Tumor-associated macrophages predict inferior outcomes in classic Hodgkin lymphoma: a correlative study from the E2496 Intergroup trial.
ABSTRACT: Increased tumor-associated macrophages (TAMs) are reported to be associated with poor prognosis in classic Hodgkin lymphoma (CHL). We investigated the prognostic significance of TAMs in the E2496 Intergroup trial, a multicenter phase 3 randomized controlled trial comparing ABVD and Stanford V chemotherapy in locally extensive and advanced stage CHL. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded tumor tissue and included 287 patients. Patients were randomly assigned into training (n = 143) and validation (n = 144) cohorts. Immunohistochemistry for CD68 and CD163, and in situ hybridization for EBV-encoded RNA were performed. CD68 and CD163 IHC were analyzed by computer image analysis; optimum thresholds for overall survival (OS) were determined in the training cohort and tested in the independent validation cohort. Increased CD68 and CD163 expression was significantly associated with inferior failure-free survival and OS in the validation cohort. Increased CD68 and CD163 expression was associated with increased age, EBV-encoded RNA positivity, and mixed cellularity subtype of CHL. Multivariate analysis in the validation cohort showed increased CD68 or CD163 expression to be significant independent predictors of inferior failure-free survival and OS. We demonstrate the prognostic significance of TAMs in locally extensive and advanced-stage CHL in a multicenter phase 3 randomized controlled clinical trial.
Project description:<h4>Background</h4>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.<h4>Methods</h4>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).<h4>Results</h4>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.<h4>Conclusions</h4>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.
Project description: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.
Project description:Tumor microenvironment and immune escape affect pathogenesis and survival in classical Hodgkin lymphoma (cHL). While tumor-associated macrophage (TAM) content has been associated with poor outcomes, macrophage-derived determinants with clinical impact have remained undefined. Here, we have used multiplex immunohistochemistry and digital image analysis to characterize TAM immunophenotypes with regard to expression of checkpoint molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1) from the diagnostic tumor tissue samples of 130 cHL patients, and correlated the findings with clinical characteristics and survival. We show that a large proportion of TAMs express PD-L1 (CD68+, median 32%; M2 type CD163+, median 22%), whereas the proportion of TAMs expressing IDO-1 is lower (CD68+, median 5.5%; CD163+, median 1.4%). A high proportion of PD-L1 and IDO-1 expressing TAMs from all TAMs (CD68+), or from CD163+ TAMs, is associated with inferior outcome. In multivariate analysis with age and stage, high proportions of PD-L1+ and IDO-1+ TAMs remain independent prognostic factors for freedom from treatment failure (PD-L1+CD68+/CD68+, HR = 2.63, 95% CI 1.17-5.88, p = 0.019; IDO-1+CD68+/CD68+, HR = 2.48, 95% CI 1.03-5.95, p = 0.042). In contrast, proportions of PD-L1+ tumor cells, all TAMs or PD-L1- and IDO-1- TAMs are not associated with outcome. The findings implicate that adverse prognostic impact of TAMs is checkpoint-dependent in cHL.
Project description:BACKGROUND:The role of tumor-associated macrophages (TAMs) in the cancer immune landscape and their potential as treatment targets or modulators of response to treatment are gaining increasing interest. TAMs display high molecular and functional complexity. Therefore their objective assessment as breast cancer biomarkers is critical. The aims of this study were to objectively determine the in situ expression and significance of TAM biomarkers (CD68, CD163, and MMP-9) in breast cancer and to identify subclasses of patients who could benefit from TAM-targeting therapies. METHODS:We measured CD68, CD163, and MMP-9 protein expression in formalin-fixed paraffin-embedded tissues of breast carcinomas represented in tissue microarray format using multiplexed quantitative immunofluorescence (QIF) in two independent Yale cohorts: cohort A-n = 398, estrogen receptor-positive (ER+) and ER- cases-and the triple-negative breast cancer (TNBC)-only cohort B (n = 160). Associations between macrophage markers, ER status, and survival were assessed. Protein expression measured by QIF was compared with mRNA expression data from the METABRIC study. RESULTS:All three macrophage markers were co-expressed, displaying higher expression in ER- cancers. High pan-macrophage marker CD68 correlated with poorer overall survival (OS) only in ER- cases of cohort A (P = 0.02). High expression of CD163 protein in TAMs was associated with improved OS in ER- cases (cohort A, P = 0.03 and TNBC cohort B, P = 0.04, respectively) but not in ER+ cancers. MMP-9 protein was not individually associated with OS. High expression of MMP-9 in the CD68+/CD163+ TAMs was associated with worse OS in ER+ tumors (P <0.001) but not in ER- cancers. In the METABRIC dataset, mRNA levels followed the co-expression pattern observed in QIF but did not always show the same trend regarding OS. CONCLUSIONS:Macrophage activity markers correlate with survival differently in ER+ and ER- cancers. The association between high co-expression and co-localization of MMP-9/CD163/CD68 and poor survival in ER+ cancers suggests that these cancers may be candidates for macrophage-targeted therapies.
Project description:BACKGROUND:Diffuse large B-cell lymphoma (DLBCL) is characterized by its clinical and biological heterogeneity. The clinical prognostic implications of tumor-associated macrophages (TAMs) in DLBCL remain controversial and the correlation between TAMs and peripheral absolute monocyte count (AMC) has not yet been elucidated. METHODS:In 221 untreated, newly diagnosed patients with DLBCL, we evaluated the prognostic value of TAMs using immunohistochemical analysis, as well as the association of TAMs and AMC. RESULTS:We found that high CD68 or high CD163 expression was correlated with clinicopathological characteristics, high CD163 expression was an adverse predictor for both overall survival (OS) [hazard ratio (HR)?=?2.265, P?=?0.005] and progression- free survival (PFS) (HR?=?1.925, P?=?0.017) in patients with DLBCL. Patients with high CD68 or high CD163 expression had significantly poorer OS and PFS than those with low CD68 or low CD163 expression, respectively (CD68: OS: P<0.001, PFS: P<0.001; CD163: OS: P<0.001, PFS: P<0.001), even in the rituximab era. Moreover, high-risk patients could be further identified by the expression of CD68 or CD163, especially in those classified as low/intermediate risk by International Prognostic Index (IPI). Furthermore, the significant positive correlation was also detected between CD68 expression or CD163 expression and AMC (r?=?0.256, P<0.001; r?=?0.303, P<0.001). CONCLUSIONS:Patients with high expression of TAMs tend to have poorer OS and PFS, even in the rituximab era, and have positive correlation with AMC. Therefore, the peripheral AMC is a useful prognostic marker reflecting the status of the tumor microenvironment (TME) in DLBCL.
Project description:Hodgkin's lymphoma (HL) is a hematological malignancy characterized by a minority of neoplastic cells outnumbered by tumor-associated macrophages (TAMs). The overexpression of the CD163 antigen by TAMs is considered to be a significant predictive biomarker for risk stratification. This is likely caused by a genetic single-nucleotide polymorphism (SNP) at the gene promoter. The aim of the present retrospective case-control study was to establish a gene expression profile of a specific biomarker for classical HL (CHL) in order to predict the outcome and survival of CHL patients in Saudi Arabia. The protein expression of CD163 on TAMs was studied using immunohistochemistry (IHC). A prognosis index was calculated for the CD163 protein to assess the risk stratification of CHL. Genotyping of selected SNPs of this antigen was performed for 100 CHL cases and controls. The analysis revealed that the CD163 protein expression level was significantly correlated with disease relapse (DR) and overall survival (OS). In addition, the CD163 index threshold (15.0) was found to be significantly correlated with the relapse rate. Among the studied CD163 SNPs, rs75608120 exhibited a significant correlation with the DR rate of CHL patients, but not with OS. The findings of the present study confirmed that CD163 is a specific marker for TAMs, and its overexpression by TAMs is significantly associated with relapse and reduced survival post-therapy. In addition, a new methodology of indexing CD163 protein expression for HL risk stratification was proposed. Thus, the present study identified a specific predictive molecular and antigenic biomarker for CHL prognosis.
Project description:Aims: Tumor associated macrophages (TAMs) play a critical role in the initiation and progression of breast cancer. However, their prognostic significance in the molecular subtype of basal-like breast cancer (BLBC) is poorly understood. The aim of this study was to investigate the extent and patterns of TAMs in BLBC and their associations with clinicopathological features and patient survival. Methods and Results: We evaluated TAMs in 200 cases of BLBC by immunohistochemistry using the M2 macrophage marker CD163 and the pan-macrophage marker CD68 in tumor nest and stroma, and assessed their prognostic significance. The study demonstrated that infiltration of CD163+ and CD68+ macrophages in tumor stroma was of clinical relevance in BLBC, but not those in tumor nest. Increased stromal infiltration of CD68+ or CD163+ macrophages correlated with larger tumor size, higher histological grade, higher 5-year recurrence and 5-year breast cancer mortality. Although both of CD68+ and CD163+ macrophages in tumor stroma were associated with poor recurrence-free survival (RFS) and overall survival (OS), multivariate analysis demonstrated that only CD163+ macrophage was an independent predictor of RFS and OS. Conclusions: Our results highlight the prognostic importance of TAMs' location in BLBC. CD163, a highly specific biomarker for M2 macrophages, is an independent prognostic marker for BLBC patients, and may serve as an indicator or potential target of macrophage-centred therapeutic strategies.
Project description:<h4>Background</h4>The roles of different subtypes of tumour-associated macrophages (TAMs) in predicting the prognosis of colorectal cancer (CRC) remain controversial. In this study, different subtypes of TAMs were investigated as prognostic and predictive biomarkers for CRC.<h4>Methods</h4>Expressions of CD68, CD86 and CD163 were investigated by immunohistochemistry (IHC) and immunofluorescence (IF), and the correlation between the expression of CD86 and CD163 was calculated in colorectal cancer tissues from 64 CRC patients.<h4>Results</h4>The results showed that high expressions of CD86<sup>+</sup> and CD68<sup>+</sup> CD86<sup>+</sup> TAMs as well as low expression of CD163<sup>+</sup> and CD68<sup>+</sup> CD163<sup>+</sup> TAMs were significantly associated with favourable overall survival (OS). The level of CD86 protein expression showed a negative correlation with CD163 protein expression. In addition, CD86 protein expression remarkably negatively correlated with tumour differentiation and tumour node metastasis (TNM) stage, while CD163 protein expression significantly positively correlated with tumour differentiation and tumour size. As an independent risk factor, high expression of CD86 TAMs had prominently favourable prognostic efficacy, while high expression of CD68<sup>+</sup> CD163<sup>+</sup> TAMs had significantly poor prognostic efficacy.<h4>Conclusions</h4>These results indicate that CD86<sup>+</sup> and CD68<sup>+</sup> CD163<sup>+</sup> TAMs as prognostic and predictive biomarkers for CRC.
Project description:<b>Background:</b> The interaction and crosstalk between tumor-associated macrophages (TAMs) and epithelial-mesenchymal transition (EMT) has been demonstrated to play a critical role in the progression and metastasis of multiple cancers. However, the roles of the M2-polarized TAMs in different tumor location in EMT and prognosis of colorectal cancer (CRC) have not been elucidated. Therefore, the present study was designed to set up a reliable ratio of CD163<sup>+</sup>/CD68<sup>+</sup> to assess M2-polarized TAMs infiltration in the tumor center (TC) and tumor invasive front (TF) and to further evaluate their prognostic value and biological effects on tumor cells in CRC. <b>Methods:</b> TAMs markers (CD68 and CD163) and EMT markers (E-cadherin and Vimentin) expression were evaluated by immunohistochemistry in 81 patients with CRC. Circulating tumor cells (CTCs) of peripheral blood from above patients was also isolated. The correlation of CD163<sup>+</sup>/CD68<sup>+</sup> ratio in different locations, EMT and CTCs counts were further analyses. Kaplan-Meier and the model analyses of univariate Cox proportional hazards were utilized to compare the survival of patients with high CD163<sup>+</sup>/CD68<sup>+</sup> ratio with those with low CD163<sup>+</sup>/CD68<sup>+</sup> ratio. Furthermore, the effects of the M2-polarized TAMs on growth, migration and invasion of CRC cells were explored <i>in vivo</i> and <i>in vitro</i> co-culture system. <b>Results</b>: The results showed that the level of CD163<sup>+</sup>/CD68<sup>+</sup> ratio in TF was significant higher than that in TC, and higher CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> ratio were closely correlated with enhanced lymphovascular invasion, tumor invasion and TNM stage. Interestingly, higher CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> ratio were also significantly associated with EMT program and CTCs counts. Meanwhile, Kaplan-Meier analysis showed that CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> was associated with both recurrence-free survival (RFS) and overall survival (OS) of patients with CRC. Multivariate Cox regression analyses demonstrated that CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> remained an independent prognostic factor for RFS and OS. Further receiver operating characteristic (ROC) curve analysis found that CD163<sup>+</sup>/CD68<sup>+</sup> <sub>TF</sub> was a better prognosticator compared with CD68<sup>+</sup> <sub>TF</sub> and CD163<sup>+</sup> <sub>TF</sub> for CRC patients. What's more, M2-polarized TAMs secreted TGF-? to facilitate the EMT, growth, proliferation and invasion of CRC cells by <i>in vivo</i> and <i>in vitro</i> experiments. <b>Conclusions:</b> Our studies preliminarily elucidated the prognostic value of CD163<sup>+</sup>/CD68<sup>+</sup> ratio in different tumor locations and the biological functions of M2-polarized TAMs in CRC progression via TGF-?.
Project description:Tumor-associated macrophages (TAMs) are correlated with the prognosis of different types of solid tumors and lymphoma, according to many clinical studies. In vitro experiments have demonstrated the roles of these cells in myeloma cell survival, angiogenesis, immunomodulation, drug resistance, and the interaction between malignant myeloma cells and the microenvironment. Here, we investigated the prognostic significance of TAMs in patients with multiple myeloma (MM). We evaluated the polarized functional status of bone marrow infiltrated by TAMs by immunohistochemical staining of CD68, iNOS, and CD163 in 240 patients with MM from January 2009 to December 2014. The overall response rates to chemotherapy were lower in patients with high CD68+ or CD163+ TAM densities than in those with low densities. Kaplan-Meier analysis showed that the progression-free survival (PFS, p = 0.001) and overall survival (OS, p < 0.001) of patients with low CD163+ TAM density were significantly higher than those of patients with high CD163+ TAM density. Furthermore, combined analysis of iNOS+ and CD163+ TAMs (iNOS/CD163 signature) exhibited greater power in predicting patient outcomes for both PFS (p < 0.001) and OS (p < 0.001). Moreover, Cox regression analysis identified iNOS+ and CD163+ TAMs as independent prognostic factors (p = 0.007, p < 0.001, respectively). These factors could be combined with the international staging system (ISS) to generate a predictive nomogram for patient outcomes. Our findings suggest that the mosaic of diametrically polarized TAMs is a novel independent prognostic factor that could be integrated into the evaluation of and therapy for MM.