Lower Expression Of CD163 Research Articles

Integrative analysis of H&E and IHC identifies prognostic immune subtypes in HPV related oropharyngeal cancer

BackgroundDeep learning techniques excel at identifying tumor-infiltrating lymphocytes (TILs) and immune phenotypes in hematoxylin and eosin (H&E)-stained slides. However, their ability to elucidate detailed functional characteristics of diverse cellular phenotypes within tumor immune microenvironment (TME) is limited. We aimed to enhance our understanding of cellular composition and functional characteristics across TME regions and improve patient stratification by integrating H&E with adjacent immunohistochemistry (IHC) images.MethodsA retrospective study was conducted on patients with Human Papillomavirus-positive oropharyngeal squamous cell carcinoma (OPSCC). Using paired H&E and IHC slides for 11 proteins, a deep learning pipeline was used to quantify tumor, stroma, and TILs in the TME. Patients were classified into immune inflamed (IN), immune excluded (IE), or immune desert (ID) phenotypes. By registering the IHC and H&E slides, we integrated IHC data to capture protein expression in the corresponding tumor regions. We further stratified patients into specific immune subtypes, such as IN, with increased or reduced CD8+ cells, based on the abundance of these proteins. This characterization provided functional insight into the H&E-based subtypes.ResultsAnalysis of 88 primary tumors and 70 involved lymph node tissue images reveals an improved prognosis in patients classified as IN in primary tumors with high CD8 and low CD163 expression (p = 0.007). Multivariate Cox regression analysis confirms a significantly better prognosis for these subtypes.ConclusionsIntegrating H&E and IHC data enhances the functional characterization of immune phenotypes of the TME with biological interpretability, and improves patient stratification in HPV( + ) OPSCC.

Tumor microenvironment and clinical efficacy of first line immunotherapy-based combinations in metastatic renal cell carcinoma

The impact of tumor microenvironment (TME) in influencing clinical response to first-line immune checkpoint inhibitor (ICI)-based treatment in advanced renal cell carcinoma (RCC) is unclear. Immunohistochemistry (IHC) could identify biomarkers related to immune checkpoints and immune cell population. This study retrospectively characterized TME from 28 RCC patients who received first line ICI-based therapy through IHC assessment of selected markers and explored preliminary evidence about their possible correlation with treatment efficacy. We found a significantly higher count of CD80+, CD163+ cells and their ratio in RCC with clear cell component compared to those without clear cell features; additionally, patients with metastatic disease at diagnosis were associated with higher expression of CD163+ cells, while higher count of CD4+ cells and CD4+/CD8+ ratio were found in RCC with sarcomatoid features. Patients achieving partial or complete response were associated with lower expression of CD163+ cells (median 28 vs 47; p = 0.049). Furthermore, lower expression of CD163+ was associated with better PFS (median PFS 20.0 vs 4.7 months; HR 0.22 p = 0.011) and OS (median OS NR vs 14.4 months; HR 0.28 p = 0.036). A longer OS was reported in PD-L1 CPS negative patients (median OS NR vs 11.8 months; HR 0.20 p = 0.024). High infiltration of CD163+ macrophages, who typically present “anti-inflammatory” M2-like phenotype, could identify a subgroup of patients with poor survival after receiving first-line ICI.

Prophylaxis of decidual CD68+/CD163+ macrophage disbalance in extracorporeal fertilized women

The demographic crisis that prevailed in Ukraine in recent years, the state of war as a result of the aggression of the Russian Federation, reproductive losses among women of childbearing age are one of the most urgent problems in modern obstetrics and gynecology. One of the most effective methods of correcting impaired reproductive function is in vitro fertilization. The purpose of this workis to develop a pharmacological complex for the prevention of imbalance of CD68+/CD163+ decidual macrophages in vitro fertilized women. Materials and methods105 pregnant women who were divided into 3 groups took part in the study. The first group included 20 women whose pregnancy occurred and is proceeding physiologically. The second group consisted of 85 women who became pregnant as a result of in vitro fertilization, including 37 pregnant women who refused prophylactic correction of the threat of premature birth, and 48 pregnant women who received prophylactic correction of the threat of premature birth: complex prescription of vitamin D3 2000 IU orally 2 times a day, micronized progesterone 200 mg 2 times a day and l-arginine aspartate 1000 mg 4 times a day, starting from 18 to 20 weeks of pregnancy. ResultsIn women who refused prophylactic correction of the threat of premature birth, a local increase in the activity of inducible NO-synthase and concentration of tumor necrosis factor-α, and a decrease in the activity of arginase and in the level of interleukin-10 were observed in the cervical mucus. They have a lower expression of CD163+ on placental decidual macrophages and an increased expression of CD68+, which indicates a shift in the polarization of macrophages from an anti-inflammatory to a pro-inflammatory phenotype. The use of prophylactic treatment brings the studied parameters closer to the results of women in whom pregnancy occurred physiologically. ConclusionsIn women who became pregnant as a result of in vitro fertilization, at 28–30 weeks of pregnancy, changes specific for pro-inflammatory phenotype of decidual macrophages were observed. Complex administration of vitamin D3, micronized progesterone and l-arginine aspartate lead to restoration of anti-inflammatory phenotype of decidual macrophages.

Airway macrophages display decreased expression of receptors mediating and regulating scavenging in early cystic fibrosis lung disease.

Cystic fibrosis (CF) airway disease is characterized by chronic inflammation, featuring neutrophil influx to the lumen. Airway macrophages (AMs) can promote both inflammation and resolution, and are thus critical to maintaining and restoring homeostasis. CF AM functions, specifically scavenging activity and resolution of inflammation, have been shown to be impaired, yet underlying processes remain unknown. We hypothesized that impaired CF AM function results from an altered expression of receptors that mediate or regulate scavenging, and set out to investigate changes in expression of these markers during the early stages of CF lung disease. Bronchoalveolar lavage fluid (BALF) was collected from 50 children with CF aged 1, 3 or 5 years. BALF cells were analyzed using flow cytometry. Expression levels of surface markers on AMs were expressed as median fluorescence intensities (MFI) or percentage of AMs positive for these markers. The effect of age and neutrophilic inflammation, among other variables, on marker expression was assessed with a multivariate linear regression model. AM expression of scavenger receptor CD163 decreased with age (p = 0.016) and was negatively correlated with BALF %neutrophils (r = -0.34, p = 0.016). AM expression of immune checkpoint molecule SIRPα also decreased with age (p = 0.0006), but did not correlate with BALF %neutrophils. Percentage of AMs expressing lipid scavenger CD36 was low overall (mean 20.1% ± 16.5) and did not correlate with other factors. Conversely, expression of immune checkpoint PD-1 was observed on the majority of AMs (mean PD-1pos 72.9% ± 11.8), but it, too, was not affected by age or BALF %neutrophils. Compared to matched blood monocytes, AMs had a higher expression of CD16, CD91, and PD-1, and a lower expression of CD163, SIRPα and CD36. In BALF of preschool children with CF, higher age and/or increased neutrophilic inflammation coincided with decreased expression of scavenger receptors on AMs. Expression of scavenging receptors and regulators showed a distinctly different pattern in AMs compared to blood monocytes. These findings suggest AM capacity to counter inflammation and promote homeostasis reduces during initiation of CF airway disease and highlight new avenues of investigation into impaired CF AM function.

Abstract 5466: Functional status of tumor-associated macrophages impacts clinical outcome of durvalumab in patients with advanced NSCLC as revealed by proteomics mass spectrometry

Abstract Introduction: Predictive biomarkers of anti‒PD-(L)1 therapies have largely focused on the tumor-T cell axis where tumor cell PD-L1 expression has demonstrated its clinical utility in predicting overall survival (OS) in patients with advanced NSCLC. In prior work using computational image analysis of multiplex immunofluorescence (mIF), we have shown the positive impact of CD68+ PD-L1+ macrophages in combination with CD8+ T cells in predicting long-term OS benefit in NSCLC patients treated with durvalumab (anti-PD-L1), highlighting the impact of the myeloid compartment on IO responses. In part, using proteomics mass spectrometry we sought to investigate further the functional impact of myeloid cells on the IO response and in particular, suppressive (M2) tumor associated macrophages (TAMs). Methods: Pre-treatment tumor samples from 66 patients with advanced NSCLC patients enrolled in durvalumab nonrandomized phase 1/2 trial (10 mg/kg Q2W, CP1108/NCT01693562) were processed for global and targeted proteomics. A pathologist determined the tumor area and a single FFPE tumor tissue section was used for laser-capture macrodissection and protein extraction followed by mass spectrometry analysis using label-free, data-independent and parallel reaction monitoring. Results: Among the immune associated proteins detected by proteomics mass spectrometry, we evaluated CD163 protein expression, a well-described marker of suppressive (M2) macrophages, and its impact on durvalumab clinical outcomes, alone and in relationship with PD-L1 and immune infiltration. While proteomics-based biomarker evaluable population (BEP) shows shorter median (m) OS compared to intended to treat population, we validated that high expression of PD-L1 as measured by IHC or proteomics associates with greater OS benefit as expected when treating with durvalumab. Building on this, the evaluation of protein associated with suppressive M2 macrophages, revealed that approximately 30% of NSCLC patients with high CD163 protein expression show poor OS benefit (5 months mOS) following durvalumab treatment. In contrast, patients with PD-L1 TC ≥50% and low CD163 expression derive greater OS benefit (13.4 months mOS) than those with high CD163 expression (5 months mOS) or with PD-L1 TC <50% regardless of level of CD163 expression (4.1-7.4 months mOS). Moreover, patients with poorer outcome have significantly higher baseline CD163 protein expression as a proportion of total CD45 protein expression. Conclusion: This analysis further confirms the importance of myeloid cell function within the tumor microenvironment (TME) in determining the outcome to T cell-directed IO therapy and highlights the utility of proteomics mass spectrometry in assessing more broadly and quantitatively the TME complementing findings based on RNAseq and IHC/mIF assays. Citation Format: Anna Margaret Hansen, Vivian Ying Wang, Andrew Chambers, David Chain, Jaime Rodriguez Canales, Jorge Blando, Steve Sweet, Zachary A. Cooper, Ashok Gupta, Carl Barrett, Yeoun Jin Kim, Ikbel Achour. Functional status of tumor-associated macrophages impacts clinical outcome of durvalumab in patients with advanced NSCLC as revealed by proteomics mass spectrometry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5466.

Macrophage polarisation in caesarean scar diverticulum

AimsTo determine immunohistochemical features and correlations between M1/M2 polarisation status with disease severity of post-caesarean scar diverticulum (CSD).MethodsHistological and immunohistological stainings were performed and inflammatory (CD16, CD163 and tumour necrosis...

Abstract 3878: Opposing CD68/CD163 tumour immune microenvironments revealed using a large multi-tumor tissue microarray (TMA) comprising cores from invasive margin (IM) and tumor center (TC)

Abstract Macrophages contribute to cancer-associated inflammation, and those with M2-like phenotype have been reported to be involved in tumor progression, immunosuppression, metastasis and angiogenesis. CD163 is considered a marker of M2-like macrophages/monocytes and high expression has been associated with poor prognosis in various tumor indications. To investigate the relative contribution of macrophage/monocyte cell populations to the tumor immune microenvironment across multiple tumor types concurrently, we have utilised a multi-tumor tissue microarray (TMA) comprising 30 different tumor indications, with approximately 12 cases per tumor type, each case represented by duplicate 1mm cores (1 from IM and 1 from TC). Serial sections of TMA slides (n=5) were stained by immunohistochemistry for CD68 and CD163, and cells/mm2/core delivered by digital image analysis of scanned images (CellProfilerTM). Comparative analysis of all tumor types regardless of core location (IM and TC combined) revealed lung, renal and cervical cancers to be the most highly infiltrated with CD68+ macrophages, while the greatest CD163 staining was observed for sarcoma and glioblastoma (GBM). Interestingly, a direct comparison of multiple tumor types simultaneously as performed here has revealed strongly opposing immune microenvironments in different tumor indications with respect to the balance of expression of CD68 and CD163. Higher frequencies of CD68+ macrophages relative to low CD163 expression was observed for prostate, endometrial, renal, cervical, thyroid, pancreatic, hepatic, small bowel and urothelial cancers. Conversely, a strong M2-like milieu exhibiting CD163+ cell frequencies exceeding CD68+ macrophages numbers was apparent for sarcoma, GBM, mesothelioma, bladder, cutaneous squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cell carcinoma, ER+ and Her2+ breast cancers. The remaining tumors, including cholangiocarcinoma, triple negative breast, lung, ovarian, gallbladder, colorectal, gastric, and esophageal cancers, all exhibited equivalent levels of infiltrating CD68+ and CD163+ cells. Analyses of IM versus TC revealed further complexities of the tumor immune microenvironment. For some disease settings, divergent CD163:CD68 ratios were observed for IM versus TC, whereas for other indications the ratio remained unchanged. In conclusion, concurrent analysis of multiple tumor indications for CD68 and CD163 has revealed immunosuppressive M2-like macrophage/monocyte tumor immune microenvironments that vary markedly between tumor types and within some tumors. These data may help to identify those patients that may benefit from therapies that target this immune pathway. Citation Format: Marie Cumberbatch, Woo Ho Kim, Lorenzo Memeo, Lorenzo Colarossi, Christopher Womack, Milan Bhagat. Opposing CD68/CD163 tumour immune microenvironments revealed using a large multi-tumor tissue microarray (TMA) comprising cores from invasive margin (IM) and tumor center (TC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3878.

Tumor-associated macrophages predict prognosis in diffuse large B-cell lymphoma and correlation with peripheral absolute monocyte count

BackgroundDiffuse 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.MethodsIn 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.ResultsWe 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).ConclusionsPatients 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.

Activated macrophages control human adipocyte mitochondrial bioenergetics via secreted factors

ObjectiveObesity-associated WAT inflammation is characterized by the accumulation and local activation of macrophages (MΦs), and recent data from mouse studies suggest that macrophages are modifiers of adipocyte energy metabolism and mitochondrial function. As mitochondrial dysfunction has been associated with obesity and the metabolic syndrome in humans, herein we aimed to delineate how human macrophages may affect energy metabolism of white adipocytes. MethodsHuman adipose tissue gene expression analysis for markers of macrophage activation and tissue inflammation (CD11c, CD40, CD163, CD206, CD80, MCP1, TNFα) in relationship to mitochondrial complex I (NDUFB8) and complex III (UQCRC2) was performed on subcutaneous WAT of 24 women (BMI 20–61 kg/m2). Guided by these results, the impact of secreted factors of LPS/IFNγ- and IL10/TGFβ-activated human macrophages (THP1, primary blood-derived) on mitochondrial function in human subcutaneous white adipocytes (SGBS, primary) was determined by extracellular flux analysis (Seahorse technology) and gene/protein expression. ResultsStepwise regression analysis of human WAT gene expression data revealed that a linear combination of CD40 and CD163 was the strongest predictor for mitochondrial complex I (NDUFB8) and complex III (UQCRC2) levels, independent of BMI. IL10/TGFβ-activated MΦs displayed high CD163 and low CD40 expression and secreted factors that decreased UQCRC2 gene/protein expression and ATP-linked respiration in human white adipocytes. In contrast, LPS/IFNγ-activated MΦs showed high CD40 and low CD163 expression and secreted factors that enhanced adipocyte mitochondrial activity resulting in a total difference of 37% in ATP-linked respiration of white adipocytes (p = 0.0024) when comparing the effect of LPS/IFNγ- vs IL10/TGFβ-activated MΦs. ConclusionOur data demonstrate that macrophages modulate human adipocyte energy metabolism via an activation-dependent paracrine mechanism.

56 Impairment of CD163 receptor are associated with increased levels of TNF- α and IL-1 β in plasma from pregnant women with preeclampsia

Introduction Preeclampsia (PE) is a pregnancy-specific syndrome characterized by a systemic inflammatory response that polarizes peripheral blood monocytes to the M1 phenotype. CD163 is a scavenger receptor expressed by monocytes/macrophages which may shed from their cell membrane after proteolytic cleavage producing soluble CD163 molecule (sCD163) that plays a role in the modulation of inflammatory response. Objectives This study aimed at evaluate expression of CD163 on monocytes and the levels of sCD163 as well as pro- and anti-inflammatory cytokines in plasma of pregnant women with the severe and mild forms of PE. Methods Fifty-six women with preeclampsia, 28 normotensive pregnant women matched for gestational age and 20 healthy non-pregnant women were included. Preeclamptic women were classified according to the disease severity as mild ( n = 28) or severe ( n = 28) PE. Plasma levels of sCD163, tumor necrosis factor-alpha (TNF- α ), Interleukin-1 beta (IL-1 β ), IL-10 and transforming growth factor beta (TGF- β 1) were determined by enzyme-linked immunosorbent assay, and CD163 expression by monocytes was assessed by flow cytometry. Differences between groups were compared by parametric analysis of variance with significance level set at 5%. Results Monocytes from preeclamptic women showed lower expression of CD163 receptor compared with normotensive pregnant and non-pregnant women. Plasma levels of sCD163 were also significantly lower in pregnant women with severe or mild PE than in normotensive pregnant women. No significant differences were detected between both groups of PE in relation to this parameter. A positive correlation was observed between sCD163 and IL-10 concentration in plasma from normotensive pregnant women ( r = 0.5829; p = 0.0111). The values of IL-1 β , TGF- β 1 and TNF- α were higher in severe PE than in mild PE and normotensive pregnant women. Both groups of preeclamptic women showed decreased plasma levels of IL-10. A negative correlation was observed between concentrations of sCD163 and IL-1 β ( r = −0.4251; p = 0.0341) and between sCD163 and TNF- α ( r = −0.5075; p = 0.0096) in the group of severe PE. Conclusions The results showed that decreased plasma levels of sCD163 could possibly be explained by the lower expression of CD163 by monocytes from preeclamptic women. The association between the pro-inflammatory cytokine profile and the lower concentration of sCD163 and IL-10 in plasma from pregnant women with severe PE suggests an impairment in the modulation of the inflammatory response in this preeclamptic group. Financial support – FAPESP 2013/12260-7.

Expression of M2-Polarized Macrophages is Associated with Poor Prognosis for Advanced Epithelial Ovarian Cancer

Macrophages are polarized into two functionally distinct forms, M1 and M2, in response to different microenvironment. Tumor-associated macrophages (TAMs) generally have M2 phenotype and promote tumor progression. Few studies to date have described the infiltration of M2-polarized macrophages in ovarian cancer. We used two macrophages markers, CD68 and CD163, to analyze the expression of TAMs and to clarify the relationship between the M2 form and survival in advanced ovarian cancer. Clinical data of 110 patients with stages III-IV epithelial ovarian cancer at Sun Yat-sen University Cancer Center between 1999 and 2007 were retrospectively reviewed. Immunohistochemical staining of CD68 and CD163 was performed. Correlations between macrophage density and patient survival were analyzed. Our data showed that no significant difference was observed in survival between patients in the high- and the low-CD68 expression groups. In contrast, the progression-free survival (PFS) rates (p = 0.003) and overall survival (OS) rates (p = 0.004) were significantly higher in the low-CD163 expression group than in the high-CD163 expression group, respectively. Similarly, we also observed significantly improved 3-year PFS (49.8% vs. 11.0%, p < 0.001) and OS (77.4% vs. 45.0%, p < 0.001) rates in patients in the low-CD163/CD68 ratio group when compared with the high-CD163/CD68 ratio group. Multivariate analysis identified the density of CD163-positive cells as well as the ratio of CD163/CD68 as negative predictors for PFS and OS, respectively. Our results show that the infiltration of CD163-positive M2 macrophages as well as activation of macrophages towards the M2 phenotype may contribute to poor survival in advanced ovarian cancer.

CXCR4 positive and angiogenic monocytes in myocardial infarction

Limited data are available on the role of monocytes in cardiac repair. In the present study, we evaluated the dynamic alterations of monocytes with reparative and angiogenic potential in patients with myocardial infarction(MI). Reparative CXCR4+ monocytes, and CD34+ and KDR+ monocytes with angiogenic potential derived from individual monocyte subsets were quantified by flow cytometry in patients with ST-elevation MI (n=50) and stable coronary artery disease (CAD, n=40). Parameters were measured on days 1, 3, 7 and 30 post MI. Monocyte subsets were defined as CD14++CD16-CCR2+ ('classical', Mon1), CD14++CD16+CCR2+ ('intermediate', Mon2), CD14+CD16++CCR2- ('non-classical', Mon3). Plasma levels of inflammatory cytokines, fibrinolytic factors and microparticles (MPs) were assessed on day 1. CXCR4+ and KDR+ monocytes were increased following MI, being more prominently associated with Mon2 (median[IQR] of CXCR4+ Mon2 60[25-126] per μl in STEMI vs. 27[21-41] per μl in stable CAD). The counts of CXCR4+ Mon2 in STEMI significantly reduced by day 30 of follow-up (27[18-47], p<0.001). Expression of the pro-reparative scavenger receptor CD163 on Mon3 was reduced in acute MI (p=0.008), and on other subsets later during the follow-up with lowest levels at day 3 post-MI (p<0.001 for Mon1, p=0.02 for Mon2). CD204 expression on Mon1 correlated with tissue type plasminogen activator levels (r=0.46, p=0.001). Interleukin(IL)6 levels correlated with counts of Mon2-derived CXCR4+ and KDR+ cells. Interleukin-1β correlated with KDR+ Mon2 counts. IL10 correlated with CXCR4+ Mon2 levels. Low count of CXCR4+ Mon2 and low CD163 expression by Mon2 were associated with higher ejection fraction six-weeks after MI. In conclusion, the Mon2 subset has the most prominent role in the observed changes in reparative monocytes in MI. The association of reparative monocytes with inflammatory/fibrinolytic markers indicates a complex interplay of these cells in the post-MI state.

Tumor-Associated Macrophages Predict Outcome in Follicular Lymphoma

AbstractAbstract 682 Background:Follicular lymphoma (FL) is the most common indolent lymphoma and is clinically characterized by slow progression and responsiveness to therapy, but almost inevitable disease recurrences. While patient outcomes are generally favorable, a substantial proportion of patients are at risk of early death due to treatment resistance, early progression or transformation. CD68 positive macrophages were shown to predict poor survival in the pre-rituximab era (Farinha et al., Blood 2005 Sep 15;106(6):2169–74), but their association with poor outcome was lost or even reversed since the introduction of rituximab into the routine management of FL (Taskinen et al, Clin Cancer Res. 2007 Oct 1;13(19):5784–9 and Canioni et al, J Clin Oncol. 2008 Jan 20;26(3):440–6). Macrophages can be broadly divided into M1 and M2 subtypes with the latter associated with pro-tumoral immunity. We hypothesize that CD68 alone insufficiently captures the various phenotypes of tumor-associated macrophages and that staining for CD163 as a marker for alternatively activated macrophages (M2) provides additional prognostic information. Methods:We constructed a tissue microarray (TMA) from formalin-fixed and paraffin-embedded tissue blocks of 187 systemic treatment-naive FL patients that were selected from the Lymphoid Cancer Database of the BC Cancer Agency and that had been uniformly treated between 2004 and 2010 with rituximab, cyclophosphamide, vincristine and prednisone (R-CVP). Since 2006, institutional guidelines recommended maintenance with rituximab for patients achieving a complete or partial remission after first-line therapy. Median follow-up of living patients was 56 months. Immunohistochemistry was performed on the TMA with antibodies against CD68 (clone KP1, DAKO) and CD163 (clone 10D6, Novocastra) and was analyzed by computer image analysis (Aperio) using the Positive Pixel Count algorithm. Disease-specific survival (DSS) served as the primary end-point, and progression-free survival (PFS) and time to transformation (TTT) as secondary endpoints. The statistical software X-tile was used to determine the optimum thresholds for CD68 and CD163 as the values that maximized the Chi-square value of the log-rank test. Results:Within the cores on the TMA, the mean percentage of positivity was 4.7% for CD68 (range 0.3–22.6%) and 1.8% for CD163 (range 0–13.7%). Above a threshold of 1.8%, high CD68 expression was associated with favorable DSS (p=0.03), but not with PFS (p=0.47) or TTT (p=0.82). Increased CD163 expression on the other hand, above a threshold of 1.1%, was significantly associated with poor DSS (p<0.01), but not with PFS (p=0.68) or time to transformation (p=0.13). When adjusting for the International Prognostic Index (IPI) in a Cox regression model, the association of high CD68 expression with favorable DSS (hazard ratio=0.42, p=0.05) and high CD163 expression with poor DSS (hazard ratio=2.93, p=0.01) remained significant. The correlation between CD68 and CD163 expression was low (Pearson correlation coefficient=0.25), suggesting that both markers define different subsets of tumor-associated macrophages. To explore whether additional prognostic information could be gained from combining these markers, we grouped CD68-high and CD163-low cases into one category (n=83) and compared them against all others (n=102). High CD68 and low CD163 expression was associated with favorable DSS (p<0.01), but not PFS (p=0.40) or TTT (p=0.21). Patients with high CD68 and low CD163 expression had excellent outcomes at 7 years of follow-up (96% DSS versus 68%). The impact on DSS remained significant after adjusting for IPI in a Cox regression model (hazard ratio=0.15, p<0.01). Conclusion:In FL patients treated with uniform induction therapy, CD163 defines a subset of tumor-associated macrophages that is associated with inferior DSS, but not with early progression or transformation, whereas the combination of CD68-high and CD163-low defines a patient category that has excellent prognosis. Our results suggest that the detrimental role of alternatively activated M2 type macrophages is not reversed by rituximab and that a population of non-tumor promoting macrophages exists that is correlated with favorable outcome in FL. Our study also illustrates the power of image analysis to provide accurate scoring, capable of delineating patient populations at variable risk of death. Disclosures:Al-Tourah:F. Hoffmann-La Roche (Roche Canada): Research Funding. Moccia:F. Hoffmann-La Roche (Roche Canada): Research Funding. Connors:F. Hoffmann-La Roche (Roche Canada): Research Funding. Sehn:F. Hoffmann-La Roche (Roche Canada): Research Funding.