FOXP3 Tumor-infiltrating Lymphocytes Research Articles

Spatial Distribution and Densities of CD103+ and FoxP3+ Tumor Infiltrating Lymphocytes by Digital Analysis for Outcome Prediction in Breast Cancer.

The evaluation of tumor-infiltrating lymphocytes (TILs) for breast cancer prognosis is now established. However, the clinical value for their spatial distributions of specific immune subsets, namely CD103+ tissue-resident memory T cells FoxP3+ regulatory T ells, have not been thoroughly examined. Representative whole sections of breast cancers were subjected to CD103 and FoxP3 double staining. Their density, ratio, and spatial features were analyzed in tumor area and tumor-stromal interface. Their associations with clinicopathological parameters and patient's prognosis were analyzed. CD103 TILs were closer to tumor nests than FoxP3 TILs in the tumor-stromal interface. Their densities were associated with high-grade disease, TNBC, and stromal TILs. High stromal FoxP3 (sFoxP3) TILs and close proximity of sCD103 TILs to tumor were independently associated with better survival at multivariate analysis. Subgroup analysis showed the high FoxP3 TILs density associated better survival was seen in HER2-OE and TNBC subtypes while the proximity of CD103 TILs to tumor nests associated better survival was seen in luminal cancers. The prognostic impact of CD103 and FoxP3 TILs in breast cancer depends on their spatial localization. High sFoxP3 TIL density and the lower distance of CD103 TILs from the tumor nests had independent favorable prognostic values.

CD3 high and FoxP3 - tumor-infiltrating lymphocytes in the invasive margin as a favorable prognostic marker in patients with invasive urothelial carcinoma of the bladder.

Tumor-infiltrating lymphocytes (TILs) have been extensively explored as prognostic biomarkers and cellular immunotherapy methods in cancer patients. However, the prognostic significance of TILs in bladder cancer remains unresolved. We evaluated the prognostic effect of TILs in bladder cancer patients. Sixty-four bladder cancer patients who underwent surgical resection between 2018 and 2020 in Zhejiang Provincial People's Hospital were analyzed in this study. Immunohistochemistry was used to evaluate CD3, CD4, CD8, and FoxP3 expression on TILs in the invasive margin of tumor tissue, and the presence of TIL subsets was correlated with the disease-free survival (DFS) of bladder cancer patients. The relationship between clinical-pathological features and DFS were analyzed. A high level of CD3 + TILs (CD3 high TILs) ( P = 0.027) or negative expression of FoxP3 TILs (FoxP3 - TILs) ( P = 0.016) was significantly related to better DFS in bladder cancer patients. Those with CD3 high FoxP3 - TILs had the best prognosis compared to those with CD3 high FoxP3 + TILs or CD3 low FoxP3 - TILs ( P = 0.0035). Advanced age [HR 4.57, (1.86-11.25); P = 0.001], CD3 low TILs [HR 0.21, (0.06-0.71); P = 0.012], CD8 low TILs [HR 0.34, (0.12-0.94); P = 0.039], and FoxP3 + TILs [HR 10.11 (1.96-52.27); P = 0.006] in the invasive margin were associated with a worse prognosis (DFS) by multivariate analysis. In conclusion, we demonstrated that CD3 high , FoxP3 - , and CD3 high FoxP3 - TILs in the invasive margin were significantly associated with better DFS. CD8 high and CD4 high TILs in the invasive margin tended to predict better DFS in bladder cancer. Patients with CD4 high CD8 high TILs in the invasive margin were likely to have a better prognosis.

The effect of chemotherapy on the tumor immune microenvironment in non-small cell lung cancer (NSCLC): A single-institution retrospective analysis.

155 Background: Platinum-doublet chemotherapy (chemo) is routinely used for treatment of early stage and metastatic NSCLC. Immunotherapy is a proven treatment in these patients as well. The presence of tumor infiltrating lymphocytes (TILs) has been shown to be prognostic of this disease; however, the effect of chemo on TILs and the tumor immune microenvironment in NSCLC has not been well studied. We tested the hypothesis that chemo results in a decrease in the presence of TILs in NSCLC. Methods: We retrospectively identified patients treated with neo-adjuvant platinum-doublet chemo who underwent surgical resection from 2007-2015 at the University of Virginia. The specimens were sectioned and IHC stained for CD8, FoxP3, and PD-L1. Two independent investigators assessed a central and a peripheral high power field of tumor for number of CD8 and FoxP3 positive TILs. An independent pathologist assessed tumor cell PD-L1 expression. The nonparametric Wilcoxon Mann Whitney test was used to compare measurements of TILs in chemo-exposed tissue to a control cohort from a database of NSCLC surgical specimens that had not received pre-operative therapy. Results: Of the 17 patients who met eligibility criteria, 11 had sufficient tissue for evaluation. Results are shown in Table 1. There was a significant decrease in CD8 and FoxP3 TILs in both the center and periphery in patients exposed to chemo. There was no difference in PD-L1 expression. Conclusions: These data suggest that platinum-doublet chemo significantly decreases the presence of cytotoxic and regulatory subsets of TILs without effecting PD-L1 expression. This study is limited by the lack of pre-treatment tumor samples to compare pre- and post-treatment TILs in individual patients. Further studies are warranted to evaluate the impact of decreased TILs in the setting of concurrent or sequential immunotherapy treatment with chemo in NSCLC. [Table: see text]

Objective measurement and clinical significance of IDO1 protein in hormone receptor-positive breast cancer

BackgroundImmunostimulatory therapies targeting immune-suppressive pathways produce durable responses in advanced solid tumors. Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting oxidoreductase that catalyzes the degradation of tryptophan to kynurenine. IDO induces immune tolerance by downregulating CD8+ and effector CD4+ T cell responses. IDO1, the most active isoform, is expressed in diverse tumor types and can be targeted using small molecule inhibitors. We used an objective, in situ assay to measure IDO1 in a collection of hormone receptor-positive breast cancers (HR+ BC).MethodsIDO1 protein was measured using quantitative immunofluorescence in 362 stage I-III HR+ BC represented in tissue microarrays. IDO1 levels were determined in the tumor and stroma, and stratified using median cut-point. Associations between IDO1, clinico-pathological features and CD3+, CD8+, CD20+ and FOXP3 tumor-infiltrating lymphocytes were examined using χ2 and Mann-Whitney tests. Survival was studied using Kaplan-Meier estimator and a proportional hazards model. All tests were two-sided.ResultsIDO1 protein was observed in 76.2% of HR+ BC. There was no association between IDO1 and major clinico-pathological characteristics. Increased IDO1 correlated with decreased CD20+ infiltration (P = 0.0004) but not with CD3+, CD8+ or FOXP3 levels. Elevated IDO1 expression was associated with worse 20-year overall survival (log-rank P = 0.02, HR = 1.39, 95% C.I.: 1.05-1.82). IDO1 scores were independently associated with outcome in multivariable analysis.ConclusionsIDO1 protein is expressed in the majority of HR+ BC and is an independent negative prognostic marker. Additionally, IDO1 expression is negatively associated with tumor B-cell infiltration. Measurement of IDO1 has the potential to identify a population that might derive benefit from IDO1 blockade.

Metastatic triple-negative breast cancers at first relapse have fewer tumor-infiltrating lymphocytes than their matched primary breast tumors: a pilot study

Tumor-infiltrating lymphocytes (TILs) convey clinically relevant information for primary breast cancers (PBCs). However, limited data characterizing the immunobiology of metastatic breast cancers (MBCs) are available. Here, we examine TILs in surgically resected MBCs relative to their matched PBCs. Tissue microarrays of PBCs and MBCs were labeled for CD3 (total T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), FoxP3 (regulatory T cells), and CD20 (B cells) to characterize TILs. Expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 (HER-2) classified the tumors as luminal (ER(+)/PR(+)/HER-2(-)), triple negative (ER(-)/PR(-)/HER-2(-)), or HER-2(+) (ER(-)/PR(-)/HER-2(+)). These analyses reveal 5 novel findings. First, MBCs overall contained fewer TILs (mean, 35.1 CD3(+) TILs/high-power field [hpf]) than their matched PBCs (mean, 23.6 CD3(+) TILS/hpf), with fewer CD20(+) cells than CD3(+) cells in PBC and MBC (P = .0247). Second, the number of CD3(+), CD8(+), CD4(+), and FoxP3 TILs was decreased in triple-negative MBCs relative to matched PBCs, whereas only CD8(+) TILs were decreased in luminal MBCs relative to matched PBCs. Third, triple-negative MBCs contain fewer TILS (mean, 16 CD3(+) TILs/hpf) than luminal MBCs (mean, 21.7 CD3(+) TILs/hpf). Fourth, brain metastases contained fewer TILs relative to MBC from other sites. Finally, in this series, a CD8(+)/FoxP3(+) T-cell ratio of 3 or greater in PBCs was associated with improved overall survival from diagnosis, whereas a CD8(+)/FoxP3(+) T-cell ratio less than 3 in MBCs at first relapse was associated with improved overall survival. These findings suggest that evaluating the immunologic microenvironment of both PBCs and MBCs may yield important clinical information to guide breast cancer prognosis and therapy.

Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple-negative breast cancer (TNBC): The immune and IGFR pathways.

1058 Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC definition by immunohistochemistry (IHC). In a phase II neoadjuvant clinical trial targeting EGFR in TNBC, we investigated various biomarkers to better identify an EGFR-sensitive population for potential further regimen development. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm (20-120); invasive ductal carcinoma: 96.7%; SBR grade III: 71.7%; complete pathological response (pCR) rate: 55.3% and 46.8% (according to Sataloff’s and Chevallier’s classifications, respectively). Paraffin-embedded and frozen tumor samples were collected before and after ST for biologic studies. Germinal BRCA1 mutations, and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs) were evaluated by IHC. Results: High CD8+ TILs was response-predictive (p=3.4.10-6). Tumor FOXP3 expression and high FOXP3 TILs tended to be predictive. High IGF-1R expressors responded better than low expressors (p = 0.028). Comparison of EGFR, IGF-1R and Her3 in biopsies versus surgical samples showed reduced EGFR levels in non-responders (p = 0.037), while Her3 (p = 0.049) and IGF-1R (p = 0.08) increased. Sequencing revealed BRCA1 mutations in 10% of pts. No difference of response (pCR) was observed between mutated patients and others. Somatic mutations of PI3K were observed in 6 pts. No mutations were observed in BRAF, KRAS, or EGFR. Conclusions: The CD8+ TIL count seems to predict the response to panitumumab. Tumor FOXP3 expression and high FOXP3 TILs also tended to be predictive. Tumor levels of IGF-1R seem to play a determinant role in TNBC response to anti-EGFR antibodies, in concordance with our observations in a head-and-neck cancer cohort. Clinical trial information: NCT00933517.

Abstract 4669: Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways.

Abstract Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC definition by immunohistochemistry (IHC) (Nielsen, 2004). In a phase II neoadjuvant clinical trial targeting EGFR in TNBC, we investigated various biomarkers to better identify an EGFR-sensitive population for potential further regimen development. Methods: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. Systemic treatment (ST) consisted of the anti-EGFR antibody panitumumab combined with FEC 100, followed by 4 cycles of docetaxel. All pts underwent surgery after ST completion. Patient characteristics: median tumor size: 40 mm (20-120); invasive ductal carcinoma: 96.7%; SBR grade III: 71.7%; complete pathological response (pCR) rate: 46.8% (Chevallier's classification). Paraffin-embedded and frozen tumor samples were collected before and after ST for biologic studies. Germinal BRCA1 mutations, and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed by NGS. EGFR, IGF-1R, MET, cytokeratins 5/6 and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs) were evaluated by IHC. Biopsies and surgical samples were analysed using Affimetrix arrays. Results : By univariate analysis, high CD8+ TILs was response-predictive (pCR rates: CD8 TILs: 84% high vs 10% low; p=3.4.10−6). Tumor FOXP3 expression and high FOXP3 TILs tended to be predictive (pCR rates: tumor FOXP3: 77% positive vs 36% negative, p=0.07; FOXP3 TILs: 66% high vs 37% low, p= 0.08). High IGF-1R expressors responded better than low expressors (80% vs 33%, respectively, p=0.028). As could be expected, a positive correlation between tumor FOXP3 expression and FOXP3 TILs was found (p = 2.7.10−4, r = 0.56). Surprisingly, a positive correlation was found between FOXP3 TILs and CD8+ TILs (p = 2.10−5, r = 0.59) and between tumor FOXP3 and CD8+ TILs (p = 6.3.10−3, r = 0.43). Comparison of EGFR, IGF-1R and Her3 in biopsies versus surgical samples showed reduced EGFR levels in non-responders (p = 0.037), while Her3 (p = 0.049) and IGF-1R (p = 0.08) increased. Sequencing revealed BRCA1 mutations in 10% of pts. No difference of response (pCR) was observed between mutated patients and others (p=0.91). Somatic mutations of PI3K were observed in 6 pts. No mutations were observed in BRAF, KRAS, or EGFR. Analysis of Affymetrix arrays for gene expression is underway and will be presented. Conclusions : Interestingly, the CD8+ TIL count seems to predict the response to panitumumab. Tumor FOXP3 expression and high FOXP3 TILs also tended to be predictive. Tumor levels of IGF-1R seem to play a determinant role in TNBC response to anti-EGFR antibodies, in concordance with our observations in a head-and-neck cancer cohort (Clin Canc Res 2012). Confirmatory and mechanistic studies of those biomarkers are warranted. Citation Format: Frederique Penault-llorca, Catherine Abrial, Marie-Melanie Dauplat, Maud Privat, Nancy Uhrhammer, Alexis Desrichard, Yannick Bidet, Nina Radosevic-Robin, Anne Cayre, Cecile Aube, Fabrice Kwiatkowski, Nassera Chalabi, Yves-Jean Bignon, Philippe Chollet, Jean-Marc Nabholtz. Response to the anti-EGFR antibody panitumumab combined with standard neoadjuvant chemotherapy in triple negative breast cancer (TNBC): the immune and IGFR pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4669. doi:10.1158/1538-7445.AM2013-4669