CD8⁺ cytotoxic T cell and FOXP3⁺ regulatory T cell infiltration in relation to breast cancer survival and molecular subtypes

The prognostic significance of tumor-associated FOXP3⁺ regulatory T cells (Tregs) and CD8⁺ cytotoxic T lymphocytes (CTLs) in invasive breast carcinomas is studied. Tregs and CTLs were assessed by immunohistochemistry in 1270 cases of invasive breast carcinoma for their associations with patient survival, histopathologic features, and molecular subtypes. Infiltrates of Tregs and CTLs were observed within tumor bed and in the tissue surrounding tumor. Within tumor bed, increased infiltration of Tregs and CTLs was significantly more common in those with unfavorable histologic features, including high histologic grade and negative ER and PR status. In addition, high density Treg infiltration was also associated with tumor HER2 overexpression, decreased overall survival (OS) and progression-free survival (PFS). In tissue surrounding tumor, in contrast, high CTL/Treg ratio was found to be significantly associated with improved OS and PFS. These prognostic associations were confirmed by multivariate analysis. Furthermore, the density of Treg infiltrates within tumors was inversely correlated with the prognosis of the molecular subtypes of tumors. The ratio of CTL/Treg infiltrates in the surrounding tissue was also significantly higher in luminal than non-luminal subtypes of carcinoma. The prognostic significances of Tregs and CTLs in breast carcinoma depend on their relative density and location. The density of intratumoral Treg infiltrates and the peritumoral CTL/Treg ratio are independent prognostic factors and correlated with the prognosis of the molecular subtypes of breast carcinoma, which may serve as potential target for stratifying immunotherapy to battle against the aggressive subtypes of breast carcinoma.     

FOXP3+ regulatory T cells affect the development and progression of hepatocarcinogenesis.

PURPOSE: Tumor-infiltrating lymphocytes represent the host immune response to cancer. CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress the immune reaction. The aim of the present study was to investigate the clinicopathologic significance and roles of Tregs and CD8+ T cells during hepatocarcinogenesis. EXPERIMENTAL DESIGN: We examined the infiltration of FOXP3+ Tregs and CD8+ T cells in the tumor stroma and nontumorous liver parenchyma using 323 hepatic nodules including precursor lesions, early hepatocellular carcinoma (HCC), and advanced HCC, along with 39 intrahepatic cholangiocarcinomas and 59 metastatic liver adenocarcinomas. We did immunohistochemical comparative studies. RESULTS: The prevalence of Tregs was significantly higher in HCC than in the nontumorous liver (P<0.001). The patient group with a high prevalence of Tregs infiltrating HCC showed a significantly lower survival rate (P=0.007). Multivariate analysis revealed that the prevalence of Tregs infiltrating HCC was an independent prognostic factor. The prevalence of Tregs increased in a stepwise manner (P<0.001) and that of CD8+ T cells decreased during the progression of hepatocarcinogenesis (P<0.001). Regardless of the presence of hepatitis virus infection or histopathologic evidence of hepatitis, the prevalence of Tregs was significantly increased in nontumorous liver bearing primary hepatic tumors. CONCLUSIONS: Tregs play a role in controlling the immune response to HCC during the progression of hepatocarcinogenesis. It has been suggested that primary hepatic cancers develop in liver that is immunosuppressed by a marked infiltration of Tregs. A high prevalence of Tregs infiltrating HCC is thought to be an unfavorable prognostic indicator.     

FOXP3-positive regulatory T lymphocytes and epithelial FOXP3 expression in synchronous normal, ductal carcinoma in situ, and invasive cancer of the breast

FOXP3-expressing T regulatory lymphocytes (Tregs) have been described as putative mediators of immune tolerance, and thus facilitators of tumor growth. When found in association with various malignancies, Tregs are generally markers of poor clinical outcome. However, it is unknown whether they are also associated with cancer progression. We evaluated quantitative FOXP3 expression in lymphocytes as well as in epithelial cells in a set of thirty-two breast tumors with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Tumors were stained for FOXP3 and CD3 expression and Tregs quantified by determining the ratio of colocalized FOXP3 and CD3 relative to 1) total CD3-expressing lymphocytes and 2) to FOXP3-expressing epithelial cells. The median proportion of FOXP3-expressing CD3 cells significantly increased with malignant progression from normal to DCIS to IDC components (0.005, 0.019 and 0.030, respectively; p ≤ 0.0001 for normal vs. IDC and p = 0.004 for DCIS vs. IDC). The median intensity of epithelial FOXP3 expression was also increased with invasive progression and most markedly augmented between normal and DCIS components (0.130 vs. 0.175, p ≤ 0.0001). Both Treg infiltration and epithelial FOXP3 expression were higher in grade 3 vs. grade 1 tumors (p = 0.014 for Tregs, p = 0.038 for epithelial FOXP3), but did not vary significantly with hormone receptor status, size of invasive tumor, lymph node status, or disease stage. Notably, Treg infiltration significantly correlated with epithelial up-regulation of FOXP3 expression (p = 0.013 for normal, p = 0.001 for IDC). These findings implicate both Treg infiltration and up-regulated epithelial FOXP3 expression in breast cancer progression.     

Association of microvessel density with infiltrating cells in human cutaneous malignant melanoma

Vascularization and host response to malignant tumors may have common molecular regulators, therefore, we analyzed the relationship between microvessel density (MVD) and tumor infiltrating cells in cutaneous malignant melanoma. Density of lymphocyte subpopulations, macrophages, dendritic cells and CD34⁺ microvessels was determined by immunohistochemistry in primary tumor samples from fifty-two patients with melanoma thicker than 1 mm. Intratumoral MVD did not show significant association with infiltration for any of these cell types. In the case of peritumoral reactive cell densities analyzed in the whole patient population, a positive correlation of MVD was found with CD3⁺ T cell density. This association was stronger in melanomas >4.0 mm and in visceral metastatic tumors. In these subgroups similar phenomenon was observed for CD8⁺ cells. We found significant correlation of MVD with CD68⁺ macrophage density only in the highest thickness category, and weak associations with B-cell and dendritic cell infiltration in visceral metastatic cases. MVD did not vary significantly in tumors categorized according to thickness, localization, ulceration or histological type. However, both intratumoral MVD and macrophage infiltration were significantly higher in male patients compared to females. The correlation of immune cell density with tumor vascularization and gender differences in vascularity and macrophage infiltration of melanoma deserve further attention.     

CD8+tumor‐infiltrating lymphocytes at primary sites as a possible prognostic factor of cutaneous angiosarcoma

Tumor‐infiltrating lymphocytes (TILs) have been reported as a prognostic factor in various cancers and are a promising target for immunotherapy. To investigate whether TILs have any impact on the prognosis of angiosarcoma patients, 55 non‐treated patients (40 patients at stage 1 with cutaneous localized tumors, 4 patients at stage 2 with lymph node metastases and 11 patients at stage 3 with distant metastases) with angiosarcoma were evaluated retrospectively by immunohistochemistry stained CD4, CD8, FOXP3 and Ki67. The Kaplan–Meier method was used to estimate overall survival with patients at stage 1. Survival differences were analyzed by the log‐rank test. Patients with higher numbers of CD8⁺ TILs in their primary tumors survived significantly longer compared with patients with lower values. Moreover, the number of CD8 in TILs was positively correlated with a distant metastasis‐free period. The total number of primary TILs (CD4 plus CD8) and CD8⁺ primary TILs of stage 3 patients with distant metastases was positively correlated with their overall survival. To evaluate whether CD8⁺ effector T cells are activated or differentiated, flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed. The percentages of CD8⁺ T cells producing IFN‐γ in PBMC were significantly higher in patients with angiosarcoma (n = 10) compared not only with that of healthy controls (n = 20) but also patients with advanced melanoma (n = 11). These results suggest that anti‐tumor immunity is clinically relevant in angiosarcoma.     

The intratumoural subsite and relation of CD8+ and FOXP3+ T lymphocytes in colorectal cancer provide important prognostic clues

Background:

To find improved tools for prognostic evaluation in patients with colorectal cancer (CRC), we have analysed how infiltration of cytotoxic T lymphocytes (CD8⁺) and regulatory T lymphocytes (FoxP3⁺) correlates to prognosis, not only according to quantity and relation, but also to subsite within tumours of different molecular characteristics (microsatellite instability and CpG island methylator phenotype status).

Methods:

CD8 and FOXP3 expression was evaluated by immunohistochemistry in 426 archival tumour tissue samples from patients surgically resected for CRC. The average infiltration of CD8⁺ and FOXP3⁺ cells was assessed along the tumour invasive front, in the tumour centre and within the tumour epithelium (intraepithelial).

Results:

We found that infiltration of CD8⁺ T lymphocytes within the tumour epithelium provided the strongest prognostic information (P<0.001). At the tumour invasive front and tumour centre, FOXP3 expression withheld the strongest association to prognosis (P<0.001), suggesting FOXP3⁺ T-lymphocyte infiltration to be a better prognostic tool than CD8⁺ T lymphocytes at these intratumoural subsites. We further analysed the possible prognostic impact of the relation between these T-cell subsets, finding that a high intraepithelial CD8 expression was associated with a better patient outcome, independent of FOXP3 infiltration. In groups of low intraepithelial CD8 expression, however, a high infiltration rate of FOXP3⁺ cells at the tumour invasive front, significantly improved prognosis.

Conclusions:

Analyses of intraepithelial infiltration of CD8⁺ T lymphocytes, infiltration of FOXP3⁺ T lymphocytes at the tumour front or centre, and the relation between these subsets, may be a valuable tool for predicting prognosis in colon cancer.     

An evaluation of the clinical significance of FOXP3<Superscript>+</Superscript> infiltrating cells in human breast cancer

Studies in mice have shown that thymic-derived CD4+ CD25+ regulatory T cells (T reg; FOXP3⁺ lymphocytes) inhibit an antitumour immune response. Additional studies have also reported that the T reg population increases in peripheral blood and tumour tissues from patients with cancer. However, the relationship between the T reg population and the patient prognosis remains controversial. Our aim was to determine the prognostic value of T reg cell density in breast cancer using immunohistochemical assessment of FOXP3, which has been shown to be the optimal marker for T regs. Tissue microarrays were used, and the density of FOXP3⁺ cells was determined in a series of 1445 cases of well-characterised primary invasive breast carcinoma cases with long-term follow up. FOXP3⁺ cell numbers were counted in tumour nests, in tumour-adjacent stroma, and in distant stroma. The total number of FOXP3⁺ cells significantly correlated with higher tumour grade (r _s = 0.37, P < 0.001) and ER negativity (Mann–Whitney U test, P < 0.001). In addition, FOXP3 infiltration positively correlated with HER2 expression and basal phenotype subclass. On univariate analysis, FOXP3⁺ cells were associated with a worse prognosis (P = 0.012, log rank = 6.36). This association was found for intratumoural FOXP3⁺ and for tumour-adjacent stromal FOXP3⁺-cells (tumour-cell associated FOXP3, P = 0.001 and log rank 10.35). However, the number of FOXP3⁺ cells was not found to be an independent prognostic factor in multivariate analysis. We therefore conclude that FOXP3⁺ infiltrating cells do not have a dominant role in breast cancer prognosis and suggest that other inflammatory cell subsets may be more critical variables.     

Increased expression of CD4+CD25+FOXP3+ regulatory T cells correlates with Epstein–Barr virus and has no impact on survival in patients with classical Hodgkin lymphoma in Brazil

The tumor microenvironment of classical Hodgkin lymphoma (cHL) is clearly responsible for the maintenance of the malignant Hodgkin-Reed?CSternberg (HRS) cells, and Epstein?CBarr virus (EBV) has been shown to play a role in this immune evasion. EBV can increase the migration of CD4⁺CD25⁺FOXP3⁺ lymphocytes, named regulatory T cells (Tregs). In this study, we assessed the distribution and biological significance of Tregs in patients with cHL. Tissue microarrays were constructed using diagnostic biopsies available in 130 cHL patients and stained with CD4, CD8, CD25, and FOXP3 antibodies. For the present study, only cHL patients whose histology could be confirmed and EBV association established were studied. From the 130 cHL patients selected for this study, 56 were classified as EBV-related and 74 EBV non-related cHL. There were no association between clinical characteristics and the expression of Tregs. However, higher levels of Tregs correlated with EBV presence on HRS cells (p?=?0.02), although it did not influence event-free survival (EFS) and overall survival (p?=?0.98 and p?=?0.59, respectively). This study demonstrates that Tregs expression correlates with EBV presence in HRS cells and has no impact on survival of patients with cHL. Further studies investigating the mechanisms in which EBV recruits Tregs to the tumor microenvironment will contribute not only to our understanding on the pathogenesis of cHL but also to the development of new therapeutic strategies.     

Prognostic value of arginase‐II expression and regulatory T‐cell infiltration in head and neck squamous cell carcinoma

Tumor‐infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3⁺ T cells, and relatively high levels of BDCA2⁺ and FOXP3⁺ cells in stromal (peripheral) regions of the tumors. Tumor‐infiltrating (intraepithelial) FOXP3⁺ T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase‐II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase‐2 by 43%, and B‐cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c⁺ myeloid dendritic cells, and high numbers of FOXP3⁺ T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease‐free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.     

Distribution and prognostic relevance of tumor-infiltrating lymphocytes (TILs) and PD-1/PD-L1 immune checkpoints in human brain metastases

The activation of immune cells by targeting checkpoint inhibitors showed promising results with increased patient survival in distinct primary cancers. Since only limited data exist for human brain metastases, we aimed at characterizing tumor infiltrating lymphocytes (TILs) and expression of immune checkpoints in the respective tumors.Two brain metastases cohorts, a mixed entity cohort (n = 252) and a breast carcinoma validation cohort (n = 96) were analyzed for CD3+, CD8+, FOXP3+, PD-1+ lymphocytes and PD-L1+ tumor cells by immunohistochemistry. Analyses for association with clinico-epidemiological and neuroradiological parameters such as patient survival or tumor size were performed.TILs infiltrated brain metastases in three different patterns (stromal, peritumoral, diffuse). While carcinomas often show a strong stromal infiltration, TILs in melanomas often diffusely infiltrate the tumors. Highest levels of CD3+ and CD8+ lymphocytes were seen in renal cell carcinomas (RCC) and strongest PD-1 levels on RCCs and melanomas. High amounts of TILs, high ratios of PD-1+/CD8+ cells and high levels of PD-L1 were negatively correlated with brain metastases size, indicating that in smaller brain metastases CD8+ immune response might get blocked. PD-L1 expression strongly correlated with TILs and FOXP3 expression. No significant association of patient survival with TILs was observed, while high levels of PD-L1 showed a strong trend towards better survival in melanoma brain metastases (Log-Rank p = 0.0537).In summary, melanomas and RCCs seem to be the most immunogenic entities. Differences in immunotherapeutic response between tumor entities regarding brain metastases might be attributable to this finding and need further investigation in larger patient cohorts.     

Prognostic value of tumor-infiltrating lymphocytes differs depending on histological type and smoking habit in completely resected non-small-cell lung cancer

BackgroundT-cell infiltration in tumors has been used as a prognostic tool in non-small-cell lung cancer (NSCLC). However, the influence of smoking habit and histological type on tumor-infiltrating lymphocytes (TILs) in NSCLC remains unclear.Patients and methodsWe evaluated the prognostic significance of TILs (CD4⁺, CD8⁺, CD20⁺, and FOXP3⁺) according to histological type and smoking habit using automatic immunohistochemical staining and cell counting in 218 patients with NSCLC.ResultsIn multivariate survival analyses of clinical, pathological, and immunological factors, a high ratio of FOXP3⁺ to CD4⁺ T cells (FOXP3/CD4) [hazard ratio (HR): 4.46, P < 0.01 for overall survival (OS); HR: 1.96, P < 0.05 for recurrence-free survival (RFS)] and a low accumulation of CD20⁺ B cells (HR: 2.45, P = 0.09 for OS; HR: 2.86, P < 0.01 for RFS) were identified as worse prognostic factors in patients with adenocarcinoma (AD). In non-AD, a low number of CD8⁺ T cells were correlated with an unfavorable outcome (HR: 7.69, P < 0.01 for OS; HR: 3.57, P < 0.02 for RFS). Regarding smoking habit in AD, a high FOXP3/CD4 ratio was poorly prognostic with a smoking history (HR: 5.21, P < 0.01 for OS; HR: 2.38, P < 0.03 for RFS), whereas a low accumulation of CD20⁺ B cells (HR: 4.54, P = 0.03 for OS; HR: 2.94, P < 0.01 for RFS) was confirmed as an unfavorable factor in non-smokers with AD.ConclusionsA low number of CD8⁺ T cells in non-AD, a high FOXP3/CD4 ratio in smokers with AD, and a low number of CD20⁺ B cells in non-smokers with AD were identified as independent unfavorable prognostic factors in resected NSCLC. Evaluating the influence of histological type and smoking habit on the immunological environment may lead to the establishment of immunological diagnosis and appropriate individualized immunotherapy for NSCLC.     

Skewed immunological balance between Th17 (CD4+IL17A+) and Treg (CD4+CD25+FOXP3+) cells in human oral squamous cell carcinoma

Background

Several studies have documented modulation of Th17 and T regulatory (Treg) cells in various human malignancies which may vary with the type and extent of the disease. However, such data in patients with oral cancer is scarce and hence the current study was designed to elaborate the immunological balance between these two T cell subsets in oral cancer.

Methods and results

We analyzed various T cell subsets in the peripheral blood of 45 oral squamous cell carcinoma (OSCC) patients and 40 healthy volunteers. We found that, compared with the healthy controls, patients had a significantly (p?<?0.0001) higher proportion of both Th17 (CD4⁺IL17A⁺) and Treg (CD4⁺CD25⁺FOXP3⁺) cells, which further showed a reciprocal balance in relation to clinico-pathological parameters in patients. We also detected a circulating CD8⁺ subset of these cells in both patients and healthy controls, although the difference between the two groups was statistically insignificant. Higher frequencies of Th17 cells were found in patients with early stages and without lymph node involvement, while an increased prevalence of Tregs was associated with higher clinical stages and lymph node involvement. Moreover, Th17 cells were quantitatively and positively correlated to CD4⁺T and CD8⁺T cells and inversely correlated with Tregs. Contrarily, Tregs showed a negative association with CD4⁺T and CD8⁺T cells.

Conclusions

Our results suggest an increase in Th17/Tregs ratio in early stages and a decrease in this ratio in higher stages of oral cancer. Such counter regulation of Th17 and Tregs may be a significant prognostic factor in oral cancer patients.     

Prognostic significance of FOXP3+ tumor-infiltrating lymphocytes in breast cancer depends on estrogen receptor and human epidermal growth factor receptor-2 expression status and concurrent cytotoxic T-cell infiltration

Introduction

The infiltration of FOXP3⁺ regulatory T cells into invasive tumors has been reported to be associated with survival in a variety of cancers. The prognostic significance of FOXP3⁺ tumor-infiltrating lymphocytes (TILs) in breast cancer, however, remains controversial.

Methods

FOXP3⁺ TILs were assessed by immunohistochemistry on tissue microarrays constructed from a well-defined cohort of 3,992 breast cancer patients linked to detailed demographic, biomarker, treatment and outcome data. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models to evaluate the association of FOXP3⁺ TILs with breast cancer-specific survival, stratified by intrinsic subtype and cytotoxic T-cell infiltration status (as defined by CD8 immunohistochemistry).

Results

The presence of high numbers of FOXP3⁺ TILs was significantly associated with young age, high grade, estrogen receptor (ER) negativity, concurrent CD8⁺ cytotoxic T-cell infiltration, and human epidermal growth factor receptor-2 positive (HER2⁺)/ER^? and core basal subtypes. On multivariate survival analysis, a high level of FOXP3⁺ TILs was significantly associated with poor survival in ER⁺ breast cancers that lacked CD8⁺ T-cell infiltrates (hazard ratio (HR)?=?1.30, 95% confidence interval (CI)?=?1.02 to 1.66). However, in ER^? breast cancers, FOXP3⁺ TILs were strongly associated with improved survival in the HER2⁺/ER^? subgroup, particularly in those with co-existent CD8⁺ T-cell infiltrates (HR?=?0.48, 95% CI?=?0.23 to 0.98), for which the presence of high levels of FOXP3⁺ TILs was independent of standard clinical prognostic factors.

Conclusions

FOXP3⁺ regulatory TILs are a poor prognostic indicator in ER⁺ breast cancer, but a favorable prognostic factor in the HER2⁺/ER^? subtype. The prognostic value of FOXP3⁺ TILs in breast cancer differs depending on ER and HER2 expression status and CD8⁺ T-cell infiltration.     

Determination of poor prognostic immune features of tumour microenvironment in non-smoking patients with lung adenocarcinoma

We have previously demonstrated that the prognostic significance of tumour-infiltrating CD8⁺ T cells significantly differs according to histological type and patient smoking habits in non-small cell lung cancer (NSCLC). This work suggested that infiltrating CD8⁺ T cells may not be activated sufficiently in the immunosuppressive microenvironment in non-smokers with adenocarcinoma. To understand the immunogenic microenvironment in NSCLC, we characterised immune cells comprehensively by performing an immunohistochemical evaluation using an alternative counting method and multicolour staining method (n = 234), and assessed immune-related gene expression by using genetic analytical approaches (n = 58). We found that high infiltration of activated CD8⁺ T cells expressing interferon gamma (IFN-γ) and granzyme was correlated with postoperative survival in patients with non-adenocarcinoma. On the contrary, CD8⁺ T-cell accumulation was identified as a worse prognostic factor in patients with adenocarcinoma, particularly in non-smokers. Infiltrating CD8⁺ T cells were significantly less activated in this microenvironment with high expression of various immunoregulation genes. Potentially immunoregulatory CD8⁺ FOXP3⁺ T cells and immunodysfunctional CD8⁺ GATA3⁺ T cells were increased in adenocarcinoma of non-smokers. CD4⁺ FOXP3⁺ regulatory T cells expressing chemokine receptor-4 (CCR4)- and chemokine ligand (CCL17)-expressing CD163⁺ M2-like macrophages also accumulated correlatively and significantly in adenocarcinoma of non-smokers. These characteristic immune cells may promote tumour progression possibly by creating an immunosuppressive microenvironment in non-smoking patients with lung adenocarcinoma. Our findings may be helpful for refining the current strategy of personalised immunotherapy including immune-checkpoint blockade therapy for NSCLC.     

Automated prognostic pattern detection shows favourable diffuse pattern of FOXP3+ Tregs in follicular lymphoma

Background:

Histopathological prognostication relies on morphological pattern recognition, but as numbers of biomarkers increase, human prognostic pattern-recognition ability decreases. Follicular lymphoma (FL) has a variable outcome, partly determined by FOXP3 Tregs. We have developed an automated method, hypothesised interaction distribution (HID) analysis, to analyse spatial patterns of multiple biomarkers which we have applied to tumour-infiltrating lymphocytes in FL.

Methods:

A tissue microarray of 40 patient samples was used in triplex immunohistochemistry for FOXP3, CD3 and CD69, and multispectral imaging used to determine the numbers and locations of CD3⁺, FOXP3/CD3⁺ and CD69/CD3⁺ T cells. HID analysis was used to identify associations between cellular pattern and outcome.

Results:

Higher numbers of CD3⁺ (P=0.0001), FOXP3/CD3⁺ (P=0.0031) and CD69/CD3⁺ (P=0.0006) cells were favourable. Cross-validated HID analysis of cell pattern identified patient subgroups with statistically significantly different survival (35.5 vs 142 months, P=0.00255), a more diffuse pattern associated with favourable outcome and an aggregated pattern with unfavourable outcome.

Conclusions:

A diffuse pattern of FOXP3 and CD69 positivity was favourable, demonstrating ability of HID analysis to automatically identify prognostic cellular patterns. It is applicable to large numbers of biomarkers, representing an unsupervised, automated method for identification of undiscovered prognostic cellular patterns in cancer tissue samples.     

Regulatory T cells and the PD-L1/PD-1 pathway mediate immune suppression in malignant human brain tumors

The brain is a specialized immune site representing a unique tumor microenvironment. The availability of fresh brain tumor material for ex vivo analysis is often limited because large parts of many brain tumors are resected using ultrasonic aspiration. We analyzed ultrasonic tumor aspirates as a biosource to study immune suppressive mechanisms in 83 human brain tumors. Lymphocyte infiltrates in brain tumor tissues and ultrasonic aspirates were comparable with respect to lymphocyte content and viability. Applying ultrasonic aspirates, we detected massive infiltration of CD4⁺FoxP3⁺CD25^high CD127^low regulatory T cells (Tregs) in glioblastomas (n = 29) and metastatic brain tumors (n = 20). No Treg accumulation was observed in benign tumors such as meningiomas (n = 10) and pituitary adenomas (n = 5). A significant Treg increase in blood was seen only in patients with metastatic brain tumors. Tregs in high-grade tumors exhibited an activated phenotype as indicated by decreased proliferation and elevated CTLA-4 and FoxP3 expression relative to blood Tregs. Functional analysis showed that the tumor-derived Tregs efficiently suppressed cytokine secretion and proliferation of autologous intratumoral lymphocytes. Most tumor-infiltrating Tregs were localized in close proximity to effector T cells, as visualized by immunohistochemistry. Furthermore, 61% of the malignant brain tumors expressed programmed death ligand-1 (PD-L1), while the inhibitory PD-1 receptor was expressed on CD4⁺ effector cells present in 26% of tumors. In conclusion, using ultrasonic tumor aspirates as a biosource we identified Tregs and the PD-L1/PD-1 pathway as immune suppressive mechanisms in malignant but not benign human brain tumors.     

Implications of NOVA1 suppression within the microenvironment of gastric cancer: association with immune cell dysregulation

Background

The neuronal splicing factor neuro-oncological ventral antigen 1 (NOVA1) is enriched in normal fibroblasts. Stromal spindle cells such as fibroblasts are major components of tissue inflammation and tertiary lymphoid structures within the microenvironment that contribute to the survival and growth of cancer cells. In the present study, we investigated changes of NOVA1 expression in tertiary lymphoid structures in early and advanced gastric cancer microenvironments in terms of tumor progression and immune regulation.

Methods

Using immunohistochemistry, we analyzed NOVA1 expression in tumor cells, T cells, and stromal spindle cells as well as infiltrating densities of CD3⁺ T cells, forkhead box P3 positive (FOXP3⁺) regulatory T cells, CD68⁺ macrophages, CD163⁺ M2 macrophages, and myeloperoxidase-positive neutrophils in 396 surgically resected gastric cancer tissues.

Results

Suppressed NOVA1 expression in tumor cells, T cells, and stromal spindle cells was closely related to decreased infiltration of FOXP3⁺ regulatory T cells, increased infiltration of CD68⁺ macrophages and CD163⁺ M2 macrophages, more advanced tumor stage, and inferior overall survival rate. In addition, low infiltration of CD3⁺ T cells and FOXP3⁺ regulatory T cells and high infiltration of CD68⁺ macrophages were associated with inferior overall survival. Specifically, weak NOVA1 expression in tumor cells was independently related to more advanced tumor stage and inferior overall survival.

Conclusions

NOVA1 suppression was frequently noted in the gastric cancer microenvironment, and attenuated NOVA1 expression in tumor cells was associated with tumor progression and poor prognosis. This finding seems to be related to immune dysfunction through changes in the immune cell composition of T cells and macrophages.

     

Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study

Introduction

The status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8⁺ TILs and FOXP3⁺ TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC).

Methods

One hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8⁺ TIL and FOXP3⁺ TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8⁺ TIL and FOXP3⁺ TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated.

Results

TNBC patients with high CD8⁺ TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8⁺ TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537–6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029–4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8⁺ TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499–9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8⁺ TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively).

Conclusions

This is the first study to demonstrate that high CD8⁺ TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0632-x) contains supplementary material, which is available to authorized users.