Immune Cells In Microenvironment Research Articles

Prognostic impact of PD-L1 expression in primary gastric and intestinal diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease and the most common gastrointestinal lymphoma. The prognostic/predictive indicators among patients with gastric and intestinal DLBCL (giDLBCL) are controversial beyond their anatomical sites. We compared giDLBCL cases and investigated the clinical utility of newly emerging indicators with an emphasis on programmed cell death ligand 1 (PD-L1) expression. This retrospective study included 174 patients with primary gastric (n = 129) or intestinal (n = 45) DLBCL treated with rituximab-containing chemotherapy between 1995 and 2018. Compared with gastric DLBCL (gDLBCL) cases, patients with intestinal DLBCL (iDLBCL) had a significantly higher rate of advanced Lugano stage (71% vs 37%, P < 0.001), perforation (13% vs. 0.8%, P = 0.001), PD-L1 expression on microenvironment immune cells (miPD-L1, 70% vs 46%, P = 0.008), CD10 positivity (47% vs 28%, P = 0.027), and CD5 positivity (9% vs 1.6%, P = 0.040). The iDLBCL patients showed significantly worse progression-free survival (PFS) and overall survival (OS) than gDLBCL cases (P = 0.0338 and P = 0.0077, respectively). PD-L1 expression on tumor cells was detected in only 3 (2%) of 174 cases with early relapse and/or an aggressive clinical course; whereas, miPD-L1-positive cases had significantly better OS than the miPD-L1-negative gDLBCL and iDLBCL cases (P = 0.0281 and P = 0.0061, respectively). Multivariate analysis revealed that miPD-L1 negativity (P = 0.030) was an independent adverse prognostic factor for OS in giDLBCL. The anatomical site of disease did not influence outcome in giDLBCL cases treated with rituximab-containing chemotherapy; while, miPD-L1 expression had a favorable impact on the outcome.

LncRNA SATB2-AS1 inhibits tumor metastasis and affects the tumor immune cell microenvironment in colorectal cancer by regulating SATB2

BackgroundEmerging studies suggest that long non-coding RNAs (lncRNAs) play crucial roles in colorectal cancer (CRC). Here, we report a lncRNA, SATB2-AS1, which is specifically expressed in colorectal tissue and is significantly reduced in CRC. We systematically elucidated its functions and possible molecular mechanisms in CRC.MethodsLncRNA expression in CRC was analyzed by RNA-sequencing and RNA microarrays. The expression level of SATB2-AS1 in tissues was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH). The functional role of SATB2-AS1 in CRC was investigated by a series of in vivo and in vitro assays. RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), chromatin isolation by RNA purification (ChIRP), Bisulfite Sequencing PCR (BSP) and bioinformatics analysis were utilized to explore the potential mechanisms of SATB2-AS1.ResultsSATB2-AS1 is specifically expressed in colorectal tissues and downregulated in CRC. Survival analysis indicates that decreased SATB2-AS1 expression is associated with poor survival. Functional experiments and bioinformatics analysis revealed that SATB2-AS1 inhibits CRC cell metastasis and regulates TH1-type chemokines expression and immune cell density in CRC. Mechanistically, SATB2-AS1 directly binds to WDR5 and GADD45A, cis-activating SATB2 (Special AT-rich binding protein 2) transcription via mediating histone H3 lysine 4 tri-methylation (H3K4me3) deposition and DNA demethylation of the promoter region of SATB2.ConclusionsThis study reveals the functions of SATB2-AS1 in CRC tumorigenesis and progression, suggesting new biomarkers and therapeutic targets in CRC.

Exploitation of Cell Mediated Immune Responses to Cancer Immunotherapy

Individual human tumors arise through a combination of genetic and epigenetic changes that facilitate immortality. Cancer occurs when there are changes to genes that control the way our cells grow and divide.. The increase in the prevalence of cancer with lack of specific treatment options became public health problem particularly in middle and low income nations. Activation of the immune system by products of viral oncogene prevents viral induced cancers. The in vitro stimulation of Dendritic cells with tumor or tumor antigen in the presence of cytokines for cross activation of CD8+ and CD4+ T cells or restoration of T cells functions by blockade of immune blockers or modification tumor microenvironment or adoptive transfer of immune cells and stem T cells are promising cancer therapies. Compared with the convectional cancer therapies, immunotherapies are most tumors targeted without harmful side effects. DOI : 10.7176/JMPB/59-03 Publication date :September 30 th 2019

Abstract 4486: Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration resistant PCa (CRPC), following failure of androgen deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa but nearly universal progression to CRPC occurs 2-3 years after ADT is initiated. Immunotherapy with checkpoint inhibitors has not been effective in most prostate cancers, perhaps because such cancers lack functional CD8 T-cells. This may be caused by infiltration of myeloid cell populations into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T cells, consistent with an immuno-suppressive or immuno-evasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets show the transcripts for TNF, as well as gene signatures for stem/progenitor tumor cells and M2 TAMs are increased in CRPC, consistent with our hypothesis that ADT drives the development of myeloid immuno-suppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Kent L. Nastiuk. Androgen-deprivation therapy promotes immune suppression in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4486.

Targeting the Immune Microenvironment in Lymphomas of B-Cell Origin: From Biology to Clinical Application.

In lymphomas of B-cell origin, cancer cells orchestrate an inflammatory microenvironment of immune and stromal cells that sustain the tumor cell survival and growth, known as a tumor microenvironment (TME). The features of the TME differ between the different lymphoma types, ranging from extremely inflammatory, such as in Hodgkin lymphoma, to anergic, leading to immune deficiency and susceptibility to infections, such as in chronic lymphocytic leukemia. Understanding the characteristic features of the TME as well as the interactions between cancer and TME cells has given insight into the pathogenesis of most lymphomas and contributed to identify novel therapeutic targets. Here, we summarize the preclinical data that contributed to clarifying the role of the immune cells in the TME of different types of lymphomas of B-cell origin, and explain how the understanding of the biological background has led to new clinical applications. Moreover, we provide an overview of the clinical results of trials that assessed the safety and efficacy of drugs directly targeting TME immune cells in lymphoma patients.

CaMKK2 in myeloid cells is a key regulator of the immune-suppressive microenvironment in breast cancer

Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8+ T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2−/− mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2−/− macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8+ T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.

1758-P: Alterations in Human Visceral Adipose Tissue Type 2 Innate Lymphoid Cells (ILC2s) with Aging: A Determinant of Insulin Resistance

Background: The obese adipose tissue (AT) immune cell microenvironment contributes to low-grade inflammation increasing the risk of multiple metabolic complications. Type 2 innate lymphoid cells (ILC2s) were recently discovered in mice to regulate metabolic health, but the clinical relevance of ILC2s in human AT is unknown. Rationale: Our goals were to characterize changes human AT ILC2s in obesity, insulin resistance, and aging. We further examined changes in AT ILC2s with aging, high fat diet (HFD), and weight loss in mice. Methods: We collected visceral AT (VAT) biopsies from 50 obese and 9 lean patients during elective surgery, isolated the adipocytes from the stromal vascular fraction, and performed flow cytometry. Results: Human VAT %ILC2s were not different between lean and obese subjects (p=0.896), but were negatively associated with insulin sensitivity (r=-0.449, p=0.005), beta-cell function (r=-0.364, p=0.024), and increasing age (r=-0.337, p=0.012). ILC2s were also positively associated with T helper type 2 cells (Th2s). All of these relationships were independent of BMI and gender, suggesting that ILC2s play a weight-independent role in human glucose homeostasis and age-related insulin resistance, possibly through an interaction with Th2s. When placed on HFD for 6 and 12 weeks, mice demonstrated a depletion in VAT ILC2s, and 12- vs. 3-month old mice had decreased VAT ILC2s, which did not recover after 12 weeks of weight loss despite normalization of body weight. Conclusions: In humans and mice, VAT ILC2s decrease with HFD and age. In humans ILC2s may be a weight-independent determinant of hyperinsulinemia and insulin resistance, provide a potential link to reduced insulin sensitivity in aging, and offer a novel therapeutic target in insulin resistance and T2D. Disclosure D. Bradley: None. A.M. Blaszczak: None. J.Z. Liu: None. A.D. Jalilvand: None. V.P. Wright: None. S. Noria: None. J. Joseph: None. W. Hsueh: None. Funding American Diabetes Association (1-16-ICTS-049 to W.H.); National Institutes of Health (KL2TR001068, HL135622)

Abstract A084: Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer

Abstract Most prostate cancer (PCa) deaths are due to castration-resistant PCa (CRPC), following failure of androgen-deprivation therapy (ADT). ADT is the standard of care for patients with advanced PCa. However, nearly universal progression to castration-resistant prostate cancer (CRPC) occurs 2-3 years after ADT is initiated. Although there have been recent improvements in the treatment of CRPC, even the most promising therapies are still not curative. One approach to this problem is to improve the initial treatment of advanced prostate cancers, by combining complementary therapies with ADT, to prevent progression of such advanced cancers to CRPC. Immunotherapy with checkpoint inhibitors (CPIs) has not been effective in prostate cancers, perhaps because such cancers are “cold” (lacking cytolytic CD8 T-cells). Some cold tumors may be caused by infiltration of myeloid cell populations (tumor associated macrophages and myeloid-derived suppressor cells) into the tumor immune cell microenvironment (TIME). Recently, we found that in a PTEN-deficient mouse PCa model, castration induces an immunosuppressive state within the tumor that is concurrent with tumor recurrence. The response to castration/ADT is tri-phasic: a pro-apoptotic regression phase when tumor shrinks, followed by selection for a residual population of resistant tumor cells and finally recurrent growth as CRPC. Using PCa cell lines to model the first two phases of the response to ADT, we have shown that ADT induces apoptosis, thereby enriching for an ADT-resistant stem/progenitor population that we propose is the in vivo source of TNF. Mechanistically, in our model system the response to ADT is driven by the soluble mediators TNF and CCL2, which facilitate communication within the TIME. Specifically, a TNF-CCL2-CCR2 paracrine loop is induced between prostate cancer cells and non-tumor cells in the microenvironment: TNF produced by tumor cells acts on myofibroblasts to induce CCL2 production, which in turn recruits CCR2+ tumor-associated macrophages (TAMs). To investigate the ADT response within the TIME in an in vivo model of prostate cancer, we employed a prostate-specific PTEN-deficient mouse model (PbCre4 x PTENf/f). Castration caused the tumors to regress, consistent with initial phase of the response that is seen in the human disease. At late times post-castration (5-6 weeks), corresponding to the selection phase, we observed a coordinate increase in the stem/progenitor tumor cell population, as well as TNF and CCL2, within the TIME. Immunohistochemical staining of tumors 5 weeks post-castration revealed an increase in TAMs, and a decrease in CD8 T-cells, consistent with an immunosuppressive or immunoevasive state. This phenotype was reversed by a soluble receptor that binds TNF (etanercept). We also observed increased myeloid-derived suppressor cells (MDSC). Thus, following ADT, TNF derived from an ADT-resistant stem/progenitor epithelial tumor cell population promotes an immunosuppressive state via CCL2 in the TIME. Analysis of public human PCa data sets shows TNF and stem/progenitor marker expression are both increased in CRPC, consistent with our hypothesis that ADT drives the development of an immunosuppressive state via a TNF-CCL2-CCR2 axis. Our results set the stage for the future development of immunotherapies that could improve the efficacy of ADT. Citation Format: John J. Krolewski, Kai Sha, Michalis Mastri, Dean Tang, Kevin Eng, Kent L. Nastiuk. Towards combining androgen deprivation and immunotherapy to prevent progression to castration-resistant prostate cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A084.

Pancreatic ductal adenocarcinoma immune microenvironment and immunotherapy prospects

The tumor components of pancreatic ductal adenocarcinoma (PDAC) are composed of immune microenvironment of extracellular matrix, fibroblasts, endothelial cells and immune cells together with a minority of malignant cells. The failure of the therapeutic measures, such as chemotherapy, targeted therapy and immunotherapy, has been linked to the specific immune microenvironment of pancreatic cancer. By analyzing the components of immune microenvironment of pancreatic cancer, the mechanism of various components in pancreatic cancer such as highly immunosuppressive, hypoxic and connective tissue hyperplasia can be clarified. New efforts in single-cell profiling will enable a better understanding of the composition of the microenvironment in primary and metastatic PDAC, as well as an understanding of how the microenvironment may respond to novel therapeutic approaches.

Advances in Research on Immunological Checkpoint Inhibitors in Immunotherapy of Liver Cancer

The current treatments of liver cancer in China are mainly comprehensive treatment and systematic treatment, with poor therapeutic effect and high recurrence rate and metastasis rate. The existence of immunosuppressive microenvironment is an important reason for liver tumor cells to escape from the host immune system, and also an important basis for the occurrence and development of liver cancer. With the recent transformation of the target of tumor therapy from tumor cells to tumor cell immune microenvironment, immunotherapy has emerged quietly. It is a new strategy for the treatment of hepatocellular carcinoma to improve the immune attack on tumor cells by changing the immunosuppressive environment of hepatocellular carcinoma cells. Immune checkpoint is the main mechanism by which liver cancer cells escape the host immune system. PD-1/PDL-1 and CTLA-4 are targeted immunocheckpoint inhibitors, which have shown good therapeutic effects and application prospects in the clinical treatment of HCC. This article reviews the latest advances in immunocheckpoint inhibitors in the immunotherapy of hepatocellular carcinoma.

Novel mRNA Detection Assay for Quantifying CD30 Expression in Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma

Introduction:Innovations in detecting and measuring antigen expression is paramount in the current era of targeted therapy and check-point inhibitors. For instance, Brentuximab Vedotin (BV), one of two FDA-approved antibody-drug conjugates, employs an anti-CD30-monomethyl auristatin E (MMAE) immunoconjugate for the treatment of refractory Hodgkin lymphoma and anaplastic large cell lymphoma with positive CD30 expression. However, a lack of correlation between objective response rate and range of CD30 expression has been previously demonstrated, and the limitations of immunohistochemistry in detecting low levels of CD30 has been implicated. Moreover, there is interest in the study of the topology of checkpoint proteins such as programmed cell death-1 ligands (PD-L1) malignant cells and surrounding PD-1 expressing immune cells, which requires assay with high sensitivity, specificity, and accurate spatial localization.In this study, we implemented a novel CD30 mRNA detection method using the BaseScopeTM assay from Advanced Cell Diagnostics to enable detection of specific CD30mRNA isoforms that correspond the different CD30 protein segments within the neoplastic cells and tumor microenvironment immune cells. Using this method, the CD30 mRNA expression patterns in classical Hodgkin lymphoma (CHL) and anaplastic large cell lymphoma (ALCL) were evaluated.Methods:Nineteen CHL and 11 ALCL cases were selected from the Roswell Park Comprehensive Cancer Institute department of pathology archives. CD30 expression by immunohistochemistry (IHC) and CD30 mRNA isoform expression by BaseScope™ assay were measured on formalin fixed paraffin embedded ( FFPE ) tissue specimens. Four probe sets spanning the exon junctions for increased specificity were used to detect domains on the protein corresponding to signal peptide (SP), transmembrane portion (TMP), cytoplasmic portion (CP), and transmembrane and cytoplasmic portion (TMP &CP) of full length CD30 protein. The hybridization, amplification, and staining of probes are based on the manufacturer's protocol. The BaseScope™ assay probe signal is detected in the form of red dots, which are localized to the neoplastic cells and tumor microenvironment immune cells. Semi-quantitative analysis of both positive and negative signals using 100X magnification was performed on 100 neoplastic cells for each probe for each case, by two hematopathologists (VN, JW). For tumor microenvironment immune cells, an average number of positive and negative cells were obtained in five 100X magnification fields.Results:In both CHL and ALCL, 100% of the neoplastic cells were positive for CD30 protein by immunohistochemistry. The table below illustrates relative expression of CD30 mRNA isoforms in CHL and ALCL neoplastic cells. Expression of CD30 mRNA in CHL the tumor microenvironment cells is also tabulated.Conclusions:Detection of CD30 mRNA isoforms using BaseScope™ assay offers a reliable and reproducible signal detection platform, as an alternative to CD30 protein expression by immunohistochemistry. The BaseScope™ assay provides a high-contrast low noise signal for easy visualization, accurate quantification, and precise positive signal localization. Different CD30 isoform patterns and signal frequency were observed within the neoplastic cells and immune cells in the tumor microenvironment. The CD30 mRNA isoform corresponding to transmembrane domain was a dominant expresser in both CHL and ALCL neoplastic cells and bystander cells in CHL. This novel mRNA-based method can potentially provide a more sensitive method for antigen expression detection and be used in new research studies. DisclosuresNo relevant conflicts of interest to declare.

Establishment of Slice Cultures as a Tool to Study the Cancer Immune Microenvironment.

Although immunotherapy is currently being widely applied to treat a variety of cancers, there is great heterogeneity in the response to these treatments. Many in the field hypothesize that this may be attributable to the characteristics of each individual tumor immune microenvironment, in addition to systemic immune factors. Therefore, understanding the immune cell microenvironment in a variety of tumors is critically important. Specifically, the interactions among immune, stromal, and cancer cells, along with other factors in tumors, may hold the key to developing rational personalized combinations of immunotherapeutic drugs. We recently developed an organotypic slice culture technique, which enables precise study of the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment. We used a Vibratome to cut fresh human tumor tissue into 250μm thick slices, and cultured slices on cell culture inserts with 0.4μm pore to produce our tumor slice culture (TSC) system. We showed that TSC maintained many elements of the original tumor microenvironment and architecture for approximately one week. Using this slice culture technique for PDA, we demonstrated that immune cells, including T cells and macrophages, cancer cells, and stromal myofibroblasts were present throughout the culture period. TSCs were functionally responsive to drug treatment. Live PDA slices could be stained for multicolor immunofluorescence imaging of each of the primary cellular constituents of the tumor. Finally, autologous CFSE-labeled splenocytes were observed to readily migrate into cocultured tumor slices.

Clinicopathological analysis of primary intestinal diffuse large B-cell lymphoma: Prognostic evaluation of CD5, PD-L1, and Epstein-Barr virus on tumor cells.

BackgroundPrimary intestinal diffuse large B‐cell lymphoma (iDLBCL) is rare. In this study, we investigated the clinicopathological features of this disease to further understand the prognostic value of CD5, programmed cell death ligand 1 (PD‐L1), and Epstein‐Barr virus (EBV) on tumor cells.MethodsTumor specimens from 62 patients consecutively diagnosed with primary iDLBCL at a single institution were analyzed.ResultsOur series consisted of EBV‐positive (EBV+) iDLBCL (n = 10), de novo CD5+ iDLBCL (n = 4), and DLBCL, not otherwise specified (DLBCL‐NOS; n = 48). Notably, seven of 10 EBV+ cases had treated lymphoma‐associated (n = 4) or iatrogenic immunodeficiency (n = 3). Two of 10 EBV+ cases expressed PD‐L1 on tumor cells, whereas the remaining eight were positive for PD‐L1 on microenvironment immune cells. Only one DLBCL‐NOS case had neoplastic PD‐L1 expression with a giant cell‐rich appearance. Both EBV‐harboring and PD‐L1 expression on tumor cells, but not CD5, were associated with worse overall survival (OS) in iDLBCL patients receiving rituximab‐containing chemotherapy (P = 0.0354, P = 0.0092, and P = 0.1097, respectively). Multivariate analysis identified PD‐L1 positivity on tumor cells (P = 0.0106), PD‐L1 negativity on microenvironment immune cells (P = 0.0193), and EBV positivity (P = 0.0324) as poor independent prognostic factors for OS. Among iDLBCL cases without any EBV association, CD5 positivity, or neoplastic PD‐L1 expression, high PD‐L1 expression (≥40%) on microenvironment immune cells predicted an extremely favorable outcome.ConclusionEBV+ iDLBCL mainly comprised immunodeficiency‐associated patients, which may highlight the specificity of the intestine. PD‐L1 expression on tumor cells or microenvironment immune cells was found to have an opposite prognostic impact in iDLBCL.

Cancer Immunotherapy and the Immune Response in Follicular Lymphoma.

Follicular lymphoma (FL) is the most frequent indolent lymphoma in the Western world and is characterized in almost all cases by the t(14;18) translocation that results in overexpression of BCL2, an anti-apoptotic protein. The entity includes a spectrum of subentities that differ from an indolent to a very aggressive growth pattern. As a consequence, treatment can include watch & wait up to intensive chemotherapy including allogeneic stem cell transplantation. The immune cell microenvironment has been recognized as a major driver of outcome of FL patients and gene expression profiling has identified a clinically relevant gene expression signature that classifies an immune response to the lymphoma cells. It is known for some time that the immune cell composition of the lymphoma microenvironment is important because high numbers of tissue-infiltrating macrophages correlate with poor outcome in patients receiving chemotherapy but not in patients receiving the combination of chemotherapy and CD20-specific monoclonal antibody rituximab. In addition, TCR signaling of tumor-infiltrating lymphocytes is dysfunctional leading to an impaired capacity to form an intact immunologic synapse. Approaches restoring local T cell function, e.g., by usage of checkpoint inhibitors has demonstrated clinical activity (ORR 40%) and can achieve long-term remissions. Ongoing trials with re-programmed autologous CART cells achieve response rates in approximately 50% of FL patients with relapsed and even refractory disease. Responses lasting for more than 6 months might be durable, indicative for a successful restoration of a functional immune system. In summary, FL is a malignant disease where the control by the immune system ultimately decides about progression and transformation rate. The advent of monoclonal antibodies has changed the way we treat FL and new approaches restoring the individual immune control will hopefully improve results further.

A prognostic model, including the EBV status of tumor cells, for primary gastric diffuse large B‐cell lymphoma in the rituximab era

EBV‐positive diffuse large B‐cell lymphoma (DLBCL), not otherwise specified (NOS), often affects the gastrointestinal tract. However, the prognostic significance of EBV associated with primary gastric DLBCL (gDLBCL) has not been established. This retrospective study included 240 patients with primary gDLBCL, diagnosed between 1995 and 2015. Tumor specimens were analyzed with EBER in situ hybridization. In 25 (10%) cases, tumor cells harbored EBV. The EBV + group more frequently exhibited programmed death‐ligand 1 (PD‐L1) expression in microenvironment immune cells, but not tumor cells, compared to the EBV − group (86% vs 43%, P = .006). Among 156 patients that received rituximab‐containing chemotherapy, the EBV + group had a significantly worse overall survival (OS) than the EBV − group (P = .0029). Multivariate analyses identified 3 independent adverse prognostic factors of OS: multiple gastric lesions (P = .002), EBER positivity (P = .003), and B symptoms (P = .018). These factors were combined to develop a gDLBCL prognostic (gDLP) model that significantly stratified the patients into 3 distinct risk groups (Scores: good = 0, intermediate = 1, and poor = 2/3, P < .0001) with 5‐year OS rates of 100%, 81%, and 39%, respectively. Patients with EBV + gDLBCL commonly exhibited microenvironmental PD‐L1 expression and showed a significantly worse prognosis than subjects with EBV − gDLBCL. Our gDLP model, which included EBV + tumor cells, provided good predictions of clinical outcome and may be useful for selecting patients in trials in the immune‐oncology era.

Microenvironment of Immune Cells Within the Visceral Adipose Tissue Sensu Lato vs. Epicardial Adipose Tissue: What Do We Know?

The chronic low-grade inflammation of the visceral adipose tissue is now fully established as one of the main contributors to metabolic disorders such as insulin resistance, subsequently leading to metabolic syndrome and other associated cardiometabolic pathologies. The orchestration of immune response and the "ratio of responsibility" of different immune cell populations have been studied extensively over the last few years within the visceral adipose tissue in general sense (sensu lato). However, it is essential to clearly distinguish different types of visceral fat distribution. Visceral adipose tissue is not only the classical omental or epididymal depot, but includes also specific type of fat in the close vicinity to the myocardium-the epicardial adipose tissue. Disruption of this type of fat during obesity was found to have a unique and direct influence over the cardiovascular disease development. Therefore, epicardial adipose tissue and other types of visceral adipose tissue depots should be studied separately. The purpose of this review is to explore the present knowledge about the morphology and dynamics of individual populations of immune cells within the visceral adipose tissue sensu lato in comparison to the knowledge regarding the epicardial adipose tissue specifically.

The Pancreatic Cancer Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is composed of a minority of malignant cells within a microenvironment of extracellular matrix, fibroblasts, endothelial cells, and immune cells. Therapeutic failures of chemotherapy, targeted therapy, and immunotherapy have all been attributed to the PDAC microenvironment. In this review, we dissect the components of the microenvironment and explain how each cell type contributes to form a highly immunosuppressive, hypoxic, and desmoplastic cancer. New efforts in single-cell profiling will enable a better understanding of the composition of the microenvironment in primary and metastatic PDAC, as well as an understanding of how the microenvironment may respond to novel therapeutic approaches.

Abstract 624: Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC

Abstract Still-emerging evidence suggests that HCC is at least moderately responsive to therapeutic agents that target tumor immune suppression pathways, specifically the programmed death 1/programmed death ligand 1 (PD-L1) pathway. Prior studies raise the possibility that PD-L1 is a prognostic biomarker in HCC, where its expression levels have been reported to correlate with tumor aggressiveness and recurrence following surgical resection. The purpose of this study was to evaluate the expression of PD-L1 in non-cirrhotic liver tissue adjacent to HCC and HCC itself in 68 procured tissue samples, as well as elucidate the immune cell composition of the HCC tumor microenvironment. All cases showed the typical morphology of HCC and were classified as low- to high-grade trabecular, pseudoglandular, or solid with the common cytoplasmic features. Immunohistochemical (IHC) staining for PD-L1 in tumor-free liver (TFL) revealed PD-L1 staining in sinusoidal lining cells (macrophages [Mϕ] and endothelial cells [EC], as confirmed by double labeling for CD68 and CD31), although there was considerable heterogeneity in the extent of PD-L1 staining. More specifically, in TFL, some but not all CD68+ Mϕ were also PD-L1+, whereas most CD31+ EC were also PD-L1+. Furthermore, we evaluated PD-L1 staining qualitatively and semiquantitatively in HCC. Only few tumor cells displayed membranous PD-L1 staining, and 62/68 of the cases (91.2%) were categorized as PD-L1- vs 6/68 (8.8%) PD-L1+ (≥1% and &amp;lt;25% = PD-L1+); instead, PD-L1 expression within the tumor microenvironment predominantly emanated from immune cell infiltrates (as confirmed by PD-L1/pan-cytokeratin double labeling). Then we assessed the immune milieu in HCC tissue specimens using quantitative IHC. We found considerable interspecimen variation in the number of CD68+ Mϕ, CD8+ cytotoxic T lymphocytes, and FoxP3+ regulatory T lymphocytes. Comparing the number of immune cells in different tumor compartments showed that the prevalence of CD68+ cells (p=.0005), CD8+ cells (p=.0004), and FoxP3+ cells (p=.05) was significantly higher within the invasive margin vs the center of the tumor. Quantitative analysis of the immune cell content also showed that there was no correlation between T lymphocyte infiltration (CD8+/FoxP3+) and Mϕ infiltration in any compartment. Taken together, our findings confirm the intertumoral heterogeneity of the immune cell microenvironment in HCC; however, future studies are needed to correlate these findings to the clinical setting with immunooncologic treatments. Citation Format: Christian Ihling, Sienna Yoast, Yue Zhang, Bartholomew Naughton, Miriam Urban, P. Alexander Rolfe, Eveline Frick-Krieger, Isabelle Dussault. Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 624. doi:10.1158/1538-7445.AM2017-624

Tumor Metabolic Features Identified by 18F-FDG PET Correlate with Gene Networks of Immune Cell Microenvironment in Head and Neck Cancer.

The importance of 18F-FDG PET in imaging head and neck squamous cell carcinoma (HNSCC) has grown in recent decades. Because PET has prognostic values, and provides functional and molecular information in HNSCC, the genetic and biologic backgrounds associated with PET parameters are of great interest. Here, as a systems biology approach, we aimed to investigate gene networks associated with tumor metabolism and their biologic function using RNA sequence and 18F-FDG PET data. Methods: Using RNA sequence data of HNSCC downloaded from The Cancer Genome Atlas data portal, we constructed a gene coexpression network. PET parameters including lesion-to-blood-pool ratio, metabolic tumor volume, and tumor lesion glycolysis were calculated. The Pearson correlation test was performed between module eigengene-the first principal component of modules' expression profile-and the PET parameters. The significantly correlated module was functionally annotated with gene ontology terms, and its hub genes were identified. Survival analysis of the significantly correlated module was performed. Results: We identified 9 coexpression network modules from the preprocessed RNA sequence data. A network module was significantly correlated with total lesion glycolysis as well as maximum and mean 18F-FDG uptake. The expression profiles of hub genes of the network were inversely correlated with 18F-FDG uptake. The significantly annotated gene ontology terms of the module were associated with immune cell activation and aggregation. The module demonstrated significant association with overall survival, and the group with higher module eigengene showed better survival than the other groups with statistical significance (P = 0.022). Conclusion: We showed that a gene network that accounts for immune cell microenvironment was associated with 18F-FDG uptake as well as prognosis in HNSCC. Our result supports the idea that competition for glucose between cancer cell and immune cell plays an important role in cancer progression associated with hypermetabolic features. In the future, PET parameters could be used as a surrogate marker of HNSCC for estimating molecular status of immune cell microenvironment.

Biological Activities of Extracellular Vesicles and Their Cargos from Bovine and Human Milk in Humans and Implications for Infants

Extracellular vesicles (EVs) in milk harbor a variety of compounds, including lipids, proteins, noncoding RNAs, and mRNAs. Among the various classes of EVs, exosomes are of particular interest, because cargo sorting in exosomes is a regulated, nonrandom process and exosomes play essential roles in cell-to-cell communication. Encapsulation in exosomes confers protection against enzymatic and nonenzymatic degradation of cargos and provides a pathway for cellular uptake of cargos by endocytosis of exosomes. Compelling evidence suggests that exosomes in bovine milk are transported by intestinal cells, vascular endothelial cells, and macrophages in human and rodent cell cultures, and bovine-milk exosomes are delivered to peripheral tissues in mice. Evidence also suggests that cargos in bovine-milk exosomes, in particular RNAs, are delivered to circulating immune cells in humans. Some microRNAs and mRNAs in bovine-milk exosomes may regulate the expression of human genes and be translated into protein, respectively. Some exosome cargos are quantitatively minor in the diet compared with endogenous synthesis. However, noncanonical pathways have been identified through which low concentrations of dietary microRNAs may alter gene expression, such as the accumulation of exosomes in the immune cell microenvironment and the binding of microRNAs to Toll-like receptors. Phenotypes observed in infant-feeding studies include higher Mental Developmental Index, Psychomotor Development Index, and Preschool Language Scale-3 scores in breastfed infants than in those fed various formulas. In mice, supplementation with plant-derived MIR-2911 improved the antiviral response compared with controls. Porcine-milk exosomes promote the proliferation of intestinal cells in mice. This article discusses the above-mentioned advances in research concerning milk exosomes and their cargos in human nutrition. Implications for infant nutrition are emphasized, where permitted, but data in infants are limited.