负性共刺激分子B7-H4在结直肠癌及其微环境中的表达及临床意义

负性共刺激分子B7-H4是新近发现的B7家族的新成员。为了检测人结直肠癌组织中肿瘤细胞及肿瘤浸润淋巴细胞(tumor-infiltrating lymphocyte,TIL)上B7-H4的表达以及分析巨噬细胞、T淋巴细胞及其亚群的浸润,进而探讨其相关临床意义,本课题采用免疫组化SP法检测98例人结直肠癌组织中肿瘤细胞及浸润淋巴细胞上B7-H4的表达以及巨噬细胞、T细胞及其亚群的浸润,统计学分析B7-H4的表达与患者临床病理参数、预后、肿瘤相关巨噬细胞(tumor-associated mac-rophage,TAM)以及CD3+T细胞、CD8+T细胞浸润程度的相关性。结果显示,结直肠癌组织中高表达B7-H4分子,而正常组织中基本不表达,并且在肿瘤细胞及浸润淋巴细胞中均检测到B7-H4的表达。统计学分析表明,浸润淋巴细胞中B7-H4的表达与患者年龄、有无淋巴结转移、是否为粘液腺癌有关(P<0.05),与患者总体生存率呈负相关(P<0.05),且B7-H4高表达组织较B7-H4低表达组织CD3+T细胞高度浸润例数显著增加(P<0.05);肠癌细胞中B7-H4的表达与患者肿瘤分期、淋巴结转移、有无远处转移以及Dukes分期有关(P<0.05)。综上所述,人结直肠癌患者的肠癌细胞及浸润淋巴细胞较正常细胞均异常表达B7-H4分子,CD3+T细胞的浸润程度与浸润淋巴细胞中B7-H4的表达呈显著相关而与肿瘤细胞上B7-H4的表达无关,而肿瘤细胞表达B7-H4与肿瘤分期、淋巴结转移、有无远处转移以及Dukes分期有关。上述结果提示肿瘤细胞表达B7-H4与浸润淋巴细胞表达B7-H4可能分别以不同机制参与了肿瘤的发生和进展,进一步分析肿瘤组织中B7-H4在不同细胞表达的生物学和临床意义具有重要的价值。     

The special immune microenvironment of tumor budding and its impact on prognosis in gastric adenocarcinoma

Recent studies showed that the tumor-infiltrating lymphocytes (TILs) are not randomly distributed, but organized to accumulate more or less densely in different regions within tumors, which have provoked new thoughts on cancer management. In this study we explored the characteristics of tumor immunemicroenvironment (TIME) for the tumor budding (TB) and lymphocytes in patients with gastric adenocarcinoma (GAC) as well as their prognostic significance. The TILs around the TB at the invasive margin were assessed by double-immunohistochemistry staining for the CD8, FOXP3, OX40 and GrB phenotypes. Results showed that there was a negative correlation between the density of TB and TILs in the budding area, tumor stroma and parenchyma. And the number of TILs around the TB was evidently reduced, compared with TILs in the non-budding region (P < 0.001). Additionally, the number of TILs in turn changed from non-budding area CD8+>FOXP3+>OX40+> GrB + T cells to FOXP3+>CD8+>OX40 + T > GrB + T cells in budding area. Survival rate was significantly lower in patients who had a higher density of TB (P < 0.001) and a lower density of TILs (P = 0.013). We concluded that TB was surrounded by a weak immune surveillance and immunosuppressive response supported the spatial heterogeneity in the TIME of gastric adenocarcinomas. The regional heterogeneity should be attached importance for identifying the influence of the TIME on cancer development and evolution.     

Tumor-infiltrating lymphocyte subsets and tertiary lymphoid structures in pulmonary metastases from colorectal cancer

The presence of tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) reflects an active inflammatory tumor microenvironment. High density of TILs as well as presence of TLS is associated with improved survival in various solid cancer types. We aimed to describe the density and distribution of TILs and TLS in pulmonary metastases (PMs) from primary colorectal cancer (CRC) and its correlation with clinicopathological variables. Fifty-seven CRC pulmonary metastasectomy specimen (PM) and 31 matched primary CRC specimen were included. Cluster of differentiation (CD)3+, CD8+, CD45RO+ and FoxP3+ TILs were evaluated by immunohistochemistry and density was scored semiquantitatively. TLS were evaluated based on morphological criteria. Survival time was defined from pulmonary metastasectomy to death or last follow up. A marked infiltration with CD3+, CD8+, CD45RO+ and FoxP3+ TILs was evident in CRC PM and matched primary CRC. Further assessment of the immune infiltrate in PM showed that a high density of FOXP3+ TILs at the invasive margin [HR 2.40 (1.11–6.96); P = 0.031] and low density of CD8+ cells in TLS [HR 0.30 (0.14–0.79); P = 0.016] were associated with a worse prognosis in univariate analysis. Moreover, a low CD8/FoxP3-ratio of TILs at the invasive margin (P = 0.042) and in TLS (P = 0.027) conferred an impaired prognosis after pulmonary metastasectomy. Our findings suggest that CRC PM harbor an immune active microenvironment. The balance of CD8+ and FoxP3+ T-cells at the tumor border and in TLS provides prognostic information in patients with CRC PM.     

Tumor-infiltrating lymphocytes and PD-L1 in breast cancer (and,what happened to medullary carcinoma?)

Tumor-infiltrating lymphocytes (TILs) and PD-L1 have emerged as important immune biomarkers in breast cancer, particularly triple negative breast carcinomas (TNBC) and human epidermal growth factor-2 positive (HER-2⁺) breast carcinomas. These components of the tumor immune microenvironment can be harnessed or targeted with immunotherapy, which represents a significant advancement in the management of TNBC. TILs are a prognostic biomarker in breast cancer, and this recognition has led to reclassification of medullary carcinoma (which were TILs rich by definition) as a pattern of invasive ductal carcinoma (no special type) rather than a distinct histologic type. PD-L1 is a predictive biomarker in TNBC, and two different PD-L1 assays have been approved as companion diagnostics for immune checkpoint inhibition in TNBC. This review will cover the roles of TILs and PD-L1 testing in breast cancer, both of which provide important clinical information to guide patient prognosis and therapy. This is a rapidly evolving and exciting field with significant implications for patient care.     

Correlation of clinicopathological features and LGR5 expression in colon adenocarcinoma

Colon cancer stem cells (CSCs) are closely related to tumorigenesis and treatment response, and LGR5 is currently the most robust and reliable CSC marker in colorectal cancer (CRC). However, LGR5 expression in CRC tumor budding (TB) is not well understood. We examined the clinicopathological and prognostic significance of LGR5 in CRC TB. LGR5 expression was evaluated by RNAscope, a newly developed RNA in situ hybridization technique, using a tissue microarray consisting of 55 patient samples of TB in colon adenocarcinoma (CA) selected from the medical archives at our hospital. Patients were stratified into negative and positive LGR5 expression groups. Tumor-infiltrating lymphocytes (TILs) and histological grade were lower in the LGR5-positive group compared with the LGR5-negative group (P = .0407 and P = .0436, respectively). There was no significant difference in overall survival between the LGR5-positive group and the LGR5-negative group (log-rank test, P = .6931). LGR5 expression did not remain a predictor of prognosis in univariate analysis (OR = 0.84, 95% CI: 0.33–2.02, P = .6928). LGR5 expression may be affected by TILs, which have been demonstrated to be associated with worse prognosis in the budding area of CA and is an important potential marker of prognosis.     

Synchronous nodal metastatic risk in screening detected and endoscopically removed pT1 colorectal cancers

BackgroundThe population screening campaigns have resulted in increasing the prevalence of endoscopically resected colorectal cancers (CRCs) invading the submucosa (pT1). Synchronous nodal involvement occurs in less than 15 % of these tumors. Histologic criteria currently used for selecting patients needing resection are imprecise and most patients could have been simply followed-up. Tumor infiltrating lymphocytes (TILs) and mismatch repair (MMR) status impact on CRC prognosis. To identify patients requiring completion surgery, the value of histologic variables, TILs and MMR status as risk factors of nodal metastasis was investigated in screening detected and endoscopically removed pT1 CRCs.MethodsIn 102 endoscopically resected pT1 CRCs, the cancer phenotype, CD3+ and CD8+ TILs, and MMR status were assessed. Univariate and multivariate analyses were used to evaluate the correlation with nodal metastasis.ResultsPositive resection margin, evidence of vascular invasion and tumor budding, wide area of submucosal invasion, and high number of CD3+ TILs were associated with nodal metastasis in univariate analyses. Vascular invasion was statistically independent in multivariate analysis. Evidence of neoplastic cells in the vessels and/or at the excision border featured 5 out of 5 metastatic tumors and 13 out of 97 non-metastatic ones.ConclusionsCompletion surgery should be recommended only in pT1 CRC with vascular invasion or with tumor cells reaching the margin. In all other cases, the treatment choice should result from a multidisciplinary discussion on the patient-centered evaluation of the risk-benefit ratio.     

The predictive and prognostic value of Foxp3+/CD25+ regulatory T cells and PD-L1 expression in triple negative breast cancer

Recent significant developments in cancer immunotherapy have led to important breakthroughs and paradigm shifts in the treatment of malignancy. Although breast cancer traditionally has been considered less immunogenic, triple-negative breast cancer (TNBC) is the most immunogenic subtype with more stromal tumor-infiltrating lymphocytes (TILs) and higher programmed death-ligand 1 (PD-L1) expression. The goal of this study is to evaluate regulatory T cells (Tregs) and PD-L1 expression in TNBC, as well as their associations with clinicopathologic features and the outcomes. Tissue microarrays (TMA) of biopsy and resection specimens of 43 TNBC patients who underwent breast biopsy, neoadjuvant chemotherapy, and mastectomy were prepared. The number of Foxp3+ Tregs, Foxp3+/CD25+ Tregs, and expression of PD-L1 in tumor cells (PD-L1 TCs) and TILs (PD-L1 TILs) were assessed by immunohistochemistry. PD-L1 expression combined positive score (PD-L1 CPS) was calculated according to the manufacturer's guidelines. PD-L1 expression was detected in 72% of the cases, and it expressed in a higher percentage and higher intensity in TILs than TCs in TNBC (p = 0.006 and 0.0005, respectively). PD-L1 TCs, PD-L1 TILs, and PD-L1 CPS were all positively associated with pathologic complete response (pCR) (p = 0.04, 0.03, and 0.02, respectively). PD-L1 TILs and PD-L1 CPS also correlated with TILs and tumor infiltrating lymphocyte volume (TILV). Foxp3+ Tregs and Foxp3+/CD25+ Tregs had strong positive correlation (r = 0.89), and they were positively associated with TILs, TILV, and PD-L1 expression. Foxp3+/CD25+ Tregs, PD-L1 TCs, and PD-L1 CPS were positively correlated with better overall survival (p = 0.04, 0.04 and 0.01, respectively).     

TCR function analysis using a novel system reveals the multiple unconventional tumor-reactive T cells in human breast cancer-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TILs) are a potent source for obtaining tumor-reactive T cell receptors (TCRs). Although comprehensive methods to analyze the TCR repertoire in TILs have been reported, the evaluation system for TCR-reactivity to endogenously expressed antigen in tumor cells remains laborious and time consuming. Consequently, very limited numbers of TCRs in TILs have been analyzed for their reactivity to tumor cells. In this study, we developed an efficient evaluation system for TCR function designated c-FIT (c omprehensive f unctional i nvestigation of T CRs) to analyze TCR reactivity. The c-FIT system enabled us to analyze up to 90 TCRs for their reactivity to tumor cells by a single assay within a month. Using c-FIT, we analyzed 70 TCRs of CD8⁺ TILs derived from two breast cancer patients and obtained 23 TCRs that reacted to tumor cells. Surprisingly, although two TCRs were HLA class I-restricted, the remaining 21 TCRs were non-HLA-restricted. Thus, c-FIT can be applied for monitoring multiple conventional and unconventional antigen-specific killer T cells in TILs, leading to the development of new designs for more effective T-cell-based immunotherapies.     

Tumor-Infiltrating Lymphocyte Therapy for Melanoma: Rationale and Issues for Further Clinical Development

Cancer immunotherapy has become an important area for the future development of cancer therapy; this includes T-cell-based therapies that involve adoptive transfer of autologous T cells derived from the tumors or peripheral blood of cancer patients, vaccines, oncolytic virus therapy, and immunomodulatory antibodies and ligands. Here, we summarize the current approaches and clinical data in the field of adoptive T-cell transfer therapy using tumor-infiltrating lymphocytes (TILs) for metastatic melanoma. We also discuss current knowledge on the mechanism of transferred TILs in mediating tumor regression and the growing need for and recent advances in the identification of predictive biomarkers to better select patients for TIL therapy. The current technical limitations of current TIL expansion methods for out-scaling are discussed as well as how these are being addressed in order to further “industrialize” this form of cell therapy. Lastly, how TIL adoptive transfer can be incorporated into the current melanoma treatment continuum, especially as combination therapy with other immunomodulators and targeted drugs, is discussed.     

Incidence and prognostic significance of occult tumor cells in lymph nodes from patients with stage IIA colorectal carcinoma

Approximately 30% of patients with lymph node (LN)-negative colorectal carcinoma (CRC) die of tumor recurrence, which can be related to the presence of tumor cells in LNs not detected by conventional histopathologic analysis. However, the prognostic significance of occult cancer cells still remains uncertain. We evaluated the incidence and the prognostic significance of occult cancer cells in LNs from 395 consecutive patients with curatively resected stage IIA CRC using immunohistochemistry for cytokeratin 20. Immunostained tumor cells were categorized as micrometastases (MCMs) or isolated tumor cells (ITCs) according to the American Joint Committee on Cancer criteria. The detection rates were compared with the clinicopathologic characteristics of the patients and with cancer-specific survival. The median follow-up time was 128 months. Micrometastases were detected in 39 patients (9.9%), whereas ITCs were found in 112 (28.4%), for an overall frequency of 38.2%. None of the clinicopathologic parameters examined was correlated with the presence of occult cancer cells. Patients with ITCs and those with negative LNs showed a similar survival rate (77.7% and 78.3%, respectively), whereas patients with MCMs had a lower survival rate (64.1%). At the univariate analysis, MCMs, tumor growth pattern, extent of tumor spread, and Crohn's-like lymphoid reaction influenced the survival rate significantly. Nevertheless, at the multivariate analysis, only the pattern of tumor growth and the extent of tumor spread were independent prognostic factors. The detection of immunostained tumor cells in the LNs of patients with stage IIA CRC occurs relatively frequently but has no significant effect on prognosis.     

Exploiting the curative potential of adoptive T-cell therapy for cancer

Adoptive T-cell therapy (ACT) is a potent and flexible cancer treatment modality that can induce complete, durable regression of certain human malignancies. Long-term follow-up of patients receiving tumor-infiltrating lymphocytes (TILs) for metastatic melanoma reveals a substantial subset that experienced complete, lasting tumor regression – and may be cured. Increasing evidence points to mutated gene products as the primary immunological targets of TILs from melanomas. Recent technological advances permit rapid identification of the neoepitopes resulting from these somatic gene mutations and of T cells with reactivity against these targets. Isolation and adoptive transfer of these T cells may improve TIL therapy for melanoma and permit its broader application to non-melanoma tumors. Extension of ACT to other malignancies may also be possible through antigen receptor gene engineering. Tumor regression has been observed following transfer of T cells engineered to express chimeric antigen receptors against CD19 in B-cell malignancies or a T-cell receptor against NY-ESO-1 in synovial cell sarcoma and melanoma. Herein, we review recent clinical trials of TILs and antigen receptor gene therapy for advanced cancers. We discuss lessons from this experience and consider how they might be applied to realize the full curative potential of ACT.     

Evolving notions on immune response in colorectal cancer and their implications for biomarker development

Introduction

Colorectal cancer (CRC) still represents the third most commonly diagnosed type of cancer in men and women worldwide. CRC is acknowledged as a heterogeneous disease that develops through a multi-step sequence of events driven by clonal selections; this observation is sustained by the fact that histologically similar tumors may have completely different outcomes, including a varied response to therapy.

Methods

In “early” and “intermediate” stage of CRC (stages II and III, respectively) there is a compelling need for new biomarkers fit to assess the metastatic potential of their disease, selecting patients with aggressive disease that might benefit from adjuvant and targeted therapies. Therefore, we review the actual notions on immune response in colorectal cancer and their implications for biomarker development.

Results

The recognition of the key role of immune cells in human cancer progression has recently drawn attention on the tumor immune microenvironment, as a source of new indicators of tumor outcome and response to therapy. Thus, beside consolidated histopathological biomarkers, immune endpoints are now emerging as potential biomarkers.

Conclusions

The introduction of immune signatures and cellular and molecular components of the immune system as biomarkers is particularly important considering the increasing use of immune-based cancer therapies as therapeutic strategies for cancer patients.

     

TIGIT,A Novel Therapeutic Target for Tumor Immunotherapy

Co-inhibitory and co-stimulatory receptors act in concert to regulate adaptive immune cell function, and the balance of these receptors is essential for the maintenance of immune homeostasis. Tumors constitute highly suppressive microenvironments in which elevated expression of co-inhibitory receptors on tumor-infiltrating lymphocytes (TILs) is often found. Functional blockade of the co-inhibitory receptors such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) have yielded encouraging outcomes in tumors, generating substantial interest in seeking for additional co-inhibitory molecules that may act as potential interfering targets. T-cell Ig and ITIM domain (TIGIT) is a newly identified co-inhibitory receptor expressed by regulatory T cells (Tregs), activated T cells, and natural killer (NK) cells. Several groups reported consistently that TIGIT expression was elevated on CD8⁺ TILs and Tregs in a variety of tumors. Moreover, TIGIT blockade has exhibited therapeutic benefits in animal models of different tumors. Therefore, TIGIT upregulation plays an important role in tumor immunity and may serve as a promising therapeutic target for tumor management.     

T-cell-mediated tumor immune surveillance and expression of B7 co-inhibitory molecules in cancers of the upper gastrointestinal tract

Tumorigenesis can induce adaptive T-cell-mediated immune responses against malignant cells. Such cellular immune responses are actively suppressed by cancer cells via mechanisms of immune tolerance. We studied T-cell responses against tumor growth by examining tumor-infiltrating lymphocytes (TILs) in upper gastrointestinal (GI) cancers. The number of T-bet(+) TILs correlates with better survival of esophageal cancer patients. Using well-defined mouse models, we have further shown that T-bet and Eomes are both required for the adaptive anti-tumor immunity by regulating T-cell trafficking into the tumor tissue and their effector functions inside the tumor microenvironment. In order to gain further insight into the tumor immune microenvironment in the upper GI cancer, we have also studied expression levels of co-inhibitory molecules such as B7-H1/PD-L1 and B7-H4 in tissue specimens of esophageal and gastric cancers. These inhibitory B7 molecules were expressed at high but variable levels by cancer cells. The overexpression of these molecules correlates with poor clinicopathological parameters and shorter patient survival time. The number of CD3(+) and CD8(+) TILs correlates inversely with expression levels of B7-H4 in samples from esophageal cancer, supporting a role of active immune suppression by inhibitory B7 molecules in the tumor microenvironment. In addition, TILs show functional exhaustion and express high levels of PD-1 and Tim-3. We propose that metabolic competition mediated by phosphatidylinositol 3-kinases (PI3Ks) characterizes the immune suppression within cancer tissues. Future tumor vaccine design should combine blockade of B7 inhibitory molecules and enhancement of T-bet and Eomes levels within the tumor microenvironment.     

Tumor apparent diffusion coefficient as an imaging biomarker to predict tumor aggressiveness in patients with estrogen‐receptor‐positive breast cancer

The purpose of this retrospective study was to evaluate whether tumor apparent diffusion coefficient (ADC) was correlated with pathologic biomarkers such as tumor cellularity, Ki67, tumor‐infiltrating lymphocytes (TILs), and peritumoral lymphocytic infiltrate (PLI) in patients with estrogen receptor (ER)‐positive breast cancer. The study was approved by the institutional review board and informed consent was waived. From July 2014 to December 2014, we reviewed 140 ER‐positive tumors in 138 consecutive patients (range, 28–77 years; mean, 52 years) who underwent preoperative breast MRI and definitive surgery. All patients underwent diffusion‐weighted imaging with a 3T scanner. Two radiologists drew the region of interest of the entire tumor and obtained the mean and pixel‐based histogram of ADC. On pathology, two pathologists reviewed tumor cellularity, Ki67, TILs, and PLI. Multiple linear regression analysis was used to determine pathologic variables independently associated with ADC. Tumors with high tumor cellularity and high Ki67 had significantly lower ADCs than those with low tumor cellularity and low Ki67 (P < 0.05 for all). Tumors without PLI had significantly higher standard deviation than those with PLI (0.23 ± 0.08 versus 0.18 ± 0.05; P < 0.001). Median ADC was negatively correlated with tumor cellularity (r = ?0.441), and Ki67 (r = ?0.382). The standard deviation of ADC was also negatively correlated with the degree of PLI (r = ?0.319). On multivariate linear regression analysis, tumor cellularity and Ki67 were independently associated with tumor ADC. Tumor ADC would be an MRI biomarker for the prediction of tumor aggressiveness indicators such as Ki67, tumor cellularity, and PLI in ER‐positive breast cancer. Copyright © 2016 John Wiley & Sons, Ltd.     

在黑色素瘤组织中免疫细胞的形态学观察

目的　探讨肿瘤浸润树突状细胞 (tumorinfiltratingdendriticcell,TIDC)和肿瘤浸润淋巴细胞(tumorinfiltratinglymphocyte ,TIL)的形态学特征。 方法　采用光镜、透射电镜和免疫组织化学方法观察 9例人皮肤黑色素瘤中TIDC和TIL的分布及形态。结果　TIDC主要分布在乳头层和癌周区及癌巢内 ,病变早期TIDC浸润比晚期明显 (P <0 0 1)。电镜下 ,TIDC体积大 ,形态不规则 ,表面有许多树枝状突起 ,核不规则 ,有切迹。胞浆内有丰富的线粒体、核糖体、内质网。TIDC具有两种形态 ,一种表现为突起多 ,细胞器丰富 ,另一种表现为突起较少 ,细胞器也较少。TIDC与肿瘤细胞、TIDC与TIL、TIL与肿瘤细胞之间存在多种接触形式。同时可见TIL与TIL ,TIDC与TIDC之间也有密切接触。还可见树突状细胞 淋巴细胞环 ,淋巴细胞环绕癌细胞。结论　TIDC和TIL存在于黑色素瘤组织 ,TIDC有两种类型。     

Relationship between T cell receptor clonotype and PD-1 expression of tumor-infiltrating lymphocytes in colorectal cancer

Adoptive T cell therapy using tumor-specific T cells or TCR-modified T cells is a promising next-generation immunotherapy. The major source of tumor-reactive T cells is PD-1⁺ tumor-infiltrating lymphocytes (TILs). In contrast, PD-1⁻ TILs have received little attention. Here, we analyzed the TCR-β repertoires of PD-1⁻ and PD-1⁺ CD8⁺ TILs derived from colorectal cancer and breast cancer. Approximately 40–60% of the PD-1⁺ population consisted of oligoclonal populations in both colorectal cancer and breast cancer. In contrast, approximately 37% of the PD-1⁻ population consisted of an oligoclonal population in colorectal cancer, whereas 14% of them were oligoclonal in breast cancer. In colorectal cancer, the TCR repertoires of PD-1⁻CD8⁺ TILs and PD-1⁺CD8⁺ TILs hardly overlapped. Interestingly, clonally expanded CD8⁺ TILs in primary tumors and the metastases expressing the same clonotypic TCR showed the same phenotype regarding the PD-1-expression. These results suggest that the intrinsic properties of TCRs determine the fate of TILs in terms of whether they become PD-1⁺ or PD-1⁻ in the tumor microenvironment. Further functional analysis of TCRs in TILs will allow us to better understand the regulatory mechanisms for PD-1 expression on TILs and may contribute to tumor immunotherapy.     

Tumor-infiltrating lymphocytes in the immunotherapy era

The clinical success of cancer immune checkpoint blockade (ICB) has refocused attention on tumor-infiltrating lymphocytes (TILs) across cancer types. The outcome of immune checkpoint inhibitor therapy in cancer patients has been linked to the quality and magnitude of T cell, NK cell, and more recently, B cell responses within the tumor microenvironment. State-of-the-art single-cell analysis of TIL gene expression profiles and clonality has revealed a remarkable degree of cellular heterogeneity and distinct patterns of immune activation and exhaustion. Many of these states are conserved across tumor types, in line with the broad responses observed clinically. Despite this homology, not all cancer types with similar TIL landscapes respond similarly to immunotherapy, highlighting the complexity of the underlying tumor-immune interactions. This observation is further confounded by the strong prognostic benefit of TILs observed for tumor types that have so far respond poorly to immunotherapy. Thus, while a holistic view of lymphocyte infiltration and dysfunction on a single-cell level is emerging, the search for response and prognostic biomarkers is just beginning. Within this review, we discuss recent advances in the understanding of TIL biology, their prognostic benefit, and their predictive value for therapy.     

Increased HIF-1alpha expression in tumor cells and lymphocytes of tumor microenvironments predicts unfavorable survival in esophageal squamous cell carcinoma patients

The expression of hypoxia-induced factor (HIF)-1α is up-regulated in tumor microenvironments under hypoxia condition. However, the prognostic significance of HIF-1α in esophageal squamous cell carcinoma (ESCC) is still elusive. We measured the HIF-1α expression by immunochemistry in tumor specimens from 136 resected ESCC; in the current study, the HIF-1α expression in tumor cells was significantly associated with tumor stage (P = 0.003) and lymph node metastasis (P = 0.006); whereas the HIF-1α expression in tumor-infiltrating lymphocytes (TILs) had no relationship with patients’ clinicopathological parameters. Patients with high HIF-1α expression in tumor cells or in TILs showed worse survival related to those with low HIF-1α expression. Multivariate analysis demonstrated that expression of HIF-1α in TILs was an independent factor for DFS (P = 0.007) and OS (P = 0.013). Additionally, the expression of HIF-1α in tumor cells was an independent factor for DFS (P = 0.037) and OS (P = 0.033) in locoregional ESCC patients, whereas the expression of HIF-1α in TILs was an independent factor for DFS (P = 0.048) and OS (P = 0.039) in metastatic ESCC patients. Correlation analysis revealed that expressions of HIF-1α in tumor cells and in TILs were positively correlated, and patients with combined high HIF-1α in both tumor cells and TILs had the worst survivals (P < 0.05). These findings suggest that the HIF-1α expressions in different cell populations of ESCC microenvironments have different clinical relevance and prognostic impact on patients.     

Lymphokine-activated killer (LAK) cell activity in tumor-infiltrating lymphocytes from non-small cell lung cancer

Tumor-infiltrating lymphocytes (TILs) are often seen in non-small cell lung cancers (NSCCs). Their functional role in the pathogenesis of lung cancer is unknown. The authors studied TILs in 27 patients with NSCC and determined the following: (1) the immunologic phenotype as defined by monoclonal antibodies against various surface markers, (2) activation state as indicated by interleukin-2 (IL-2) receptor expression and the kinetics of proliferation response to IL-2, and (3) the ability to develop lymphokine-activated killer (LAK) type cytotoxicity against both natural killer (NK)-resistant tumor cell targets (M14) and fresh autologous tumor cells. The authors' results show TILs from NSCCs to be a heterogeneous population composed of T-cells, B-cells, monocytes, and NK cells in frequencies similar to those found in peripheral blood lymphocytes (PBLs). TILs demonstrated increased IL-2 receptor expression and a more rapid proliferative response to IL-2 than PBLs, implying activation of TILs by the tumor milieu. Finally, TILs generated cytotoxicity against NK-sensitive (K562) and NK-resistant (M14) cell line targets consistently after in vitro treatment with IL-2 but were less consistent in their ability to lyse fresh autologous tumor cells and less effective than PBL LAK cells in lysing all targets. Comparison with LAK cells generated from normal volunteers suggests that decreased killing of autologous tumor cells only partially results from an inherent resistance to lysis by fresh NSCC targets. It appears, therefore, that tumor cells taken from NSCCs are not readily killed by the immune cells that infiltrate the tumor stroma and that this failure does not result from nonspecific immune deficiency in TILs.