Single-cell sequencing of tumour infiltrating T cells efficiently identifies tumour-specific T cell receptors based on the T cell activation score.

Adoptively transferred T cell receptor-engineered T cells are a promising cancer treatment strategy, and the identification of tumour-specific TCRs is essential. Previous studies reported that tumour-reactive T cells and TCRs could be isolated based on the expression of activation markers. However, since T cells with different cell states could not respond uniformly to activation but show a heterogeneous expression profile of activation and effector molecules, isolation of tumour-reactive T cells based on single activation or effector molecules could result in the absence of tumour-reactive T cells; thus, combinations of multiple activation and effector molecules could improve the efficiency of isolating tumour-specific TCRs. We enrolled two patients with lung adenocarcinoma and obtained their tumour infiltrating lymphocytes (TILs) and autologous tumour cells (ATCs). TILs were cocultured with the corresponding ATCs for 12 h and subjected to single-cell RNA sequencing. First, we identified three TCRs with the highest expression levels of IFNG and TNFRSF9 mRNA for each patient, yet only the top one or two recognized the corresponding ATCs in each patient. Next, we defined the activation score based on normalized expression levels of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF1 mRNA for each T cell and then identified three TCRs with the highest activation score for each patient. We found that all three TCRs in each patient could specifically identify corresponding ATCs. In conclusion, we established an efficient approach to isolate tumour-reactive TCRs based on combinations of multiple activation and effector molecules through single-cell RNA sequencing.

Pharmacologic inhibition of IL11/STAT3 signaling increases MHC-I expression and T cell infiltration.

BACKGROUND: Recent studies have discovered an emerging role of IL11 in various colitis-associated cancers, suggesting that IL11 mainly promotes tumor cell survival and proliferation in regulating tumorigenesis. Herein we aimed to reveal a novel function of IL-11 through STAT3 signaling in regulating tumor immune evasion. METHODS: AOM/DSS model in Il11-/- and Apcmin/+/Il11-/- mice were used to detect tumor growth and CD8+ T infiltration. STAT1/3 phosphorylation and MHC-I, CXCL9, H2-K1 and H2-D1 expression were detected in MC38 cells and intestine organoids treated with/without recombinant IL11 to explore effect of IL11/STAT3 signaling, with IL11 mutein used to competitively inhibit IL11 and rescue inhibited STAT1 activation. Correlation between IL11 and CD8+ T infiltration was analyzed using TIMER2.0 website. IL11 expression and survival prognosis was analyzed in clinical data of patient cohort from Nanfang Hospital. RESULTS: IL11 is highly expressed in CRC and indicates unfavorable prognosis. IL11 knockout increased CD8+ T cell infiltration and reduced intestinal and colon formation. Tumors were significantly suppressed while MHC-I and CXCL9 expression for CD8+ T infiltration were remarkably increased in the tumor tissues of Apcmin/+/Il11-/- mice or Il11-/- mice induced by AOM/DSS. IL11/STAT3 signaling downregulated MHC-I and CXCL9 by inhibiting IFNγ-induced STAT1 phosphorylation. IL11 mutein competitively inhibit IL11 to upregulate CXCL9 and MHC-I in tumor and attenuated tumor growth. CONCLUSIONS: This study ascribes for a new immunomodulatory role for IL11 during tumor development that is amenable to anti-cytokine based therapy of colon cancer.

TCR extracellular domain genetically linked to CD28, 2B4/41BB and DAP10/CD3ζ -engineered NK cells mediates antitumor effects.

NK cells, especially FDA-approved NK-92 cells, could be used for TCR engineering owing to their specialized cytotoxicity against tumors, safety profile and potential use as an off-the-shelf cellular therapy. The TCR complex requires assembly of TCR- α/ ß chains with CD3 molecules (CD3δ, CD3γ, CD3ε, CD3ζ) to be correctly expressed at the cell membrane, and yet NK cells lack expression of these CD3 subunits besides CD3ζ. Since transmembrane regions of TCR α and ß chains are involved in TCR complex assembly, transmembrane regions of TCR replaced by CD28 transmembrane domain could result in the expression of TCR independent of its companion CD3 subunits. However, since the absence of CD3 signaling components can influence the transmission of TCR signals to NK cells, it is necessary to add the signaling molecules of NK cells followed by CD28 transmembrane domain. Both CD3ζ and DAP10 play an important role in the activation and cytotoxicity of NK cells; moreover, 2B4 and 4-1BB are the main costimulatory molecules in NK cells. Therefore, we designed a chimeric TCR that consisted of the extracellular domains of the TCR α and ß chains specific for NYESO-1 fused to the CD28 transmembrane domain followed by the 41BB and CD3ζ signaling domains as well as the 2B4 and DAP10 signaling domain, respectively. The chimeric TCR genetically engineered NK-92 cells exhibit antigen-specific recognition and lysis of tumor cells both in vitro and in vivo. In addition, TCR-28-2B10/BBζ can be feasibly expressed in primary NK cells and exhibit antigen-reactive recognition and effect function. The overall encouraging data highlight the value of NK-92 cells and primary NK cells engineered to express therapeutic chimeric TCR for adoptive immunotherapies.

Autophagic flux restoration of senescent T cells improves antitumor activity of TCR-engineered T cells.

Objectives: Although adoptive cell therapy with T-cell receptor-engineered T cells (TCR-Ts) has mediated effective antitumor responses in several cancers, senescence of T cells could impair the therapeutic effect of TCR-Ts. Thus, it is essential to elucidate the characteristics of senescent TCR-Ts and how to subsequently improve their antitumor effect. Here, we focused on the influence of autophagy on TCR-Ts, since autophagy is tightly associated with the regulation of T-cell activation, proliferation and differentiation. Methods: We first evaluated autophagy level of senescent TCR-Ts, and then the senescent TCR-Ts were expanded in vitro for 7 days with and without spermidine treatment, respectively. Furthermore, the proliferative potential, phenotypical characteristics and functionality of the propagated senescent TCR-Ts were analysed in vitro and in vivo after 7-day ex vivo expansion. Results: We found that autophagic flux of senescent TCR-T cells was significantly impaired. The restoration of autophagic flux via spermidine treatment reduced the expression of inhibitory immunoreceptors (PD-1, TIM-3 or LAG-3), enhanced proliferation and effector functions and subsequently demonstrated the superior in vitro and in vivo antitumor activity of TCR-Ts. Conclusion: These data suggest that spermidine treatment presents an opportunity to improve the antitumor effect of TCR-Ts for the treatment of solid tumors.

Autophagic flux restoration enhances the antitumor efficacy of tumor infiltrating lymphocytes.

BACKGROUND: Although adoptive cell therapy with tumor infiltrating lymphocytes (TILs) has mediated effective antitumor responses in several cancers, dysfunction and exhaustion of TILs significantly impair the therapeutic effect of TILs. Thus, it is essential to elucidate the exhausted characteristics of TILs and improve the antitumor effect of TILs by reversing their exhaustion. Here, we focused on the influence of autophagy on TILs in terms of T-cell activation, proliferation, and differentiation in vitro and in vivo. METHODS: We first evaluated autophagy level of TILs and influence of spermidine treatment on autophagy levels of TILs. Furthermore, we assessed the proliferative potential, phenotypical characteristics, T cell receptor (TCR) repertoire and antitumor activity of TILs with and without spermidine treatment. RESULTS: We found that autophagic flux of TILs, especially exhausted TILs that express inhibitory immunoreceptors and have impaired proliferative capacity and decreased production of cytotoxic effector molecules, was significantly impaired. The restoration of autophagic flux via spermidine treatment resulted in increased diversity of the TCR repertoire, reduced expression of inhibitory immunoreceptors (PD1, TIM3, or LAG3), enhanced proliferation and effector functions, which subsequently demonstrated the superior in vitro and in vivo antitumor activity of TILs. Our findings unveil that spermidine, as an autophagy inducer, reverses dysfunction and exhaustion of TILs and subsequently improves the antitumor activity of TILs. CONCLUSIONS: These data suggest that spermidine treatment presents an opportunity to improve adoptive TIL therapy for the treatment of solid tumors.

Blockade of IL-6 inhibits tumor immune evasion and improves anti-PD-1 immunotherapy.

Long-standing inflammatory bowel disease predisposes to the development of colorectal cancer (CRC). Interleukin (IL) -6, a pivotal link between chronic inflammation and tumor progression, has recently been recognized as a potential therapeutic target. The effect of IL-6 on proliferation and metastasis of CRC by activating the STAT3 pathway has been widely demonstrated in recent years, but few on mediating tumor immune evasion. In this study, we found that IL-6 was remarkably overexpressed in CRC and its elevation was associated with a poor prognosis. We studied CRC tumorigenesis in vivo by inoculating MC38 tumors and induced-CRC model via AOM/DSS (azoxymethane/dextransulfate sodium) in IL-6 deficient (IL-6-/-) and wild-type (WT) mice and found that IL-6-/- mice were less susceptible to develop tumors, compared to WT mice. We detected CD8+ T cells via immunofluorescence and found they exhibit high expression in tumor of IL-6-/- mice. High level of IL-6 was found in colitis model, with down-regulation of MHC-I molecules. In in vitro experiments, we found that IL-6 may act as a negative regulator in IFNγ-STAT1-MHC-I signaling. In addition, vivo trials also confirmed that MHC-I mRNA level was negatively related to the existence of IL-6. Furthermore, the blockade of IL-6 also activated CD8+T-cell accumulation and led to the high PD-L1 expression in CRC, which can sensitize animals to anti-PD-1 therapy. Our study provides a research basis for the significant role of IL-6 in tumor evasion and highlights a novel target to improve the efficacy of immunotherapy.

Clonal Distribution and Intratumor Heterogeneity of the TCR Repertoire in Papillary Thyroid Cancer With or Without Coexistent Hashimoto's Thyroiditis.

The intratumor heterogeneity (ITH) of the amount and TCR repertoires of tumor infiltrating lymphocytes (TILs) in PTC with and without coexistent Hashimoto's thyroiditis (HT) are unclear. Here, we investigated the amount of T cells in tumor and corresponding normal tissues by immunohistochemical staining on 80 tumor samples and 40 normal samples from 40 patients. The immune repertoire of T cells was identified on 24 tumor samples and 12 normal samples from 12 patients using TCR high-throughput sequencing. The results demonstrated that the numbers of CD3+, CD4+ and CD8+ T cells in PTC without coexistent HT (PTC-WO) were significantly lower than those in PTC with existing HT (PTC-W). In PTC-W, the density of CD4+ TILs were generally higher when compared with CD8+ TILs. Furthermore, we found that the numbers of CD3+ T cells and their CD4+, CD8+ subtypes in tumor samples were generally higher than those in normal tissue in PTC-WO and moreover, the number of CD3+ T cells was negatively associated with TCR clonality in PTC-WO. In addition, although ITH of the TCR repertoire truly existed in PTC-W and PTC-WO, the TCR repertoires between distinct regions of the non-adjacent tumor foci were presented with a higher degree of similarity than those between tumor and matched normal tissue in PTC-WO, yet the similarity of intratumor repertoires was not significantly higher than those between tumor and corresponding normal samples in PTC-W. This research comprehensively delineated the quantity and TCR repertoire ITH of T cells in PTC-W and PTC-WO, suggesting that TILs might be reactive to tumor antigens in PTC-WO. Moreover, multiregion biopsies should be performed to precisely identify the immune background in PTC-W and PTC-WO.

Metabolic reprogramming by ex vivo glutamine inhibition endows CAR-T cells with less-differentiated phenotype and persistent antitumor activity.

The inadequate in vivo persistence of chimeric antigen receptor (CAR)-modified T cells has been shown to lead to poor therapeutic efficacy and disease recurrence. In vivo persistence is associated with the differentiation subsets infused, with less differentiated TN or TCM conferring superior renewal capacity and antitumor immunity compared to TEM or TEFF. However, ex vivo expanded CAR-T cells exhibit phenotypic heterogeneity with majority of TEM or TEFF subsets and very low populations of TN and TCM. The transition of differentiation subsets is closely correlated with T cell metabolism fitness. Effector T cell differentiation from TN or TCM requires glutamine uptake and metabolism. Using a CD19-specific CAR, we demonstrated that glutamine inhibition by adding the glutamine antagonist 6-Diazo-5-oxo-l-norleucine (DON) into the culture endows CAR-T cells with enhanced mitochondrial OXPHOS utilizing fatty acids and reduced glycolytic activity, and retains more TN or TCM subsets. DON- pretreated CAR-T cells exhibited stronger cytotoxic lysis in vitro and more robust elimination of tumor burdens in vivo. This study suggests that glutamine inhibition ex vivo would be a potential approach for modulating metabolism and differentiation state to improve the efficacy of CAR-T cell therapy.

Induction of EBV latent membrane protein-2A (LMP2A)-specific T cells and construction of individualized TCR-engineered T cells for EBV-associated malignancies.

BACKGROUND: Latent membrane protein-2A (LMP2A)-specific TCR-engineered T cells could be a promising treatment approach to Epstein-Barr virus-associated malignancies. However, previous studies mainly reported LMP2A-reactive TCRs only focusing on specific HLA subtypes and corresponding epitopes, and thus, they were only suitable for patients with specific HLA. METHODS: Due to hugely varied HLA subtypes and presented LMP2A epitopes in different individuals, our study attempted to develop an individualized approach, based on the weekly in vitro stimulation of peripheral T cells for 2 weeks with autologous dendritic cells (DCs) pulsed with a pool of LMP2A peptides covering LMP2A whole protein and combination analysis of high throughput TCRß sequencing of prestimulated and poststimulated T cells and single-cell TCR sequencing of poststimulated T cells, and to identify LMP2A-specific TCRs of which poststimulated frequencies significantly increased than corresponding prestimulated frequencies. RESULTS: Following this approach, multiple LMP2A-reactive TCRs were identified, optimized and cloned into lentiviral vector, and then transduced into peripheral T cells. These engineerd T cells were demonstrated to specifically recognize the LMP2A presented by autologous DCs and lymphoblastoid cell lines in vitro and in vivo. CONCLUSIONS: This approach provides an efficient procedure to isolate individualized LMP2A-specific TCRs for basic and translational research, as well as for clinical applications.

CD19 CAR-T expressing PD-1/CD28 chimeric switch receptor as a salvage therapy for DLBCL patients treated with different CD19-directed CAR T-cell therapies.

CD19-targeted chimeric antigen receptor T (CAR T) cell therapy is a promising option to treat relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). However, the majority of CAR T-treated patients will eventually progress and require salvage treatment, for which there is no current standard. In this study, we analyzed data from 6 patients with R/R DLBCL who experienced progression following CD19-CAR T therapy, and then received CD19-specific CAR T cells that express a PD-1/CD28 chimeric switch-receptor (CD19-PD-1/CD28-CAR T) as salvage therapy at our institution. After the second infusion of CAR T cells, 3 of 6 patients achieved complete remissions and the duration of the response of responsive patients ranged from 8 to 25 months. One patient showed a stable disease. In contrast, 2/6 patients died on 60 days because of progression disease. Importantly, no severe neurologic toxicity or cytokine release syndrome was observed. These data suggest that CD19-PD-1/CD28-CAR-T cells, a novel anti-CD19 CAR-T cell therapy, elicit a potent and durable anticancer response, and can be used in the post-CD19-CAR T failure setting.

CD19-specific CAR T Cells that Express a PD-1/CD28 Chimeric Switch-Receptor are Effective in Patients with PD-L1-positive B-Cell Lymphoma.

PURPOSE: CD19-specific chimeric antigen receptor (CAR) T-cell therapy is effective against refractory or relapsed (R/R) B-cell lymphoma, but the efficacy is hindered by the existence of PD-1/PD-L1 pathway. PATIENTS AND METHODS: Here, we generated a novel anti-CD19 CAR-expressing PD-1/CD28 chimeric switch-receptor (CD19-PD-1/CD28-CAR). We then conducted a phase Ib study to evaluate safety and efficacy of CD19-PD-1/CD28-CAR T cells in the treatment of PD-L1+ B-cell lymphoma. RESULTS: We found that CD19-PD-1/CD28-CAR T cells had superior T-cell proliferation, cytokine production, and sequentially capability of killing PD-L1+ B-cell lymphoma cells in vitro and in vivo relative to the prototype, CD19-CAR T cells. Among 17 adult patients with R/R lymphoma who received the CAR T therapy, 10 patients had objective response (58.8%), including seven patients with complete remission (41.2%). At a median follow-up 15 months, median overall survival for all patients was not reached. Remarkably, no severe neurologic toxicity or cytokine release syndrome was observed. CONCLUSIONS: This first-in-human study demonstrates the tolerability, safety, and encouraging efficacy of CD19-PD-1/CD28-CART in PD-L1+ large B-cell lymphoma.

Pre-depletion of TRBC1+ T cells promotes the therapeutic efficacy of anti-TRBC1 CAR-T for T-cell malignancies.

Targeting T cell receptor ß-chain constant region 1 (TRBC1) CAR-T could specifically kill TRBC1+ T-cell malignancies. However, over-expressed CARs on anti-TRBC1 CAR transduced TRBC1+ T cells (CAR-C1) bound to autologous TRBC1, masking TRBC1 from identification by other anti-TRBC1 CAR-T, and moreover only the remaining unoccupied CARs recognized TRBC1+ cells, considerably reducing therapeutic potency of CAR-C1. In addition, co-culture of anti-TRBC1 CAR-T and TRBC1+ cells could promote exhaustion and terminal differentiation of CAR-T. These findings provide a rationale for pre-depleting TRBC1+ T cells before anti-TRBC1 CAR-T manufacturing.

Characterization of Microbiota in Cancerous Lung and the Contralateral Non-Cancerous Lung Within Lung Cancer Patients.

BACKGROUND: The functional role of lung microbiota has attracted an accumulating attention recently, but the profile and functional role of the lung microbiota in patients with lung cancer remained largely unknown. METHODS: To evaluate the association of the microbiota with lung cancer, we performed comparative analysis of the lung microbiota using 16S rRNA amplicon sequencing approach in the paired bronchoalveolar lavage fluid (BALF) samples (paired samples from cancerous lung and the contralateral non-cancerous lung) from 50 cancer patients with unilateral lobar masses. RESULTS: We found that the relative abundance of phylum Tenericutes, its class Mollicutes, its order Entomoplasmatales, its family Spiroplasmataceae, and its genus Spiroplasma was significantly increased in cancerous lung, but the relative abundance of phylum Bacteroidetes, its class Bacteroidia, and its order Bacteroidales was significantly decreased in cancerous lung. In addition, the relative abundance of family Leuconostocaceae and its genus Weissella was significantly increased in cancerous lung. CONCLUSION: Our findings provide insights into a change of lung microbiota community associated with the development of lung cancer.

Oral microbiome and risk of malignant esophageal lesions in a high-risk area of China: A nested case-control study.

OBJECTIVE: We aimed to prospectively evaluate the association of oral microbiome with malignant esophageal lesions and its predictive potential as a biomarker of risk. METHODS: We conducted a case-control study nested within a population-based cohort with up to 8 visits of oral swab collection for each subject over an 11-year period in a high-risk area for esophageal cancer in China. The oral microbiome was evaluated with 16S ribosomal RNA (rRNA) gene sequencing in 428 pre-diagnostic oral specimens from 84 cases with esophageal lesions of severe squamous dysplasia and above (SDA) and 168 matched healthy controls. DESeq analysis was performed to identify taxa of differential abundance. Differential oral species together with subject characteristics were evaluated for their potential in predicting SDA risk by constructing conditional logistic regression models. RESULTS: A total of 125 taxa including 37 named species showed significantly different abundance between SDA cases and controls (all P<0.05 & false discovery rate-adjusted Q<0.10). A multivariate logistic model including 11 SDA lesion-related species and family history of esophageal cancer provided an area under the receiver operating characteristic curve (AUC) of 0.89 (95% CI, 0.84-0.93). Cross-validation and sensitivity analysis, excluding cases diagnosed within 1 year of collection of the baseline specimen and their matched controls, or restriction to screen-endoscopic-detected or clinically diagnosed case-control triads, or using only bacterial data measured at the baseline, yielded AUCs>0.84. CONCLUSIONS: The oral microbiome may play an etiological and predictive role in esophageal cancer, and it holds promise as a non-invasive early warning biomarker for risk stratification for esophageal cancer screening programs.

Isolation of T cell receptor specifically reactive with autologous tumour cells from tumour-infiltrating lymphocytes and construction of T cell receptor engineered T cells for esophageal squamous cell carcinoma.

BACKGROUND: T cell receptor-engineered T cells (TCR-Ts) therapy is a promising cancer treatment strategy. Nowadays, most studies focused on identification of high-avidity T cell receptors (TCRs) directed against neoantigens derived from somatic mutations. However, few neoantigens per patient could induce immune response in epithelial cancer and additionally many tumor-specific antigens could be derived from noncoding region. Autologous tumor cells (ATCs) could be unbiased stimulators in activating and enriching tumor-reactive T cells. However, it's unknown if T cells engineered to express TCRs isolated from tumor-reactive T cells enriched by ATCs have strong antitumor response. METHODS: In this study, multiple TIL fragments obtained from a patient with esophageal squamous cell carcinoma (ESCC) were screened for specific recognition of ATCs. Tumor-reactive TILs were enriched by in vitro repeated stimulation of ATCs and isolated based on CD137 upregulation. Subsequently, tumor-reactive TCR was obtained by single-cell RT-PCR analysis and was introduced into peripheral blood lymphocytes to generate TCR-Ts. RESULTS: We found that phenotype and effect function of TIL fragments derived from different tumor sites were spatially heterogeneous. Of four TIL fragments, only TIL-F1 could specifically identify ATCs. Subsequently, we isolated CD8+ CD137+ T cells from pre- and post-stimulated TIL-F1 co-cultured with ATCs, and identified their most dominant TCR. This TCR was introduced into PBLs to generate TCR-Ts, which specifically identified and killed ATCs in vivo and in vitro. CONCLUSION: This strategy provides the means to generate tumor-reactive TCR-Ts for ESCC, which is especially important for patients without prior knowledge of specific epitopes and might be applied for other cancers.

Effectiveness of Intensive Endoscopic Screening for Esophageal Cancer in China: A Community-Based Study.

Evidence is required to evaluate the effectiveness of population-level endoscopic screening for esophageal cancer (EC). In this study, 5,632 permanent residents aged 25-65 years from 6 villages in Hua County, Henan Province, China, were defined as the screening cohort and were offered intensive endoscopic screening. Residents of all 914 remaining villages in Hua County were included as the control cohort, and age-sex standardization was used to calculate the expected numbers of EC and upper gastrointestinal (GI) tract cancer cases and deaths in the screening cohort. The effectiveness of screening was assessed by comparing observed numbers of cases and deaths with expected numbers after 9-year follow-up of these screened subjects (2007-2016). In the screening cohort, 23 upper GI cancers (including 16 ECs) and 10 upper GI cancer deaths (including 5 EC deaths) were identified, and 47% (standardized incidence ratio = 0.53, 95% confidence interval (CI): 0.33, 0.87) and 66% (standardized mortality ratio = 0.34, 95% CI: 0.14, 0.81) reductions in cumulative EC incidence and mortality were found. For upper GI cancers, incidence and mortality were lowered by 43% (standardized incidence ratio = 0.57, 95% CI: 0.38, 0.86) and 53% (standardized mortality ratio = 0.47, 95% CI: 0.25, 0.88), respectively. This study showed that upper GI tract endoscopy is an effective population-level screening test for EC in high-risk regions.

Efficacy of endoscopic screening for esophageal cancer in China (ESECC): design and preliminary results of a population-based randomised controlled trial.

OBJECTIVE: Description of the design and preliminary results of baseline recruitment and screening in the endoscopic screening for esophageal cancer in China (ESECC), the first randomised controlled trial (RCT) assessing efficacy and cost-effectiveness of endoscopic screening for esophageal squamous cell carcinoma (ESCC). DESIGN: ESECC trial is a cluster RCT, and 668 villages in rural Hua County, Henan Province, a high-incidence area of ESCC in China, were randomised into two arms at a ratio of 1:1. Screening arm participants were screened by Lugol chromoendoscopy; no screening was performed in the control arm. ESCC-specific and all-cause mortality, incidence of advanced ESCC and cost-effectiveness of screening will be evaluated in the next 10-year follow-up. Here, we report the performance of baseline recruitment and randomisation, prevalence of upper GI lesions and risk factors for ESCC. RESULTS: A total of 17 151 and 16 797 participants were enrolled in screening and control arms from January 2012 to September 2016. The truncated prevalence (aged 45-69 years) of oesophageal and overall upper GI high-grade lesions was 744.0/100 000 and 902.0/100 000. 69.9% of the 113 patients with high-grade oesophageal lesions were of early stage. Risk factors for severe oesophageal dysplasia and more severe lesions in this population included higher age, family history of ESCC, lower body mass index, eating rapidly and frequent ingestion of leftovers. CONCLUSION: This ESECC trial met the predesigned recruitment and randomisation requirements. Age, family history, undernutrition and unhealthy dietary habits increased the risk for high-grade oesophageal lesions in this high-risk population. TRAIL REGISTRATION NUMBER: NCT01688908; Pre-results.

TCR repertoire intratumor heterogeneity of CD4+ and CD8+ T cells in centers and margins of localized lung adenocarcinomas.

Intratumor heterogeneity (ITH) of T cell receptor (TCR) repertoire in different T-cell subsets and locations in lung adenocarcinomas was unclear. Here, we investigated percentages and TCR repertoire of freshly isolated CD4+ and CD8+ tumor infiltrating lymphocytes (TILs) in tumor centers and margins by flow cytometry on 80 tumor samples from 20 patients and high-throughput TCR sequencing on 27 and 25 samples of CD4+ and CD8+ TILs from seven patients. Our results demonstrated that amount and TCR repertoire diversity of CD4+ TILs were significantly higher than those of CD8+ TILs and moreover substantial ITH regarding amount and TCR repertoire of CD4+ and CD8+ TILs were observed. Additionally, ITH of CD4/CD8 T-cell ratio and CD8+ TIL repertoire across center regions was lower than that across margin regions. The amount and TCR repertoire ITH of CD4+ and CD8+ TILs and mean clonality of CD8+ TILs in tumor centers were associated with relapse. Our study provides insights into amount and TCR repertoire ITH of CD4+ and CD8+ TILs in tumor centers and margins as well as corresponding association with prognosis in lung adenocarcinoma patients, suggesting potential clinical significance of TCR repertoire.

T cell receptor ß-chain repertoire analysis of tumor-infiltrating lymphocytes in pancreatic cancer.

Pancreatic cancer is lethal due to lack of perceptible symptoms and effective treatment methods. Immunotherapy may provide promising therapeutic choices for malignant tumors like pancreatic cancer. Tumor-infiltrating lymphocytes (TIL) in tumor mesenchyme could recognize peptide antigens presented on the surface of tumor cells. The present study aimed to test the relationship between the T cell receptor (TCR) ß repertoire of the tumor and peripheral blood, and also to investigate the intra-tumor spatial heterogeneity of the TCR ß repertoire in pancreatic cancer. To the best of our knowledge, this is the first study to evaluate the clonal composition of TCR ß repertoire in TIL across the spatial extent of pancreatic cancer. In this study, we studied 5 patients who were diagnosed with primary pancreatic cancer. Ultra-deep sequencing was used to assess the rearrangement of the TCR ß-chain (TCR ß) gene. HE staining and immunohistochemistry of CD3, CD4, CD8 and HLA class I were used to show histopathology and immune conditions macroscopically. TIL repertoire showed that different regions of the same tumor showed a greater number of repertoire overlaps between each other than between peripheral blood, which suggested that T cell clones in pancreatic cancer might be quite different from those in peripheral blood. In contrast, intra-tumoral TCR ß repertoires were spatially homogeneous between different regions of a single tumor tissue. Based on these results, we speculated that the cellular adaptive immune response in pancreatic cancer was spatially homogeneous; this may pave the way for immunotherapy for the treatment of pancreatic cancer patients.