Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profi ling for the fi rst time the phenotype of the Hodgkin lymphoma-specifi c immune microenvironment at single-cell resolution. Single-cell expression profi ling identifi ed a novel Hodgkin lymphoma-associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3 + T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3 + T cells in the direct vicinity of MHC class II-defi cient tumor cells. Our fi ndings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. SIGNIFICANCE:We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifi cally, we identifi ed a regulatory T-cell-like immunosuppressive subset of LAG3 + T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

Periodontal diseases can lead to chronic inflammation affecting the integrity of the tooth supporting tissues. Recently, a striking association has been made between periodontal diseases and primary cancers in the absence of a mechanistic understanding. Here we address the effect of periodontal inflammation (PI) on tumor progression, metastasis, and possible underlining mechanisms. We show that an experimental model of PI in mice can promote lymph node (LN) micrometastasis, as well as head and neck metastasis of 4T1 breast cancer cells, both in early and late stages of cancer progression. The cervical LNs had a greater tumor burden and infiltration of MDSC and M2 macrophages compared with LNs at other sites. Pyroptosis and the resultant IL-1β production were detected in patients with PI, mirrored in mouse models. Anakinra, IL-1 receptor antagonist, limited metastasis, and MDSC recruitment at early stages of tumor progression, but failed to reverse established metastatic tumors. PI and the resulting production of IL-1β was found to promote CCL5, CXCL12, CCL2, and CXCL5 expression. These chemokines recruit MDSC and macrophages, finally enabling the generation of a premetastatic niche in the inflammatory site. These findings support the idea that periodontal inflammation promotes metastasis of breast cancer by recruiting MDSC in part by pyroptosis-induced IL-1β generation and downstream CCL2, CCL5, and CXCL5 signaling in the early steps of metastasis. These studies define the role for IL-1β in the metastatic progression of breast cancer and highlight the need to control PI, a pervasive inflammatory condition in older patients.

Most patients with rectal cancer receive neoadjuvant radiochemotherapy (RCT), causing a variable decrease in tumor mass. We evaluated the prognostic impact of pathologic parameters reflecting tumor response to RCT, either directly or indirectly. Seventy-six rectal cancer patients receiving neoadjuvant RCT between 2006 and 2009 were included. We studied the association between disease-free survival (DFS) and the "classical" clinicopathologic features as well as tumor deposits, circumferential resection margin (CRM), Dworak regression grade, and tumor and nodal downstaging. Patients with tumor downstaging had a longer DFS (p = 0.05), indicating a more favorable prognosis when regression was accompanied by a decrease in tumor infiltrative depth, referred to as tumor shrinkage. Moreover, tumor downstaging was significantly associated with larger CRM and nodal downstaging (p = 0.02), suggesting that shrinkage of the primary tumor was associated with a decreased nodal tumor load. Higher Dworak grade did not correlate with tumor downstaging, nor with higher CRM or prolonged DFS. This implies that tumor mass decrease was sometimes due to fragmentation rather than shrinkage of the primary tumor. Lastly, the presence of tumor deposits was clearly associated with reduced DFS (p = 0.01). Assessment of tumor shrinkage after RCT via tumor downstaging and CRM is a good way of predicting DFS in rectal cancer, and shrinkage of the primary tumor is associated with a decreased nodal tumor load. Assessing regression based on the amount of tumor in relation to stromal fibrosis does not accurately discern tumor fragmentation from tumor shrinkage, which is most likely the reason why Dworak grade had less prognostic relevance.

Neoadjuvant radio(chemo)therapy is increasingly used in rectal cancer and induces a number of morphologic changes that affect prognostication after curative surgery, thereby creating new challenges for surgical pathologists, particularly in evaluating morphologic changes and tumour response to preoperative treatment. Surgical pathologists play an important role in determining the many facets of rectal carcinoma patient care after neoadjuvant treatment. These range from proper handling of macroscopic specimens to accurate microscopic evaluation of pathological features associated with patients' prognosis. This review presents the well-established pathological prognostic indicators and discusses challenging features in order to provide both surgical pathologists and treating physicians with a checklist that is useful in a neoadjuvant setting.

The aim of this study was to investigate murine double minute-2 (MDM2) gene copy number changes in colon carcinoma and to correlate these findings with an immunohistochemical analysis of MDM2 protein expression and histopathologic prognostic indicators of the tumors. The study included 80 cases of sporadic colon carcinomas. MDM2 protein expression was assessed by immunohistochemistry, and MDM2 gene status by fluorescence in situ hybridization. MDM2 gene amplification was detected in 18% of the 80 cases examined. A strong correlation was found between MDM2 gene amplification and the presence, intensity, and staining proportion of cytoplasmic MDM2 protein expression (p = 0.01). No correlation was found between MDM2 gene amplification and the well-established histopathologic prognostic factors. Given the correlation with gene amplification, we clearly demonstrated that cytoplasmic expression of MDM2 protein is true and relevant and that this finding has to be taken into account when immunohistochemistry would be used as a screening for MDM2 gene amplification in the near future. Targeting MDM2 could be a new approach in colon cancer therapy. The amplification status could be a predictive factor of the response to MDM2-targeted therapy.

Multiplexed immune cell profiling of the tumor microenvironment (TME) in cancer has improved our understanding of cancer immunology, but complex spatial analyses of tumor-immune interactions in lymphoma are lacking. Here, we used imaging mass cytometry (IMC) on 33 cases of diffuse large B-cell lymphoma (DLBCL) to characterize tumor and immune cell architecture and correlate it to clinicopathological features such as cell of origin, gene mutations, and responsiveness to chemotherapy. To understand the poor response of DLBCL to immune checkpoint inhibitors (ICI), we compared our results to IMC data from Hodgkin lymphoma, a cancer highly responsive to ICI, and observed differences in the expression of PD-L1, PD-1, and TIM-3. We created a spatial classification of tumor cells and identified tumor-centric subregions of immune activation, immune suppression, and immune exclusion within the topology of DLBCL. Finally, the spatial analysis allowed us to identify markers such as CXCR3, which are associated with penetration of immune cells into immune desert regions, with important implications for engineered cellular therapies. This is the first study to integrate tumor mutational profiling, cell of origin classification, and multiplexed immuno-phenotyping of the TME into a spatial analysis of DLBCL at the single-cell level. We demonstrate that, far from being histopathologically monotonous, DLBCL has a complex tumor architecture, and that changes in tumor topology can be correlated with clinically relevant features. This analysis identifies candidate biomarkers and therapeutic targets such as TIM-3, CCR4, and CXCR3 that are relevant for combination treatment strategies in immuno-oncology and cellular therapies.

BackgroundMultiplexed ion beam imaging (MIBI) combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) with metal labeled antibodies to image 40+ proteins in a single scan at subcellular spatial resolution. Here, we show that the recently released MIBIscope provides improved sensitivity for detecting immune checkpoint markers and offers greater throughput at higher resolution than the alpha instrument.MethodsSerial sections from three FFPE NSCLC samples, in addition to a control slide consisting of various unremarkable tissues, were stained with a panel of 25 metal labeled antibodies. The tissue was imaged at subcellular resolution using the MIBIscope and the alpha instrument. Masses of detected species were assigned to target biomolecules given the unique label of each antibody and multi-step processing was used to create images. Cell classification was performed using two complementary methods that differed in the need for cell segmentation to phenotypically characterize the tissue environments and quantify marker expression.ResultsReplicate regions of interest (ROIs) were collected on both instruments with similarly sized ROIs acquired in 17 minutes with the MIBIscope compared to 280 minutes with the alpha instrument. Fourier Ring Correlation (FRC) showed the resolution to be greater on the MIBIscope as compared to the alpha instrument with FRC also demonstrating uniform resolution across an ROI 2.5X greater in size. Even with the 16X greater speed of the MIBIscope, the signal of the 25 markers across replicate ROIs was increased (y=x^1.07) and showed similar expression patterns to those observed on the alpha instrument (figure 1). This resulted in greater sensitivity to markers with low expression, such as checkpoint markers. Eleven cell populations were classified across the ROIs utilizing two methods, with both methods showing a similar frequency of tumor cells and B, T, and myeloid cell subsets between instruments. Segmentation enabled the number of cells within a population to be calculated but defining boundaries is laborious and signal from neighboring cells can result in misclassification. Performing classification at the pixel level, without segmentation, enabled the fraction of the tissue that is tumor or any other cell type to be rapidly determined.Abstract 48 Figure 1Comparison of images acquired between instrumentsThe signal intensity is greater on the MIBIscope and shows a similar staining pattern as achieved by the alpha instrument. Shown are 3 overlays from a single scan from replicate ROIs of an NSCLC sample displayed with the same contrast settings.ConclusionsThe MIBIscope enables the phenotypic characterization of tumor and non-tumor microenvironments. Co-expression of markers can be used to classify tumor and immune populations and to quantify the expression of markers associated with immune suppression. The increased sensitivity and throughput of the MIBIscope, in combination with the 40-parameter capability and subcellular resolution, provides a platform uniquely suited to understanding the complex tumor immune landscape.