry influences the antitumor immune response in murine and human melanoma

Abstract Molecular mimicry is known to be one of the leading mechanisms by which infectious agents may induce autoimmunity. However, whether a similar mechanism triggers anti-tumor immune response is unexplored, and the role of anti-viral T-cells infiltrating the tumor has remained anecdotal. To address this question, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we showed that, in mice, viral pre-existing immunity enhanced the efficacy of cancer immunotherapy via molecular mimicry. Specifically, when treated with a cancer vaccine consisting of peptides with a high degree of homology with specific viral peptides, the mice with induced pre-existing immunity to these viral peptides showed significantly better anti-tumor response.To understand whether this mechanism could partly explain immunotherapy-response in humans, we analyzed a cohort of melanoma patients undergoing PD1 treatment with high IgG titer for Cytomegalovirus (CMV). In this cohort of patients, we showed that high level of CMV-antibodies was associated with a prolonged progression free survival, and found that in some cases PBMCs could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell TCR sequencing revealed expansion of the same CD8+ T-cell clones, when PBMCs were pulsed with tumor- or homologous viral peptides.In conclusion, we have demonstrated that pre-existing immunity and molecular mimicry could explain part of the response observed in immunotherapy. Most importantly, we have developed a tool able to identify tumor antigens and neoantigens based on their similarity to pathogen antigens, in order to exploit molecular mimicry and cross-reactive T-cells in cancer vaccine development. Citation Format: Jacopo Chiaro, Henna Kasanen, Thomas Whalley, Cristian Capasso, Sara Feola, Mikaela Grönholm, Firas Hamdan, Karita Peltonen, Micaela Hernberg, Siru Mäkelä, Hanna Karhapää, Paul Brown, Beatriz Martins, Manlio Fusciello, Erkko Ylösmäki, Anna Kreutzman, Satu Mustjoki, Barbara Szomolay, Vincenzo Cerullo. Viral molecular mimicry influences the antitumor immune response in murine and human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1488.

Influence of DM-sensitivity on immunogenicity of MHC class II restricted antigens

BackgroundGraft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted antigens depending on their behavior towards HLA-DM....

Abstract 1747: Oncolytic Vesicular Stomatitis Virus expressing a species relevant interferon-beta is safe and provides a survival benefit when added as neoadjuvant to the standard of care for dogs with naturally occurring bone cancer

Abstract The goal of this study was to evaluate safety, efficacy, and immunological activity of Vesicular Stomatitis Virus (VSV) expressing a species relevant (canine) interferon-beta (IFN-β) and the sodium iodide symporter (NIS), henceforth called VSV, in companion dogs with naturally occurring appendicular osteosarcoma. The hypothesis was that VSV would track to the tumor, induce oncolysis, and lead to enhanced infiltration of immune cells, expansion of virus- and tumor specific T-cell clones, and improved survival outcomes.The first 15 dogs enrolled in the VSV Immunotherapy and Genomics of Osteosarcoma Research (VIGOR) study received a single iv dose of VSV (1x109 TCID50/kg) 10 days before amputation and adjuvant carboplatin chemotherapy. The final 13 dogs were randomized in a double-blinded fashion to receive either a single dose of VSV or placebo. Outcome data were compared to those from an historical control group of 57 dogs with appendicular osteosarcoma treated between 2006 and 2018 using the standard of care of alone. VSV treatment was associated with mild systemic effects (fever and inflammation), which were self-limiting and caused no symptomatology. Alterations in the tumor microenvironment (micronecrosis, increased fibrosis, and increased inflammation) were more common and more pronounced in VSV treated dogs than in placebo-treated dogs.Reduction in the expression of co-regulated cell cycle gene clusters in tumors after VSV-treatment was observed in dogs with longer survival. Conversely, increased expression of co-regulated immune/inflammatory gene clusters was observed in both VSV-treated dogs and in placebo-treated dogs, suggesting this might be, at least partly, due to the biopsy procedures. Assessment of changes in the clonal composition of T cells within the tumors, draining lymph nodes, and systemic circulation is in progress. No differences were evident in median event-free or overall survival times among the different groups, although aspects of the study design and sample size could obscure differences. Intriguingly, a “tail” of nine dogs (41%) that were long-term survivors (>450 days) was apparent in the VSV-treated group. All but one of these long-term survivors had evidence of inflammation in the pre-treatment tumor samples or showed increased inflammation in the post-treatment samples. We conclude that neoadjuvant VSV has an excellent safety profile and provides a survival benefit for a subset of dogs with spontaneous bone cancer. Ongoing work seeks to establish distinguishing characteristics that identify those individuals most likely to benefit from this therapy, as well as to evaluate the potential to enhance this benefit through the addition of novel immunomodulatory agents as part of our efforts to translate this approach to human cancer patients. Citation Format: Kelly M. Makielski, Aaron L. Sarver, Aishwarya Sathyanarayan, Michael S. Henson, Kathleen M. Stuebner, Alexandru-Flaviu Tabaran, Ingrid Cornax, Gerard O'Sullivan, Andrea Chehadeh, Donna Groschen, Kelly Bergsrud, Lauren J. Mills, Amber L. Winter, Sara Pracht, Milcah C. Scott, Michael A. Farrar, Gary R. Cutter, Joseph S. Koopmeiners, Stephen J. Russell, Shruthi Naik, Jaime F. Modiano. Oncolytic Vesicular Stomatitis Virus expressing a species relevant interferon-beta is safe and provides a survival benefit when added as neoadjuvant to the standard of care for dogs with naturally occurring bone cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1747.

Abstract 619: Integrated multi-platform profiling of early-stage non-small cell lung cancer identifies relationship between disease recurrence and decreased native immune response in treatment-naïve resected NSCLC

Abstract Introduction: Differences in the host immune environment are thought to mediate heterogeneous treatment responses in non-small cell lung cancer (NSCLC). Unlike individual platform analyses, integrative analysis of multi-platform profiling allows for the discovery of novel interactions that expand our understanding of the disease. Utilizing the ImmunogenomiC prOfiling of NSCLC patient cohort (ICON), a prospective multi-omics protocol of operable early-stage NSCLC tumors with integrated immune, genomic, and clinical data, we hypothesized that multi-platform analyses would identify differences in the immune-genomic landscape that are associated with disease recurrence. Methods: Tumor and tumor-adjacent uninvolved lung was collected at resection; blood was collected before and after surgery. Tissue samples underwent WES, RNAseq, TCR sequencing (TCRseq), multiplex immunofluorescence (mIF), and RPPA profiling; tissue and blood (PBMC) samples were analyzed by flow cytometry. An integrated, inter-modality network was built using Spearman correlations between measurement pairs from different data modalities. Multivariate analysis was performed to adjust for stage and histology. Results: A total of 89 treatment-naïve patients with Stage 1-3 resected NSCLC (Squamous: 19; Non-squamous: 70) and 24 months of follow-up were analyzed (recurrence N = 24; no recurrence N = 65). The data network includes over 4,000 measurements linked by over 50,000 correlations. InfoMap, a community detection approach, was used to extract sub-network modules, which were used to contextualize the results of multivariate analysis. Tumors from patients with recurrence demonstrated decreased immune cell infiltration and activation including decreased cytotoxic CD8 T-cells (CD8+PD1+; fold-change (FC) = 0.898, p = 0.018; flow cytometry), decreased T-cell clonality (FC = 0.954, p = 0.017; TCRseq), and decreased tumor-associated macrophages (CD68+PD-L1+; FC = 0.426, p = 0.011; mIF). Furthermore, circulating CD8+ICOS+ activated T cells were decreased in patients with recurrence suggesting an impaired systemic anti-tumor immune response (FC = 0.552, p = 0.042; PBMC Flow). Finally, tumor-adjacent uninvolved lungs showed distinct T-cell phenotypes with accumulation of inactive CD8 T-cells (CD8+PD1-TIM3-) in patients with recurrence and increased populations of activated CD8 T-cells (CD8+PD1+) in patients without recurrence. Conclusion: Integrative multi-omic analysis suggests preserved anti-tumor immune surveillance in patients who are disease-free after 2 years from surgical resection with curative intent for treatment of NSCLC relative to patients with disease recurrence. Further analysis is ongoing to interrogate genomic and immune variables that are associated with disease recurrence. Citation Format: Neal Akhave, Stephanie Schmidt, Alexandre Reuben, Tina Cascone, Jianhua Zhang, Jun Li, Junya Fujimoto, Lauren A. Byers, Beatriz Sanchez-Espiridion, Lixia Diao, Jing Wang, Lorenzo Federico, Marie-Andree Forget, Daniel J. McGrail, Annikka Weissferdt, Shiaw-Yih Lin, Younghee Lee, Carmen Behrens, Ignacio I. Wistuba, Andrew Futreal, Ara Vaporciyan, Boris Sepesi, John V. Heymach, Chantale Bernatchez, Cara Haymaker, Jianjun Zhang, Christopher A. Bristow, Marcelo V. Negrao, Don L. Gibbons. Integrated multi-platform profiling of early-stage non-small cell lung cancer identifies relationship between disease recurrence and decreased native immune response in treatment-naïve resected NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 619.

Mutational signatures in T-lymphocytes of rats treated with N-propyl-N-nitrosourea and procarbazine.

We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T-cells of rats treated in vivo with N-propyl-N-nitrosourea (PNU) or procarbazine (PCZ). The signatures from the treated rats were different from the signature of background mutations. The main component of the spontaneous T-cell mutational signature was C➔T transition with all other single base substitutions evenly distributed. The PNU-induced mutational signature showed relatively equal contributions from C➔T and T➔C transitions, and T➔A transversions. The PCZ-induced signature was characterized by T➔C transitions, T➔A and, to a smaller extent, T➔G transversions. C➔G transversions were infrequent in either the PNU or PCZ signatures. WGS not only allowed mutational signature detection, but also measured quantitative responses to mutagen treatment: 10-40× increases in the number of mutations per clone were detected in T-cell clones from treated rats. The overall strand specificity of induced mutations for annotated rat genes was comparable to the strand specificity of mutations determined previously for the endogenous X-linked Pig-a gene. Our results provide valuable reference data for future applications of WGS in safety research and risk assessment.

T-Cell Receptor Gene Rearrangement Clonality, Flow Cytometry Status, and Associated Outcomes in Early-Stage Cutaneous T-Cell Lymphoma

The prognostic significance of clonal T-cell receptor (TCR) rearrangement or low-level blood involvement as assessed by flow cytometry for patients with early-stage cutaneous T-cell lymphoma (CTCL) is not clear. To assess the association of low-level blood involvement by TCR clonality and flow cytometry with outcomes for patients with early-stage CTCL. A retrospective cohort analysis was conducted from September 1, 2019, to February 29, 2020, of 322 patients with early-stage (I-IIA) CTCL seen at the Winship Cancer Institute of Emory University and Grady Memorial Hospital. T-cell receptor gene rearrangement and flow cytometry records from the peripheral blood were documented at initial assessment. T-cell receptor clonality and peripheral blood flow cytometry. Univariate and multivariable models and Kaplan-Meier assessments were analyzed for overall survival (OS) and time to next treatment. The primary outcome was OS from diagnosis and time to next treatment, and the hypotheses were formulated prior to data collection. A total of 322 patients (166 female patients [51.6%]; median age at diagnosis, 53.8 years [range, 8.6-87.4 years]) with early-stage CTCL diagnosed from 1990 to 2018 were identified; of these, 258 had data available for both flow cytometry and TCR. Positive results for both TCR clonality and flow cytometry were associated with inferior OS in early-stage CTCL compared with both having negative results (hazard ratio [HR], 2.86; 95% CI, 1.02-8.06; P = .046). Positive results for only TCR clonality or only flow cytometry were not associated with OS (TCR clonality: HR, 1.31; 95% CI, 0.70-2.47; P = .40; flow cytometry: HR, 1.21; 95% CI, 0.58-2.52; P = .61) or time to next treatment (TCR clonality: HR, 1.05; 95% CI, 0.77-1.43; P = .76; flow cytometry: HR, 0.74; 95% CI, 0.47-1.16; P = .12). However, positive flow cytometry results were associated with reduced OS in the stage IIA subgroup (n = 94; HR, 1.17; 95% CI, 1.18-8.74; P = .02). Covariates associated with reduced survival included advanced age at diagnosis, male sex, and higher disease stage. This cohort study of patients with early-stage CTCL suggests that low-level blood involvement as indicated by positive results for both TCR gene rearrangement and flow cytometry was associated with inferior OS, whereas positive results for either flow cytometry or TCR clonality was not. More precise measurements of blood involvement in CTCL and larger multi-institutional cohorts are needed to validate the prognostic significance of low-level blood involvement in early-stage CTCL.

Deciphering Adverse Drug Reactions: In Vitro Priming and Characterization of Vancomycin-Specific T Cells From Healthy Donors Expressing HLA-A*32:01.

Drug rash with eosinophilia with systemic symptoms (DRESS) is a serious adverse event associated with use of the glycopeptide antibiotic vancomycin. Vancomycin-induced drug rash with eosinophilia with systemic symptoms is associated with the expression of human leukocyte antigen (HLA)-A*32:01, suggesting that the drug interacts with this HLA to activate CD8+ T cells. The purpose of this study was to utilize peripheral blood mononuclear cell from healthy donors to: (1) investigate whether expression of HLA-A*32:01 is critical for the priming naïve of T cells with vancomycin and (2) generate T-cell clones (TCC) to determine whether vancomycin exclusively activates CD8+ T cells and to define cellular phenotype, pathways of drug presentation and cross-reactivity. Dendritic cells were cultured with naïve T cells and vancomycin for 2 weeks. On day 14, cells were restimulated with vancomycin and T-cell proliferation was assessed by [3H]-thymidine incorporation. Vancomycin-specific TCC were generated by serial dilution and repetitive mitogen stimulation. Naïve T cells from HLA-A*02:01 positive and negative donors were activated with vancomycin; however the strength of the induced response was significantly stronger in donors expressing HLA-A*32:01. Vancomycin-responsive CD4+ and CD8+ TCC from HLA-A*32:01+ donors expressed high levels of CXCR3 and CCR4, and secreted IFN‐γ, IL-13, and cytolytic molecules. Activation of CD8+ TCC was HLA class I-restricted and dependent on a direct vancomycin HLA binding interaction with no requirement for processing. Several TCC displayed cross-reactivity with teicoplanin and daptomycin. To conclude, this study provides evidence that vancomycin primes naïve T cells from healthy donors expressing HLA-A*32:01 through a direct pharmacological binding interaction. Cross-reactivity of CD8+ TCC with teicoplanin provides an explanation for the teicoplanin reactions observed in vancomycin hypersensitive patients.

Differential Impact of Signaling Inhibitor Treatment Versus Standard Chemoimmunotherapy on Chronic Lymphocytic Leukemia T Cells: Clinical Implications

Studies by us and others have documented the clonal architecture of the T cell repertoire in chronic lymphocytic leukemia (CLL), with T cell clones which persist and expand over time in untreated patients, appear disease-specific and may be shared among different patients, thereby indicating selection by restricted antigens. Here, we investigated T cell repertoire dynamics in CLL after different types of treatment by employing high-throughput next-generation sequencing in order to comprehensively assess changes in relation to the type of treatment as well as the clinical response. We analyzed 42 pre- and 3-month post-treatment blood samples from 16 CLL patients who received (i) standard chemoimmunotherapy (FCR regimen, n=5), (ii) ibrutinib (IB, n=9), and/or (iii) rituximab-idelalisib (R-ID, n=7). At the post-treatment sampling, all patients who received FCR had achieved complete remission (CR), and all patients on IB or R-ID had achieved partial remission (PR). Two patients, one on IB and one on R-ID, who later achieved CR were analyzed at that timepoint, as well. We excluded patients who developed rituximab-related late-onset neutropenia after FCR, since it is known to be mediated by cytotoxic T-cell clones. Starting material was PB mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw NGS reads were processed through a previously published, purpose-built bioinformatics pipeline designed for paired-read stitching, sequence curation, clonotype computation and repertoire analysis. Only productive TRBV-TRBD-TRBJ rearrangements were included in the analysis (n= 7,494,236, 85.3% of filtered-in sequences, median 178,063/sample). For repertoire analysis, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence) were considered (median 11,290 distinct clonotypes/sample). The TRBV gene repertoire remained relatively stable after treatment across all treatment groups. All cases displayed significant clonal T cell expansions both pre- and post-treatment (median cumulative frequency of the 10 most expanded T cell clonotypes/sample 30.3% versus 38.6%, respectively). However, differences were noted between the three treatment subgroups. More specifically, clonality (measured as the median cumulative frequency of the 10 most expanded clonotypes/sample) significantly increased after FCR (20.8% to 39.0%, p=0.03) and R-ID (33.0% to 41.1%, p=0.001), whereas it tended to decrease after IB (36.1% to 31.0%, p=0.34). Notably, achieving deeper clinical response (from PR to CR) correlated with a significant increase of clonality in the R-ID case (41.0% to 56.2%), but had no impact in this respect for the IB case (48.3% to 48.1%). The increased clonality in the R-ID case culminated from further expansion of 5 major clonotypes which also dominated the pre-treatment repertoire, suggesting that they may correspond to T-cell clones with anti-tumor properties activated by treatment. Overall, FCR resulted in reconstitution of the T-cell repertoire (median of 2/10 most frequent T-cell clones pre-treatment remained significantly expanded post-treatment), whereas pre-treatment major clones persisted after both IB and R-ID (8/10 and 7/10, respectively). In conclusion, NGS immunoprofiling in CLL documents the differential impact of various treatments on T cells. Particularly, chemoimmunotherapy increases T cell clonality likely through an ablative mechanism, in contrast to signaling inhibitors which retain T cell clones that may have developed in response to tumor antigens and possibly activate them, at least as indicated for the R-ID case, with obvious clinical implications. DisclosuresAgathangelidis:Gilead Sciences: Research Funding. Stavroyianni:Gilead Sciences: Honoraria; Takeda: Honoraria; Janssen Pharmaceuticals: Honoraria. Stamatopoulos:Janssen Pharmaceuticals: Honoraria, Research Funding; Novartis SA: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

Clonal T-Lymphocyte Populations in Healthy Donors

IntroductionT-cell clonality analysis based on T-cell receptor gamma chain (TCRG) genes rearrangements in peripheral blood could be useful for T-cell neoplasms diagnosis. Monoclonal products that exceed polyclonal background more than 3 times are often seen in lymphoid malignancies. Doubtful clonal results might be a possible feature of a relatively low tumor burden as well as a sign of a benign reactive process or reduction of the repertoire of lymphocytes associated with age. T-cell clonality in peripheral blood has been described in autoimmune diseases and viral infections. Examination of T-cell clonality in healthy people is necessary to define normal values of clonal variation and to develop algorithms for differential diagnosis of reactive and neoplastic conditions. Cytotoxic T-lymphocytes are the intended substrate of reactive processes. CD8 and CD57 are the characteristic surface markers of the cell population. Selection of CD8+ and CD57+ cells followed by determination of clonality based on TCRG genes rearrangements used to correlate clonal products with a specific functional population of lymphocytes and thereby to understand the basis of clonal expansion.Thus the aim of the research was to study the frequency of T-cell clonality and possible features of the clonal expansion of T-lymphocytes in healthy adults.Materials and methodsThe study included 25 healthy donors with a median age of 40 years (from 19 to 62 years). Samples of peripheral blood were obtained at the blood transfusion facility of the National Research Center for Hematology. All donors gave informed consent for participation in the research.CD8+ and CD57+ lymphocytes were isolated by means of immunomagnetic selection on Miltenyi Biotec beads. T-cell clonality was assessed by TCRG gene rearrangements using multiplex PCR with BIOMED-2 primer system and subsequent GeneScan analysis.ResultsBulk lymphocyte population in 2 of 25 donors (8%) was monoclonal, in 5 (20%) donors -doubtful monoclonal, in 2 donors (8%) - oligoclonal and in 16 of 25 donors (64%) - polyclonal (table 1). Thus there was the predominance of reactive clonal populations in the bulk population of cells in 28% (7/25) of donors.No correlation of the presence of clonal products with the age of donors has been demonstrated (Spearman test). According to the literature the frequency of clonal expansion of lymphocytes increases after 60 years. On the contrary, all participants in our study with the sole exception were younger. The median age of donors was 40 years. We suggest than at least in our donor sample T-cell clonality might be associated with individual characteristics of the donors rather than age related T-cell repertoire narrowing.Clonal products dominant in bulk population of lymphocytes, were attributed to CD8+ and CD57+ cells by means of magnetic selection. Therefore, the association of reactive clonal products with cytotoxic lymphocytes (figure 1, #8, #13) might be assumed.Additionally, in the case of donor #7 doubtful clonal product was discovered in the CD8- population, not previously dominant in the bulk population of lymphocytes and not identified in the CD8+ population (figure 1, #7).Since clear monoclonality of donor case #12 (figure 2) additional blood tests were run. No deviations in blood formula were found. Clinical evaluation confirmed the woman to be healthy, without any symptoms or history of chronic viral or autoimmune diseases.ConclusionsThe presence of clonal expansion of T-lymphocytes in peripheral blood should not be taken as a specific feature of lymphoid malignancy when seen in the absence of clinical or pathological signs of the disease. The typical cause of such conditions is the accumulation of clonal cytotoxic CD8+ CD57+ cells. The nature of clonal products in CD8- populations of lymphocytes are currently not clear and requires more extensive investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Skewed HSC Clonal Output of T-Cells in an Aged Rhesus Macaque after CD3e-Immunotoxin Treatment

Background:Lymphodepletion is often required for cancer therapy, organ transplantation, and the treatment of severe immunological diseases. People of advanced age (>55 years old) are at a higher risk for such diseases and show significantly increased morbidity and mortality with delayed and/or incomplete immune recovery following cytotoxic therapies. Our understanding of hematopoietic homeostasis and why the risks of lymphodepleting treatments are exacerbated in the elderly remains incomplete. Recent retroviral tagging studies, including our own, have shown that hematopoietic homeostasis is maintained through complex yet well-orchestrated polyclonal repopulation by heterogeneous HSC and progenitors, but clonal repopulation under stress conditions (such as lymphodepletion) has not yet been well characterized. Here we demonstrate hematopoietic stem cell (HSC) clonal behaviors prior to and after CD3e-immunotoxin treatment in one of our nonhuman primates (animal 95E132) that we have followed for up to 18 years (equivalent to approximately 60-70 human years) post-transplant.Methods:After 16 years of follow-up after HSC transplant, animal 95E132 was treated with 8 doses (25 ug/kg/dose) of CD3e-immunotoxin (CD3e-IT: monoclonal antibodies against CD3e-conjugated with Diphtheria toxin). Hematopoietic recovery was monitored for an additional 19 months by a complete blood cell count, flow cytometry, T-cell receptor V beta (TCRvb) spectratyping, and quantitative vector integration site (VIS) sequencing (Kim et al, Journal of Virology, 2010; Suryavanshi et al, JoVE, 2017). HSC clonal profiles in T-cell, B-cell, Granulocytes, Monocytes, and NK cells in the peripheral blood were analyzed over time. Clonal profiles in lymph nodes, spleen, bone marrow, thymus, and gut tissues were analyzed after necropsy. Two treatment-naïve animals (RA1209 and RA1174) were also treated with 8 doses (25 ug/kg/dose) of CD3e-IT to assess the effects of CD3e-IT on various blood lineages, including T-cell, B-cell, Granulocytes, Monocytes, and NK Cells, in different organs. Peripheral blood, bone marrow, and lymph nodes were collected at both one month before and 3 days after the CD3e-IT. Other lymphoid and non-lymphoid organs were collected after necropsy.Results:CD3e-IT treatment in the control animals, RA1209 and RA1174, showed effective T-cell depletion in the peripheral blood and bone marrow. The levels of T-cell depletion in different lymph nodes and other test organs varied notably. After CD3e-IT treatment in animal 95E132, the T-cell fraction rebounded to its normal level in 2 months, with T-cell recovery was achieved by repopulation of a few dominant clones. Such clonal dominance was maintained in peripheral T-cells until necropsy (19 months post-immunotoxin). HSC clonal profiles of T-cells after CD3e-IT shared similar patterns with the clonal profiles in bone marrow and spleen. Interestingly, the clonal profiles of thymus and lymph node T-cells were similar to those of pre-treatment blood T-cells and granulocytes (Pearson's r value of 0.75-0.91). The TCRvb repertoire was severely skewed after CD3e-IT. The recovery of naïve T-cells was slow but measurable at 19 months post-IT. B-cell, monocytes, and NK cells showed significant clonal fluctuations over time, whereas granulocytes remained highly stable (r > 0.90).Conclusion:Our data suggest that peripheral expansion of a subset of T-cell clones is the first means by which T-cells repopulate following CD3e-IT treatment in aged primates. Thymic contribution occurs, but is slow and limited. T-cell repopulation in some lymphoid organs differs from that in blood and may indicate segregated homeostatic expansion. Those T-cell clones that repopulate and dominate are a very small subset of all T-cell clones, but it is unclear whether the fates of the dominant clones are predetermined. Despite the systemic and severe perturbation occurred in the T-cell pool, there was no notable sign of oligoclonal repopulation or skewed clonality in other blood lineages. DisclosuresDunbar:Novartis/GSK to institute: Research Funding. Chen:Calimmune Inc: Equity Ownership.

FVIII Proteins with a Modified Immunodominant T-Cell Epitope Exhibit Reduced Immunogenicity and Normal Procoagulant Activity

Factor VIII (FVIII) neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 (legacy FVIII numbering, sequence: SYFTNMFATWSPSKARLHLQ) has been shown to contain an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells isolated from HA subjects. The aim of this study was to identify amino acid substitutions to de-immunize this epitope while retaining pro-coagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The minimal DRB1*01:01-binding motif was mapped to FVIII2194-2205 by quantitative peptide-MHC Class II (MHCII) binding assays. Further binding assays with sequence-modified FVIII peptides established that the residues important for DRB1*01:01 affinity were F2196, M2199, A2201, and S2204, corresponding to MHCII anchor positions 1, 4, 6 and 9. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation and cytokine secretion of five clones specific for FVIII2194-2213. The substitution F2196A was then engineered into a recombinant (r)FVIII-C2 protein. T-cell clones that were strongly stimulated by wild-type rFVIII-C2 did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic synthetic peptide includes a naturally processed T-cell epitope. The measured and predicted effects of additional amino acid substitutions within this binding motif were further evaluated by peptide-MHCII binding assays, CD4+ T-cell proliferation assays, and MHCII-binding prediction algorithms. Amino acid homologs within this binding motif were evaluated with sequence homologies to FVIII and FV proteins from different species. On the basis of all these results, six rB-domain-deleted (BDD)-FVIII proteins with amino acid substitutions F2196A, F2196L, F2196K, M2199A, M2199W or M2199R, respectively, were designed and produced; all but the F2196A variant had specific activities comparable to that of rWT-BDD-FVIII. The F2196A variant also had the lowest expression level among the rBDD-FVIIII proteins produced. Peptide-MHCII binding assays confirmed that FVIII2194-2205 peptides with substitutions F2196A, F2196K, M2199A and M2199R did not bind to DRB1*01:01 or to an additional 9 HLA-DR proteins tested, with the exception of the M2199A peptide binding to DRB1*09:01. The predicted effects of these FVIII sequence modifications on binding to additional MHCII alleles included in reference sets representing ~99% of the human population HLA diversity were evaluated using ProPred and Immune Epitope Database algorithms. These programs correctly predicted that the substitutions would abrogate binding to DRB1*01:01 and also indicated that affinities for additional MHCII would be reduced by each substitution. The substitutions were predicted to increase peptide-MHCII affinities for a smaller number of MHCII alleles, indicating a possible risk of creating neo-epitopes restricted to these specific HLA alleles. Importantly, proliferation of patient-derived T-cell clones in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A proteins was dramatically reduced compared with responses to rWT-BDD-FVIII. We have previously demonstrated that a severe HA inhibitor subject had an oligoclonal T-cell response to only one T-cell epitope within the FVIII A2, C1 and C2 domains and that one epitope was FVIII2194-2213 (Ettinger et al., Blood 2106; 128(16):2043). This observation, together with the results presented here, further suggests that modification of only a small number of immunodominant epitopes may be sufficient to significantly de-immunize FVIII and decrease FVIII inhibitors in HA patients. The present results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients. DisclosuresEttinger:Bloodworks NW: Patents & Royalties: Inventor on FVIII patents. James:Bloodworks NW: Patents & Royalties: inventor on FVIII patent(s). Pratt:Bloodworks NW: Patents & Royalties: inventor on FVIII patents.

CD68+ CD163+ M2-like Macrophages and Myeloid-Derived Suppressor Cells in Primary Immune Thrombocytopenia

BackgroundClinical investigations for the pathogenesis of immune thrombocytopenia (ITP) have focused on the aberrations related to adaptive immunity including platelet autoantibodies, T-cell clonality, and T helper differentiation. Recently, impaired myeloid-derived suppressor cells (MDSCs) have been implicated to participate in the pathogenesis of ITP, which could also act as a prognostic marker for treatment responses. However, another myeloid-derived cell population signified as M2 macrophages, has not been investigated properly in ITP patients. M2 macrophages, predominantly characterized as CD68+ (pan macrophages) and CD163+ (M2 specific), could promote immune modulation and tissue remodeling. Interestingly, a bidirectional cross-talk has been revealed between M2 macrophages known as “tumor associated macrophages” and MDSCs via high level of IL-10 in tumor microenvironments, which could result in facilitating tumor progression. These immunemodulation properties of MDSCs and M2 macrophages are posited to be severe disadvantages in tumor-bearing patients, while they might be beneficial in autoimmunity. In the present study, we intended to determine the features of circulating M2-like macrophages, to examine its relationship with MDSCs, and to explore their prognostic values in ITP.MethodsPBMC were isolated from the freshly collected peripheral blood of 10 healthy controls and 28 primary ITP patients. The circulating M2-like macrophages were defined as CD68+CD163+ (intracellular staining), and the phenotypic features of the circulating MDSCs were CD11b+CD33+HLA-DR- (surface staining). Stained cells were tested by FACS Aria II flow cytometer.ResultsThere are 17 patients in CR, and 11 patients in NR group. The population of circulating MDSCs expanded in newly diagnosed patients with ITP (p < 0.001), especially among the CR group (p < 0.001), but no significant difference was found in circulating M2-like macrophages. Positive linear correlation was verified between percentages of circulating M2-like macrophages and MDSCs (r= 0.15, p= 0.045). The same correlation was also determined in the CR group (r= 0.30, p= 0.021) while not in the NR group (r= 0.11, p= 0.313). Compared with those before the treatment, the percentages of both M2-like macrophages (p = 0.039) and MDSCs (p = 0.036) increased significantly after the treatment among patients in CR group, however, decreased number of MDSCs was found in NR group patients (p = 0.035).ConclusionsThe present investigation indicated critical roles of both circulating M2-like macrophages and MDSCs in the pathogenesis of ITP. The positive correlation between them might be related to some bidirectional interaction or partially due to their similar background patterns during differentiation. Further studies are required to demonstrate the cytokine profile related to the expansion of both MDSCs and M2 macrophages population in ITP patients. DisclosuresNo relevant conflicts of interest to declare.

Germline Mutations in CTLA4 and Spectrum of Bone Marrow Features Overlapping with Aplastic Anemia and Large Granular Lymphocyte Lymphocytosis

Introduction: CTLA4 haploinsufficiency due to germline heterozygous mutations in the cytotoxic T-lymphocyte antigen-4 gene (CTLA4) results in severe systemic immune dysregulation; infiltrative lymphocytic lesions of lungs (e.g. lymphocytic pneumonitis), GI tract (e.g.lymphocytic colitis), and CNS; and a spectrum of autoimmunity, immunodeficiency and peripheral blood cytopenias. We describe the hematologic, bone marrow (BM), and flow cytometry (FC) features of CTLA4 haploinsufficiency of a cohort of 14 patients, which show overlapping morphologic features with acquired aplastic anemia (acAA), and large granular lymphocyte lymphocytosis (LGL) in a subset of patients. We compared the findings to those in a separate cohort of 20 age matched patients diagnosed with acAA. There were significant differences between acAA and CTLA4 deficiency based on analysis of BM lymphocyte subsets and marrow lymphocyte infiltration patterns.Materials and Methods: Peripheral blood (PB), BM and FC analysis of patients with germline mutations in CTLA4, and acAA was performed along with review of clinical, molecular and laboratory data. Statistical comparisons were made using Mann Whitney test, with p < 0.05 for significance.Results: Median age at initial evaluation for patients with CTLA4 haploinsufficiency was 23.5 years (range 11-71 years), with a male-to-female ratio of 11:3. 11 out of 14 patients had cytopenias. Of those with cytopenias 2/11 had pancytopenia, 4/11 had bicytopenia and 5/11 had unilineage cytopenia. Hypogammaglobulinemia was common (13/14). In some cases cytopenias were recognized as autoimmune in etiology with immune thrombocytopenic purpura and autoimmune hemolytic anemia, among the most common hematologic manifestations. Three patients met criteria for a diagnosis of moderate AA, 1 for severe AA, and 1 for LGL. Overall bone marrow biopsies were hypocellular for age (10/14) with a subset (29% [4/14]) showing severe trilineage hypoplasia with ≤20% cellularity. Atypical interstitial CD8-positive T-cell infiltrates with prominent non-paratrabecular CD4-positive T-cell aggregates were evident by immunohistochemistry in most cases (10/14), accompanied by a near absence of B-cells. T-cell clonality was assessed by T-cell receptor (TCR) gamma PCR and showed abnormal oligoclonal T-cell populations in 3/8, and one case with a clonal T-cell receptor gene rearrangement. B-cell IgH PCR reactions were polyclonal. In all cases, cytogenetic studies were either negative or showed only minor abnormalities (i.e. loss of Y chromosome). Given the morphologic overlap of a subset of CTLA4 cases with acAA, we compared bone marrow flow cytometry analysis of 8 CTLA4 cases with 20 acAA cases. CTLA4 patients showed >5 fold reduction in the median percentage of B-cells in comparison to acAA (p=0.0005). While acAA cases demonstrated relative preservation the B-cell compartment with intact B-cell maturation sequence; in contrast CTLA4 cases showed abnormal B-cell maturation patterns in half of cases characterized by few detectable early B-cell precursors, and progressive loss of intermediate and mature forms. T-cell populations in CTLA4 showed inverted CD4:CD8 ratios with relatively increased CD57+ T-cells, and atypical dim expression of CD20 on T-cells. Morphologically, when the markedly hypocellular CTLA4 cases were compared to acAA on core biopsies, little difference in marrow cellularity was seen; however, the CTLA4 cases showed multiple large atypical T-cell aggregates and near total absence of B-cells, which was not seen in acAA; acAA cases showed scattered background T-cells and B-cells.Conclusions: CTLA-4 deficiency presents with cytopenias, autoimmunity, and immunodeficiency with a subset of patients that have features overlapping with bone marrow failure, specifically AA and LGL. Herein we demonstrate a combination of abnormal BM findings, previously unreported, comprising hypocellularity, CD8-positive interstitial T-cell infiltrates with atypical CD4-positive T-cell aggregates, marked B-cell lymphopenia, increased LGLs and abnormal T-cell clonality patterns by PCR. These features may help identify patients harboring CTLA4 mutations for early detection and optimal therapeutic management. DisclosuresYoung:Novartis/GSK to institute: Research Funding. Townsley:Novartis/GSK to institute: Research Funding.

Highly Sensitive Viral RNA-Seq Is a Powerful Tool to Analyze the Viral Transcripts in HTLV-1-Infected Individuals

IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is a retrovirus which causes two major pathologies in infected individuals, Adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There are tens of thousands of different infected clones in asymptomatic HTLV-1 carriers (AC) and each clone can be identified by the viral integration site. Monoclonal integration of HTLV-1 provirus is observed in the host genomic DNA in ATL cells, suggesting that these originate from a single virus-infected clone. It is well known that Tax and HBZ, encoded in the sense and antisense strands of HTLV-1 provirus, respectively, have the ability to induce oncogenesis. The question is how a certain infected clone, among various different ones, becomes a malignant clone. One hypothesis is that a malignant clone may express viral gene transcripts, associated with oncogenesis, more abundantly than a non-malignant clone. Another hypothesis is that HTLV-1 integration may induce an abnormal transcript in the host cell, such as the aberrant expression of the host gene nearby the integration site or the generation of a chimeric transcript between the virus and the host. To analyze HTLV-1 transcript or explore the presence of virus-host chimeric transcripts efficiently, we aimed to establish a method with higher resolution than the conventional RNA-seq by using DNA probe targeting for the entire HTLV-1 sequence. And then, we applied the protocol to characterize the pattern of transcription from HTLV-1 provirus and analyzed virus-host chimeric transcripts in patients' samples.MethodsTo check the efficiency and possible bias of the highly sensitive viral RNA-seq analysis, we analyzed viral transcript of an HTLV-1-infected clone, TBX-4B. Subsequently, mRNA-seq was performed with PBMCs from 6 AC, 4 HAM/TSP, and 5 ATL patients. After cDNA libraries synthesis, HTLV-1 sequences were enriched by the probe-based capture method as we reported previously for DNA-seq (Miyazato et al, Sci Rep 2016). The enriched libraries were sequenced in an Illumina NextSeq device.ResultsWe analyzed HTLV-1 transcript of a virus-infected T-cell clone both before and after the enrichment by viral-specific DNA probes. The proportion of viral sequences within the total number of reads was significantly increased from 0.05% to 80.62% (1600-fold) by the enrichment method. We found that most of the viral regions were significantly and similarly enriched by the additional enrichment step, suggesting the DNA probe-based enrichment of HTLV-1 sequences is applicable to RNA-seq. Next, we applied the protocol to analyze PBMC samples from HTLV-1-infected individuals. The results showed that the level of anti-sense HTLV-1 transcription was significantly higher than that of the sense transcription, not only in ATL patients, which is consistent with previous reports by other groups, but also in AC and HAM/TSP patients. Average proportion of sense and antisense signals were 3.42% and 96.46%, respectively. Even after the enrichment step, tax expression, a sense viral transcript, was undetectable in most of the samples except for one AC and one ATL patient, while an anti-sense transcript HBZ was detectable in all infected individuals. There was no significant difference in the expression level of HBZ between different clinical states. We identified more abundant virus-host chimeric transcripts in ATL samples than in HAM/TSP patients or AC. In ATL cases, HTLV-1 generated chimeric transcripts with the following host genes, HSD17B11, COLEC12, AHSA2, EEF2, TAOK1, KIAA0100, and KIF1B . The presence of abundant chimeric transcripts was validated by RT-PCR.DiscussionWe have established a highly sensitive viral RNA-seq for HTLV-1 transcript analysis. That significantly increased the sensitivity of detection of proviral and chimeric transcripts between virus and the host genome. The result was not consistent with the hypothesis that a malignant clone may express viral gene transcripts more abundantly than a non-malignant clone. Thus, the presence of chimeric transcripts could play a role in cell proliferation and help us reveal the mechanism of clonal selection for HTLV-1-infected cells. We are now going to analyze virus-host chimeric transcripts detected by the high sensitive RNA-seq in more patients' samples to clarify their association with oncogenesis. [Display omitted] DisclosuresYamano:Daiichi Sankyo: Research Funding; Ono Pharma: Research Funding. Utsunomiya:Celgene: Honoraria; Novartis Pharma: Honoraria; Roche Diagnostics: Honoraria; Japan Blood Products Organization: Honoraria; Daiichi Sankyo: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria; Chugai Pharma: Honoraria; Bristol-Myers Squibb: Honoraria; Siemens: Honoraria; Nippon Shinyaku: Honoraria; HUYA Bioscience International: Honoraria; Pfizer: Honoraria.

Impact of Post-Transplant High-Dose Cyclophosphamide on Reconstitution in Bone Marrow Recipients

Introduction: Hematopoetic stem cell transplantation (HSCT) is the only curative therapy for many patients with hematologic malignancies. Occurrence of complications and mortality after allo HSCT is still high and it's strongly associated with immune reconstitution. Despite the wide-spread of Post-Transplant High-Dose Cyclophosphamide (PTCy) immune reconstitution and immunological safety of this method is still poorly understood.Aim: to evaluate immune reconstitution profile in patients who was underwent HSCT with a bone marrow as a stem cell source and with or without PTCy.Patients and methods: In National Research Center for Hematology 41patients after allogeneic HSCT were analyzed in 2 groups; patients with PTCy (n=17) and without PTCy (n=24). The total cohort had 12 males and 29 females, and had a median age of 33 years (range 19-61). All patients were diagnosed with a hematological malignancy. 14 patients were underwent myeloablative conditioning and 27 patients - non-myeloalative prior HSCT.The GVHD prophylaxis consisted of ATG-PTCy-CsA-MMF (n=10), ATG-PTCy (n=5), Mono-PTCy (n=1), ATG-CsA-MMF-MTX (n=20), CsA-MTX (n=2), ATG-CsA-MTX (n=2), CsA-MMF-MTX(n=1). The patients who only had received ATG were included on the analysis.Immune reconstitution profile was tested via serial flow cytometry analysis of peripheral blood on day +14, +30, +60 and +90 after allo-HSCT. Anti-CD3 FITC, anti-CD16PE, anti-CD56 PE, anti-CD45 Per-CP-CYTM5.5, anti-CD4 PE-CyTM7, anti-CD19 APC, anti-CD8 APC-Cy7; anti-CD62L FITC (BD Biosciences, USA); anti-CD14PE, anti-CD16PE, anti-HLA-DR APC (eBiosciences, USA) were used to define white blood cell subsets.Results: Reported detailed data is presented in Table 1. In bone marrow recipients the number of CD4+- cells was significantly lower on day +14 and +30, NK-cells on day +30 and CD8+-cells on day +60 in case of using PTCy versus immunosuppression without PTCy.The number of classical monocytes on day +30 and patrol monocytes on day +30 and +90 was significantly lower in case of using PTCy versus a standard protocol.Conclusion:Lymphocyte and monocyte reconstitution was impaired for the PTCy groups in the immediate post-HSCT period but quickly recovered. The mechanism of tolerance induction with PTCy on day+ 3, + 4 is not limited only to deletion of alloreactive T-cell clones, but also affects other leukocyte subpopulations (B cells, monocytes, granulocytes). The use of PTCy on day+ 3, + 4 is an immunologically safe method for prevention of GVHD.Table 1. Short-term reconstitution in BM recipients with and without PTCy [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Next-Generation Sequencing Based Monitoring of Circulating-Tumor DNA in Untreated Peripheral T-Cell Lymphoma

Background: Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with a wide range of clinical outcomes. Overall prognosis is worse for PTCL than B-cell lymphomas, but pts who achieve remission can be cured. After initial therapy, controversy exists regarding consolidation with autologous stem cell transplant, and clinical decisions are largely empirical. Circulating-tumor DNA (ctDNA) is emerging as a promising non-invasive biomarker that may aid clinical decision-making. Quantitative baseline levels of ctDNA and kinetics of ctDNA clearance during therapy may provide prognostic information. Further, novel methods that detect disease relapse prior to imaging may improve outcomes. We studied a high-throughput DNA sequencing method of detecting T-cell receptor (TCR) β and γ gene sequences in serum with a sensitivity of 1 lymphoma molecule per million leukocytes. Using this platform, we determined the predictive ability of monitoring ctDNA in pts undergoing frontline therapy for PTCL.Methods: All pts had PTCL undergoing frontline EPOCH-based chemotherapy between May 1993 through December 2015. Pre-treatment FFPE biopsy samples and/or baseline serum samples were sent for VDJ amplification and TCR sequencing calibration. High-throughput TCR sequencing of the CDR3s of TCRβ/TCRγ genes was employed to provide a comprehensive and quantitative analysis of distinct T-cell clones within each sample, the relative frequency of each clone, and the exact unique nucleotide sequences of each clone's CDR3 regions. Pts with a calibrating TCRβ/TCRγ sequence had all available serum samples sent for identification and quantification. Prospectively collected serum sample time points included baseline, start of each cycle, and each follow-up visit until disease progression. Clinic visits after therapy included paired contrast CT scans performed at 3, 4, 6, 12 and 12 mos. intervals for the first 5 years, respectively. All analyses for ctDNA were performed blinded to clinical outcomes.Results: Fifty PTCL pts had a pre-treatment FFPE or serum sample available for calibration. Nineteen of 23 (83%) pts with pre-treatment FFPE had a calibrating rearrangement; Fourteen of 27 (52%) pts with only pre-treatment serum available had a calibrating rearrangement; 1 pt who did not calibrate on FFPE had a dominant sequence identified in serum. Overall, tumor-specific clonotypes were identified in 34 (68%) of 50 pts tested. Ten of 18 (56%) pts with both FFPE and serum available had dominant clones in both compartments. Of the 44 calibrating pre-treatment samples, 1 calibrated for TCRβ only, 15 for TCRγ only, and 28 for both loci. Pt characteristics shown in Table 1. Of 34 study pts, 20 (59%) progressed and 14 (41%) were non-progressors. Quantitative baseline ctDNA levels were significantly higher in pts with stage IV disease compared to stages I-III (p=0.014) but did not correlate with LDH levels (p=0.53).We analyzed the predictive ability of ctDNA as a measure of minimal residual disease (MRD) within 1 month of completing therapy in 28 pts. We compared pts who became MRD- to those who remained MRD+ and identified a trend towards improved PFS in those who became MRD- (median, NR vs. 208 days, p=0.07) (Figure 1). In 20 pts who achieved remission, we studied the use of ctDNA monitoring as a surveillance tool. Of the 10 pts who progressed after remission, ctDNA was detected prior to clinical relapse in 6 cases with a median lead-time of 13.2 mos. (range, 2.7 to 88.5) and 4 with detectable ctDNA > 12 mos. prior to clinical relapse. Four pts progressed without detectable ctDNA prior to clinical relapse. Interestingly, of the 10 non-progressors followed in surveillance, 5 pts developed low-level ctDNA positivity without clinical progression. Results of interim ctDNA kinetics and TCRβ testing is ongoing and will be presented at the meeting.Conclusions: Next-generation based sequencing of the TCR can identify tumor-specific clonotypic sequences in the majority of pts from both FFPE and serum. As a marker of MRD, monitoring TCR sequences in serum may identify pts who do not benefit from treatment intensification, but further studies are needed. Surveillance monitoring of ctDNA in serum identifies TCR sequences months before clinical progression but not all pts with clonotypic sequences progressed clinically in our series. Further studies are needed to investigate surveillance monitoring methods and optimize clinical utility. [Display omitted] DisclosuresJacob:Adaptive Biotechnologies: Employment, Equity Ownership. Chan:Adaptive Biotechnologies: Employment. Vignali:Adaptive Biotechnologies: Employment.

The Orally Available Selective Axl Inhibitor BGB324 Induces Diversification of the Immune Repertoire and Specific Changes in Plasma Biomarker Profiles

Introduction: The RTK Axl represents a novel therapeutic target in AML promoting proliferation and therapy resistance. In addition, Axl suppresses immune responses by decreasing antigen presentation and inhibiting NK cells. BGB324 is a first-in-class, orally available, selective Axl kinase inhibitor which is being explored as a therapy for solid tumours and myeloid malignancies. Selective blockade of Axl by BGB324 in AML is currently being explored in the Phase 1/2 trial BGBC003 (NCT02488408). Until now, n=25 patients have been treated in a classical 3+3 dose escalation design and treatment was generally well-tolerated (n=22 with AML and n=3 with MDS). Steady-state levels of BGB324 were achieved between three and six days after initiation of treatment. One AML patient achieved a CRi of five months, a second achieved a PR and another achieved clearance of circulating blasts accompanied by peripheral blood count recovery > three months (ORR 10%). Four additional AML patients (25%) experienced disease stabilisation for more than four months. Two MDS patients experienced a PR.Here, we report the spectrum of plasma proteins modulated by BGB324 in patients with AML/MDS and their relationship to treatment outcome and compound exposure. Furthermore, we analysed the B- and T- cell repertoire in order to assess potential effects of BGB324 on the immune response in AML patients.Methods: We used the DiscoveryMap panel (Myriad RBM) to measure plasma protein biomarker levels in a selection of patients pre-dose and at C2D1 and performed a cross-comparison with samples from an ongoing NSCLC study with BGB324 (BGBC004, NCT02424617) to identify candidates specifically modulated in AML. The TCRß repertoire was quantified by Next Generation Sequencing of DNA isolated from peripheral blood MNCs using an Illumina MiSeq sequencer. TCRß gene containing the entire Vß, Dß and Jß segments were amplified with BIOMED2-TCRß-A and -B primer pools. The IGH repertoire was analysed using BIOMED2-FR1/-FR3 primer pools. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß and IGH data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa, which relies on MiXCR analysis tools.Results: Treatment with BGB324 significantly modulated Gas6, soluble Axl (sAxl), Angpt2, CXCL1 (GROalpha), SCGB1A1 (uteroglobin, club cell 10kDa protein) and PLAU (uPAR) levels in patient plasma. Of these, Gas6 was modulated after treatment with BGB324 in AML but not in lung cancer patients while CXCL1 and SCGB1A1 were only influenced in lung cancer patients. sAxl levels correlated with BGB324 exposure indicating that the shed Axl receptor represents a novel biomarker of Axl kinase inhibition.Furthermore, we studied the effects of BGB324 on the T- and B- cell receptor repertoire in n=5 available matched PB samples. We could demonstrate diversification of the TCR repertoire in PB by higher abundance of small T-cell clones at C2D1 with BGB324 compared to pre-treatment levels in three out of five analysed patients.The analyses of the IGH repertoire in the PB indicates higher diversity at C2D1 in two out of five available patients.Thus, treatment with BGB324 can increase the diversity of the IGH and TCRb repertoire in the PB of AML patients.Conclusion: BGB324 is well tolerated in AML patients, exhibits anti-leukemic activity and induces exposure-related modulation of biomarker candidates. Furthermore, BGB324 can induce a diversification of the TCR repertoire in AML patients and might hold potential as an immune-activating drug. DisclosuresLoges:BerGenBio ASA: Research Funding. Ben-Batalla:BerGenBio ASA: Research Funding. Micklem:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Brown:BerGenBIo ASA: Employment. Lorens:BerGenBIo ASA: Employment, Equity Ownership, Patents & Royalties, Research Funding. Yule:BerGenBIo ASA: Employment. Fiedler:Amgen: Patents & Royalties; Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen, Pfizer: Research Funding; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance. Cortes:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding.