Purpose: Thyroid cancer recurrence following curative thyroidectomy is associated with increased morbidity and mortality, but current surveillance strategies are inadequate for early detection. Prior studies indicate that tissue glycosylation is altered in thyroid cancer, but the utility of serum glycosylation in thyroid cancer surveillance remains unexplored. We therefore assessed the potential utility of altered serum glycomic profile as a tumor-specific target for disease surveillance in recurrent thyroid cancer. Experimental design: We employed banked serum samples from patients with recurrent thyroid cancer post thyroidectomy and healthy controls. N-glycans were enzymatically released from serum glycoproteins, labeled via permethylation, and analyzed by MALDI-TOF mass spectrometry. Global level and specific subtypes of glycan structures were compared between patients and controls. Results: We evaluated 28 independent samples from 13 patients with cancer recurrence and 15 healthy controls. Global features of glycosylation, including N-glycan class and terminal glycan modifications were similar between groups, but three of 35 individual glycans showed significant differences. The three glycans were biosynthetically related biantennary core fucosylated N-glycans that only varied by the degree of galactosylation (G0F, G1F, and G2F; G: galactose, F: fucose). The ratio of G0F:G1F that captures reduced galactosylation was observed in patients samples but not in healthy controls (p = 0.004) and predicted thyroid cancer recurrence (AUC = 0.82, CI 95% = 0.64–0.99). Conclusions: Altered N-glycomic profile was associated with thyroid cancer recurrence. This serum-based biomarker would be useful as an effective surveillance tool to improve the care and prognosis of thyroid cancer after prospective validation.
Background
Reliable predictive biomarkers are required to address the challenge of disease recurrence following thyroid cancer surgery. We assessed the association of cellular- and serum-based immunologic mediators with thyroid cancer recurrence.
Methods
Leukocyte subset counts and immune regulatory cytokines were determined in peripheral blood samples using multiparameter flow cytometry and 51-panel multiplex ELISA (Luminex) assays, respectively. Functional activity of circulating B-, T- and NK lymphocytes was assessed ex vivo. Differences in mean biomarker levels between defined subject groups and correlations between biomarkers and cancer recurrence were assessed by t-test or Wilcoxon test and by univariate and multivariate analyses with Cox model. Optimal cut-off values of significantly correlated biomarkers that best predict recurrence post surgery were established by receiver operating characteristics and validated by the optimal cut-point determination algorithm.
Results
We enrolled 35 patients (median age - 49.4 years; 24 females; 15 patients with disease recurrence) and 21 controls. Patients without recurrence had higher levels of soluble FAS ligand (sFASL), TGF-b, T-regs and PD-1/PD-L1 expressing leukocytes. sFASL (HR:0.60; 95%CI:0.38–0.95; p=0.031) and IFN-α (HR:1.55; 95%CI:1.03–2.34; p=0.038) showed significant association with cancer recurrence. There was a significant difference in PFS between patient groups stratified by sFASL optimal cut-point of 15 pg/ml (Logrank p=0.0009).
Conclusions
sFASL and IFN-α showed significant correlation with thyroid cancer recurrenceand may be useful for risk-adapted surveillance strategies for thyroid cancer.
Background Immune checkpoint blockade (ICB) targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein 4 has achieved modest clinical activity as salvage therapy in relapsed small cell lung cancer (SCLC). We conducted this signal-finding study to assess the efficacy of ICB with or without radiation in relapsed SCLC. Methods Patients with relapsed SCLC and ≤2 previous lines of therapy were randomized to (1) arm A: durvalumab (D) 1500 mg/tremelimumab (T) 75 mg (intravenously every 4 weeks without stereotactic body radiation therapy (SBRT)) or (2) arm B: immune-sensitizing SBRT to one selected tumor site (9 Gy × 3 fractions) followed by D/T. Treatment continued until progression or a maximum of 12 months. The co-primary endpoints of the study were overall response rate (ORR) and progression-free survival (PFS). We evaluated circulating lymphocyte repertoire in serial peripheral blood samples and tumor infiltrating lymphocytes (TILs) from on-treatment biopsies as pharmacodynamic markers. Results Eighteen patients were randomized to arms A and B (n=9 each): median age 70 years; 41.2% women. The median PFS and ORR were 2.1 months and 0% in arm A and 3.3 months and 28.6% in arm B. The median overall survival (OS) was 2.8 months in arm A and 5.7 months in arm B (p=0.3772). Pooled efficacy of D/T±SBRT in 15 Response evaluation criteria in solid tumors (RECIST) evaluable patients across both arms showed the best ORR in terms of partial response in 13.3%, stable disease in 26.6% and progressive disease in 60.0%; the overall median PFS and OS were 2.76 and 3.9 months. The most common adverse events were grade 1 fatigue (66%) and grade 1 elevated amylase (56%) in arm A, and grade 1 fatigue (56%) and pain (44%) in arm B. There was a significant increase in activated CD8(+)ICOS+ T cells (p=0.048) and a reduction in naïve T cells (p=0.0454) in peripheral blood following treatment, along with a significant amount of activated CD8+ICOS+ T cells in TILs from responders. Conclusions The D/T combination with and without SBRT was safe but did not show sufficient efficacy signal in relapsed SCLC. Changes in peripheral blood lymphocyte and TILs were consistent with an immunologic response.
Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ~10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.
Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1-mediated cellular immunity. We previously reported that VIP-knockout (VIP-KO) mice have enhanced cellular immune responses and increased survival following murine cytomegalovirus (mCMV) infection in C57BL/6 mice. In this study, we tested whether treatment with a VIP receptor antagonistic peptide protects C57BL/6 and BALB/c mice from mCMV-infection. One week of daily subcutaneous injections of VIPhyb was non-toxic and did not alter frequencies of immune cell subsets in non-infected mice. VIPhyb administration to mCMV-infected C57BL/6 and BALB/c mice markedly enhanced survival, viral clearance, and reduced liver and lung pathology compared with saline-treated controls. The numbers of effector/memory CD8+ T-cells and mature NK cells were increased in VIPhyb-treated mice compared with PBS-treated groups. Pharmacological blockade of VIP-receptor binding or genetic blockade of VIP-signaling prevented the up-regulation of PD-L1 and PD-1 expression on DC and activated CD8+ T-cells, respectively, in mCMV-infected mice, and enhanced CD80, CD86, and MHC-II expression on conventional and plasmacytoid DC. VIPhyb-treatment increased type-I IFN synthesis, numbers of IFN-γ- and TNF-α-expressing NK cells and T-cells, and the numbers of mCMV-M45 epitope-peptide-MHC-I tetramer CD8+ T-cells following mCMV infection. VIP-treatment lowered the percentage of Treg cells in spleens compared with PBS-treated WT mice following mCMV infection, while significantly decreasing levels of serum VEGF induced by mCMV-infection. The mice in all treated groups exhibited similar levels of anti-mCMV antibody titers. Short-term administration of a VIP-receptor antagonist represents a novel approach to enhance innate and adaptive cellular immunity in a murine model of CMV infection.
Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Post-transplant immunosuppressive drugs incompletely control GvHD and increase susceptibility to opportunistic infections. In this study we used flagellin, a TLR5 agonist protein (~50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GvHD in allogeneic HSCT recipient. Based upon the radio-protective effects of flagellin, we hypothesized that flagellin could ameliorate GvHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 hrs. before irradiation and 24 hrs. after HSCT) reduced GvHD and led to better survival in both H-2b → CB6F1 and H-2k → B6 allogeneic HSCT models while preserving over 99% donor T cells chimerism. Flagellin treatment preserved long-term post-transplant immune reconstitution characterized by more donor thymic-derived CD4+CD25+foxp3+ regulatory T cells (Tregs) and significantly enhanced anti-viral immunity following murine cytomegalovirus (mCMV) infection model. The proliferation index and activation status of donor spleen-derived T cells, and serum concentration of pro-inflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 days post-transplant compared with the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host non-hematopoietic cells were required to reduce GvHD. Thus, the peri-transplant administration of flagellin is a novel therapeutic approach control GvHD while preserving post-transplant donor immunity.
Vasoactive intestinal peptide (VIP) induces regulatory dendritic cells (DC) in vitro that inhibit cellular immune responses. We tested the role of physiological levels of VIP on immune responses to murine cytomegalovirus (mCMV) using VIP-knockout (VIP-KO) mice and radiation chimeras engrafted with syngenic VIP-KO hematopoietic cells. VIP-KO mice and had less weight loss and better survival following mCMV infection compared with wild-type littermates (WT). MCMV-infected VIP-KO mice had lower viral loads, faster clearance of virus, with increased numbers of IFN-γ+ NK and NKT cells, and enhanced cytolytic activity of NK cells. Adaptive anti-viral cellular immunity was increased in mCMV-infected VIP-KO mice compared with WT mice, with more Th1/Tc1 polarized T-cells, fewer IL-10+ T-cells, and more mCMV-M45 epitope peptide-MHC class I-tetramer+ CD8+ T-cells (tetramer+ CD8 T-cells). MCMV-immune VIP-KO mice had enhanced ability to clear mCMV-peptide pulsed target cells in vivo. Enhanced anti-viral immunity was also seen in WT transplant recipients engrafted with VIP-KO hematopoietic cells, indicating that VIP synthesized by neuronal cells did not suppress immune responses. Following mCMV infection there was a marked up-regulation of MHC class II (MHC-II) and CD80 co-stimulatory molecule expression on DC from VIP-KO mice compared with DC from WT mice, while PD-1 and PD-L1 expression were up-regulated in activated CD8+ T-cells and DC, respectively, in WT mice but not in VIP-KO mice. Since the absence of VIP in immune cells increased innate and adaptive anti-viral immunity by altering co-stimulatory and co-inhibitory pathways, selective targeting of VIP-signaling represents an attractive therapeutic target to enhance anti-viral immunity.
Background: Purpose: The combination of a mammalian target of rapamycin inhibitor and lenalidomide showed enhanced preclinical cytotoxicity. We conducted a phase 1 study in advanced solid tumour patients to assess safety, efficacy and pharmacodynamic (PD) outcomes. Methods: We employed a 3+3 dose escalation design to establish the safety and recommended phase 2 doses (RP2D) of daily everolimus and lenalidomide in patients with advanced solid tumours. The starting doses were 5 and 10 mg, respectively, with planned escalation to maximum single-agent doses of 10 and 25 mg in the absence of dose-limiting toxicity. PD endpoints of lymphocyte subsets and immune cytokines were assessed in peripheral blood using multiparameter flow cytometry and LUMINEX assay. Efficacy was evaluated by cross-sectional imaging after every two cycles of treatment. Results: The study enrolled 44 patients, median age of 58 years and 28 males (63.6%). The RP2D was established as 10 and 25 mg daily continuously for everolimus and lenalidomide. Common (>5%) grade ≥3 adverse events included rash (19%), neutropenia (19%), hypokalaemia (11%) and fatigue (9%). Best efficacy outcomes in 36 evaluable patients were partial response in 5 (13.8%), stable disease in 24 (55.8%) and progressive disease in 7 (19.4%) patients. PD assessment revealed significant association of cytokine levels (interleukin-2 (IL2), IL21 and IL17), baseline activated and total CD8+ lymphocytes and change in B cell lymphocytes and activated NK cells with clinical benefit. Conclusions: The study demonstrated the safety of everolimus and lenalidomide with promising efficacy signal in thyroid and adenoid cystic cancers. Clinical Trial Registration: NCT01218555
The expression of checkpoint blockade molecules PD-1, PD-L1, CTLA-4, and foxp3+CD25+CD4+ T cells (Tregs) regulate donor T cell activation and graft-vs-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT). Detailed kinetics of PD-1-, CTLA-4-, and PD-L1 expression on donor and host cells in GvHD target organs have not been well studied. Using an established GvHD model of allo-HSCT (B6 → CB6F1), we noted transient increases of PD-1- and CTLA-4-expressing donor CD4+ and CD8+ T cells on day 10 post transplant in spleens of allo-HSCT recipients compared with syngeneic HSCT (syn-HSCT) recipients. In contrast, expression of PD-1- and CTLA-4 on donor T cells was persistently increased in bone marrow (BM) of allo-HSCT recipients compared with syn-HSCT recipients. Similar differential patterns of donor T cell immune response were observed in a minor histocompatibility (miHA) mismatched transplant model of GvHD. Despite higher PD-1 and CTLA-4 expression in BM, numbers of foxp3+ T cells and Tregs were much lower in allo-HSCT recipients compared with syn-HSCT recipients. PD-L1-expressing host cells were markedly decreased concomitant with elimination of residual host hematopoietic elements in spleens of allo-HSCT recipients. Allo-HSCT recipients lacking PD-L1 rapidly developed increased serum inflammatory cytokines and lethal acute GvHD compared with wild-type (WT) B6 allo-HSCT recipients. These data suggest that increased expression of checkpoint blockade molecules PD-1 and CTLA-4 on donor T cells is not sufficient to prevent GvHD, and that cooperation between checkpoint blockade signaling by host cells and donor Tregs is necessary to limit GvHD in allo-HSCT recipients.
The competence of cellular immunity depends on a diverse T-cell receptor (TCR) repertoire arising from thymic output. Normal thymopoiesis arises from marrow-derived CD3(-)CD4(-)CD8(-) triple-negative T-cell progenitors (TN), which develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive, DP) transiently, leading to de novo T-cell production. Interleukin-7 (IL7) is a singularly important common γ-chain IL involved in normal thymic development. Our previous work has demonstrated that γc cytokines fused with granulocyte-macrophage colony stimulating factor (GMCSF) at the N-terminus acquire unheralded biological properties. Therefore, to enhance thymopoiesis, we developed a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 leads to cortical thymic hyperplasia including the specific expansion of CD44(int)CD25(-) double-negative 1 (DN1) thymic progenitors. During murine cytomegalovirus (mCMV) infection of aged animals, GIFT7-mediated neo-thymopoiesis led to increased absolute numbers of viral-specific CD8(+) T cell. Our work demonstrated that thymic precursors can be therapeutically repopulated and its reconstitution leads to meaningful central and peripheral T-cell neogenesis, correcting immune dysfunction arising from age-associated thymic atrophy.