Clonality of T cell repertoire in the tumor (TME) and peripheral blood of regionally advanced melanoma patients (pts) treated with neoadjuvant ipilimumab (ipi) and high dose interferon-α (HDI).

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 9585-9585
Author(s):  
Priyanka Vallabhaneni ◽  
Tala Achkar ◽  
Marissa Vignali ◽  
Sharon Benzeno ◽  
Julie Rytlewski ◽  
...  
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Pathologic Complete Response ◽  
Advanced Melanoma ◽  
Cell Fraction ◽  
High Dose ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Primary Tumors ◽  
Significant Difference

9585 Background: Pts with regionally advanced melanoma were treated with neoadjuvant ipi and HDI in a reported study ( Tarhini. J Clin Oncol suppl, 2016; abstr 9585). Pathologic complete response (pCR) was found in 32% of pts. Clonality of T cell repertoire was investigated in TME and peripheral blood mononuclear cells (PBMC). Methods: Pts were randomized to neoadjuvant ipi 3 or 10 mg/kg combined with HDI. Tumor biopsies were evaluable for testing at pretreatment (N = 20 pts) and definitive surgery (week 6-8; N = 25). When available, primary (N = 24) and relapse tumors (N = 6) were tested. PBMC: pretreatment (N = 29), 6 weeks (wk) (N = 24), then 3 (N = 23), 6 (N = 21), 12 (N = 14) months. T cell receptor beta chain (TCRB) repertoire was immunosequenced in PBMC and TME to determine repertoire clonality and T cell fraction in blood and TME (TIL; fraction of all nucleated cells identified as T cells). Results: PBMC T cell fraction when measured early on-treatment (6 wks) was significantly higher in pts who had pCR or microscopic residual disease vs. gross disease at the 6-8 wks surgery (p = 0.047). PBMC clonality was significantly lower at 12 wks (p = 0.025) for pts who continued to be relapse free (NED) long term vs. those who eventually relapsed. In TME, except for trends no significant difference in clonality was seen, but in pts with pCR TIL fraction was significantly higher when measured in primary tumors (p = 0.033). The number of tumor-associated clones that were expanded in blood post-treatment was strongly correlated with both TIL fraction (Rho 0.7299, p = 0.0003) and TIL clone diversity (Rho 0.882, p = 2.7-7). Conclusions: Higher T cell fraction and lower clonality in PBMC when measured early on-treatment, and higher TIL fraction in primary tumor constituted promising biomarkers of response. Pts with higher TIL fractions were more likely to have tumor-associated clones detectable in blood, suggesting these may be useful for tracking the immune response. These findings warrant validation in an independent cohort and exploration with other immunotherapeutics. Clinical trial information: NCT01608594.

Clonality of T-cell repertoire in the tumor microenvironment (TME) and peripheral blood of metastatic melanoma patients treated with CTLA4 blockade and interferon-α (IFN).

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 9-9
Author(s):  
Ahmad A. Tarhini ◽  
Aya Agha ◽  
Zahra Rahman ◽  
Sharon Benzeno ◽  
Erik Yusko ◽  
...  
Keyword(s):  
T Cell ◽  
Metastatic Melanoma ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Interferon Α ◽  
High Dose ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clonality ◽  
Cell Clonality

9 Background: Patients with metastatic melanoma were treated with tremelimumab and IFN in a previously reported study (Tarhini. J Clin Oncol. 2012). Clonality of T-cell repertoire was analyzed in terms of clinical response both in TME and in peripheral blood. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks. High dose IFN (HDI) was administered concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). T-cell receptor beta chain (TCRB) repertoire was immunosequenced in peripheral blood mononuclear cells (PBMC) (N=33 patients) and tumor (N=18) utilizing Adaptive Biotechnologies immunoSEQ platform to determine repertoire clonality and T-cell fractions at pre-treatment (tumor, PBMC), one month (PBMC), and 3 months (PBMC). The clonality metric quantitates, the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. Values range from 0 to 1; values approaching 1 indicate a nearly monoclonal population. Results: In pretreatment TME, T-cell clonality was significantly (p = 0.0008) different and greater in patients who achieved disease control (CR, PR, SD) versus those with PD. Further, there was a significant (p = 0.044) difference between the increased TCR fraction in TME in responders (CR, PR) and non-responders (SD, PD). There was a trend towards association between pretreatment TME T-cell clonality and overall survival (OS) (p = 0.24) and progression free survival (PFS) (p = 0.18) not reaching significance. Within the circulation (PBMC), no significant associations were found by examining the pretreatment samples. However, early on-treatment (day 29) there was significant association and decrease in T-cell clonality and OS (p = 0.005) and PFS (p = 0.003). Conclusions: T-cell clonality in the TME pretreatment is a promising biomarker of immunotherapeutic benefit in our study. While baseline PBMC clonality was not associated with clinical benefit, early on-treatment (day 29) was significantly associated. These findings require validation in an independent cohort and exploration in relation to other immunotherapeutics. Clinical trial information: NCT00610857.

Evolution of the Donor T Cell Repertoire In Allogeneic Stem Cell Transplant Recipients In the Second Decade After Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 831-831
Author(s):  
Robert Q. Le ◽  
J. Joseph Melenhorst ◽  
Brenna Hill ◽  
Sarfraz Memon ◽  
Minoo Battiwalla ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Significant Difference

Abstract Abstract 831 After allogeneic stem cell transplantation (SCT), donor T lymphocyte immune function is slowly re-established in the recipient through reconstruction of the donor's post-thymic T cell repertoire and from T cell neogenesis in the thymus. Although long-term survivors from SCT appear healthy, their immune repertoire and differences from that of their donors have not been characterized. We studied 38 healthy patients surviving more than 10 years from a myeloablative SCT for hematological malignancy (median follow-up 12 years, range 10–16 years). T cell and natural killer (NK) cell repertoires in these patients were compared with cells from their stem cell donors cryopreserved at time of transplant and from the same donors at 10 year after SCT. The median age of both recipients and their sibling donors at time of transplant was identical (36 years). Patients received cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Only one patient was on continued immunosuppressive treatment at the time of study. Compared with the donor pre-transplant counts there was no significant difference in the absolute lymphocyte, neutrophil, monocyte, CD4+ and CD8+ T cell, NK cell, and B cell subset counts. However, compared to their donors, recipients had a) significantly fewer naïve CD4+ and CD8+ T cells; b) lower T cell receptor excision circles levels; c) fewer CD4+ central memory T cells; d) more effector CD8+ T cells; e) and more FOXP3+ regulatory T cells. These data suggest that the patient had a persistent deficiency on T cell neogenesis. Molecular examination of the T cell receptor Vbeta (TCRBV) repertoire by spectratype analysis showed that there was no significant difference in total complexity score, defined as the sum of the number of discrete peaks for each Vbeta subfamily, between the patients and their donors. TCRBV subfamily spectratyping profiles of patients and donors, however, had diverged, with both gains and losses of peaks identifiable in both patient and donor. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a T cell repertoire that reflects expansion of the donor-derived post thymic T cell compartment, with a limited contribution by new T cell generation and persistently increased Tregs. It therefore appears that a diverse TCRBV repertoire predominantly derived from the memory T cell pool is compatible with good health. Disclosures: No relevant conflicts of interest to declare.

High-Throughput Sequencing Of T Cell Receptors Detects Signatures Of T Cell Repertoire That Predict MRD Status In Patients With Mantle Cell Lymphoma Undergoing Immunotransplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4251-4251
Author(s):  
Malek Faham ◽  
Joshua Brody ◽  
Holbrook E Kohrt ◽  
Debra K Czerwinski ◽  
Ronald Levy
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Vaccine ◽  
Cell Transplant ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Blood Samples ◽  
Post Transplant ◽  
Before And After

Background A clinical trial is ongoing at Stanford for MCL patients in first remission that interdigitates an autologous CpG-stimulated tumor cell vaccine with autologous peripheral blood stem cell transplant (PSCT) (NCT00490529). In this trial, blood samples collected before and after vaccination and serially post-transplant are assayed for minimal residual disease (MRD) and for T cell repertoire using the LymphoSIGHT™ sequencing method (Faham et al., Blood 2012). We identified a set of T cell clones that appear to be responding to the vaccine, and therefore we investigated whether the number of these clonotypes was correlated with MRD status. Methods Using universal primer sets, we amplified rearranged IgH variable (V), diversity, and joining (J) gene segments from genomic DNA. Amplified products were sequenced to obtain >1 million reads. The B cell tumor-specific sequence was identified for each patient based on its high frequency in the original tumor biopsy. The presence of the tumor cells was then monitored in serial blood samples with a sensitivity of 1 cell per million leukocytes. The same blood samples were used for amplification, sequencing and analysis of the entire TCRβ repertoire. To facilitate identification of tumor vaccine-induced TCRβ clonotypes, we sequenced the TCRβ repertoire immediately before and after the administration of both the priming vaccination and a booster vaccination. We developed a metric called the vaccine response score (V score). This metric is calculated for each clonotype and reflects the increase in frequency after the initial vaccination AND after the boost. The formula for calculating V score is: V = F1 x F2 x square root [1/ (|F1 – F2| + 1)], where F1 and F2 represent the fold-change of the priming and boost vaccinations, respectively. Clonotypes with a V score >10 were deemed to be vaccination-induced by virtue of these frequency changes. Results In a series of 12 vaccinated patients, the number of clonotypes with V score ≥ 10 ranged between 0 and 262, with a median of 57. We utilized an antigen-specific analysis to validate that clones with high V scores (≥ 10) were in fact tumor-specific. For this analysis, we incubated peripheral blood mononuclear cells (PBMCs) with the tumor and then sequenced the TCRβ repertoire from cells obtained after culture. Clones that were enriched after culture compared to pre-stimulation PBMCs were deemed to be antigen-specific. These clones that are antigen-specific are highly likely to have a high V score compared to a random frequency-matched set of clones (P two tailed = 1.8 x 10-10), providing further evidence that clones with a high V score are tumor-specific. We then analyzed the relationship between V score and clinical outcome. Patients could be stratified into two groups with “high” (> 25) or “low” (<25) numbers of vaccine-responsive clonotypes. Patients in the high V score group, who had larger numbers of putative tumor-specific T cells, were more likely to have sustained molecular remission during the first-year post-transplant compared with patients in the low V score group (P = 0.018) (Figure 1). Conclusions T cell repertoire analysis identified clonotypes responding to the vaccination, and the presence of these vaccine-specific clonotypes correlates with MRD positivity at the important landmark of one year post-PSCT. Further analysis of additional patients enrolled on the MCL trial is ongoing. This data underscores the prognostic relevance of the sequencing-based V score metric and provides a novel approach for assessment of cancer immunotherapy responses. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Lymphoid Reconstitution After Autologous PBSC Transplantation With FACS-Sorted CD34+ Hematopoietic Progenitors

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2588-2600 ◽  
Author(s):  
Catherine Bomberger ◽  
Meeta Singh-Jairam ◽  
Glenn Rodey ◽  
Anastasia Guerriero ◽  
Andrew M. Yeager ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Peripheral Blood ◽  
Pcr Amplification ◽  
Cell Receptor ◽  
High Dose ◽  
Normal Numbers ◽  
Hematopoietic Progenitors ◽  
Cell Repertoire

Abstract T-cell and B-cell reconstitution was studied in nine patients who received fluorescence activated cell sorter (FACS)-sorted autologous CD34+ hematopoietic progenitor cells (HPC). The mean numbers of T cells (CD3+), B cells (CD19+) and CD34+ HPC administered to each patient were .004, .002, and 1.8 × 106 cells/kg, respectively. After high-dose myeloablative chemotherapy (busulfan, cyclophosphamide, etoposide) CD34+ HPC were infused and lymphoid reconstitution was monitored using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VDJ T-cell receptor (TcR) sequences. Restoration of normal numbers of peripheral blood T cells and B cells among recipients of FACS-sorted CD34+ HPC was delayed compared to recipients of non-T-cell–depleted PBSC autografts. In both patient groups, the circulating T cells were primarily CD4−, CD8+, αβ TcR+, and CD45RO+, CD45RA− during the first 2 months after transplant. Subsequent increases in the frequency of CD45RA+ CD45RO− T cells occurred at 2 to 3 months after transplant, suggesting maturation of CD34+hematopoietic progenitors to “naive” T cells. Analysis of the TcR repertoire after hematopoietic reconstitution demonstrated decreased diversity of Vβ TcR expression associated with global decreases in the absolute number of total peripheral blood T cells and most Vβ TcR+ subsets. Three of nine recipients of FACS-sorted CD34+ HPC demonstrated significant increases in the percentage of γδ+ peripheral T cells and CD5+ B cells at 3 to 9 weeks after transplantation, and all patients had transient oligoclonal expansions of T cells expressing specific Vβ TcR. Transplantation with highly purified CD34+ HPC results in reduced diversity of the peripheral T-cell repertoire during the early post-transplant period compared with patients receiving unmanipulated or MoAb-depleted transplants.

Clonal expansion of tumor infiltrating lymphocytes (TILs) in the peripheral blood of metastatic melanoma patients is significantly associated with response to CTLA4 blockade-based immunotherapy.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 2541-2541
Author(s):  
Arjun Khunger ◽  
Julie Rytlewski ◽  
Erik C. Yusko ◽  
Ahmad A. Tarhini
Keyword(s):  
T Cell ◽  
Clinical Response ◽  
Clin Oncol ◽  
Peripheral Blood ◽  
Clonal Expansion ◽  
Post Treatment ◽  
Response To Therapy ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Morisita Index

2541 Background: Patients with metastatic melanoma were treated on a clinical trial with tremelimumab and High Dose Interferon-Alfa (HDI) (Tarhini. J Clin Oncol. 2012). We previously reported that patients who achieved disease control and clinical response had significantly greater T-cell clonality (p = 0.0008) and T-cell fraction (p = 0.044) respectively in their pretreatment tumor biopsy samples (Tarhini. J Clin Oncol. 2017). In this study, we further characterize T-cell repertoire clonality and clonal expansion in the peripheral blood at different time points to evaluate the association between repertoire features and clinical response. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks and HDI was given concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 33) were obtained at baseline, day 29, and day 85 (following tremelimumab-HDI treatment); tumor samples at baseline were also obtained (N = 18). The T-cell receptor beta chain (TCRB) repertoire of PBMCs and tumor samples was immunosequenced using the immunoSEQ assay (Adaptive Biotechnologies), and repertoire clonality was assessed at baseline, day 29, and day 85. Differential abundance analysis was used to detect and quantify peripheral clonal expansion pre- versus post-treatment and identify the subset of peripheral clones also detected in the tumor repertoire. The Morisita Index of repertoire similarity was also calculated to compare global repertoire changes between pre- and post-treatment PBMC samples. Results: T-cell repertoire turnover, as measured by the Morisita Index, showed a trend towards responders (CR/PR) having greater turnover (lower Morisita Index) post-treatment than non-responders (SD/PD). Similarly, the total number of clones expanding in the peripheral repertoire varied over time within an individual (p = 0.034) but was not significantly affected by response to therapy (p = 0.275) or by on-treatment time point (p = 0.768). When the analysis was restricted to peripherally expanded clones that were also found in the tumor repertoire, responders had significantly more TILs expanded in the periphery at day 29 than non-responders (p = 0.036). Conclusions: Our analysis of the peripheral T-cell repertoire following treatment showed that detection of TILs in early peripheral clonal expansion correlates with response to therapy.

Restricted T-cell repertoire diversity in peripheral blood and tissue infiltrating lymphocytes in omenn's syndrome

1998 ◽  
Vol 16 ◽  
pp. S171
Author(s):  
Ph. Bahadoran ◽  
F. Le Deist ◽  
F. Rieux-Laucat ◽  
N. de Saint-Sauveur ◽  
N. Brousse ◽  
...  
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Repertoire Diversity ◽  
Infiltrating Lymphocytes

scholarly journals Characterization of the T-cell Repertoire after Autologous HSCT in Patients with Ankylosing Spondylitis

Acta Naturae ◽  
2018 ◽  
Vol 10 (2) ◽  
pp. 48-57
Author(s):  
E. A. Komech ◽  
I. V. Zvyagin ◽  
M. V. Pogorelyy ◽  
I. Z. Mamedov ◽  
D. A. Fedorenko ◽  
...  
Keyword(s):  
Ankylosing Spondylitis ◽  
T Cell ◽  
Peripheral Blood ◽  
Structural Changes ◽  
Clonal Diversity ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
The Impact ◽  
Autologous Hsct

Autologous hematopoietic stem cell transplantation (HSCT), a safer type of HSCT than allogeneic HSCT, is a promising therapy for patients with severe autoimmune diseases (ADs). Despite the long history of medical practice, structural changes in the adaptive immune system as a result of autologous HSCT in patients with various types of ADs remain poorly understood. In this study, we used high-throughput sequencing to investigate the structural changes in the peripheral blood T-cell repertoire in adult patients with ankylosing spondylitis (AS) during two years after autologous HSCT. The implementation of unique molecular identifiers allowed us to substantially reduce the impact of the biases occurring during the preparation of libraries, to carry out a comparative analysis of the various properties of the T-cell repertoire between different time points, and to track the dynamics of both distinct T-cell clonotypes and T-cell subpopulations. In the first year of the reconstitution, clonal diversity of the T-cell repertoire remained lower than the initial one in both patients. During the second year after HSCT, clonal diversity continued to increase and reached a normal value in one of the patients. The increase in the diversity was associated with the emergence of a large number of low-frequency clonotypes, which were not identified before HSCT. Efficiency of clonotypes detection after HSCT was dependent on their abundance in the initial repertoire. Almost all of the 100 most abundant clonotypes observed before HSCT were detected 2 years after transplantation and remained highly abundant irrespective of their CD4+ or CD8+ phenotype. A total of up to 25% of peripheral blood T cells 2 years after HSCT were represented by clonotypes from the initial repertoire.

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