Raoul A. Droeser

High frequency of tumor-infiltrating FOXP3+ regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients†

Regulatory T cells (Treg) inhibit the generation of host‐versus‐tumor immunity via suppression of tumor‐specific effector T‐cell responses and development of immune tolerance to neoplastic cells. The transcription factor forkhead box P3 (FOXP3) is an intracellular key molecule for Treg development and function and is considered to represent the most specific Treg cell marker. The aim of this study was to analyze the frequency and prognostic impact of tumor‐infiltrating FOXP3+ Treg in colorectal cancer (CRC) stratified by mismatch‐repair (MMR) status. Using the tissue microarray technique, 1,420 tumor samples were immunohistochemically stained for FOXP3 and stratified into 1,197 MMR‐proficient and 223 MMR‐deficient CRCs. Additionally, the 1,197 MMR‐proficient CRCs were randomized into 2 subgroups (Test Groups 1 and 2; n = 613 and 584, respectively). In both MMR‐proficient CRC subgroups high frequency tumor‐infiltrating FOXP3+ Treg was associated with early T stage (p = 0.001 and <0.001), tumor location (p = 0.01 and 0.045) and increased 5‐year survival rate (p = 0.004 and <0.001), whereas in MMR‐deficient CRCs an association between FOXP3+ Treg and absence of lymph node involvement (p = 0.023), absence of vascular invasion (p = 0.023) and improved 5‐year survival rate (p = 0.029) could be detected. In a multivariable analysis including age, gender, T stage, N stage, tumor grade, vascular invasion, and tumor border configuration, a high FOXP3+ Treg frequency was an independent prognostic factor in both MMR‐proficient CRC subsets (p = 0.019 and p = 0.007), but not in the MMR‐deficient CRCs (p = 0.13). Therefore, high frequency of tumor‐infiltrating FOXP3+ Treg is associated with early T stage and independently predicts improved disease‐specific survival in MMR‐proficient CRC patients.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer.

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P = 0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P = 0.0001) and validation (P = 0.03) sets. A similar trend (P = 0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n = 42) were found to be significantly associated (r = 0.33, P = 0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Tumor infiltration by FcγRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan–Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearmans rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6–0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7–0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3–0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64− HLA‐DR‐myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.

High Myeloperoxidase Positive Cell Infiltration in Colorectal Cancer Is an Independent Favorable Prognostic Factor

Background Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG). Methods A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets. Results MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO−. Conclusions High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC.

Differential pattern and prognostic significance of CD4+, FOXP3+ and IL-17+ tumor infiltrating lymphocytes in ductal and lobular breast cancers

BackgroundClinical relevance of tumor infiltrating lymphocytes (TILs) in breast cancer is controversial. Here, we used a tumor microarray including a large series of ductal and lobular breast cancers with long term follow up data, to analyze clinical impact of TIL expressing specific phenotypes and distribution of TILs within different tumor compartments and in different histological subtypes.MethodsA tissue microarray (TMA) including 894 ductal and 164 lobular breast cancers was stained with antibodies recognizing CD4, FOXP3, and IL-17 by standard immunohistochemical techniques. Lymphocyte counts were correlated with clinico-pathological parameters and survival.ResultsCD4+ lymphocytes were more prevalent than FOXP3+ TILs whereas IL-17+ TILs were rare. Increased numbers of total CD4+ and FOXP3+ TIL were observed in ductal, as compared with lobular carcinomas. High grade (G3) and estrogen receptor (ER) negative ductal carcinomas displayed significantly (p < 0.001) higher CD4+ and FOXP3+ lymphocyte infiltration while her2/neu over-expression in ductal carcinomas was significantly (p < 0.001) associated with higher FOXP3+ TIL counts. In contrast, lymphocyte infiltration was not linked to any clinico-pathological parameters in lobular cancers. In univariate but not in multivariate analysis CD4+ infiltration was associated with significantly shorter survival in patients bearing ductal, but not lobular cancers. However, a FOXP3+/CD4+ ratio > 1 was associated with improved overall survival even in multivariate analysis (p = 0.033).ConclusionsDuctal and lobular breast cancers appear to be infiltrated by different lymphocyte subpopulations. In ductal cancers increased CD4+ and FOXP3+ TIL numbers are associated with more aggressive tumor features. In survival analysis, absolute numbers of TILs do not represent major prognostic indicators in ductal and lobular breast cancer. Remarkably however, a ratio > 1 of total FOXP3+/CD4+ TILs in ductal carcinoma appears to represent an independent favorable prognostic factor.

NK cells and T cells cooperate during the clinical course of colorectal cancer

Recent evidence suggests that natural killer (NK) cells are typically defective in infiltrating solid tumors, with the exception of gastrointestinal stromal tumors (GIST). Interestingly, however, infrequently infiltrating NK cells do not appear to have a direct effect on tumor progression. Here, prompted by the recent evidence that NK cell and T cell crosstalk may trigger, or enhance, tumor antigen-specific immune responses, we have tested the clinical significance of this reciprocal signaling. To this end, a tissue microarray constructed with 1410 colorectal carcinoma (CRC) patient specimens was stained with NK and T cell antigen-specific monoclonal antibodies, utilizing the immunoperoxidase staining technique. Cut-off scores for positive (>4 NK cells) and negative (≤4 NK cells) NK cell CRC patient samples were determined using receiver operating characteristic curve analysis. Using this approach, NK cells were detected in 423 (30%) of the 1410 CRC specimens evaluated. The number of NK cells was >4 in only 132 (9%) of CRC samples. Correlation of the immunohistochemical staining results together with analysis of the clinical course of the disease revealed that the infiltration of colorectal tumors with both NK cells and CD8+ T cells is associated with prolonged patient survival. In contrast, infiltration of tumors with NK cells in combination with CD3+ and CD4+ T lymphocytes had no detectable effect on the clinical course of the disease. These results suggest that NK cell and CD8+ T cell crosstalk in the tumor microenvironment may benefit patient outcome and further, that the enumeration of infiltrating NK and CD8+ T cells in CRC tumors may provide useful prognostic information.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

Background The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. Objective To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. Methods IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. Results IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. Conclusions Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

The interplay between neutrophils and CD8+ T cells improves survival in human colorectal cancer.

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer–infiltrating CD66b+ neutrophils and their crosstalk with CD8+ T cells. Experimental Design: CD66b+ and CD8+ cell infiltration was analyzed by IHC on a tissue microarray including >650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b+ cells were evaluated by flow cytometry. CD66b+/CD8+ cells crosstalk was investigated by in vitro experiments. Results: CD66b+ cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8+ T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8+ T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8+ cell stimulation in the presence of CD66b+ cells results in increasing numbers of cells expressing CD45RO/CD62L “central memory” phenotype. Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with significantly better prognosis, as compared with CD8+ T-cell infiltration alone. Conclusions: Neutrophils enhance the responsiveness of CD8+ T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8+ T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 23(14); 3847–58. ©2017 AACR.

Long-term follow-up of a randomized controlled trial of Lichtenstein's operation versus mesh plug repair for inguinal hernia.

Objective:To compare long-term results of Lichtensteins operation versus mesh plug repair for open inguinal hernia repair. Background:The technique of best choice in open prosthetic inguinal hernia repair remains a subject of ongoing debate. Methods:In this prospective, randomized controlled multicenter trial, patients with primary or recurrent inguinal hernias were randomized to undergo either Lichtensteins operation or mesh plug repair. The primary endpoint was the long-term recurrence rate. Secondary endpoints included chronic pain, sensibility disorders, and reoperation rate. Results:In total, 697 hernias in 594 patients were randomized (297 patients per group). At a median follow-up of 6.5 years, 528 (76%) operated hernias in 444 (75%) patients were clinically evaluated. The recurrence rate was similar in both groups [mesh plug: 21/268 hernias = 7.8%; Lichtenstein: 21/260 hernias = 8.1%; adjusted odds ratio (OR): 0.92; 95% confidence interval (CI): 0.51, 1.68; P = 0.795]. We did not find a significant difference for chronic pain (Visual Analog Scale score >3) (OR: 0.58; 95% CI: 0.31, 1.09; P = 0.088) and sensory testing (17% vs 20% of patients; OR: 0.53; 95% CI: 0.21, 1.37; P = 0.190) between the 2 groups. There were less reoperations in the mesh plug than in the Lichtensteins operation group (OR: 0.43; 95% CI: 0.22, 0.85; P = 0.016). Conclusions:The long-term results of this trial indicate not enough evidence for differences in recurrence, chronic pain, and sensibility disorders between mesh plug repair and Lichtensteins operation but a lower likelihood for reoperation for mesh plug repair. Estimates for all endpoints were statistically not significant or based on large CIs. Clinical Trials Registration:ClinicalTrials.gov Identifier: NCT01637818.

Nuclear Expression of Snail Is an Independent Negative Prognostic Factor in Human Breast Cancer

Background. Snail is a key regulator of epithelial-mesenchymal transition of tumor cells. Several studies have shown nuclear Snail expression to be a negative prognostic factor in human cancer, where it is generally associated with more aggressive tumor behavior and worse survival. Objectives and Methods. To further explore the role of Snail expression in breast cancer, we conducted a study on a tissue microarray, encompassing 1043 breast cancer cases. Results. A total of 265 (25.4%) breast cancers were positive for Snail. Snail expression was significantly associated with greater tumor size, higher tumor stage and grade, positive lymph node status, and hormone receptor negative status and was differently expressed in the intrinsic subtypes of breast cancer, being the highest in the basal-like subtype and the lowest in the luminal A subtype. In multivariate analysis, Snail proved to be an independent negative prognostic factor for OS. In the intrinsic subtypes, Snail expression was a negative prognostic factor for OS in the luminal B HER2−, the luminal B HER2+, and the basal-like subtype. Conclusions. This is the first study demonstrating that nuclear Snail expression is an independent negative predictor of prognosis in breast cancer, thus suggesting that it may represent a potential therapeutic target.