CDDO-Me Redirects Activation of Breast Tumor Associated Macrophages

Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer.     

Polarisation of Tumor-Associated Macrophages toward M2 Phenotype Correlates with Poor Response to Chemoradiation and Reduced Survival in Patients with Locally Advanced Cervical Cancer

Objective

we investigate the prognostic role of pre-treatment ratio between Type 1 (M1) and Type 2 (M2) tumor-associated macrophages (TAMs) in locally advanced cervical cancer (LACC) patients treated with chemoradiation (CT/RT).

Methods

84 consecutive LACC patients treated with cisplatin-based CT/RT for a total dose of 50.0 Gy, followed by radical surgery were analysed. Double-staining immunohistochemistry of CD163/p-STAT, CD68/pSTAT1, CD163/c-MAF, and CD68/c-MAF was performed on tumor samples taken at the time of diagnosis. TAMs with CD163+pSTAT1+, or CD68+pSTAT1+ were defined M1; CD163+c-MAF+ or CD68+c-MAF+ defined the M2 phenotype. The number of M1 and M2 cells was counted at low magnification by evaluating for each case the same tumour area. The ratio between M1 and M2 (M1/M2) was finally calculated.

Results

At diagnosis, we observed a direct correlation between the number of circulating monocytes and of TAMs (p-value = 0.001). Patients with high M1/M2 experienced more frequently complete pathologic response (no residual tumor) to CT/RT, compared to cases with low M1/M2 (55.0% Vs 29.5%; p-value = 0.029). At multivariate analysis M1/M2 (OR = 2.067; p-value = 0.037) emerged as independent predictor of pathologic response to CT/RT. Women with high M1/M2 showed a longer 5-yrs Disease-free (67.2% Vs. 44.3%; p-value = 0.019), and 5-yrs Overall (69.3% Vs. 46.9%; p-value = 0.037) survival, compared to cases with low M1/M2. The presence of a high M1/M2 ratio was independently associated with an unfavourable survival outcome in multivariate analysis.

Conclusions

polarisation of TAMs toward a M2 phenotype, as reflected by a lower M1/M2 ratio, is an independent predictor of poor response to CT/RT, and shorter survival in LACC.     

Prognostic value of polarized macrophages in patients with hepatocellular carcinoma after curative resection

As the most predominant tumour‐infiltrating immune cells, tumour‐associated macrophages (TAMs) are significant for fostering tumour growth, progression and metastasis. CD68‐positive TAMs display dissimilarly polarized programmes comprising CD11c‐positive pro‐inflammatory macrophages (M1) and CD206‐positive immunosuppressive macrophages (M2). The aim of this study is to determine the prognostic significance of diametrically polarized TAMs in hepatocellular carcinoma (HCC) and their application to risk stratification of patients according to their specific prognostic values. This study included 80 consecutive patients with HCC, and we evaluated diametrically polarized functional status of macrophages by immunohistochemical staining of CD68, CD11c and CD206. Prognostic values and clinicopathologic features were assessed in these patients. High versus low CD11c‐positive TAM density (P = 0.005) and low versus high CD206‐positive TAM density (P = 0.002) were associated with better overall survival, whereas CD68‐positive TAM density had no prognostic significance (low versus high, P = 0.065). Furthermore, the presence of these positive staining macrophages did not show any prognostic significance for recurrence‐free survival (all P > 0.05). Multivariate Cox regression analysis identified CD11c‐positive and CD206‐positive TAMs as an independent prognostic factor (P < 0.001, P = 0.031, respectively). Intratumoural infiltration of diametrically polarized TAMs, a novel identified independent prognostic factor for survival in patients with HCC, could be combined with the TNM stage and the Barcelona Clinic Liver Cancer stage to improve a risk stratification system.     

The Clinical Significance of the CD163+ and CD68+ Macrophages in Patients with Hepatocellular Carcinoma

Our previous study has found that the abundance of peritumoral CD68⁺ macrophages was associated with poor prognosis in hepatocellular carcinoma (HCC) after resection. However, CD68 staining could not discriminate the protumoral or tumoricidal subpopulations from pan-macrophages. CD163 is a marker of alternatively activated macrophages. In this study, the clinical significance of CD163⁺ cells in tumors and peritumoral liver tissues was evaluated in a cohort of 295 patients with HCC after curative resection. We found that the density of CD163⁺ cells was well correlated with that of CD68⁺ cells in both tumors and peritumoral liver tissues but was much more. Immunostaining on consecutive sections and flow cytometry assay on surgical resected specimens further supported the findings that the CD163⁺ cells was more abundant than CD68⁺ cells. The density of peritumoral CD68⁺ cells was associated with poor recurrence-free survival (RFS) and poor overall survival (OS) (P = 0.004 and P = 0.001, respectively), whereas the CD163⁺ cells have no prognostic values either in tumors or in peritumoral liver tissues. In another cohort of 107 HCC patients, preoperative plasma concentration of soluble form of CD163 (sCD163) was associated with active hepatitis-related factors but not associated with the markers of tumor invasion. In conclusion, both the CD163⁺ cells local infiltration and plasma sCD163 were of limited significance in HCC, and they were more likely markers related to active hepatitis rather than tumor progression.     

Identification of miR-30e* Regulation of Bmi1 Expression Mediated by Tumor-Associated Macrophages in Gastrointestinal Cancer

Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3′ untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.     

CD163 Expression Was Associated with Angiogenesis and Shortened Survival in Patients with Uniformly Treated Classical Hodgkin Lymphoma

Background

Recent studies have reported the prognostic value of tissue-associated magrophages (TAMs) in classical Hodgkin lymphoma (cHL). In addition, TAMs are implicated in the tumor angiogenesis. In this study, we examined the prognostic relevance of TAMs in relation to vascular endothelial growth factor (VEGF) expression and angiogenesis in uniformly treated cases of cHL.

Methods

Diagnostic tissue from 116 patients with ABVD-treated cHL was evaluated retrospectively by immunohistochemical analysis for CD68, CD163 and VEGF expression and for CD31 expression as a measure of microvessel density (MVD).

Results

High CD163 expression (≥35% of cellularity) correlated with VEGF expression (Pearson’s Chi-square test, P = 0.008) and MVD (Spearman correlation coefficient 0.310, P<0.001). High CD163 expression was associated with inferior event-free survival (EFS, P = 0.005) and overall survival (OS, P<0.001) in univariate analysis. In multivariate analysis, high CD163 expression was strongly associated with inferior EFS (P = 0.043) and OS (P = 0.008). Patients with high MVD had a lower OS than those with low MVD, but the difference was not significant (P = 0.071, respectively). While high expression of CD68 was also associated with inferior EFS (P = 0.007), it showed no correlation with VEGF or MVD.

Conclusions

Our data confirms that CD163 expression provides independent prognostic information in cHL. The correlation of CD163 with VEGF expression and MVD suggests the role of CD163-positive cells in tumor angiogenesis of cHL.     

The correlation between tumor-associated macrophage infiltration and progression in cervical carcinoma

Tumor microenvironment (TME) plays a particularly important role in the progression, invasion and metastasis of cervical carcinoma (CC). Tumor-associated macrophages (TAMs) are significant components of the tumor microenvironment in CC. However, the results of studies on the correlation between TAMs and progression in CC are still controversial. This research aimed to investigate the relationship between TAMs infiltration and progression in CC. A total of 100 patients with CC were included in the study. The correlation between TAMs and clinicopathologic features was studied. Besides, a systematic literature search was conducted from legitimate electronic databases to specifically evaluate the role of TAMs in TME of cervical carcinoma. In the meta-analysis, high stromal CD68⁺ TAMs density was relevant to lymph node metastasis (WMD = 11.89, 95% CI: 5.30–18.47). At the same time, CD163⁺ M2 TAM density was associated with lymph node metastasis (OR = 2.42, 95% CI: 1.09–5.37; WMD = 39.37, 95% CI: 28.25–50.49) and FIGO stage (WMD = -33.60, 95% CI: -45.04 to -22.16). This was further confirmed in the experimental study of 100 tissues of cervical cancer. It supported a critical role of TAMs as a prospective predictor of cervical cancer. In conclusion, CD68⁺ TAM and CD163⁺ M2 TAM infiltration in CC were associated with tumor progression. And CD163⁺ M2 TAM infiltration was associated with more advanced FIGO stage and lymph node metastasis in CC.     

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.     

The Distribution of Macrophages with a M1 or M2 Phenotype in Relation to Prognosis and the Molecular Characteristics of Colorectal Cancer

High macrophage infiltration has been correlated to improved survival in colorectal cancer (CRC). Tumor associated macrophages (TAMs) play complex roles in tumorigenesis since they are believed to hold both tumor preventing (M1 macrophages) and tumor promoting (M2 macrophages) activities. Here we have applied an immunohistochemical approach to determine the degree of infiltrating macrophages with a M1 or M2 phenotype in clinical specimens of CRC in relation to prognosis, both in CRC in general but also in subgroups of CRC defined by microsatellite instability (MSI) screening status and the CpG island methylator phenotype (CIMP). A total of 485 consecutive CRC specimens were stained for nitric oxide synthase 2 (NOS2) (also denoted iNOS) as a marker for the M1 macrophage phenotype and the scavenger receptor CD163 as a marker for the M2 macrophage phenotype. The average infiltration of NOS2 and CD163 expressing macrophages along the invasive tumor front was semi-quantitatively evaluated using a four-graded scale. Two subtypes of macrophages, displaying M1 (NOS2⁺) or M2 (CD163⁺) phenotypes, were recognized. We observed a significant correlation between the amount of NOS2⁺ and CD163⁺ cells (P<0.0001). A strong inverse correlation to tumor stage was found for both NOS2 (P<0.0001) and CD163 (P<0.0001) infiltration. Furthermore, patients harbouring tumors highly infiltrated by NOS2⁺ cells had a significantly better prognosis than those infiltrated by few NOS2⁺ cells, and this was found to be independent of MSI screening status and CIMP status. No significant difference was found on cancer-specific survival in groups of CRC with different NOS2/CD163 ratios. In conclusion, an increased infiltration of macrophages with a M1 phenotype at the tumor front is accompanied by a concomitant increase in macrophages with a M2 phenotype, and in a stage dependent manner correlated to a better prognosis in patients with CRC.     

Higher numbers of T-bet+ intratumoral lymphoid cells correlate with better survival in gastric cancer

In the present study, we studied the expression of T-bet, a key marker for type 1 immune responses, within the tumor microenvironment of gastric cancer, and analyzed its association with clinicopathological parameters. One hundred and fifty-two archival paraffin-embedded gastric tumor tissues were collected, and the expression of T-bet in these cancer tissue specimens was examined by immunohistochemistry. T-bet⁺ tumor-infiltrating lymphocytes (TILs) in some gastric cancer tissues were further characterized by flow cytometric analysis. The density of T-bet⁺ TILs in gastric cancer tissues in relation to patient’s clinicopathological parameters and postoperative prognosis has been analyzed. Herein, we have found significant increases in T-bet⁺ lymphocytes in tumor tissues as compared with normal stomach tissues, gastritis tissues or gastric polyp specimens. T-bet⁺ cells mainly consisted of CD4⁺, CD8⁺ and CD56⁺ TILs. In addition, lower numbers of T-bet⁺ TILs were associated with poor clinicopathological parameters such as invasion to muscular layer, larger tumor size and advanced cancer stages. Moreover, patients with higher numbers of T-bet⁺ TILs have longer disease-free survival and overall survival. Thus, our study supports the idea that tumor growth elicits spontaneous type 1 cellular immune responses and tumor progression is associated with suppression of antitumor immunity. T-bet expression within tumor can serve as a prognostic indicator for gastric cancer and a potential biomarker for immunotherapy.     

Integration of OV6 expression and CD68+ tumor-associated macrophages with clinical features better predicts the prognosis of patients with hepatocellular carcinoma

Background: Reliable prognostic indicators for accurately predicting postoperative outcomes in Hepatocellular carcinoma (HCC) patients are lacking. Although cancer stem-like cells (CSCs) and tumor-associated macrophages (TAMs) in tumor microenvironment are implicated in the occurrence and development of HCC, whether the combination of CSC biomarkers and TAM populations could achieve better performance in predicting the prognosis of patients with HCC has been rarely reported.Methods: A total of 306 HCC patients were randomly divided into the training and validation cohorts at a 1:1 ratio, and the expression of OV6 and CD68 was assessed using immunohistochemistry in HCC samples. The prognostic value of these biomarkers for post-surgical survival and recurrence were evaluated by the curve of receiver operating characteristic and multivariate Cox regression analyses.Results: The density of OV6⁺ CSCs was positively correlated with the infiltration of CD68⁺ TAMs in HCC. Both high OV6 expression and CD68⁺ TAM infiltration was closely associated with poor overall survival (OS) and progression-free survival (PFS) of HCC patients. Moreover, overexpression of OV6 and infiltration of CD68⁺ TAMs were identified as independent prognostic factors for OS and PFS after liver resection. The integration of OV6 and CD68 with tumor size and microvascular invasion exhibited highest C-index value for survival predictivity in HCC patients than any other biomarkers or clinical indicators alone.Conclusion: Incorporating intratumoral OV6 expression and CD68⁺ TAMs infiltration with established clinical indicators may serve as a promising prognostic signature for HCC, and could more accurately predict the clinical outcomes for HCC patients after liver resection.     

Prognostic Value of Tumor-Associated Macrophages According to Histologic Locations and Hormone Receptor Status in Breast Cancer

Tumor-associated macrophages (TAMs) are involved in tumor progression by promoting epithelial-mesenchymal transition (EMT), tumor cell invasion, migration and angiogenesis. However, in breast cancer, the clinical relevance of the TAM infiltration according to distinct histologic locations (intratumoral vs. stromal) and hormone receptor status is unclear. We investigated the significance of the levels of TAM infiltration in distinct histologic locations in invasive breast cancer. We also examined the relationship of the TAM levels with the clinicopathologic features of tumors, expression of EMT markers, and clinical outcomes. Finally, we analyzed the prognostic value of TAM levels according to hormone receptor status. High levels of infiltration of intratumoral, stromal and total TAMs were associated with high histologic grade, p53 overexpression, high Ki-67 proliferation index and negative hormone receptor status. Infiltration of TAMs was also correlated with overexpression of vimentin, smooth muscle actin and alteration of β-catenin. Overall, a high level of infiltration of intratumoral TAMs was associated with poor disease-free survival, and was found to be an independent prognostic factor. In subgroup analyses by hormone receptor status, a high level of infiltration of intratumoral TAM was an independent prognostic factor in the hormone receptor-positive subgroup, but not in the hormone-receptor negative subgroup. Our findings suggest that intratumoral TAMs play an important role in tumor progression in breast cancer, especially in the hormone receptor-positive group, and the level of TAM infiltration may be used as a prognostic factor and even a therapeutic target in breast cancer.     

Prognostic Significance of Interleukin-8 and CD163-Positive Cell-Infiltration in Tumor Tissues in Patients with Oral Squamous Cell Carcinoma

Purpose

We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC).

Experimental Design

Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens.

Results

We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p = 0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p = 0.033 and p = 0.038, respectively), and they were associated with poor clinical outcome (p = 0.007 and p = 0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10.

Conclusions

These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.     

胃癌中TAMs与血管生成及转移相关性的分析

目的：研究胃癌中肿瘤相关巨噬细胞（tumor associated macrophages,TAMs）的表达及其与肿瘤血管生成的关系,进而分析其与转移的相关性。方法：利用免疫组化双染方法同时标记TAMs（抗CD68抗体）和内皮细胞（抗CD34抗体）,检测90例胃癌中TAMs、微血管密度（microvessel density,MVD）。结果：①TAMs与胃癌的大小、淋巴结转移、远处转移呈显著正相关（P均〈0.05）;②MVD与胃癌的远处转移呈显著正相关（P均〈0.05）;③在胃癌组织中TAMs与MVD的表达呈显著的正相关（r=0.325,P=0.002）。结论：TAMs可能通过促进胃癌血管的生成,进而促进胃癌细胞发生血管内渗,最终实现胃癌细胞的转移。     

Effective Predictor of Colorectal Cancer Survival Based on Exclusive Expression Pattern Among Different Immune Cell Infiltration

Tumor-infiltrating immune/inflammatory cells, the important components of the tumor microenvironment (TME), remarkably affect the progression of human cancers. To understand the actual conditions within the TME of colorectal cancer (CRC), the interrelationship among tumor-infiltrating neutrophils, M2 macrophages, and regulatory T-cells (T_regs) was systematically analyzed. The infiltration conditions of CD66b⁺ neutrophils, CD163⁺ M2 macrophages, and FOXP3⁺ T_regs in tissue microarrays including 1021 cases of CRC were determined by immunohistochemical analysis. The prediction power of these immune cells for CRC prognosis was evaluated by subgroup analysis of the CRC cohort. Results revealed the existence pattern of infiltrating neutrophils, and T_regs/M2 macrophages fulfilled a “X-low implies Y-high” Boolean relationship, indicative of a mutually exclusive correlation between neutrophils and M2 macrophages, and between neutrophils and T_regs in the TME of CRC. What’s more, the tumor-infiltrating M2 macrophages and T_regs were associated with adverse prognostic factors, whereas neutrophils were corelated with favorable factors. The high infiltration of neutrophils predicted longer survival and better chemotherapeutic response. Nonetheless, high infiltration of M2 macrophages and T_regs predicted poor prognosis. The combination of these tumor-infiltrating immune cells can serve as an effective predictor for the survival of CRC and for the chemotherapeutic outcomes of stage II–III patients.      

The prognostic value of CD3+ tumor-infiltrating lymphocytes for stage II colon cancer according to use of adjuvant chemotherapy: A large single-institution cohort study

BackgroundHigh tumor infiltrating lymphocytes (TILs) density was previously shown to be associated with favorable prognosis for patients with colon cancer (CC). However, the impact of TILs on overall survival (OS) of stage II CC patients who received adjuvant chemotherapy (ADJ) or not (no-ADJ) is unknown. We assessed the prognostic value of CD3+ TILs in stage II CC patients according to whether they had ADJ or not.MethodsPatients treated with curative surgery for stage II CC (2002–2013) were selected from the Santa Maria alle Scotte Hospital registry. TILs at the invasive front, center of tumor, and stroma were determined by immunohistochemistry and manually quantified as the rate of TILs/total tissue areas. High TILs (H-TILs) was defined as >20%. Patients were categorized as high or low TILs (L-TILs) and ADJ or no-ADJ.ResultsOf the 678 patients included, 137 (20%) received ADJ and 541 (80%) did not. The distribution of the 4 groups were: 16% (L-TIL/ADJ), 64% (L-TIL/no-ADJ), 5% (H-TIL/ADJ), 15% (H-TIL/no-ADJ). Compared to H-TILs/no-ADJ, ADJ patients showed a significantly increased OS (P<.01) regardless of the TILs rate whereas L-TILs/no-ADJ had significantly decreased OS and higher risk of death (HR=1.41; 95% CI, 1.06–1.88; P<.0001). On multivariable analysis, the unfavorable prognostic value of L-TILs (vs. H-TILs) for no-ADJ patients was confirmed (HR=1.36; 95% CI 1.02, 1.82; P=.0373).ConclusionLow CD3+ TILs rate was associated with shorter OS in those with stage II colon cancer who did not receive adjuvant therapy. Low CD3+ TILs could be considered an additional risk factor for still ADJ-untreated stage II CC patients, which could facilitate clinical decision making.     

Tumor-associated macrophages (TAMs) form an interconnected cellular supportive network in anaplastic thyroid carcinoma

Background

A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC.

Methodology/Principal Findings

Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a “microglia-like” appearance on these cells which we termed “Ramified TAMs” (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells.

Conclusion

ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined.     

Analysis of FoxP3+ T-Regulatory Cells and CD8+T-Cells in Ovarian Carcinoma: Location and Tumor Infiltration Patterns Are Key Prognostic Markers

Purpose

Tumor infiltrating CD4+CD25+FoxP3+ regulatory immune cells (Treg) have been associated with impaired anti- tumor immune response and unfavorable prognosis for patients affected by ovarian carcinoma, whereas CD8+ T-cells have been found to positively influence survival rates in a large panel of solid tumors. Recently, density, location and tumor infiltration patterns of the respective immune cell subtypes have been identified as key prognostic factors for different types of tumors.

Patients and Methods

We stained 210 human ovarian carcinoma samples immunhistochemically for FoxP3 and CD8 to identify the impact different immune cell patterns have on generally accepted prognostic variables as well as on overall survival.

Results

We found that FoxP3+ cells located within lymphoid aggregates surrounding the tumor were strongly associated with reduced survival time (P = 0.007). Central accumulation of CD8+ effector cells within the tumor bed shows a positive effect on survival (P = 0,001).

Conclusion

The distribution pattern of immune cells within the tumor environment strongly influences prognosis and overall survival time of patients with ovarian carcinoma.     

CD4-Positive T Cells and M2 Macrophages Dominate the Peritoneal Infiltrate of Patients with Encapsulating Peritoneal Sclerosis

Background

Encapsulating peritoneal sclerosis (EPS) is a severe complication of peritoneal dialysis (PD). Previously, it has been shown that infiltrating CD4-positive T cells and M2 macrophages are associated with several fibrotic conditions. Therefore, the characteristics of the peritoneal cell infiltrate in EPS may be of interest to understand EPS pathogenesis. In this study, we aim to elucidate the composition of the peritoneal cell infiltrate in EPS patients and relate the findings to clinical outcome.

Study Design, Setting, and Participants

We studied peritoneal membrane biopsies of 23 EPS patients and compared them to biopsies of 15 PD patients without EPS. The cellular infiltrate was characterized by immunohistochemistry to detect T cells(CD3-positive), CD4-positive (CD4+) and CD8-positive T cell subsets, B cells(CD20-positive), granulocytes(CD15-positive), macrophages(CD68-positive), M1(CD80-positive), and M2(CD163-positive) macrophages. Tissues were analysed using digital image analysis. Kaplan-Meier survival analysis was performed to investigate the survival in the different staining groups.

Results

The cellular infiltrate in EPS biopsies was dominated by mononuclear cells. For both CD3 and CD68, the median percentage of area stained was higher in biopsies of EPS as opposed to non-EPS patients (p<0.001). EPS biopsies showed a higher percentage of area stained for CD4 (1.29%(0.61-3.20)) compared to CD8 (0.71%(0.46-1.01), p = 0.04), while in the non-EPS group these cells were almost equally represented (respectively 0.28%(0.05-0.83) versus 0.22%(0.17-0.43), p = 0.97). The percentage of area stained for both CD80 and CD163 was higher in EPS than in non-EPS biopsies (p<0.001), with CD163+ cells being the most abundant phenotype. Virtually no CD20-positive and CD15-positive cells were present in biopsies of a subgroup of EPS patients. No relation was found between the composition of the mononuclear cell infiltrate and clinical outcome.

Conclusions

A characteristic mononuclear cell infiltrate consisting of CD4+ and CD163+ cells dominates the peritoneum of EPS patients. These findings suggest a role for both CD4+ T cells and M2 macrophages in the pathogenesis of EPS.