Tumor-infiltrating FOXP3 Research Articles

Insight into the significance of Foxp3 + tumor-infiltrating lymphocytes in squamous cell lung cancer

PurposeAlthough developing a better understanding of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) might provide essential knowledge to predict response to immunotherapy and prognosis, our current knowledge about Foxp3 + TILs is inadequate. This study investigated the prognostic significance of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) in squamous cell lung cancer (SQ-LC) objectively.MethodsAmong patients with SQ-LC surgically resected in our institution between 2011 and 2017, those with pathological stage IA3-IIIA were immunohistochemically studied to evaluate Foxp3 + TILs in their tumor stroma. The impact of Foxp3 + TILs on relapse-free survival (RFS) was analyzed with Kaplan–Meier survival analysis and multivariate analysis using a Cox proportional hazards model/Fine-Gray model.ResultsThis study analyzed 100 patients. Multivariate analysis showed that a large number of Foxp3 + TILs in the stroma does not associate with a poor prognosis, rather that a large number of Foxp3 + TILs (≥ 64 cells) tend to be associated with a more favorable prognosis than a small number of Foxp3 + TILs (< 64 cells) (large vs small number: HR, 0.56; 95% CI, 0.17–1.83; P = 0.34). Exploratory analysis also showed that in the two populations divided by a difference in Foxp3 expression levels, similar trends to the main analysis were observed.ConclusionOur results showed that a large number of Foxp3 + TILs in the stroma may not associate with a poor prognosis in SQ-LC. To use the seemingly complicated information of Foxp3 + TILs as biomarkers, better understanding the diversity and heterogeneity of Foxp3 + TILs and analyzing their subpopulations that increase in the TME may be needed.

Cancer-associated fibroblasts-induced remodeling of tumor immune microenvironment via Jagged1 in glioma

Tumor immunosuppression are prominent characteristics of brain glioma. Current standard modality including surgical resection and chemoradiotherapy do not significantly improve clinical outcomes. Cancer-associated fibroblasts (CAFs) that regard as important stromal cells in tumor microenvironment have been confirmed to play crucial roles in tumor development. However, the effects of CAFs on tumor immunosuppression in glioma are not well expounded. In this study, we report that CAFs contributes to the formation of glioma immunosuppressive microenvironment. Specifically, we found that glioma-derived Jagged1 enhanced the proliferation and PD-L1 expression of CAFs in vitro. Importantly, we discovered that Notch1, c-Myc and PD-L1 expression were significantly increased in high Jagged1-expressing gliomas, moreover, we further confirmed that Notch1 and PD-L1 expression located on the CAFs in glioma tissues. We also found that glioma-derived Jagged1 promotes the increase of tumor-infiltrating macrophages, M2 macrophages and Foxp3 Treg cells, as well as no significance of M1 macrophages and CD8+ T cells, indicating potential immunosuppression. This study opens up novel therapeutic strategies reversing CAF immunosuppression for gliomas.

Tumor-infiltrating lymphocytes and levels of PD-L1 and BRCA protein expression may identify patients with breast cancer with a higher rate of BRCA1 mutations

Introduction. Breast cancer (BC) is a heterogeneous disease, treated as per the predictive role of immunohistoche­mistry (IHC) identifiers as estrogen/progesterone and HER2 receptor proteins. Deeper molecular classification (MC) identifies molecular subtypes according to the gene-expression profiles, with different molecular genetic alterations and biological features, present in the different subtype. An overlap between IHC and MC exists, even if somewhat incomplete. We aimed to identify the overlap between IHC and MC, and identify patients with basal-like subtype of BC. We hypothesized that the rates of tumor expression of breast cancer-related protein 1 (BRCA1), the type of tumor-infiltrating lymphocytes, and the expression of programmed death ligand 1 (PD-L1) by immune cells vary among different subtypes of BC. Material and methods. Parafin-embedded samples from 100 patients with primary invasive BC were analyzed and expression levels of estrogen and progesterone receptors, HER2 status, and Ki-67 were assessed via IHC, defining four groups – luminal A-like, luminal B-like (LumA, LumB), HER2-positive non-luminal, and triple negative (TN). The pri­mary endpoint of our study was to identify via IHC with CK5/6 and 17 basal-like subtypes of BC amongst others, and to describe specific clinicopathological features together with protein expression of BRCA1 and PD-L1 and tumor-infiltrating lymphocytes, using CD20, CD3, CD4, CD8, and FoxP3. Results. Basal-like BC were predominantly characterized as triple negative by IHC (p &lt; 0.05) and were more frequently seen among special BC subtypes as compared to no special type (NST), with p = 0.036. Their immune response was represented mostly by high concentration of intratumoral cytotoxic CD8 (+) T-lymphocytes (p &lt; 0.05) and stromal PD-L1-positive immune cells (p = 0.008). In these tumors, absence of expression of BRCA1 protein was more frequent (p &lt; 0.001). Basal-like subtype of BC with absent expression of BRCA1 is associated with poorer &lt;5-year survival (p = 0.001 and p = 0.017, respectively). Conclusions. The use of IHC can establish basal-like BC, the type of its immune response and possible dysfunction in the BRCA -gene, reflected in the lack of expression in the BRCA-related protein.

Immunohistochemical expression of CD8+ tumor-infiltrating lymphocytes and Foxp3 expression in colorectal carcinoma

Background/aim Colorectal cancer (CRC) is third among diagnosed tumors (6.1%) and second according to mortality (9.2%). Disease prognosis is determined not only by the histologic and molecular features of the tumor but also by the host response. Histologic distributions of tumor-infiltrating lymphocytes (TIL) in the microenvironment can be correlated with staging and prognosis of CRC patients. Abundance of CD8+ T lymphocytes has been associated with good prognosis in different types of solid tumors. The association between tumor cell expression of FoxP3 and tumor infiltration by FoxP3-expressing T lymphocytes with prognosis is still controversial. The aim of this study is to evaluate CD8+ TILs and expression of FoxP3 in CRC and to correlate their expression with patients’ clinicopathological parameters. Materials and methods Tumor paraffin blocks and clinicopathological data of 60 patients with CRC were obtained from the Pathology Department at Cairo University. The density of CD8 TILs and FoxP3 expression was assessed immunohistochemically and evaluated by image analysis in CRC specimens using area percentage parameter. The CD8+ cell tumor infiltrate and FoxP3 expression were classified into scanty, moderate, and abundant. Results CD8+ TIL in the present study was insignificantly correlated with the clinicopathological parameters, and no correlation was detected between FoxP3 and CD8 expression (P&gt;0.05). However, FOXP3 expression was significantly correlated with tumor grade, nodal status, distant metastasis, tumor stage, and Dukes’ classification (P&lt;0.01). Conclusion The presence of FoxP3 expression in CRC correlates with favorable pathological prognostic parameters. Cancer colon progression is influenced by host immune response. More studies are needed to assess the role of tumor microenvironment in CRC prognosis.

Abstract 6122: Different characteristics of tumor immune microenvironment between LUSC and LUAD

Abstract Background: Patients with lung squamous cell carcinoma (LUSC) have higher tumor mutation burden (TMB) and PD-L1 expression than those with lung adenocarcinoma (LUAD). Several studies showed that tumor immune microenvironment (TIME), mainly the tumor-infiltrating immune cells, may affect the efficacy of immunotherapy. However, the comparison of tumor immune microenvironment between LUAD and LUSC is little known. Methods: 14 patients with LUSC and 21 patients with LUAD were enrolled in the retrospective analysis of multiple immunofluorescence staining to assess the number of immune cells. Genomic profiling of DNA was performed on formalin-fixed paraffin-embedded tumor samples from 3752 LUAD and 459 LUSC patients by NGS with 733 cancer-related genes panel. TMB is defined as the total counts of single nucleotide variants and indels in the exon region. Results: Overall, as the most abundant tumor-infiltrating immune cells in both groups, CD3+ cells are more in LUAD [median (number/mm2 in LUSC vs in LUAD): 312 vs 918, p = 0.02]. However, there were no significant differences in CD8+ T cells (median: 48 vs 36, p = 0.74), indicating higher infiltration of T lymphocytes rather than cytotoxic T lymphocytes (CTLs) in LUAD than that in LUSC. Unlike in tumor, LUAD patients had both more T lymphocytes (p &amp;lt; 0.001) and CTLs (p &amp;lt; 0.01) in stroma. Besides, LUSC was correlated with fewer M2 tumor-associated macrophages (TAMs) and PD-1+ immune cells (p &amp;lt; 0.001; p = 0.02), while no significant differences were found in tumor-infiltrating FOXP3+ cells (p = 0.41) and B lymphocytes (p = 0.61). In addition, natural killer cells were the fewest among the tumor-infiltrating lymphocytes detected in both groups (median: 2 vs 13). Similar to the previous studies, TMB level was higher in LUSC (median: 8.94 vs 3.36, p &amp;lt; 0.0001). Although LUSC patients had a larger proportion of positive PD-L1 expression (45% vs 30.5%, p &amp;lt; 0.0001) and strong PD-L1 expression in tumor cells (20.9% vs 10.7%, p &amp;lt; 0.0001), the number of CTLs and TAMs expressing PD-L1 is significantly lower than that in LUAD ( p = 0.04; p = 0.0001). Conclusion: LUAD had more immune invasion, while LUSC was related to higher TMB and PD-L1 expression. It suggested that the mechanisms affecting the benefits of immunotherapy may be different. The immune microenvironment of tumor and stroma should be considered simultaneously to explore the mechanism of immunotherapy and the respective thresholds should be selected according to the types of immune cells. Citation Format: Na Li, Mengna Hu, Ding Zhang, Xiaoyu Ji. Different characteristics of tumor immune microenvironment between LUSC and LUAD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6122.

The ratio of CD8\u2009+\u2009lymphocytes to tumor-infiltrating suppressive FOXP3\u2009+\u2009effector regulatory T cells is associated with treatment response in invasive breast cancer

PurposeFOXP3 + and CD8 + are recognized markers of tumor-infiltrating lymphocytes (TILs) for breast cancer. FOXP3 + TILs are composed of effector Tregs (eTregs) and other subpopulations that are classified by their differences in suppressive function. In this prospective study, we evaluated Treg subpopulations and CD8 + TILs in breast cancer.Methods84 patients with breast cancer were enrolled. Fresh TILs were extracted andTregs were classified into eTregs (CD4+FOXP3highCD45RA−), other FOXP3+ Treg subsets (naïve and non-Tregs), and total CD8+CD4− TILs using flow cytometry. The suppression strength of each Treg subpopulation was analyzed. The association between TIL subpopulations, clinicopathological characteristics, and response to chemotherapy was evaluated.ResultsThe mean CD8/eTreg ratio value was 7.86 (interquartile range: 4.08–12.80). The proliferation function of eTregs was significantly suppressed compared with that of the other subpopulations (proliferation rates: control: 89.3%, + naiiveTreg: 64.2%, + non-Treg: 78.2% vs eTreg 1.93%; all P < 0.05). The patients with high with a high CD8 + /eTreg ratio achieved excellent pathological complete response (pCR) rate of neoadjuvant chemotherapy (90.2%) and the CD8/eTreg ratio were independent predictive factors for pCR (odds ratio:18.7(confidence interval 1.25–279) P < 0.05). A detailed assessment of the CD8/eTreg ratio for each patient who underwent NAC revealed that high CD8/eTreg ratio showed a significantly higher pCR rate compared to patients with a low CD8/FOXP3 ratio (39.6% vs 13.3, P < 0.05) in triple negative subtype patients with stromal TILs < 50%.ConclusionsA high CD8/eTreg ratio enhances pCR rate in patients with invasive breast cancer.

The prognostic value of intratumoral and peritumoral tumor-infiltrating FoxP3+Treg cells in of pancreatic adenocarcinoma: a meta-analysis

BackgroundTumor-infiltrating lymphocytes (TILs) are major participants in the tumor microenvironment. The prognostic value of TILs in patients with pancreatic cancer is still controversial.MethodsThe aim of our meta-analysis was to determine the impact of FoxP3+Treg cells on the survival of pancreatic cancer patients. We searched for related studies in PubMed, EMBASE, Ovid, and Cochrane Library from the time the databases were established to Mar 30, 2017. We identified studies reporting the prognostic value of FoxP3+Treg cells in patients with pancreatic cancer. Overall survival (OS) and disease-free survival (DFS)/progression-free survival (PFS)/relapse-free survival (RFS) were investigated by pooling the data. The pooled hazard ratios (HRs) with 95% confidence intervals (95% CI) were used to evaluate the association between FoxP3+Treg cells and survival outcomes of pancreatic cancer patients. A total of 972 pancreatic cancer patients from 8 studies were included in our meta-analysis.ResultsHigh levels of infiltration with FoxP3+Treg cells were significantly associated with poor OS (HR=2.13; 95% CI 1.64–2.77; P<0.05) and poor DFS/PFS/RFS (HR=1.70; 95% CI 1.04 ~ 2.78; P< 0.05). Similar results were also observed in the peritumoral tissue; high levels of FoxP3+Treg cells were associated with poor OS (HR =2.1795% CI, CI 1.50–3.13).ConclusionThis meta-analysis indicated that high levels of intratumoral or peritumoral FoxP3+Treg cell infiltration could be recognized as a negative factor in the prognosis of pancreatic cancer.

Tumor-infiltrating FoxP3+ T cells are associated with poor prognosis in oral squamous cell carcinoma.

ObjectivesSquamous cell carcinoma is the most common malignancy in the oral cavity. Moreover, human papillomavirus (HPV) infection has been recently implicated in the onset of oral squamous cell carcinoma (OSCC). Regulatory T cells (Tregs) are Forkhead box P3 (FoxP3) positive and are normally involved in the mechanism by which organisms escape attacks from their own immune system; however, in tumors, these cells are known to suppress antitumor immunity and block the attack against tumors. The present study evaluated the associations of the number of Tregs and HPV infection with prognoses in patients with OSCC.Material and methodsSamples from 106 patients diagnosed with OSCC were evaluated by immunohistochemical staining for the identification of FoxP3+ Tregs and HPV. The relationship between the observed number of Foxp3‐positive cells, the presence/absence of HPV infection and associations with clinicopathological indicators were analyzed.ResultsTissues were classified into high (High) and low (Low) Treg count groups, with 69 patients classified as High and 37 classified as Low. The prognoses were significantly better in the Low group compared with the High group (p = 0.04). FoxP3 expression may have had some effect on nodal metastases (p = 0.09). HPV antigens were detected in 65 patients, but there were no significant associations with prognosis (p = 0.34). HPV‐infected tumors were more common in the gums and tongues than in the lips, cheeks, and floor of the mouth (p = 0.05).ConclusionsThese results indicate that Tregs in tumor sites are associated with worsened prognoses of patients with OSCC and suggest potential therapies targeting Tregs in OSCC.

Pathological Complete Remission of Liver Metastases Correlates With Elimination of Tumor-infiltrating Tregs in Gastric Cancer.

Tumor-infiltrating Foxp3+ regulatory T-cells (Ti-Tregs) promote tumor progression and contribute to poor prognosis in gastric cancer, but the relationship between Ti-Tregs and response to chemotherapy for liver metastases from gastric cancer (LMGC) is unclear. We estimated the correlation between pathological response to chemotherapy and Ti-Tregs in LMGC. Ti-Tregs were analyzed with immunohistochemistry as CD3+ Foxp3+ cells in patients with synchronous LMGC. Of 53 patients with LMGC, 49 received chemotherapy as initial treatment and 10 underwent R0 resection. LMGC disappeared pathologically in 5 resected cases despite radiologically residual disease. Ti-Tregs were found frequently in residual LMGC and primary lesions but rarely in tumor scar tissue. There was no relationship between frequency of CD8+ cells and pathological response. Marked reduction in Ti-Tregs correlates with pathological complete remission of LMGC. Ti-Tregs may be a biomarker to predict the effects of chemotherapy when used in combination with radiological findings.

Relevance of CD4+ and CD8+ tumor-infiltrating lymphocytes and Foxp3 as prognostic factors in papillary thyroid carcinoma

Background There is increasing evidence supporting the prognostic significance of tumor-infiltrating lymphocytes as CD4, CD8, and forkhead box P3 (Foxp3)-expressing regulatory T cells in different cancers, however, their significance in thyroid cancer remains uncertain. Aims We investigated the expression of CD4, CD8, and Foxp3 in papillary thyroid carcinoma (PTC) and assessed its prognostic implications. Study design It is a retrospective study carried out on 43 PTC cases that were retrieved from archives of pathology lab of Oncology Center, Mansoura University, Egypt. Materials and methods Immunostaining for CD4, CD8, and Foxp3 was done. Results We found that CD4/CD8 expression was significantly higher among patients with higher lymph-node stage and more extranodal extension (P=0.038 and 0.043, respectively). Disease-free survival was found to be shorter among patients with high CD4/CD8 ratio compared with patients with low ratio (P=0.014). As regards Foxp3, there was a difference in its expression between neoplastic and nonneoplastic tissues. Both disease-free survival and overall survival were found to be shorter among patients with positive Foxp3 in tumor and tumor-infiltrating lymphocytes compared with patients with negative Foxp3 in both. Conclusions CD4/CD8 ratio and Foxp3 could be used as predictive markers and as a target therapy for PTC.

Spatial Distribution and Prognostic Implications of Tumor-Infiltrating FoxP3- CD4+ T Cells in Biliary Tract Cancer.

PurposeThe clinical implications of tumor-infiltrating T cell subsets and their spatial distribution in biliary tract cancer (BTC) patients treated with gemcitabine plus cisplatin were investigated.Materials and MethodsA total of 52 BTC patients treated with palliative gemcitabine plus cisplatin were included. Multiplexed immunohistochemistry was performed on tumor tissues, and immune infiltrates were separately analyzed for the stroma, tumor margin, and tumor core.ResultsThe density of CD8+ T cells, FoxP3− CD4+ helper T cells, and FoxP3+ CD4+ regulatory T cells was significantly higher in the tumor margin than in the stroma and tumor core. The density of LAG3- or TIM3-expressing CD8+ T cell and FoxP3− CD4+ helper T cell infiltrates was also higher in the tumor margin. In extrahepatic cholangiocarcinoma, there was a higher density of T cell subsets in the tumor core and regulatory T cells in all regions. A high density of FoxP3− CD4+ helper T cells in the tumor margin showed a trend toward better progression-free survival (PFS) (p=0.092) and significantly better overall survival (OS) (p=0.012). In multivariate analyses, a high density of FoxP3− CD4+ helper T cells in the tumor margin was independently associated with favorable PFS and OS.ConclusionThe tumor margin is the major site for the active infiltration of T cell subsets with higher levels of LAG3 and TIM3 expression in BTC. The density of tumor margin-infiltrating FoxP3− CD4+ helper T cells may be associated with clinical outcomes in BTC patients treated with gemcitabine plus cisplatin.

Prognostic value of FoxP3 and CTLA-4 expression in patients with oral squamous cell carcinoma.

Tumor-infiltrating lymphocytes include tumor-reactive lymphocytes and regulatory T-cells. However, the prognostic value of tumor-infiltrating lymphocytes in oral squamous cell carcinoma (OSCC) remains unclear. We used immunohistochemistry to evaluate the presence of tumor-infiltrating FoxP3⁺ T-cells and CTLA-4⁺ cells in four distinct histological compartments (tumor parenchyma and stroma at the tumor center, and parenchyma and stroma at the invasive front) and assessed the association between the prevalence of these cells and the histopathological status of 137 patients with OSCC. Five-year overall survival, disease-specific survival, and recurrence-free survival were favorable in patients with high numbers of FoxP3⁺ T-cells in the parenchyma of the invasive front. Recurrence-free survival and metastasis-free survival were decreased in patients with high numbers of CTLA-4⁺ cells in the parenchyma of the invasive front. The presence of FoxP3⁺ T-cells in the parenchyma of the invasive front may be a useful prognostic factor. Our results indicate that FoxP3⁺ T-cells may exert site-specific anti-tumor effects but may not play an immunosuppressive role in OSCC. In addition, our results suggest that CTLA-4+ cells suppress the function of FoxP3+ T-cells and promote anti-tumor immunity in OSCC.

Foxp3+ Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs.

Regulatory T cells (Tregs) can be targeted in cancer immunotherapy. A previous study has shown that the chemokine CCL17 and the receptor CCR4 play a role in Treg recruitment in canine urothelial carcinoma. Here, we describe the association of tumor-infiltrating Tregs with CCL17/CCR4 expression in dogs with other carcinomas. In this study, we investigated 23 dogs with mammary carcinoma, 14 dogs with oral squamous cell carcinoma, 16 dogs with pulmonary adenocarcinoma, and 8 healthy control dogs. Immunohistochemistry showed that Foxp3+ Tregs and CCR4+ cells were increased in the tumor tissues of mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, when compared with the healthy tissues. The number of CCR4+ cells was associated with that of Foxp3+ Tregs. Double immunofluorescence labeling confirmed that most tumor-infiltrating Foxp3+ Tregs expressed CCR4. In vitro, canine carcinoma cell lines expressed CCL17 mRNA. Quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) showed that CCL17 mRNA expression in canine carcinomas was increased approximately 10- to 25-fold relative to that of healthy tissues. These results suggest that the CCL17/CCR4 axis may drive Treg recruitment in a variety of canine carcinomas. CCR4 blockade may be a potential therapeutic option for tumor eradication through Treg depletion.

A New Immunological Prognostic Model Based on Immunohistochemistry for Extranodal Natural Killer/T-Cell Lymphoma Patients After Non-Anthracycline-Based Chemotherapy.

PurposeProgrammed death ligand 1 (PD-L1) has been proposed as an important prognostic factor in many types of cancer. However, the role of predicting the prognosis of PD-L1 in extranodal natural killer/T-cell lymphoma (ENKTL) was controversial. Combining other biomarkers might enhance its predictive power. This study aims to evaluate the prognostic value of PD-L1 in conjunction with tumor-infiltrating FoxP3+Tregs for ENKTL after non-anthracycline-based chemotherapy.Patients and MethodsA total of 81 patients with ENKTL were included in this study. Clinicopathological characteristics were collected, and prognostic significance of PD-L1 in neoplastic cells (nPD-L1) and tumor-infiltrating FoxP3+Tregs were evaluated.ResultsPatients with nPD-L1-positive had significantly inferior overall survival (OS) and progression-free survival (PFS) compared with nPD-L1-negative (3-year OS, 37.2% vs 67.3%, p = 0.014; 3-year PFS, 31.0% vs 61.8%, p =0.010, respectively). Patients who had low FoxP3+Tregs had significantly inferior OS and PFS compared with high FoxP3+Tregs (3-year OS, 36.4% vs 63.0%, p = 0.004; 3-year PFS, 31.7% vs 56.3%, p = 0.020, respectively). The results of multivariate analysis showed that nPD-L1 positivity (HR 6.629, 95% CI 1.966–22.350, p=0.002) and low FoxP3+Tregs (HR 7.317, 95% CI 2.154–24.855, p=0.001) were independent predictors of inferior OS. Using these 2 variables, we constructed a new prognostic model that singled out 3 groups with different risk profiles: group 1, no adverse factors; group 2, 1 adverse factor; and group 3, 2 adverse factors. The 3-year OS rates of group 1, group 2, and group 3 were 93.3%, 46.6% and 20.8%, respectively (p<0.001), and the 3-year PFS rates were 86.7%, 40.8% and 15.0%, respectively (p=0.001).ConclusionThis study is the first to validate the prognostic value of nPD-L1 and tumor-infiltrating FoxP3+Tregs in ENKTL; the new immunological prognostic model might be used to stratify ENKTL patients in clinical trials for new therapeutic strategies.

Biomarker recommendation for PD-1/PD-L1 immunotherapy development in pediatric cancer based on digital image analysis of PD-L1 and immune cells.

Anti‐PD‐1/PD‐L1 immunotherapy could offer an alternative to traditional chemo‐ and/or radiotherapy to treat pediatric cancer patients. To unveil the potential benefit of this new therapeutic approach, the prevalence of PD‐L1 and other relevant immune markers using quantitative digital image analysis (DIA) could help to clarify this point. A bridging study was first conducted using commercially available normal formalin‐fixed paraffin‐embedded (FFPE) tonsils to compare immunostaining patterns and intensities from PD‐L1, tumor infiltrating lymphocyte (TIL) markers CD3, CD8, FoxP3, CD45RO, and macrophage marker CD68 in adult (n = 5) and pediatric (n = 10) samples. Then, commercially available pediatric FFPE tumor samples from five prevalent pediatric solid tumor indications: ganglioneuroblastoma (n = 7); neuroblastoma (n = 23); nephroblastoma (n = 30); osteosarcoma (n = 24); and rhabdomyosarcoma (n = 25) were immunostained and their images (n = 654) digitally analyzed using predefined algorithms. The qualitative analysis of staining patterns and intensities in all 15 tonsils for all 6 biomarkers was similar regardless of age category. Quantitative DIA showed that PD‐L1 values varied across cancer‐types, nephroblastoma having the lowest counts. PD‐L1 counts in ganglioneuroblastoma, our pediatric indication with the highest average value, was approximately 12‐times lower than in a similar nonsmall cell lung cancer study, an indication approved for anti‐PD‐1/PD‐L1 immunotherapies. Variable values were measured for the TIL markers CD3, CD8, and CD45RO. FoxP3 was scant across all indications. The macrophage marker CD68 showed highest values in ganglioneuroblastoma, with lowest levels in nephroblastoma. In conclusion, the low PD‐L1 levels uncorrelated with TIL values from the present biomarker morphological study suggest that a PD‐L1 immunohistochemistry patient selection strategy used for anti‐PD‐1/PD‐L1 monotherapy in adult tumors may not succeed in these pediatric indications.

Potent immunomodulatory effects of an anti-CEA-IL-2 immunocytokine on tumor therapy and effects of stereotactic radiation

ABSTRACT While anti-CEA antibodies have no direct effect on CEA-positive tumors, they can be used to direct potent anti-tumor effects as an antibody-IL-2 fusion protein (immunocytokine, ICK), and at the same time reduce the toxicity of IL-2 as a single agent. Using a fusion protein of humanized anti-CEA with human IL-2 (M5A-IL-2) in a transgenic murine model expressing human CEA, we show high tumor uptake of the ICK to CEA-positive tumors with additional lymph node targeting. ICK treated CEA-positive tumors exhibit significant tumor eradication. Analysis of tumor-infiltrating lymphocytes shows a high frequency of both CD8+ and CD4+ T cells along with CD11b positive myeloid cells in ICK treated mice. The frequency of tumor-infiltrating FoxP3+ CD4+ T cells (Tregs) is significantly reduced vs anti-CEA antibody-treated controls, indicating that ICK did not preferentially stimulate migration or proliferation of Tregs to the tumor. Combination therapy with anti-PD-1 antibody did not improve tumor reduction over ICK therapy alone. Since stereotactic tumor irradiation (SRT), commonly used in cancer therapy has immunomodulatory effects, we tested combination SRT+ICK therapy in two tumor model systems. Use of fractionated vs single high dose SRT in combination with ICK resulted in greater tumor inhibition and immunity to tumor rechallenge. In particular, tumor microenvironment and myeloid cell composition appear to play a significant role in the response rate to ICK+SRT combination therapy.

Genetic Subtypes of Systemic Anaplastic Large Cell Lymphoma Show Distinct Differences in PD-L1 Expression and Regulatory and Cytotoxic T Cells in the Tumor Microenvironment.

Anaplastic large cell lymphomas (ALCL) encompass several subgroups that differ in their clinical presentation, genetic features, and prognosis. We characterized the genetic subgroups of 74 patients with ALCL and correlated programmed death ligand 1 (PD-L1) protein expression and compared the densities and ratios of FOXP3+ T regulatory cells and CD8+ tumor-infiltrating lymphocytes (TILs) in tumor cells and the immune microenvironment. The subgroups included anaplastic lymphoma kinase (ALK)-positive (ALK+) ALCL and ALK-negative (ALK-) ALCL and DUSP22-rearranged and nonrearranged ALK- ALCL. None of our cases represented the TP63-rearrangement ALK- ALCL subgroup. Our results showed that ALK+ ALCL had a higher expression of PD-L1 in the tumor cells, in contrast to ALK- ALCL, which expressed high PD-L1 in tumor-associated macrophages (TAMs). DUSP22-rearranged ALK- ALCL lacked PD-L1 expression in the tumor cells and instead expressed PD-L1 only in TAMs. There was a significant positive correlation of PD-L1 expression between tumor and TAMs in ALK+ ALCL with a negative correlation in ALK- ALCL. Systemic ALCL subgroups had similar densities of CD8+ tumor-infiltrating lymphocytes and FOXP3 T regulatory cells, but differences were observed in the ratio of CD8/FOXP3. Our results suggest that alterations in tumor microenvironment and immune responses exist among systemic ALCL subgroups and these features may account for different clinical behavior and prognosis.

Relationship between prognosis of Lumina B breast cancer and tumor infiltration of forxhead box protein 3+ regulatory T lymphocytes and CD8+ cytotoxic T lymphocytes

Objective To detect the expression level of forxhead box Protein 3 (FoxP3) and CD8 in invasive ductal carcinoma of breast Lumina B, investigate the relevance between FoxP3+ regulatory T lymphocyte (Treg) and CD8+ cytotoxic T lymphocyte (CTL) as well as prognosis of breast cancer patients. Methods Immunohistochemistry was used to detect the expression level of FoxP3 and CD8 in the paraffin-embedded breast cancer samples. One-sample t test was applied to analyze the relationship among the expression levels of FoxP3 and CD8 of patient’s age, tumor size, nodal status, and histophathologic grade. Kaplan meier Log-rank was used to analyze the correlation between the expression level of CD8 and FoxP3 and the overall survival and disease-free survival of breast cancer patients. Results The expression of CD8 is associated with tumor diameter ≤2 cm, lymph node negative metastasis, and with lower histopathologic grade (gradeⅠ/Ⅱ) (P 2 cm, lymph node metastasis (P<0.01). High expression level of FoxP3 infiltrating in tumor was could reduce the desease-free survival of breast cancer patients (P<0.05), while low expression of CD8 prolonged desease-free survival (P<0.01). Conclusion Tumor-infiltrating CD8+ CTLs are associated with good prognosis, whereas tumor-infiltrating FoxP3+ Tregs are associated with adverse outcomes. Key words: Invasive ductal carcinoma of breast; Tumor-infiltrating T lymphocyte; Regulatory T lymphocyte; Cytotoxic T lymphocytes; Forxhead box protein 3; CD8

Granzyme B-expressing treg cells are enriched in colorectal cancer and present the potential to eliminate autologous T conventional cells

In addition to expressing inhibitory cytokines and suppressive molecules, Treg cells could downplay inflammation by releasing cytotoxic molecules and eliminating proinflammatory immune cells. Colorectal cancer (CRC) is a common malignancy that has led to many cancer-related deaths. In this study, we investigated the cytotoxic aspect of Treg cells in CRC patients. Data showed that tumor-infiltrating FOXP3+ Treg cells expressed granzyme B immediately following resection, indicating that granzyme B-expressing Treg cells were present directly ex vivo. In the tumor-associated lymph nodes (LNs) and circulating lymphocytes, however, granzyme B-expressing Treg cells were only scarcely found. We then attempted to stimulate granzyme B expression in circulating Treg cells. Granzyme B upregulation in Treg cells could not be activated by standard T cell receptor (TCR) activation through anti-CD3/CD28 and IL-2 but required stimulation with bacterial products, such as with heat-killed Staphylococcus aureus. Interestingly, granzyme B expression was highly concentrated in TIM-3+ Treg cells, a Treg subset previously shown to be enriched in the tumor microenvironment and presented increased suppressive capacity. These TIM-3+ Treg cells presented higher cytolytic capacity toward autologous T conventional cells than the TIM-3− Treg cells, in a manner that was dependent on granzyme B but not TIM-3. Overall, we found that granzyme B-expressing Treg cells were enriched in the tumors from CRC patients and had the potential to eliminate autologous T conventional cells.

Potential prognostic value of PD-L1 and FOXP3 as predictors of relapse in breast cancer

Background: Expression of PD-L1 detected by immunohistochemistry can represent a new hope for cancer management. The role of PD L1 in breast cancer is still unclear. Similarly, is the role of tumor-infiltrating FOXP3 +ve regulatory T (Treg) cells where literature data are conflicting. Our study aimed to evaluate the immunohistochemical expression of PD L1 and FOXP3 in breast cancer, correlate them with clinicopathological parameters as well as evaluating their relation.Methods: This is a retrospective study carried out on 136 breast cancer specimens. Only cases with proved pathological diagnosis of infiltrating duct carcinoma of no special type (NST) were included. Tissue microarray blocks were constructed and immunostained with the polyclonal antibody for PDL1 and monoclonal antibody for FOXP3.Results: Statistically significant correlation was found between high FOXP3 and nearly all adverse prognostic factors including; grade III tumors (p = .003), basal-like subtype(p = .001), high Ki67(p = .001), negative ER status(p = .001), negative PR(p = .028), HER2 expression(p = .04), advanced stage (p = .001), and LN metastases(p = .001). For PDL1, only statistically significant correlation with high Ki67 (p = .018) and advanced stage(p = .03) was found. A statistically significant positive correlation was found between PD L1 and FOXP3(p= .001). No statistically significant correlation was found between both PDL1 and FOXP3 in relation to disease-free survival (DFS) (p = .054). PDL1, age (≥ 50 years), nodal metastases were significant predictors of relapse in breast cancer.Conclusion: The current study supports PDL1 as a predictor of relapse in breast cancer. Additionally, it highlights the synergistic role between PDL1 and FOXP3 in breast cancer microenvironment. Each can be considered as a poor prognostic marker in breast cancer. This raises a concern about the benefit of breast cancer patients from blocking of PDL1 pathway.