The Immune Infiltration in HNSCC and Its Clinical Value: A Comprehensive Study Based on the TCGA and GEO Databases.

BACKGROUND: Being potential field of research for tumor immunological therapy, the head and neck squamous cell carcinoma (HNSCC) is one of most discussed types of tumor. Recently, some clinical trials have also used immunological therapy and demonstrated a subset of HNSCC patients who have shown a clear longer survival time. OBJECTIVE: To conduct further studies and deeper research in the immunological oncology of HNSCC, a more detailed description and comprehending of the complicated landscape of immune infiltrative may be required. METHODS: Firstly, we have described the fraction of different infiltrating immune cells in the HNSCC tumor and then compared it to the normal tissue, and secondly, we have explored the clinical implications of various infiltrated immune cell fractions meticulously. The gene expression profiles of HNSCC tissue were obtained from databases of TCGA and GEO and utilized the deconvolution algorithm (CIBERSORT) to presume the fractions of 22 several immune sensitive cells. RESULTS: Our results indicated that the immune infiltrating cell fractions were considerably different between HNSCC tumor tissue and paired normal tissue, but at the same time, we found a potential internal correlation among the immune cells and also showed the association between immune infiltrating cells and their clinical characteristics. It is worth noting that the resting dendritic cells and M1 macrophages were linked with a favorable prognosis, while the CD4+ T cells with a poorer outcome. CONCLUSION: Fractions of immune cell percentage were also associated with tumors' pathological grade, age, and TNM stage.

Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer.

T cells are important in tumour immunity but a better understanding is needed of the differentiation of antigen-specific T cells in human cancer1,2. Here we studied CD8 T cells in patients with human papillomavirus (HPV)-positive head and neck cancer and identified several epitopes derived from HPV E2, E5 and E6 proteins that allowed us to analyse virus-specific CD8 T cells using major histocompatibility complex (MHC) class I tetramers. HPV-specific CD8 T cells expressed PD-1 and were detectable in the tumour at levels that ranged from 0.1% to 10% of tumour-infiltrating CD8 T lymphocytes (TILs) for a given epitope. Single-cell RNA-sequencing analyses of tetramer-sorted HPV-specific PD-1+ CD8 TILs revealed three transcriptionally distinct subsets. One subset expressed TCF7 and other genes associated with PD-1+ stem-like CD8 T cells that are critical for maintaining T cell responses in conditions of antigen persistence. The second subset expressed more effector molecules, representing a transitory cell population, and the third subset was characterized by a terminally differentiated gene signature. T cell receptor clonotypes were shared between the three subsets and pseudotime analysis suggested a hypothetical differentiation trajectory from stem-like to transitory to terminally differentiated cells. More notably, HPV-specific PD-1+TCF-1+ stem-like TILs proliferated and differentiated into more effector-like cells after in vitro stimulation with the cognate HPV peptide, whereas the more terminally differentiated cells did not proliferate. The presence of functional HPV-specific PD-1+TCF-1+CD45RO+ stem-like CD8 T cells with proliferative capacity shows that the cellular machinery to respond to PD-1 blockade exists in HPV-positive head and neck cancer, supporting the further investigation of PD-1 targeted therapies in this malignancy. Furthermore, HPV therapeutic vaccination efforts have focused on E6 and E7 proteins; our results suggest that E2 and E5 should also be considered for inclusion as vaccine antigens to elicit tumour-reactive CD8 T cell responses of maximal breadth.

Comprehensive analysis of immune cell enrichment in the tumor microenvironment of head and neck squamous cell carcinoma.

Head and neck squamous carcinoma (HNSCC) is highly infiltrated by immune cells, including tumor-infiltrating lymphocytes and myeloid lineage cells. In the tumor microenvironment, tumor cells orchestrate a highly immunosuppressive microenvironment by secreting immunosuppressive mediators, expressing immune checkpoint ligands, and downregulating human leukocyte antigen expression. In the present study, we aimed to comprehensively profile the immune microenvironment of HNSCC using gene expression data obtained from public database. We calculated enrichment scores of 33 immune cell types based on gene expression data of HNSCC tissues and adjacent non-cancer tissues. Based on these scores, we performed non-supervised clustering and identified three immune signatures-cold, lymphocyte, and myeloid/dendritic cell (DC)-based on the clustering results. We then compared the clinical and biological features of the three signatures. Among HNSCC and non-cancer tissues, human papillomavirus (HPV)-positive HNSCCs exhibited the highest scores in various immune cell types, including CD4+ T cells, CD8+ T cells, B cells, plasma cells, basophils, and their subpopulations. Among the three immune signatures, the proportions of HPV-positive tumors, oropharyngeal cancers, early T tumors, and N factor positive cases were significantly higher in the lymphocyte signature than in other signatures. Among the three signatures, the lymphocyte signature showed the longest overall survival (OS), especially in HPV-positive patients, whereas the myeloid/DC signature demonstrated the shortest OS in these patients. Gene set enrichment analysis revealed the upregulation of several pathways related to inflammatory and proinflammatory responses in the lymphocyte signature. The expression of PRF1, IFNG, GZMB, CXCL9, CXCL10, PDCD1, LAG3, CTLA4, HAVCR2, and TIGIT was the highest in the lymphocyte signature. Meanwhile, the expression of PD-1 ligand genes CD274 and PDCD1LG2 was highest in the myeloid/DC signature. Herein, our findings revealed the transcriptomic landscape of the immune microenvironment that closely reflects the clinical and biological significance of HNSCC, indicating that molecular profiling of the immune microenvironment can be employed to develop novel biomarkers and precision immunotherapies for HNSCC.

Multi-resolution characterization of molecular taxonomies in bulk and single-cell transcriptomics data.

As high-throughput genomics assays become more efficient and cost effective, their utilization has become standard in large-scale biomedical projects. These studies are often explorative, in that relationships between samples are not explicitly defined a priori, but rather emerge from data-driven discovery and annotation of molecular subtypes, thereby informing hypotheses and independent evaluation. Here, we present K2Taxonomer, a novel unsupervised recursive partitioning algorithm and associated R package that utilize ensemble learning to identify robust subgroups in a 'taxonomy-like' structure. K2Taxonomer was devised to accommodate different data paradigms, and is suitable for the analysis of both bulk and single-cell transcriptomics, and other '-omics', data. For each of these data types, we demonstrate the power of K2Taxonomer to discover known relationships in both simulated and human tissue data. We conclude with a practical application on breast cancer tumor infiltrating lymphocyte (TIL) single-cell profiles, in which we identified co-expression of translational machinery genes as a dominant transcriptional program shared by T cells subtypes, associated with better prognosis in breast cancer tissue bulk expression data.

Tumor infiltrating NK cell (TINK) subsets and functional molecules in patients with breast cancer.

INTRODUCTION: NK cells have been introduced as the main innate arm of immunity against malignancies. Recent advances introduced new subsets of, and new effector molecules on NK cells suggesting new paradigms for NK cell functions in tumor immunity. Considering these new paradigms, in the current research we investigated the frequency of tumor infiltrating NK cell (TINK) subsets and their functional molecules in breast tumor tissues by flowcytometry method. METHODS: Breast tumor tissues were obtained from 32 untreated patients with breast cancer. The tissues were then minced mechanically to acquire a single cell suspension and surface-stained with monoclonal antibodies against CD3, CD56, CD11b, CD27, NKG2A, NKG2D and CXCR3. For intracellular staining (ICS), the surface-stained cells were then fixed, permeabilized and stained with anti-Perforin and anti-Granzyme B antibodies. The samples were run and the data were acquired on a four-color flowcytometer. RESULTS: The cell suspension derived from tumor tissue encompassed 3.10 ± 0.52 % CD3-CD56+(bright/dim) total NK cells. Based on the conventional classification the percentages of cytotoxic (CD3- CD56dim) and regulatory (CD3- CD56bright) NK cells were respectively 1.74 ± 0.24 % and 1.36 ± 0.48 %. According to the new classification the percentages of cytotoxic (CD3- CD56+ CD11b+ CD27-), regulatory (CD3-CD56+ CD11b+/- CD27+) and tolerant (CD3-CD56+ CD27- CD11b-) NK cells were respectively 0.48 ± 0.07, 1.55 ± 0.34 and 1.15 ± 0.51. A significant higher frequency of total NK cells (CD3-CD56+ (bright/dim)) in the breast tumor tissues of the patients whose tumor draining lymph nodes (TDLNs) has not been yet involved by tumor cells (LN- patients) compared with the ones with lymph nodes involvement (LN+) (5.91 ± 1.79 % Vs. 2.20 ± 0.20 %, P < 0.004). Furthermore, NK cells with overexpressed activating receptor; NKGD2 (CD3- CD56+(bright/dim) NKG2D+ NK cells) was observed to be elevated in LN- patients compared with the LN+ ones (70.01 ± 7.96 Vs. 42.5 ± 4.81, P < 0.011). Correlation analysis revealed the percentages of conventional regulatory NK cells (CD3- CD56bright) in breast tumor tissue to be in positive correlation with the tumor size (R = 0.380, P < 0.04). The mean percentage of this cell subset was also observed to be higher in patients with T3 tumor size compared with smaller T1 tumor size (1.61 ± 0.20 % vs. 0.75 ± 0.15 %, P < 0.023. CONCLUSION: Our observations suggest that accumulation of NK cells as well as the expression of activating NKG2D receptor by TINKs may play roles in breast tumor regression especially in the LN- patients. As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. These observations may have implications in cancer NK cell-based immunotherapy.

Landscape of toll-like receptors expression in tumor microenvironment of triple negative breast cancer (TNBC): Distinct roles of TLR4 and TLR8.

TNBC is the most aggressive and hormone receptor-negative subtype of breast cancer with molecular heterogeneity in bulk tumors hindering effective treatment. Toll-like receptors (TLRs) have the potential to ignite diverse immune responses in the tumor microenvironment (TME). This encouraged us to screen their transcript expression in the publically available TCGA datasets. Reported molecular subtypes of TNBC may represent different TMEs and we observed differentially expressed TLRs (DETs) i.e. TLR3/4/6/8/9 have unique expression pattern in the TNBC subtypes, particularly in Immunomodulatory (IM) TNBC subtype. We then dissected expression of the DETs in immune and other components of the TME. TLR4 and TLR8 showed significant (p-value ≤ 0.05) negative partial correlation with tumor purity compared to other DETs. Interestingly, TLR4 and TLR8 expression showed a significant (adjusted p-value ≤ 0.05) correlation with different subsets of immune infiltrating cells having the highest correlation with monocytes/macrophage/dendritic cell populations mediating both innate and adaptive response in TNBC. The co-expression network identified genes correlated with these immune cells. Further, GSEA analysis of co-expressed genes showed a significant association of TLR8 partners with 'Peptide ligand binding', 'Gά-signaling', and 'Cytokine-cytokine interaction' while TLR4 associated genes correlated with 'Adaptive immune system' and 'Systemic lupus erythematosus' interactome. Finally, the expression of TLR4 protein was validated in a panel of TNBC cell lines. TLR4 expression in chemoresponsive TNBC was also validated in TNBC cell lines upon Paclitaxel (PTX) treatment. Collectively, the present study identified specific DETs in TNBC and discovered a prospective role of TLR4 and TLR8 in the maintenance of tumor-immune-microenvironment.

A prognostic immune risk score for diffuse large B-cell lymphoma.

We constructed a prognostic score for persons with diffuse large B-cell lymphoma (DLBCL) based on infiltrating immune cells. Data of 956 consecutive subjects were retrieved from the Gene Expression Omnibus database and assigned to training (GSE10846, n = 305) or validation (GSE87371 n = 206 and GSE117556 n = 445 combined) cohorts. Proportions of non-lymphoma cells in the sample were inferred using the ESTIMATE algorithm. An immune risk score was constructed comprised of eight types of non-lymphoma immune cells calculated using the CIBERSORT algorithm. Five-year survival of subjects with an immune risk score ≤ 0·45 in the training cohort was better than that of subjects with a score > 0·45 (hazard ratio [HR] = 3·99; 95% confidence interval [CI] = 2·74, 5·82; P < 0·001). HR in the validation cohort was HR = 2·17 (1·47, 3·21; P < 0·001). Enrichment analyses indicated correlations with genes controlling immune-related biological processes and pathways. A nomogram comprised of the immune risk score and most covariates including age, lactate dehydrogenase concentration (LDH), lymphoma-type (germinal centre B cell [GCB] versus non-GCB), Eastern Cooperative Oncology Group performance status (ECOG-PS) and rituximab therapy had a C-statistic of 0·76 compared with C-statistics of 0·69 and 0·69 for the International Prognostic Index (IPI) and Revised International Prognostic Index (R-IPI). These data indicate the immune risk score is an accurate, independent survival predictor in persons with DLBCL.

Prognostic value of the common tumour-infiltrating lymphocyte subtypes for patients with non-small cell lung cancer: A meta-analysis.

BACKGROUND: Many previous studies have revealed that tumour-infiltrating lymphocytes (TILs) are significantly associated with prognosis in various tumours. However, this finding remains controversial in non-small cell lung cancer (NSCLC). We performed this meta-analysis systematically to evaluate the prognostic value of TILs in NSCLC. METHODS: The references were collected by searching the PubMed, EMBASE and Web of Science databases. The pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were summarized using random or fixed effects models to evaluate the association between TILs and NSCLC survival outcomes. RESULTS: A total of 45 interrelated studies were eligible that included 11,448 patients. Pooled analysis showed that a high density of TILs indicated a better overall survival (HR = 0.80, 0.70-0.89) and progression-free survival (HR = 0.73, 0.61-0.85) for patients with NSCLC; a high density of CD3+ TILs in the tumour nest indicated a better overall survival (HR = 0.84, 0.69-0.99) and disease-specific survival (HR = 0.57, 0.34-0.80); a high density of CD4+ TILs in the tumor nest indicated a favourable overall survival (HR = 0.86, 0.76-0.96); a high density of CD8+ TILs indicated a favourable overall survival (HR = 0.995, 0.99-1.0), progression-free survival (HR = 0.52, 0.34-0.71), disease-free survival (HR = 0.64, 0.43-0.85), relapse/recurrence-free survival (HR = 0.42, 0.18-0.67) and disease-specific survival (HR = 0.56, 0.35-0.78); and a high density of CD20+ TILs in the tumour nest indicated a favourable overall survival (HR = 0.65, 0.36-0.94). However, a high density of Foxp3+ TILs in the tumour stroma indicated a worse relapse/recurrence-free survival (HR = 1.90, 1.05-2.76) in NSCLC. CONCLUSIONS: Our meta-analysis confirmed that high densities of TILs, CD3+TILs, CD4+TILs, CD8+TILs and CD20+TILs in the tumour nest are favourable prognostic biomarkers for patients with NSCLC, and Foxp3+TILs in the tumour stroma are a poor prognostic biomarker.

Immune Infiltration Landscape in Lung Squamous Cell Carcinoma Implications.

Intrinsic cancer cells and the tumor-infiltrating immune cells (TIICs) recruited to the immune microenvironment define the malignant phenotype of lung squamous cell carcinoma (LUSC). Understanding more about the immune microenvironment of LUSC enables the selection of high-risk patients who would derive benefit from immunotherapy. Based on large public LUSC cohorts obtained from TCGA and GEO datasets, 22 types of infiltrating immune cell subgroups were evaluated by CIBERSORT. Meta-analysis, principal component analysis (PCA), single-sample gene set enrichment analysis (ssGSEA), and hierarchical clustering analysis were used to evaluate specific immune responses of LUSC. The distribution of TIICs of LUSC was entirely different from normal. TIIC subpopulations were also found to be closely associated with clinical features and molecular subtypes. Unsupervised clustering analysis revealed that three distinct TIIC subgroups existed with different survival patterns. TIICs are extensively implicated in the pathogenesis and development of LUSC. Characterizing the composition of TIICs influences the metabolism, pathological stage, and survival of tumor patients. It is hoped that this immune landscape could provide a more accurate understanding of the development and immunotherapy of LUSC.

Immune phenotyping of diverse syngeneic murine brain tumors identifies immunologically distinct types.

Immunotherapy has emerged as a promising approach to treat cancer, however, its efficacy in highly malignant brain-tumors, glioblastomas (GBM), is limited. Here, we generate distinct imageable syngeneic mouse GBM-tumor models and utilize RNA-sequencing, CyTOF and correlative immunohistochemistry to assess immune-profiles in these models. We identify immunologically-inert and -active syngeneic-tumor types and show that inert tumors have an immune-suppressive phenotype with numerous exhausted CD8 T cells and resident macrophages; fewer eosinophils and SiglecF+ macrophages. To mimic the clinical-settings of first line of GBM-treatment, we show that tumor-resection invigorates an anti-tumor response via increasing T cells, activated microglia and SiglecF+ macrophages and decreasing resident macrophages. A comparative CyTOF analysis of resected-tumor samples from GBM-patients and mouse GBM-tumors show stark similarities in one of the mouse GBM-tumors tested. These findings guide informed choices for use of GBM models for immunotherapeutic interventions and offer a potential to facilitate immune-therapies in GBM patients.

The Immune Profile of Pituitary Adenomas and a Novel Immune Classification for Predicting Immunotherapy Responsiveness.

CONTEXT: The tumor immune microenvironment is associated with clinical outcomes and immunotherapy responsiveness. OBJECTIVE: To investigate the intratumoral immune profile of pituitary adenomas (PAs) and its clinical relevance and to explore a novel immune classification for predicting immunotherapy responsiveness. DESIGN, PATIENTS, AND METHODS: The transcriptomic data from 259 PAs and 20 normal pituitaries were included for analysis. The ImmuCellAI algorithm was used to estimate the abundance of 24 types of tumor-infiltrating immune cells (TIICs) and the expression of immune checkpoint molecules (ICMs). RESULTS: The distributions of TIICs differed between PAs and normal pituitaries and varied among PA subtypes. T cells dominated the immune microenvironment across all subtypes of PAs. The tumor size and patient age were correlated with the TIIC abundance, and the ubiquitin-specific protease 8 (USP8) mutation in corticotroph adenomas influenced the intratumoral TIIC distributions. Three immune clusters were identified across PAs based on the TIIC distributions. Each cluster of PAs showed unique features of ICM expression that were correlated with distinct pathways related to tumor development and progression. CTLA4/CD86 expression was upregulated in cluster 1, whereas programmed cell death protein 1/programmed cell death 1 ligand 2 (PD1/PD-L2) expression was upregulated in cluster 2. Clusters 1 and 2 exhibited a "hot" immune microenvironment and were predicted to exhibit higher immunotherapy responsiveness than cluster 3, which exhibited an overall "cold" immune microenvironment. CONCLUSIONS: We summarized the immune profile of PAs and identified 3 novel immune clusters. These findings establish a foundation for further immune studies on PAs and provide new insights into immunotherapy strategies for PAs.

Levels of different subtypes of tumour-infiltrating lymphocytes correlate with each other, with matched circulating lymphocytes, and with survival in breast cancer.

PURPOSE: Breast cancer tumour-infiltrating lymphocytes associate with clinico-pathological factors, including survival, although the literature includes many conflicting findings. Our aim was to assess these associations for key lymphocyte subtypes and in different tumour compartments, to determine whether these provide differential correlations and could, therefore, explain published inconsistencies. Uniquely, we also examine whether infiltrating levels merely reflect systemic lymphocyte levels or whether local factors are predominant in recruitment. METHODS: Immunohistochemistry was used to detect tumour-infiltrating CD20+ (B), CD4+ (helper T), CD8+ (cytotoxic T) and FoxP3+ (regulatory T) cells in breast cancers from 62 patients, with quantification in tumour stroma, tumour cell nests, and tumour margins. Levels were analysed with respect to clinico-pathological characteristics and matched circulating levels (determined by flow-cytometry). RESULTS: CD4+ lymphocytes were the most prevalent subtype in tumour stroma and at tumour edge and CD8+ lymphocytes were most prevalent in tumour nests; FoxP3+ lymphocytes were rarest in all compartments. High grade or hormone receptor negative tumours generally had significantly increased lymphocytes, especially in tumour stroma. Only intra-tumoural levels of CD8+ lymphocytes correlated significantly with matched circulating levels (p < 0.03), suggesting that recruitment is mainly unrelated to systemic activity. High levels of stromal CD4+ and CD20+ cells associated with improved survival in hormone receptor negative cases (p < 0.04), while tumour nest CD8+ and FoxP3+ cells associated with poor survival in hormone receptor positives (p < 0.005). CONCLUSIONS: Lymphocyte subtype and location define differential impacts on tumour biology, therefore, roles of tumour-infiltrating lymphocytes will only be unravelled through thorough analyses that take this into account.

Clinicopathologic Characteristics of Breast Cancer According to the Infiltrating Immune Cell Subtypes.

The clinical significance of immune cell subtypes in breast cancer remains poorly understood. To identify tumor-infiltrating immune cell subtypes in breast cancer and investigate their implications, tissue microarrays were constructed using 334 cases of invasive ductal carcinoma (luminal A type: 162 (48.5%), luminal B type: 96 (28.7%), HER-2 type: 21 (6.3%), and triple negative breast cancer: 55 (16.5%)). Hormone receptors (ER, PR, and HER-2), Ki-67, and immune cell subtype-related proteins (STAT4, STAT6, FOXP3, CD8, CD68, and CD163) were assessed immunohistochemically. The proportion of highly expressed STAT6, FOXP3, CD8, CD68, and CD163 proteins was found to be lowest in luminal A type but highest in the HER-2 type. Additionally, high-level STAT6, FOXP3, CD68, and CD163 protein expression was associated with higher histologic grade. ER negativity was associated with high STAT6, FOXP3, and CD163 expression levels, whereas PR negativity and high Ki-67 labeling index were associated with high CD163 expression. Univariate (p = 0.003) and multivariate Cox (hazard ratio: 2.435, 95% CI: 1.110-5.344, p = 0.049) analyses showed that high CD8 expression is an independent factor associated with shorter disease-free survival. Immune cell subtype-related protein expression is dependent on breast cancer molecular subtypes, and CD8 expression is associated with patient prognosis.

Genomic analysis and clinical implications of immune cell infiltration in gastric cancer.

The immune infiltration of patients with gastric cancer (GC) is closely associated with clinical prognosis. However, previous studies failed to explain the different subsets of immune cells involved in immune responses and diverse functions. The present study aimed to uncover the differences in immunophenotypes in a tumor microenvironment (TME) between adjacent and tumor tissues and to explore their therapeutic targets. In our study, the relative proportion of immune cells in 229 GC tumor samples and 22 paired matched tissues was evaluated with a Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts (CIBERSORT) algorithm. The correlation between immune cell infiltration and clinical information was analyzed. The proportion of 22 immune cell subsets was assessed to determine the correlation between each immune cell type and clinical features. Three molecular subtypes were identified with 'CancerSubtypes' R-package. Functional enrichment was analyzed in each subtype. The profiles of immune infiltration in the GC cohort from The Cancer Genome Atlas (TCGA) varied significantly between the 22 paired tissues. TNM stage was associated with M1 macrophages and eosinophils. Follicular helper T cells were activated at the late stage. Monocytes were associated with radiation therapy. Three clustering processes were obtained via the 'CancerSubtypes' R-package. Each cancer subtype had a specific molecular classification and subtype-specific characterization. These findings showed that the CIBERSOFT algorithm could be used to detect differences in the composition of immune-infiltrating cells in GC samples, and these differences might be an important driver of GC progression and treatment response.

The Immune Microenvironment in Breast Carcinoma: Predictive and Prognostic Role in the Neoadjuvant Setting.

The prognostic value of the immune cell infiltrate in the breast carcinoma microenvironment is still uncertain. We reviewed published articles analysing the infiltration of inflammatorycells in the microenvironment of breast carcinoma. Data revealed the importance of infiltration of these immune cells in the prognosis of breast carcinoma, particularly the triple-negative and HER2-positive phenotypes. Tumour-infiltrating lymphocytes and their subtypes play a fundamental role in predicting the pathological complete response (pCR) to neoadjuvant chemotherapy. More research aiming to dissect a complex network of communication between cancer cells and other cellular components of the tumour microenvironment is necessary to develop more effective therapeutic approaches.

Immune infiltration in nasopharyngeal carcinoma based on gene expression.

Immune infiltration of nasopharyngeal carcinoma (NPC) is closely associated with the patients' prognosis. However, previous studies have not interpreted the difference of infiltrating immune cells in NPC.We comprehensively analyzed the tumor-infiltrating immune cells present in NPC for the first time, which was based on a scientific deconvolution algorithm (CIBERSORT) and the gene expression data of GSE64634. The fractions of 22 immune cells were assessed to reveal the associations between normal samples and NPC samples.Profiles of immune infiltration vary significantly between normal samples and NPC samples, and the variation could characterize the individual differences. NPC samples contained a higher proportion for M1 macrophages, whereas memory B cells and CD4 memory resting T cells were relatively lower.Our data suggest that the differences in the infiltrating immune cells in NPC and these differences would probably facilitate patient consultation and individualized treatment.

Tumor-Infiltrating Leukocyte Composition and Prognostic Power in Hepatitis B- and Hepatitis C-Related Hepatocellular Carcinomas.

BACKGROUND: Tumor-infiltrating leukocytes (TILs) are immune cells surrounding tumor cells, and several studies have shown that TILs are potential survival predictors in different cancers. However, few studies have dissected the differences between hepatitis B- and hepatitis C-related hepatocellular carcinoma (HBV-HCC and HCV-HCC). Therefore, we aimed to determine whether the abundance and composition of TILs are potential predictors for survival outcomes in HCC and which TILs are the most significant predictors. METHODS: Two bioinformatics algorithms, ESTIMATE and CIBERSORT, were utilized to analyze the gene expression profiles from 6 datasets, from which the abundance of corresponding TILs was inferred. The ESTIMATE algorithm examined the overall abundance of TILs, whereas the CIBERSORT algorithm reported the relative abundance of 22 different TILs. Both HBV-HCC and HCV-HCC were analyzed. RESULTS: The results indicated that the total abundance of TILs was higher in non-tumor tissue regardless of the HCC type. Alternatively, the specific TILs associated with overall survival (OS) and recurrence-free survival (RFS) varied between subtypes. For example, in HBV-HCC, plasma cells (hazard ratio [HR] = 1.05; 95% CI 1.00-1.10; p = 0.034) and activated dendritic cells (HR = 1.08; 95% CI 1.01-1.17; p = 0.03) were significantly associated with OS, whereas in HCV-HCC, monocytes (HR = 1.21) were significantly associated with OS. Furthermore, for RFS, CD8+ T cells (HR = 0.98) and M0 macrophages (HR = 1.02) were potential biomarkers in HBV-HCC, whereas neutrophils (HR = 1.01) were an independent predictor in HCV-HCC. Lastly, in both HBV-HCC and HCV-HCC, CD8+ T cells (HR = 0.97) and activated dendritic cells (HR = 1.09) had a significant association with OS, while γ delta T cells (HR = 1.04), monocytes (HR = 1.05), M0 macrophages (HR = 1.04), M1 macrophages (HR = 1.02), and activated dendritic cells (HR = 1.15) were highly associated with RFS. Conclusions: These findings demonstrated that TILs are potential survival predictors in HCC and different kinds of TILs are observed according to the virus type. Therefore, further investigations are warranted to elucidate the role of TILs in HCC, which may improve immunotherapy outcomes.

Assessment of TILs, IDO-1, and PD-L1 in resected non-small cell lung cancer: an immunohistochemical study with clinicopathological and prognostic implications.

Several cancers, especially non-small cell lung cancer (NSCLC), are able to escape the immunosurveillance of tumor-infiltrating lymphocytes (TILs); among the molecules involved, the indoleamine 2,3-dioxygenase 1 (IDO-1) and the programmed cell death ligand-1 (PD-L1) play a crucial role. These aspects are of great interest in the current immunotherapeutic era, therefore the current study analyses the TILs, IDO-1, and PD-L1 interactions and their correlations with clinicopathological parameters and prognosis in NSCLC. One hundred ninety-three NSCLC surgical specimens, formalin-fixed, and paraffin-embedded were assessed for TILs density, TILs localization, IDO-1 (clone 4.16H1), and PD-L1 (clone E1L3N) immunohistochemical expressions. This data was correlated with clinicopathological parameters, disease free, and overall survivals. IDO-1 and PD-L1 high expressions were related to the solid pattern of adenocarcinomas (respectively p = 0.036 and p = 0.026); high PD-L1 expression was correlated with squamous histotype (p = 0.048). IDO-1 overexpression correlated with former smokers (p = 0.041), higher adenocarcinoma stages (p = 0.039), and with both higher TILs density and PD-L1 expression (respectively p = 0.025 and p = 0.0003). A better prognosis was associated with TILs intratumoral or mixed localizations (p = 0.029). TILs localization affects NSCLC prognosis; the higher expression of IDO-1 and PD-L1 in poorly differentiated and more aggressive lung adenocarcinomas, as well as the correlation between high PD-L1 expression and squamous cell histotype, confirm the more efficient immunoescaping of these NSCLC subgroups.

Prognostic value of tumor-infiltrating lymphocytes in hepatocellular carcinoma: A meta-analysis.

BACKGROUND: In patients with hepatocellular carcinoma (HCC), the prognostic role of tumor-infiltrating lymphocytes (TILs) for survival is still controversial. A meta-analysis was performed to investigate the prognostic effect of TILs in HCC. METHODS: We identify studies from PubMed, Embase, and the Cochrane Library to evaluate the prognostic value of TILs in patients with HCC. A meta-analysis was conducted to estimate overall survival and disease-free survival. The hazard ratio (HR) and 95% confidence interval (CI) were calculated employing fixed-effect or random-effect models depending on the heterogeneity of the included trials. RESULTS: A total of 7905 patients from 46 observational studies were enrolled. For TILs subsets, the density of CD8+, FOXP3+, CD3+, and Granzyme B+ lymphocytes was significantly associated with improved survival (P < .05). The density of FOXP3+ TILs in intratumor (IT) was the most significant prognostic marker (pooled HR = 1.894; 95% CI = 1.659-2.164; P < .001). Patients with high infiltration of CD8+ TILs in IT (pooled HR = 0.676; 95% CI = 0.540-0.845; P = .001) or in margin of tumor (MT) (pooled HR = 0.577; 95% CI = 0.437-0.760; P < .001) had better OS. The pooled analysis revealed that high density of Granzyme B+ T-lymphocytes in IT was statistically significant associated with better OS (pooled HR = 0.621; 95% CI = 0.516-0.748; P < .001) and DFS (pooled HR = 0.678; 95% CI = 0.563-0.815; P < .001). It was interesting that high density of CD3+ in IT foreboded worse OS (pooled HR = 1.008; 95% CI = 1.000-1.015; P = .037), but better DFS (pooled HR = 0.596; 95% CI = 0.374-0.948; P = .029). CONCLUSION: Our findings suggested that some TIL subsets could serve as prognostic biomarkers in HCC. High-quality randomized controlled trials are needed to determine if these TILs could serve as targets for immunotherapy in HCC.

Multispectral imaging for quantitative and compartment-specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients.

Semiquantitative assessment of immune markers by immunohistochemistry (IHC) has significant limitations for describing the diversity of the immune response in cancer. Therefore, we evaluated a fluorescence-based multiplexed immunohistochemical method in combination with a multispectral imaging system to quantify immune infiltrates in situ in the environment of non-small-cell lung cancer (NSCLC). A tissue microarray including 57 NSCLC cases was stained with antibodies against CD8, CD20, CD4, FOXP3, CD45RO, and pan-cytokeratin, and immune cells were quantified in epithelial and stromal compartments. The results were compared with those of conventional IHC, and related to corresponding RNA-sequencing (RNAseq) expression values. We found a strong correlation between the visual and digital quantification of lymphocytes for CD45RO (correlation coefficient: r = 0.52), FOXP3 (r = 0.87), CD4 (r = 0.79), CD20 (r = 0.81) and CD8 (r = 0.90) cells. The correlation with RNAseq data for digital quantification (0.35-0.65) was comparable to or better than that for visual quantification (0.38-0.58). Combination of the signals of the five immune markers enabled further subpopulations of lymphocytes to be identified and localized. The specific pattern of immune cell infiltration based either on the spatial distribution (distance between regulatory CD8+ T and cancer cells) or the relationships of lymphocyte subclasses with each other (e.g. cytotoxic/regulatory cell ratio) were associated with patient prognosis. In conclusion, the fluorescence multiplexed immunohistochemical method, based on only one tissue section, provided reliable quantification and localization of immune cells in cancer tissue. The application of this technique to clinical biopsies can provide a basic characterization of immune infiltrates to guide clinical decisions in the era of immunotherapy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.