CD204-positive Tumor-associated Macrophages Research Articles

Intrahepatic cholangiocarcinoma with arterial phase hyperenhancement and specialized tumor microenvironment associated with good prognosis after radical resection: A single-center retrospective study

BackgroundThis single-center retrospective study aimed to clarify the clinical and pathologic background of mass-forming intrahepatic cholangiocarcinomas. MethodsA total of 53 patients with mass-forming intrahepatic cholangiocarcinomas were selected from 2007 to 2021 and analyzed based on several parameters, including the preoperative computed tomography pattern (enhancement in the arterial phase of dynamic contrast-enhanced computed tomography), clinical data, and tumor microenvironment evaluated by immunohistochemistry. The hyperenhancement (n = 13) and hypoenhancement (n = 40) groups were defined using the 50% cutoff of tumors with higher attenuation than the liver parenchyma. ResultsThe hyperenhancement group was characterized by a better overall survival than the hypoenhancement group (5-year survival: 86% vs 27%, respectively; P < .001) and by a higher infiltration of peritumoral (92% vs 58%; P = .020) and intratumoral CD3-positive T lymphocytes (85% vs 35%; P = .002). Conversely, the hypoenhancement group was characterized by a higher infiltration versus peritumoral CD163-positive tumor-associated macrophages (60% vs 8%; P = .001), peritumoral pentraxin 3–positive tumor-associated macrophages (50% vs 15%; P = .024), and intratumoral α–smooth muscle actin–positive cancer-associated fibroblasts (15% vs 68%; P = .001). A multiple regression analysis was performed to predict overall survival from the microenvironment, and the independent poor predictor factors were low intratumoral CD3-positive T lymphocytes (hazard ratio = 2.75), high peritumoral (hazard ratio = 2.38), and intratumoral CD163-positive tumor-associated macrophages (hazard ratio = 2.81) (all P values < 0.05). ConclusionCompared with hypovascular, hypervascular mass-forming intrahepatic cholangiocarcinomas have better tumor immunity and prognosis.

Serum-Soluble CD163 Levels as a Prognostic Biomarker in Patients with Diffuse Large B-Cell Lymphoma Treated with Chemoimmunotherapy.

The majority of patients with Diffuse Large B-cell Lymphoma (DLBCL) will respond to first-line treatment and be cured. However, the disease is heterogeneous, and biomarkers able to discriminate patients with suboptimal prognosis are needed. M2 CD163-positive tumor-associated macrophages (TAMs) were shown to be implicated in DLBCL disease activity regulation. Serum-soluble CD163 (sCD163) functions as a scavenger receptor for haptoglobin-hemoglobin complexes and is mostly expressed by monocytes and macrophages. Its levels are used to determine macrophage activation. We aimed to determine serum sCD163 in a sample of DLBCL patients and study eventual correlations with parameters of disease activity or survival. Serum sCD163 levels were measured in 40 frozen sera from patients diagnosed with DLBCL and 30 healthy individuals (HIs) using an enzyme-linked immunosorbent assay (ELISA). Statistical analyses were performed using SPSS version 28. The results showed that patients who achieved complete response after standard-of-care immunochemotherapy and were alive and disease-free after 12 months of follow-up but had elevated sCD163 levels (above median) at diagnosis presented a significantly worse overall survival compared to those with initial serum sCD163 levels below the median (p = 0.03). Consequently, serum sCD163 levels in patients with DLBCL may constitute a marker of long-term response to chemoimmunotherapy.

Histopathological analysis of tumor microenvironment in adrenocortical carcinoma: Possible effects of in situ disorganized glucocorticoid production on tumor immunity

Adrenocortical carcinoma (ACC) patients with glucocorticoid excess have been reported to be associated with decreased tumor-infiltrating immune cells, but the effects of in situ glucocorticoid production on tumor immunity have remained unknown. In addition, ACC was also known to harbor marked intra-tumoral heterogeneity of steroidogenesis or disorganized steroidogenesis. Therefore, in this study, we immune-profiled tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) and pivotal steroidogenic enzymes of glucocorticoid biosynthesis (CYP17A and CYP11B1) to explore the potential effects of in situ glucocorticoid production and intra-tumoral heterogeneity/disorganized steroidogenesis on tumor immunity of ACC. We also studied the correlations of the status of tumor immunity with that of angiogenesis and tumor grade to further explore the tumor tissue microenvironment of ACC. TILs (CD3, CD4, CD8, and FOXP3), TAMs (CD68 and CD163), key steroidogenic enzymes of glucocorticoid (CYP17A and CYP11B1), angiogenesis (CD31 and vasohibin-1 (VASH-1)), tumor grade (Ki-67 and Weiss score) were immunohistochemically evaluated in 34 ACCs. Increased CYP17A immunoreactivity in the whole tumor area was significantly positively correlated with FOXP3-positive TILs (p = 0.021) and negatively with CD4/CD3 ratio (p = 0.001). Increased CYP11B1 immunoreactivity in the whole tumor area was significantly positively correlated with CD8/CD3 (p = 0.039) and CD163/CD68 ratios (p = 0.006) and negatively with CD4-positive TILs (p = 0.036) and CD4/CD3 ratio (p = 0.001). There were also significant positive correlations between CYP17A and CD8 (r = 0.334, p < 0.001) and FOXP3-positive TILs (r = 0.414, p < 0.001), CD8/CD3 ratio (r = 0.421, p < 0.001), and CD68-positive TAMs (r = 0.298, p < 0.001) in randomly selected areas. Significant positive correlations were also detected between CYP11B1 and CD8/CD3 ratio (r = 0.276, p = 0.001) and negative ones detected between CYP11B1 and CD3- (r = –0.259, p = 0.002) and CD4-positive TILs (r = –0.312, p < 0.001) in those areas above. Increased micro-vessel density (MVD) -VASH-1 was significantly positively correlated with CD68- (p = 0.015) and CD163-positive TAMs (p = 0.009) and CD163/CD68 ratio and the high VASH-1 with CD163-positive TAMs (p = 0.042). Ki-67 labeling index was significantly positively correlated with MAD-VASH-1 (p = 0.006) and VASH-1 (p = 0.006) status. Results of our present study indicated that in situ glucocorticoid production did influence the status of tumor immunity in ACC. In particular, increased levels of CYP17A and CYP11B1, both involved in glucocorticoid producing immunoreactivity played different effects on tumor immunity, i.e., reflecting the involvement of intra-tumoral heterogeneity and disorganized steroidogenesis of ACC, which also did indicate the importance of in situ approaches when analyzing tumor immunity of ACC.

A Case Report of Aggressive Fumarate Hydrase-deficient Renal Cell Carcinoma With Loss of HLA Antigens.

Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is a rare RCC subtype, and FH-deficient RCC may be misdiagnosed as another type of RCC, such as type 2 papillary RCC or collecting duct carcinoma. FH and 2-succinocysteine (2SC) are useful diagnostic markers for FH-deficient RCC and can be measured using immunohistochemistry (IHC). A 30-year-old female with 3-month history of fatigue and left-flank mass was diagnosed with a 20×13×10 cm left-side renal mass with massive inferior vena cava (IVC) tumor thrombus that extended into the right atrium. She underwent nephrectomy and IVC thrombectomy, and a pathological diagnosis of type 2 papillary RCC was made. Four months after the surgery, computed tomography scan showed multiple liver metastases not observed immediately after surgery. Systemic treatment with sorafenib was initiated; however, she did not respond and died 3 months after treatment. Subsequent re-review of hematoxylin and eosin-stained sections indicated morphologic characteristics consistent with FH-deficient RCC, and IHC staining was negative for FH but positive for 2SC, indicating a diagnosis of FH-deficient RCC. Further immunological analyses revealed the loss of HLA-class I, b2 microglobulin, and HLA-DR antigens in cancer cells. In addition, a few CD8-positive cytotoxic T cells and CD163-positive tumor-associated macrophages were noted. An immunosuppressive tumor microenvironment that facilitates cancer immune evasion might be associated with the rapid progression and poor prognosis in our patient. Further investigation of the tumor immune microenvironment in patients with FH-deficient RCC is warranted.

High-grade B-cell lymphomas with MYC and BCL2 translocations lack tumor-associated macrophages and PD-L1 expression: A possible noninflamed subgroup.

We investigated the intratumoral source of PD-L1 expression and the infiltration of tumor-associated macrophages (TAMs) in large B-cell lymphomas (LBCLs) with or without MYC-translocation, as well as possible correlations to BCL2-and BCL6-translocations and cell of origin (COO). One-hundred and twenty-six patient samples were studied in a cohort enriched for MYC-translocated tumors with 34 samples carrying this translocation. Demonstration of intratumoral distribution and cellular source of PD-L1 was enabled by immunohistochemical (IHC) dual staining specifically highlighting PD-L1 expression in lymphoma B-cells with antibodies against PD-L1 and PAX5. Additional IHC with antibodies against CD68 and CD163 identified TAMs. We found that CD68-positive TAMs were the main source of PD-L1 protein expression in contrast to lymphoma B cells which rarely expressed PD-L1. Semiquantitative IHC demonstrated a significant correlation between CD68 and PD-L1 protein expression. Unsupervised hierarchical analysis of PD-L1, CD68, and CD163 IHC data subsequently demonstrated three potential clusters defined by expression of the three biomarkers. Cluster A consisted of patient samples with significantly lower expression of PD-L1, CD68, and CD163, but also significantly higher prevalence of BCL2-translocation and MYC-BCL2-double-hit (DH) compared to the other two clusters. In cluster C we found a significant accumulation of BCL6 translocated tumors. This cluster in contrast had the highest protein expression of PD-L1, CD68, and CD163. Cluster B tumors had an intermediate expression of the three biomarkers, but no accumulation of the specific genetic translocations. Our data, which were based on morphological analysis, immunophenotyping and genotyping by fluorescence in situ hybridization were in line with new concepts of LBCL taxonomy integrating genetic, phenotypical, and immunological characteristics with identification of new subgroups where MYC translocation and MYC-BCL2 DH may identify a noninflamed subtype. These findings may furthermore hold significant predictive value especially regarding immune checkpoint blockade therapy, but further molecular characterization should be done to substantiate this hypothesis.

Prognostic impact of the tumor immune microenvironment in pulmonary pleomorphic carcinoma

IntroductionPulmonary pleomorphic carcinoma (PC) is a rare non-small cell lung carcinoma (NSCLC) and is characterized by sarcomatoid and NSCLC components. This study aimed to characterize the association between immune microenvironmental factors and clinicopathological characteristics of PC. MethodsEighty consecutive PC patients who had undergone complete surgical resection were enrolled. We calculated the immunohistochemical staining scores for E-cadherin, vimentin, programmed death ligand 1 (PD-L1), and carbonic anhydrase IX in cancer cells and counted the numbers of CD204-positive tumor-associated macrophages (TAMs) and Foxp3-, CD8-, and CD20-positive tumor-infiltrating lymphocytes (TILs). We also examined the association between these scores and the prognostic outcomes. ResultsThe staining score for PD-L1 in cancer cells and the number of CD204-positive TAMs in the sarcomatoid component were significantly higher than those in the NSCLC component; E-cadherin score in the sarcomatoid component was significantly lower. Patients with high PD-L1 expression in the NSCLC component had significantly longer overall survival (OS) and recurrence-free survival (RFS) than those with low PD-L1 expression in the NSCLC component (OS: p = 0.001, RFS: p = 0.038). Multivariate analysis revealed that high PD-L1 expression in the NSCLC component was an independent favorable prognostic factor for OS (p = 0.018), whereas high PD-L1 expression in the sarcomatoid component was not. The number of CD8-positive TILs was significantly higher in the high PD-L1 expression group than in the low expression group (NSCLC components: p < 0.001). ConclusionHigh PD-L1 expression in the NSCLC component may be associated with a favorable prognostic value in pulmonary PC.

High-Grade B-Cell Lymphomas with MYC and BCL2 Translocations Lack Tumor-Associated Macrophages and PD-L1 Expression: A Possible Noninflamed Subgroup

Extensive molecular analysis of large B-cell lymphomas (LBCL) have provided basis for new concepts of lymphoma taxonomy. These include stages of differentiation, phenotype (cell of origin) as well as genotype (including, but not confined to gene rearrangements) and immune microenvironment. The importance of the tumor microenvironment has recently regained focus due to development of immune checkpoint-inhibitors such as PD1/PD-L1 inhibitors, and LBCL may according to the new subtypes be stratified as microenvironment "inflamed" or "non-inflamed", which may hold prognostic as well as predictive value. We investigated the composition of the tumor microenvironment in LBCL with or without MYC-translocation regarding PD-L1 expression and the infiltration of tumor-associated macrophages (TAMs). 126 patient samples were studied in a cohort enriched for MYC translocated tumors with 34 samples carrying this translocation. Demonstration of intratumoral distribution and cellular source of PD-L1 was enabled by immunohistochemical (IHC) dual staining specifically highlighting PD-L1 expression in lymphoma B-cells with antibodies against PD-L1 and Pax-5. Additional IHC with antibodies against CD68 and CD163 identified tumor associated macrophages (TAMs). All IHC stainings were quantitated independently and blinded by two consultant hematopathologists in 10% intervals of all tumor cells both neoplastic B-cells, immune cells, and stromal cells. We found that CD68 positive TAMs were the main source of PD-L1 protein expression in contrast to lymphoma B-cells which rarely expressed PD-L1 (Fig.1). Quantitative IHC demonstrated a significant correlation between CD68 and PD-L1 protein expression. CD68 positive TAMS and thereby also PD-L1 expression was significantly lower in MYC translocated lymphomas compared to non-translocated. The correlation was significant for both MYC single-hit (SH) and MYC double-hit (DH) with concurrent BCL2 and/or BCL6 translocation, the latter now termed High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH). For a more detailed analysis we next performed unsupervised cluster analysis of PD-L1, CD68 and CD163 quantitative IHC data, which demonstrated three significant clusters (Clusters 1-3) defined by expression of the three biomarkers (Fig. 2). Cluster 3 consisted of patient samples with significantly lower expression of PD-L1, CD68 and CD163, but also significantly higher prevalence of BCL2-translocation and MYC/BCL2-DH compared to the other two clusters. In cluster 1 we found a significant accumulation of BCL6 translocated tumors. This cluster in contrast had the highest protein expression of PD-L1, CD68 and CD163. Our data which were based on morphological analysis, immunophenotyping and genotyping by FISH, were in line with new concepts of LBCL taxonomy integrating genetic, phenotypical and immunological characteristics with identification of new subgroups where MYC-translocation and MYC/BCL2 DH may identify a noninflamed subtype. These findings may furthermore hold significant predictive value especially regarding immune checkpoint blockade therapy, but further molecular characterization should be done to substantiate this hypothesis. Disclosures No relevant conflicts of interest to declare.

Tumor Cells Interleukin-22 Expression Associates with Elevated Tumor-Associated Macrophages Infiltrating and Poor Prognosis in Patients with Breast Cancer.

Interleukin-22 (IL-22), secreted by tumor infiltrated lymphocytes, is identified as a tumor-promoting factor in certain cancers, which was secreted by tumor infiltrated lymphocytes. However, the role of IL-22 in breast cancer remains conflicting. In this study, we assessed the expression of IL-22, IL-22 receptor 1 (IL-22R1), CD4, CD8, FOXP3, and CD68 in breast cancer by immunohistochemistry. IL-22 expression was exhibited in 105 (69.1%) cases in tumor cells (tIL-22), whereas only 24 (15.8%) samples displayed IL-22 expression in stromal cells. Multivariate analysis showed that tIL-22 expression was a poor prognostic factor for overall survival (OS) (p = 0.04). Meanwhile, IL-22R1 was predominantly presented in tumor cells (84.9%), which was associated with tIL-22 expression. The CD68-positive tumor-associated macrophages (TAMs) displayed the highest infiltration rate (50.7%) compared with CD4-, CD8-, and FOXP3-positive cells. Kaplan-Meier analysis confirmed patients with high TAM infiltration displayed significantly worse relapse-free survival (RFS) compared with low TAMs group (p = 0.017). TAM infiltration was also positively associated with tIL-22 and IL-22R1 expression. Furthermore, tIL-22 expression together with high TAM infiltration displayed the worst prognosis outcomes both in OS (p = 0.039) and RFS (p = 0.008). Instead of lymphocytes, our data indicated that tumor cells express IL-22 in breast cancer that is associated with IL-22R1, high TAM infiltrating, and poor prognosis.

Abstract 1597: The landscape of PD1-PD-L1 axis in the context of immune cells: Tumors versus stromal compartments

Abstract Introduction: Immune response, tolerance, suppression, and resistance are the result of a complex dynamic interaction comprising PD1-PD-L1 axis in the context of immune cells (ICs) involving tumor cells, and anti/pro-tumor immune-cells of the stromal compartment. Aim: Here we present the landscape of PD1-PD-L1 axis in the context of CD3, CD4, CD8, PD1, FOXP3, and IL12RB2 positive ICs in the tumor (T) and the tumor-adjacent normal (TAN) tissues from patients with ovarian cancers (OC), and lung cancers (LC). Methods: The study was approved by Avera IRB. Surgically resected paired T and TAN tissue samples (from patients with OC and LC) were used to identify CD3 (ab16669), CD4 (ab133616), CD8 (ab85792), FOXP3 ([236A/E7], PD1 (ab137132), IL12RB2 (NBP1-85983), CD68 (Dako. #M0876), CD163 (Cell Signaling #93498) in ICs and PD-L1 (Clone 22C3; Dako) in T cells &amp; tumor-associated macrophages (TAMs). Tonsil tissue was used for the IHC validation. Paired specimens were morphologically evaluated by H&amp;E stain &amp; Ki-67, cl-caspase 3. Anatomic pathological evaluation for the diagnosis of the disease was carried out by a Pathologist. Results: Expression of CD3, CD4, CD8, PD1, and FOXP3 was heterogeneous, with each tumor presenting an individual pattern of distribution of ICs. However in most tumor samples, the distribution of ICs was of tumor-cooperating phenotypes. PD-L1 expression in tumors did not correlate with any other ICs of the tumor-microenvironment. In TAN tissues from OC, PD1-PD-L1 staining was absent. While macrophages in TAN tissue from LC were moderately positive for PD-L1. In T tissue from OC and LC, PD1-PD-L1 staining was only present in the stroma, lymphocytes, and absent in the tumor compartments while PD-L1 staining was identified only in the macrophages. Tumoricidal cells were rarely observed within the tumor compartment of T tissues from OC. The tumors cells from LC was only positive for PD-L1. Conclusion: Using dual stain of PD1 and PD-L1, we present for the first time, the co-distribution pattern of PD1-PD-L1 axis in the context of the expression of CD3, CD4, CD8, FOXP3 T cells as well as CD68, and CD163 positive TAMs in the paired sample of a T tissue and its TAN tissue in LC and OC. The relationship between the PD1-PD-L1 axis and the expression of IL12 Receptor in the context of immune-landscape is being worked out and will be presented in the meeting. Citation Format: Xiaoqian Lin, Raed Sulaiman, Jennifer C. Aske, Paul Meyer, Luis RojasEspaillat, David Starks, Pradip De, Nandini Dey. The landscape of PD1-PD-L1 axis in the context of immune cells: Tumors versus stromal compartments [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 1597.

Abundance of CD163-Positive Tumor-Associated Macrophages in the Early Gastric Cancer Predicts the Recurrence after Curative Resection

Background: We aimed to investigate the prognostic value of M2 macrophages to predict the recurrence of early gastric cancer (EGC). Methods: A retrospective analysis was carried out among EGC patients (95 non-recurrence and 78 recurrence) who underwent surgery at Luhe People’s Hospital of Nanjing. A 5-year recurrence status was utilized to classify the patients into the recurrence group and non-recurrence group. CD163 and proliferating cell nuclear antigen were utilized as markers to detect M2 macrophages and proliferation. Cumulative tumor recurrence curve was adopted to analyze the association between the number of tumor-associated macrophages (TAMs) and the recurrence of EGC. Colony formation and invasion abilities of MKN45 (JCRB0254, human gastric epithelial cell line) with or without M2 macrophage coculture were detected in vitro, and the xenograft model was utilized to detect in vivo effect of M2 macrophages on tumor growth. Results: The number of CD163⁺ macrophages and expression of transforming growth factor-β1, matrix metallopeptidase 9, and vascular endothelial growth factor A were significantly different between the EGC recurrence and non-recurrence group. The cumulative tumor recurrence rate was found to be dependent on the infiltration number of TAMs. M2 macrophages promoted the proliferation and invasion of human MKN45 cells in vitro, as well as tumor growth in the xenograft model. Conclusion: The abundance of CD163⁺-positive TAMs in EGC predicts the recurrence after curative resection.

CD68- and CD163-positive tumor-associated macrophages in triple negative cancer of the breast

Tumor-associated macrophages (TAMs) have recently been reported as an important factor in tumor growth and the progression of cancer. The prognostic significance of localizations and densities of TAMs in triple negative cancer (TNC) of the breast is not well understood. The aim of this study was to assess the localizations and densities of the TAMs subtype in TNC and examine their clinicopathological features. The study was based on 107 TNC cases operated on at Dokkyo Medical University Hospital using the pan-macrophage marker CD68 and the M2 macrophage marker CD163 in the tumor stroma (TS) and tumor nest (TN), respectively, and examined the clinicopathological significance. Multivariate Cox regression analyses revealed that age and CD163+ TAMs in both the TS and TN were independent prognostic factors for relapse-free survival and overall survival. No correlation was found between the number of CD68+ cells or the CD163/CD68 ratio either in TS or TN, or clinicopathological features. Our study found that infiltration of CD163+ TAMs, rather than CD68+, in both TS and TN was associated with poor prognosis in TNC patients by multivariate analysis. This suggests that CD163+ TAMs may affect the prognosis of TNC by not only regulating the immune reaction by TAMs in TS, but also because of their direct influence on TN.

THE ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN THE PATHOGENESIS OF ORAL SQUAMOUS CELL CARCINOMA CORRELATED WITH THE CLINICOPATHOLOGICAL PARAMETERS

Introduction: Oral cancer is a major health problem, causing high morbidity and mortality rates. Oral squamous cell carcinoma (OSCC) accounts for 90-95% of all oral malignancies. During the last decade, significant evidence has suggested that inflammatory tumor microenvironment (TME) plays an important role in tumorigenesis. The inflammatory cells and their signals are indispensable participants in the neoplastic process, fostering proliferation, survival, and migration of cancer cells. The macrophages are the most abundant and important stromal cells in the TME, which orchestrate the inflammatory response. They control the cellular proliferation and survival by stimulating the immune cells and by promoting integrated processes of inflammation and tissue repair. Therefore, the tumor associated macrophages (TAMs) may be of great prognostic significance in different types of tumors. Objectives: This study was conducted to assess the TAMs density in OSCC using CD163 in metastasizing and non-metastasizing OSCC. Moreover, the correlation of TAM density with the lymph node status and the different histopathological grades of OSCC was assessed. Materials and methods: The density of TAM was calculated in 30 surgical specimens taken from OSCC patients. Biopsies were taken from the primary tumor of 15 cases with lymph node metastasis and 15 cases with no lymph node metastasis. Fifteen normal mucosal tissues were taken from healthy individuals indicated for alveoloplasty as a control group. Immunohistochemical staining using the CD163 antibody was performed, using Labeled Strept-Avidin Biotin complex method. Positive cells were counted using the Image J free software package. Results:CD163 was expressed in human OSCC and the TAMs count was significantly correlated with lymph node metastasis and with the tumor differentiation. Higher density was detected in metastatic tumors and in the poorly differentiated OSCC than in the well and moderately differentiated cases. Conclusions: CD163 positive TAMs could be a prognostic factor in OSCC cases as TAM density was significantly correlated with the lymph node status and the grade of differentiation of OSCC.

Correlation between maximum standardized uptake values on FDG-PET and microenvironmental factors in patients with clinical stage IA radiologic pure-solid lung adenocarcinoma

ObjectivesThe purpose of this study was to investigate whether fluorodeoxyglucose (FDG) accumulation is associated with the expression of microenvironmental factors in radiological pure-solid lung adenocarcinoma. MethodsWe selected 50 cases involving patients with clinical stage IA radiological pure-solid lung adenocarcinoma who were examined with 18 F-FDG positron emission tomography (18 F-FDG PET) prior to surgery and whose FDG–PET maximal standardized uptake values (SUVmax) were calculated. Tumor specimens were analyzed by immunohistochemistry (IHC) for phosphorylated AKT (pAKT), glucose transporter type 1 (GLUT-1), carbonic anhydrase IX (CA IX), podoplanin-positive cancer associated fibroblasts (PDPN + CAFs), and CD204-positive tumor-associated macrophages (CD204+ TAMs). We compared the clinicopathological characteristics and the immunophenotypes between two groups with high and low SUVmax. ResultsA multivariate analysis revealed that SUVmax was an independently significant prognostic factor (P = .03). The 5-year overall survival (OS) and recurrence free survival (RFS) rates of the SUV max high and low groups were 68.0% versus 100% ((P = .002; OS) 54.3% versus 90.8% (P < .001; RFS)), respectively. Vascular invasion, pleural invasion, and the prevalence of solid predominant subtype tumors were more frequent in the SUVmax high group. Additionally, the expression levels of GLUT-1 and pAKT in cancer cells were significantly higher in this group (P < .001, and P < .001 respectively). Furthermore, the numbers of the tumor-promoting stromal cells, i.e., PDPN + CAFs and CD204+ TAMs, were also significantly higher in the SUVmax high group (P = .001, and P < .001 respectively). ConclusionOur results indicated that a close association exists between the SUVmax and expressions of not only metabolism associated markers in cancer cells but also of tumor promoting markers in stromal cells among patients with clinical stage IA adenocarcinoma with radiologically pure-solid nodules.

CD204-Positive Tumor-associated Macrophages Relate to Malignant Transformation of Colorectal Adenoma.

Colorectal adenoma is well known as a precursor lesion of colorectal adenocarcinoma (ADC). We recently reported the significance of CD204 (+) tumor-associated macrophages (TAMs), a vital component of the tumor microenvironment, in the carcinoma development of gastric adenoma. The aim of the present study was to clarify the roles of TAM in the malignant transformation of colorectal adenoma. We immunohistochemically assessed the TAM number in 88 tubular or tubulovillous adenomas that were classified into L (low-grade adenomas) or H (high-grade adenomas). Larger adenoma size, higher frequency of villous structure, loss of proliferation polarity, p53 expression, larger TAM numbers and larger microvessel density (MVD) were detected in Group H than in Group L adenomas. Positive relations were observed between TAM and MVD, proliferation polarity and the expression of p53. CD204 (+) TAM is a novel component in the malignant transformation of colorectal adenoma.

Relationship between stromal cells and tumor spread through air spaces in lung adenocarcinoma.

BackgroundThe mechanism underlying tumor spread through air spaces (STAS) has not been well studied. We investigated the role of tumor stromal cells in the pathogenesis of STAS from a pathological perspective and evaluated the prognostic significance of tumor stromal cells and STAS in postoperative patients with lung adenocarcinoma.MethodsWe retrospectively analyzed 208 postsurgical patients with stage I–IIIA lung adenocarcinoma. The presence of STAS was evaluated by hematoxylin and eosin staining. The expression of α‐smooth muscle actin (SMA)‐positive cancer‐associated fibroblasts (CAFs) and CD204‐positive tumor‐associated macrophages (TAMs) was analyzed by immunohistochemistry. A logistic regression model was applied to confirm the predictive factors of STAS. Survival analysis was performed to evaluate the effect of α‐SMA‐positive CAFs, CD204‐positive TAMs, and STAS on prognosis. A nomogram was generated to evaluate the prognosis of postoperative patients.ResultsLogistic regression suggested that the expression of α‐SMA‐positive CAFs (P < 0.001) and the number of CD204‐positive TAMs (P < 0.001) were related to the presence of STAS. The multivariate Cox proportional hazards model suggested that STAS (P = 0.004), α‐SMA‐positive CAFs (P < 0.001), and CD204‐positive TAMs (P < 0.001) were independent risk factors for prognosis. Harrell's c‐indexes for overall and recurrence‐free survival prediction based on nomograms were 0.84 (95% confidence interval 0.76–0.91) and 0.82 (95% confidence interval 0.76–0.89), respectively.ConclusionsThe presence of STAS was associated with high expression of α‐SMA and CD204 in lung adenocarcinoma. Nomograms including STAS and stromal cells as variables are recommended as practical models to evaluate the prognosis of lung adenocarcinoma patients.

CD68- and CD163-positive tumor infiltrating macrophages in non-metastatic breast cancer: a retrospective study and meta-analysis.

Studies have indicated the significance of tumor associated macrophages (TAMs) in breast cancer; however, inconsistent results still exist. We retrospectively reviewed the macrophage distribution in 1579 breast cancer specimens with anti-CD68 or anti-CD163 immunohistochemical staining, and further analyzed the overall survival data. Furthermore, we performed a retrospective study and systematic review of the published studies on CD68- and CD163-positive macrophages in non-metastatic breast cancer. 13 studies with 5116 patients were included in this meta-analysis. Our own data revealed a high density of both CD68- and CD163-positive TAMs that was significantly related to lymph node metastasis (CD68, P = 0.003; CD163, P < 0.001); high Ki67 (CD68, P = 0.026; CD163, P < 0.001), poor histological grade (CD68, P < 0.001; CD163, P < 0.001) and hormonal receptor negativity (CD68, P < 0.001; CD163, P < 0.001); only CD163-positive TAMs were associated with poor overall survival (P = 0.003). Nonetheless, the meta-analysis only found that CD68- and CD163-positive TAMs were associated with high Ki67 [CD68, Relative risk (RR): 1.18, 95% confidence interval (CI): 1.09-1.28; CD163, RR: 1.75, 95% CI: 1.39-2.20], advanced histological grade (CD68, RR: 1.72, 95% CI: 1.46-2.03; CD163, RR: 1.99, 95% CI: 1.35-2.94) and low hormonal receptor levels (CD68, RR: 0.75, 95% CI: 0.69-0.82; CD163, RR: 0.82, 95% CI: 0.74-0.90), but not lymph node metastasis and HER2 expression. This meta-analysis further supports the clinical significance of TAMs in breast cancer, and both CD68- and CD163-positive TAMs could be prognostic markers in non-metastatic breast cancer.

Link between tumor-promoting fibrous microenvironment and an immunosuppressive microenvironment in stage I lung adenocarcinoma

ObjectivesPodoplanin-positive cancer-associated fibroblasts [PDPN (+) CAFs] play an important role in cancer progression in non-small-cell lung cancer. The aim of this study was to clarify the correlation between a fibrous microenvironment containing PDPN (+) CAFs and an immune microenvironment. Materials and methodsA total of 174 patients with pathological stage I lung adenocarcinoma were analyzed. We evaluated PDPN (+) CAFs and immune-related cells, CD 204-positive tumor-associated macrophages [CD204 (+) TAMs], CD8-positive T cells, and FOXP3-positive T cells, in cancer stroma by using immunohistochemical staining. We compared the expression levels of immune-regulatory cytokines between the PDPN high and low expression groups by analyzing the gene expression profiles of lung adenocarcinoma (n = 442). ResultsPresence of PDPN (+) CAFs was a risk factor for recurrence (P = 0.042). The number of CD204 (+) TAMs was significantly higher (P < 0.001) and the CD8/FOXP3 T cell ratio was significantly lower in PDPN (+) CAFs cases than in PDPN (-) CAFs cases (P = 0.027). Within the same tumor, the number of CD 204 (+) TAMs was significantly higher (P < 0.001) and CD8/FOXP3 T cell ratio tended to be lower (P = 0.062) in PDPN (+) CAF areas. Microarray analysis revealed that the PDPN expression-high group had significantly higher gene expression levels of cytokines that inducing M2 macrophage polarization and suppressing immune-related lymphocytes. ConclusionThe current results show that lung adenocarcinoma with PDPN (+) CAFs is typified by the immunosuppressive microenvironment, suggesting a close link between the tumor-promoting fibrous microenvironment and the immunosuppressive microenvironment.

P3.09-06 The Link Between Tumor Promoting Fibrous Microenvironment and Immunosuppressive Microenvironment in Stage I Lung Adenocarcinoma

Podoplanin-positive cancer-associated fibroblasts (PDPN (+)CAFs) play an important role in cancer progression in non-small-cell lung cancer (NSCLC). The aim of this study is to clarify the correlation between fibrous microenvironment containing PDPN (+) CAFs and immune microenvironment. 174 cases with stage I primary lung adenocarcinoma were analyzed in this study. We evaluated PDPN (+) CAFs and immune-related cells; CD 204-positive tumor-associated macrophages (CD204 (+) TAMs); CD8-positive T cells; and FOXP3-positive T cells in cancer stroma by immunohistochemical staining method. By analyzing the gene expression profiles of lung adenocarcinoma (n=442), we compared the expression level of immune-regulatory cytokines between PDPN expression-high group and PDPN expression-low group. Presence of PDPN (+) CAFs was a risk factor for recurrence (P = 0.012). The number of CD204 (+) TAMs was significantly higher in PDPN (+) CAFs cases than in PDPN (+) CAFs cases (P < 0.001). Furthermore, CD8 (+)/FOXP3 (+) T cell ratio was significantly lower in PDPN (+) CAFs cases (P = 0.027). Within the same tumor, the number of CD 204 (+) TAMs was significantly higher in PDPN (+) CAFs area than in PDPN (-) CAFs area (P < 0.001). Moreover, CD8 (+)/FOXP3 (+) T cell ratio tend to be lower in PDPN (+) CAFs area than in PDPN (+) CAFs area (P = 0.062). Microarray analysis revealed PDPN expression-high group had significant higher levels of M-CSF; a cytokine inducing M2 macrophage polarization, and TGFβ1, IDO, VEGFA and galectin 1; immunosuppressive cytokines. The current results revealed that lung adenocarcinoma with PDPN (+) CAFs is typified by an immunosuppressive microenvironment, suggesting the close link between tumor promoting fibrous microenvironment and immune microenvironment.

Origin of cancer-associated fibroblasts and tumor-associated macrophages in humans after sex-mismatched bone marrow transplantation

Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in tumor stroma play a key role in disease progression. Recent studies using mice models suggest that CAFs are partly derived from bone marrow and TAMs primarily originate from bone marrow-derived inflammatory monocytes. However, the origin of these cells in humans remains unclear. Hence, we investigated their human origin, using specimens from human secondary tumors that developed after sex-mismatched bone marrow transplantation, by modified immunofluorescent in situ hybridization analysis and triple immunostaining. We observed that most of the α-smooth muscle actin (αSMA)-positive CAFs in the mammary gland, liver, and oral mucosa specimens obtained 3–19 years after bone marrow transplantation are recipient-derived cells. In contrast, the majority of the peritumoral αSMA-negative fibroblast-like cells are actually bone marrow-derived HLA-DR-positive myeloid cells, such as macrophages and dendritic cells. Furthermore, almost all CD163-positive TAMs and macrophages present in the non-tumor areas are derived from bone marrow.

Differences of tumor microenvironment between stage I lepidic-positive and lepidic-negative lung adenocarcinomas

ObjectiveLepidic growth is a noninvasive component of lung adenocarcinoma. Many adenocarcinoma cases contain coexistent lepidic and nonlepidic (invasive) components (lepidic-growth positive [Lep+] adenocarcinoma); however, some cases comprise only nonlepidic components (lepidic-growth negative [Lep-] adenocarcinoma). The aim of this study was to investigate the biological differences between the invasive components of Lep+ and Lep- adenocarcinoma. MethodsWe investigated the clinicopathologic characteristics of 232 adenocarcinomas (116 size-matched tumor pairs from Lep+ and Lep- adenocarcinomas). We then evaluated the cancer cell–specific expression levels of cancer stem cell, hypoxia, and invasion molecules in these lesions. The number of tumor-promoting stromal cells, including podoplanin-positive cancer-associated fibroblasts and CD204-positive tumor-associated macrophages, was also analyzed. ResultsAmong cases with size-matched invasive components, significant differences were shown in total tumor size and predominant subtype in invasive component between Lep+ and Lep- adenocarcinomas. The expression levels of hypoxia-related molecules were significantly lower in Lep+ adenocarcinomas (glucose transporter 1: 0 vs 10, P < .01; carbonic anhydrase IX: 0 vs 0 [mean, 4.7 vs 14.1], P = .01). The number of podoplanin-positive cancer-associated fibroblasts and CD204-positive tumor-associated macrophages was significantly lower in Lep+ adenocarcinomas (podoplanin-positive cancer-associated fibroblasts: 0 vs 0 [mean: 1.6 vs 11.6], P < .01; CD204-positive tumor-associated macrophages: 8.7 vs 24.7, P < .01). ConclusionsOur results indicated that lower cancer cell–specific expression levels of hypoxia markers and a smaller number of tumor-promoting stromal cells in invasive component were characteristic features of Lep+ adenocarcinomas.