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Figure S1. The relative mRNA expression of DKK4 and CTNNB1 in human KIT+ cells.

Primary untreated KIT+ vs. Metastatic resistant KIT+, pathways with P< 0.05.

Supplementary Table 2: Primary untreated (Prim/UT) KIT+ ( n = 8 ) vs. Metastatic resistant (Met/Res) KIT+ ( n = 4). Significant genes with a False Discovery Rate of < 0.05 and a fold change > 2.0

Image cytometry methods overview

Purpose:To characterize changes in the soft-tissue sarcoma (STS) tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design.Experimental Design:Paired pre- and postneoadjuvant therapy specimens were retrospectively identified for 32 patients with STSs and analyzed by three modalities: multiplexed IHC, NanoString, and RNA sequencing with ImmunoPrism analysis.Results:All 32 patients, representing a variety of STS histologic subtypes, received neoadjuvant radiotherapy and 21 (66%) received chemotherapy prior to radiotherapy. The most prevalent immune cells in the tumor before neoadjuvant therapy were myeloid cells (45% of all immune cells) and B cells (37%), with T (13%) and natural killer (NK) cells (5%) also present. Neoadjuvant therapy significantly increased the total immune cells infiltrating the tumors across all histologic subtypes for patients receiving neoadjuvant radiotherapy with or without chemotherapy. An increase in the percentage of monocytes and macrophages, particularly M2 macrophages, B cells, and CD4+ T cells was observed postneoadjuvant therapy. Upregulation of genes and cytokines associated with antigen presentation was also observed, and a favorable pathologic response (≥90% necrosis postneoadjuvant therapy) was associated with an increase in monocytic infiltrate. Upregulation of the T-cell checkpoint TIM3 and downregulation of OX40 were observed posttreatment.Conclusions:Standard neoadjuvant therapy induces both immunostimulatory and immunosuppressive effects within a complex sarcoma microenvironment dominated by myeloid and B cells. This work informs ongoing efforts to incorporate immune checkpoint inhibitors and novel immunotherapies into the neoadjuvant setting for STSs.

Live microscopy analysis of CD8+ T cell distribution in slice cultures

Purpose: Gastric cancers may harbor a subset of cells with cancer stem cell (CSC) properties, including chemotherapy resistance, and CD44 is a gastric CSC marker. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. Here, we examine the role of HH signaling in CD44(+) gastric cancer cells.Experimental Design: Gastric cancer cell lines, tumor xenografts, and patient tumors were examined.Results: Gastric cancer cell lines AGS, MKN-45, and NCI-N87 grown as spheroids or sorted for CD44(+) were found to have upregulation of HH pathway proteins. HH inhibition using Smoothened (Smo) shRNA or vismodegib (VIS) decreased spheroid formation and colony formation. CD44(+) cells, compared with unselected cells, were also resistant to 5-fluorouracil and cisplatin chemotherapy, and this resistance was reversed in vitro and in xenografts with Smo shRNA or VIS. CD44(+) cells also had significantly more migration, invasion, and anchorage-independent growth, and these properties could all be blocked with HH inhibition. Clinical tumor samples from a phase II trial of chemotherapy with or without VIS for advanced gastric cancer were analyzed for CD44 expression. In the chemotherapy alone group, high CD44 expression was associated with decreased survival, whereas in the chemotherapy plus VIS group, high CD44 expression was associated with improved survival.Conclusions: HH signaling maintains CSC phenotypes and malignant transformation phenotypes in CD44(+) gastric cancer cells, and HH inhibition can reverse chemotherapy resistance in CD44(+) cells. Gastric cancer is a heterogeneous disease, and the strategy of combining chemotherapy with HH inhibition may only be effective in tumors with high CD44 levels. Clin Cancer Res; 20(15); 3974–88. ©2014 AACR.

Supplementary Data from Neoadjuvant Therapy Induces a Potent Immune Response to Sarcoma, Dominated by Myeloid and B Cells

Supplementary Figure from Neoadjuvant Therapy Induces a Potent Immune Response to Sarcoma, Dominated by Myeloid and B Cells

PDA tumor slice culture platform utilized for multimodal analysis before and after ex vivo immune modulation

Supplementary Figure 5 - Cisplatin chemotherapy combined with Hedgehog pathway inhibition with vismodegib in MKN-45 xenografts.

PDF file - 35KB, Supplemental Figure S2. Biodistribution of 99mTc-Sestamibi in KitV558del/+ mice.

Purpose: Tyrosine kinase inhibitors are effective in gastrointestinal stromal tumors (GISTs) but often are of transient benefit as resistance commonly develops. Immunotherapy, particularly blockade of the inhibitory receptor programmed death 1 (PD-1) or the ligand programmed death ligand 1 (PD-L1), has shown effectiveness in a variety of cancers. The functional effects of PD-1/PD-L1 blockade are unknown in GISTs.Experimental Design: We analyzed tumor and matched blood samples from 85 patients with GISTs and determined the expression of immune checkpoint molecules using flow cytometry. We investigated the combination of imatinib with PD-1/PD-L1 blockade in KitV558Δ/+ mice that develop GISTs.Results: The inhibitory receptors PD-1, lymphocyte activation gene 3, and T-cell immunoglobulin mucin-3 were upregulated on tumor-infiltrating T cells compared with T cells from matched blood. PD-1 expression on T cells was highest in imatinib-treated human GISTs. Meanwhile, intratumoral PD-L1 expression was variable. In human GIST cell lines, treatment with imatinib abrogated the IFNγ-induced upregulation of PD-L1 via STAT1 inhibition. In KitV558Δ/+ mice, imatinib downregulated IFNγ-related genes and reduced PD-L1 expression on tumor cells. PD-1 and PD-L1 blockade in vivo each had no efficacy alone but enhanced the antitumor effects of imatinib by increasing T-cell effector function in the presence of KIT and IDO inhibition.Conclusions: PD-1/PD-L1 blockade is a promising strategy to improve the effects of targeted therapy in GISTs. Collectively, our results provide the rationale to combine these agents in human GISTs. Clin Cancer Res; 23(2); 454–65. ©2016 AACR.

Clinicopathological features of the patients with GIST in this study.

TCR overlap analysis

PDF file - 119KB, Supplemental Figure S1. Additional PLX3397-mediated immune effects.

Purpose:Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy.Experimental Design:Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization.Results:mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis.Conclusions:Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA.See related commentary by Medina and Miller, p. 3747

Patient demographic and tumor pathology

Image cytometry infiltration results by layer

Purpose: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and a model of targeted molecular therapy. GIST depends on oncogenic KIT signaling and responds to the tyrosine kinase inhibitor imatinib. However, imatinib is rarely curative. We hypothesized that PLX3397, which inhibits KIT and colony-stimulating-factor-1 receptor (CSF1R), would be more efficacious than imatinib in GIST by also depleting tumor-associated macrophages, which are generally thought to support tumor growth.Experimental Design: We treated KitV558del/+ mice that develop GIST or mice with subcutaneous human GIST xenografts with imatinib or PLX3397 and analyzed tumor weight, cellular composition, histology, molecular signaling, and fibrosis. In vitro assays on human GIST cell lines were also performed.Results: PLX3397 was more effective than imatinib in reducing tumor weight and cellularity in both KitV558del/+ murine GIST and human GIST xenografts. The superiority of PLX3397 did not depend on depletion of tumor-associated macrophages, because adding CSF1R inhibition did not improve the effects of imatinib. Instead, PLX3397 was a more potent KIT inhibitor than imatinib in vitro. PLX3397 therapy also induced substantial intratumoral fibrosis, which impaired the subsequent delivery of small molecules.Conclusions: PLX3397 therapy has greater efficacy than imatinib in preclinical GIST models and warrants study in patients with GIST. The resultant intratumoral fibrosis may represent one of the barriers to achieving complete tumor eradication. Clin Cancer Res; 20(9); 2350–62. ©2014 AACR.

Combination immunotherapy tumor slice culture experiment replicates

Purpose: To characterize changes in the soft-tissue sarcoma (STS) tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design. Experimental Design: Paired pre- and postneoadjuvant therapy specimens were retrospectively identified for 32 patients with STSs and analyzed by three modalities: multiplexed IHC, NanoString, and RNA sequencing with ImmunoPrism analysis. Results: All 32 patients, representing a variety of STS histologic subtypes, received neoadjuvant radiotherapy and 21 (66%) received chemotherapy prior to radiotherapy. The most prevalent immune cells in the tumor before neoadjuvant therapy were myeloid cells (45% of all immune cells) and B cells (37%), with T (13%) and natural killer (NK) cells (5%) also present. Neoadjuvant therapy significantly increased the total immune cells infiltrating the tumors across all histologic subtypes for patients receiving neoadjuvant radiotherapy with or without chemotherapy. An increase in the percentage of monocytes and macrophages, particularly M2 macrophages, B cells, and CD4+ T cells was observed postneoadjuvant therapy. Upregulation of genes and cytokines associated with antigen presentation was also observed, and a favorable pathologic response (≥90% necrosis postneoadjuvant therapy) was associated with an increase in monocytic infiltrate. Upregulation of the T-cell checkpoint TIM3 and downregulation of OX40 were observed posttreatment. Conclusions: Standard neoadjuvant therapy induces both immunostimulatory and immunosuppressive effects within a complex sarcoma microenvironment dominated by myeloid and B cells. This work informs ongoing efforts to incorporate immune checkpoint inhibitors and novel immunotherapies into the neoadjuvant setting for STSs.

Immune cell infiltration into solid tumors, their movement within the tumor microenvironment (TME), and interaction with other immune cells are controlled by their directed migration towards gradients of chemokines. Dysregulated chemokine signaling in TME favors the growth of tumors, exclusion of effector immune cells, and abundance of immunosuppressive cells. Key chemokines directing the migration of immune cells into tumor tissue have been identified. In this review, we discuss well-studied chemokine receptors that regulate migration of effector and immunosuppressive immune cells in the context of cancer immunology. We discuss preclinical models that have described the role of respective chemokine receptors in immune cell migration into TME and review preclinical and clinical studies that target chemokine signaling as standalone or combination therapies.

Background: Extraskeletal myxoid chondrosarcoma (EMCS) is a rare tumor that typically has an indolent course but high rate of recurrence. We queried the Surveillance, Epidemiology, and End Results (SEER) database to assess factors associated with metastasis, treatment, and survival. Methods: We queried the SEER 1973–2016 database for patients with myxoid chondrosarcoma (ICD-O-3: 9231/3). Kaplan–Meier analyses and Cox proportional hazard models assessed effects on overall survival (OS) of demographics and clinical characteristics. Logistic regression assessed associations between tumor location and distant disease. Primary analysis was a complete case analysis; multiple imputation (MI) was used in a sensitivity analysis. Results: Locoregional disease (LRD) was found in 373 (85%) of patients. In univariate analysis with LRD, surgery correlated with superior OS [HR = 0.27; 95% confidence interval (CI), 0.16–0.47]; chemotherapy and radiotherapy associated with inferior OS (HR = 1.90; 95% CI, 1.11–3.27 and HR = 1.45; 95% CI, 1.03–2.06, respectively). No treatment modality associated with OS in the adjusted, complete case model. In the adjusted sensitivity analysis, surgery associated with superior outcomes (HR = 0.36; 95% CI, 0.19–0.69). There was no OS difference by primary tumor site. 10-year OS with distant disease was 10% (95% CI, 2%–25%). Conclusions: Surgery in LRD associated with improved OS in univariate analysis and adjusted models correcting for missing data. There was no OS benefit with chemotherapy or radiotherapy. Impact: This represents the largest report of EMCS with long-term follow-up. Despite the reputedly indolent nature of EMCS, outcomes with metastatic disease are poor. We provide OS benchmarks and guidance for stratification in future prospective trials.

Objectives: Factors associated with nodal involvement in chondrosarcoma and outcomes based on treatment modality were analyzed using the Surveillance, Epidemiology, and End Results (SEER) database. Methods: Chondrosarcomas involving axial and appendicular parts of the body diagnosed from 2004 to 2015 were identified in SEER. Clinical, pathologic, and treatment parameters were compared with respect to nodal status at initial diagnosis by Fisher’s exact or Student’s t-test. Disease-specific survival (DSS) and overall survival (OS) were evaluated by Kaplan-Meier analyses, and by Cox regression models. Results: Synchronous regional nodal metastases were present in 1.3% of chondrosarcoma patients. Lymph node involvement was associated with primary tumor location in extraskeletal tissue and the pelvis, and with distant metastasis and larger primary tumor size at diagnosis. Patients with nodal involvement had 5-year DSS of 48% [95% confidence interval (CI): 28%–65%], versus 82% (95%CI: 80%–84%) for those without (log-rank P Conclusions: The nodal disease is uncommon at presentation in chondrosarcoma. Greater clinical vigilance for regional nodal metastases may be warranted for those with specific risk factors, including extraskeletal or pelvic primary sites, myxoid, mesenchymal, or dedifferentiated histologies, and large size. Surgical excision of regional nodes is associated with improvedDSS/OS. This analysis suggests a therapeutic effect of surgical treatment, rather than selection for favorable underlying biological factors.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome, and high-dimensional analyses of patient samples reveal the heterogeneity of the immune TME. Macrophages infiltrate TMEs and generally associate with tumor progression, but the underlying mechanisms are incompletely understood. Because experimental systems are needed to elucidate the functional properties of these cells, we developed a humanized mouse model reconstituted with human immune cells and human melanoma. We used two strains of recipient mice, supporting or not supporting the development of human myeloid cells. We found that human myeloid cells favored metastatic spread of the primary tumor, thereby recapitulating the cancer-supportive role of macrophages. We next analyzed the transcriptome of human immune cells infiltrating tumors versus other tissues. This analysis identified a cluster of myeloid cells present in the TME, but not in other tissues, which do not correspond to canonical M2 cells. The transcriptome of these cells is characterized by high expression of glycolytic enzymes and multiple chemokines and by low expression of gene sets associated with inflammation and adaptive immunity. Compared with humanized mouse results, we found transcriptionally similar myeloid cells in patient-derived samples of melanoma and other cancer types. The humanized mouse model described here thus complements patient sample analyses, enabling further elucidation of fundamental principles in melanoma biology beyond M1/M2 macrophage polarization. The model can also support the development and evaluation of candidate antitumor therapies.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome. High dimensional analyses of patient samples can reveal the composition and heterogeneity of the immune TME. In particular, macrophages are known for their cancer-supportive role, but the underlying mechanisms are incompletely understood, and experimental in vivo systems are needed to test the functional properties of these cells. We characterized a humanized mouse model, reconstituted with a human immune system and a human melanoma, in which: (1) human macrophages support metastatic spread of the tumor; and (2) tumor-infiltrating macrophages have a specific transcriptional signature that faithfully represents the transcriptome of macrophages from patient melanoma samples and is associated with shorter survival. This model complements patient sample analyses, enabling the elucidation of fundamental principles in melanoma biology, and the development and evaluation of candidate therapies.

ObjectiveProgrammed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures.DesignWe created organotypic slice cultures from human CRLM (n=38 patients’ tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy.ResultsαIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function.ConclusionNeutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

BackgroundDedifferentiated liposarcoma (DDLPS) is one of the most common soft tissue sarcoma subtypes and is devastating in the advanced/metastatic stage. Despite the observation of clinical responses to PD-1 inhibitors, little is known about the immune microenvironment in relation to patient prognosis.MethodsWe performed a retrospective study of 61 patients with DDLPS. We completed deep sequencing of the T-cell receptor (TCR) β-chain and RNA sequencing for predictive modeling, evaluating both immune markers and tumor escape genes. Hierarchical clustering and recursive partitioning were employed to elucidate relationships of cellular infiltrates within the tumor microenvironment, while an immune score for single markers was created as a predictive tool.ResultsAlthough many DDLPS samples had low TCR clonality, high TCR clonality combined with low T-cell fraction predicted lower 3-year overall survival (p=0.05). Higher levels of CD14+ monocytes (p=0.02) inversely correlated with 3-year recurrence-free survival (RFS), while CD4+ T-cell infiltration (p=0.05) was associated with a higher RFS. Genes associated with longer RFS included PD-1 (p=0.003), ICOS (p=0.006), BTLA (p=0.033), and CTLA4 (p=0.02). In a composite immune score, CD4+ T cells had the strongest positive predictive value, while CD14+ monocytes and M2 macrophages had the strongest negative predictive values.ConclusionsImmune cell infiltration predicts clinical outcome in DDLPS, with CD4+ cells associated with better outcomes; CD14+ cells and M2 macrophages are associated with worse outcomes. Future checkpoint inhibitor studies in DDLPS should incorporate immunosequencing and gene expression profiling techniques that can generate immune landscape profiles.

BACKGROUND: Immune checkpoint inhibition is effective in a subset of patients with advanced gastric cancer. Genomic profiling has revealed the heterogeneity of gastric adenocarcinomas, but the immune microenvironment and predictors of immunotherapy response remain poorly understood. We aimed to better characterize the underlying immune response to gastric cancer. METHODS: Retrospective review of a prospectively maintained institutional database was performed to identify patients who underwent curative intent resection of gastric adenocarcinoma from 2006–2016. Tumors were classified according to modified TCGA subtype: Epstein-Barr virus-associated (EBV), microsatellite instability-high (MSI), intestinal as a surrogate for chromosomal instability (CIN), diffuse as a surrogate for genomically stable (GS). Tumor infiltrating leukocytes (TIL) were measured using immunohistochemistry. RESULTS: Forty-three patients were identified: 6 EBV, 11 MSI, 14 intestinal, 12 diffuse. The most prevalent TIL were CD8(+) T lymphocytes and CD68(+) macrophages, comprising 15% and 13% of all tumor cells. EBV and MSI tumors were the most infiltrated, harboring 30–50% T cells and 20% macrophages. Intestinal tumors contained fewer T cells but disproportionately more macrophages. Diffuse tumors were the least infiltrated. Programmed cell death protein 1 (PD1) was most frequently expressed in intestinal tumors, whereas 70% of EBV and MSI tumors expressed programmed death-ligand 1 (PDL1). CONCLUSION: We herein demonstrate a heterogeneous immune response to gastric cancer, which varies by tumor subtype and has implications for future immunotherapy trials. Checkpoint inhibition is unlikely to be effective as single-agent therapy against intestinal and diffuse tumors lacking prominent T cell infiltration or substantial PDL1 expression.

CD4(+) T cells that recognize tumor antigens are required for immune checkpoint inhibitor efficacy in murine models but their contributions in human cancer are unclear. We used single cell RNA sequencing and T cell receptor sequences to identify signatures and functional correlates of tumor specific CD4(+) T cells infiltrating human melanoma. Conventional CD4(+) T cells that recognize tumor neoantigens express CXCL13 and are subdivided into clusters expressing memory and T follicular helper markers, and those expressing cytolytic markers, inhibitory receptors, and IFN-γ. The frequency of CXCL13(+) CD4(+) T cells in the tumor correlated with the transcriptional states of CD8(+) T cells and macrophages, maturation of B cells, and patient survival. Similar correlations were observed in a breast cancer cohort. These results identify phenotypes and functional correlates of tumor specific CD4(+) T cells in melanoma and suggest the possibility of using such cells to modify the tumor microenvironment.

Background Dedifferentiated chondrosarcoma is a chondrosarcoma subtype associated with high rates of recurrence and a poor prognosis. Others have proposed treatment of dedifferentiated chondrosarcoma using osteosarcoma protocols, including perioperative chemotherapy. However, the rarity of this condition poses difficulties in undertaking single- institution studies of sufficient sample size. Question/purpose Is perioperative chemotherapy associated with improved overall survival in patients with dedifferentiated chondrosarcoma? Methods We queried the Surveillance, Epidemiology, and End Results (SEER) 1973 to 2016 database for patients with a diagnosis of dedifferentiated chondrosarcoma (n = 308). As dedifferentiated chondrosarcoma was only classified as a distinct entity in SEER starting in 2000, only patients treated in 2000 and later were included. We excluded from our analyses those patients with distant disease at diagnosis, a primary site of disease other than bone or joints, and those who did not receive cancer-directed surgery. These criteria yielded 185 dedifferentiated chondrosarcoma patients for inclusion. We used Kaplan-Meier analyses and Cox proportional hazards models to assess the association of clinical, demographic, and treatment characteristics on overall survival (OS). Results After controlling for confounding variables, including age, sex, tumor size, stage, grade, location, and radiation treatment status, and after adjusting for missing data, no overall survival benefit was associated with receipt of chemotherapy in patients with dedifferentiated chondrosarcoma (hazard ratio 0.75 [95% confidence interval 0.49 to 1.12]; p = 0.16). Conclusion Chemotherapy treatment of dedifferentiated chondrosarcoma was not associated with improved OS. These results must be viewed cautiously, given the limited granularity of information on chemotherapy treatment, the concerns regarding chemotherapy misclassification in SEER data, and the small sample of patients with dedifferentiated chondrosarcoma, all of which limit the power to detect a difference. Our findings are nevertheless consistent with those of prior reports in which no benefit of chemotherapy could be detected. Lack of clear benefit from perioperative chemotherapy in dedifferentiated chondrosarcoma argues that it should be used only after careful consideration, and ideally in the context of a clinical trial. Level of evidence Level III, therapeutic study.

Background: Colorectal cancer liver metastasis (CRLM) causes major morbidity and mortality. Improved systemic therapies are crucial. We previously reported that macrophages infiltrated CRLM and expressed the immunosuppressive cytokine interleukin-10 (IL-10). In patient-derived CRLM tumor slice cultures, we found that a neutralizing antibody against IL-10 (anti-IL-10) caused tumor apoptosis. Here, we investigated the efficacy and immune-dependent mechanisms of IL-10 blockade in a larger cohort of CRLM patients. Methods: Tumor specimens were obtained from consenting patients at the time of surgery and cut into 250mm-thick tumor slice cultures as previously described. Tumor slices were treated with control or neutralizing antibodies against programmed cell death protein 1 (PD-1, EH12.1), IL-10 (JES3-9D7), IL-10 receptor alpha (IL-10RA, 3F9), major histocompatibility complex (MHC) class I (G46-2.6) or class II (Tu39). After 4-6 days of treatment, tumor apoptosis was evaluated by cleaved caspase-3 (CC3) immunohistochemistry (IHC) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Tumors were evaluated by IHC and in situ hybridization (ISH) for immune markers. Student's t-test, paired t-test, or 1-way ANOVA was used as indicated. p < 0.05 was defined as significant. Results: We generated tumor slice cultures from 34 unique CRLM patients, 76% (26/34) of whom received preoperative chemotherapy, and all of whom had either microsatellite stable tumors (76%, 26/34) or unknown microsatellite status. Anti-PD-1 did not generate tumor apoptosis (n = 4 patients' tumors). In contrast, anti-IL-10 caused nearly 2-fold increased tumor apoptosis compared with control, in the majority of 34 CRLM patients' tumors evaluated (median 50.1% versus 27.4% apoptotic cells, p < 0.0001). IL-10 receptor blockade generated a similar but non-significant increase in apoptosis. Tumors treated with anti-IL-10 demonstrated increased frequency of CD8+ T cells (median 17.8% versus 7.0% of total cells, n = 5 patients' tumors, p = 0.02) and a non-significant increase in activated PD-1+CD3+ T cells. IL-10 blockade also increased tumor IFNG expression (median 2.9 versus 2.3 counts per total cell number, n = 6 patients' tumors, p < 0.05). Macrophage frequency did not increase, but MHC class II expression nearly doubled (median 19.5% versus 11.5% of total cells, n = 5 patients' tumors, p = 0.02). To test which immune cells were required for anti-IL-10 effect, we treated human CRLM slices with anti-IL-10 +/- blocking antibodies against MHC class I or II. In the 3 of 4 patients' tumors that responded to anti-IL-10, the effect was nearly completely reversed by aMHC-I or aMHC-II (p = 0.0005). Conclusion: IL-10 is a dominant and reversible mechanism of immune evasion in human CRLM. IL-10 blockade nearly doubled tumor apoptosis in a heterogeneous cohort of CRLM patients' tumors, through a mechanism that required intact CD8+ T cell and antigen-presenting cell function. IL-10 is a compelling immunotherapeutic target for CRLM. Citation Format: Teresa S. Kim, Kevin Sullivan, Xiuyun Jiang, Cynthia Hsu, Kevin Labadie, Karan Kohli, Heidi Kenerson, Sara Daniel, Arezou Abbasi, Raymond Yeung, Venu Pillarisetty. Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P037.

BackgroundTumor-antigen specific CD4+ T cells are crucial for the efficacy of antibodies that block immune checkpoint proteins in mouse tumor models, but their activities in human tumor immunity are less clear. CD8+ T cells infiltrating human tumors, including those specific for tumor antigens, have been studied using single cell profiling techniques and exist in a variety of dysfunctional states. The transcriptional states of tumor-specific CD4+ T cells present in tumors and their potential contributions to the tumor microenvironment are less well understood.MethodsWe used targeted single cell RNA sequencing and matching of T cell receptor (TCR) sequences to identify phenotypic signatures that discriminated tumor antigen- and viral antigen-specific CD4+ T cells infiltrating human melanoma tumors in four patients. The presence of CD4+ T cells with these signatures was correlated with the number and phenotype of other immune cells in the tumor microenvironment in an extended cohort of 20 patients.ResultsWe identified 259 CD4+ T cells representing 40 different TCR clonotypes specific for 13 neoantigens and 108 cells representing 14 TCR clonotypes specific for self-antigens in four melanoma patients. High expression of CXCL13 defined conventional CD4+ T cells that recognize tumor associated neoantigens and self-antigens from bystander and viral antigen-specific CD4+ T cells. Tumor-reactive CD4+ T cells could be subdivided into clusters expressing memory and T follicular helper markers, and those expressing cytolytic markers and IFN-g. In an extended cohort of 20 patients with melanoma, the frequency of CXCL13+ CD4+ T cells in the tumor microenvironment correlated with the presence and proliferation of CD8+ T cells, the presence and maturation of B cells, the activation of interferon responsive genes in tumor associated macrophages, and patient survival. CD4+ T cells with similar transcriptional signatures were identified in data sets from breast and non-small cell lung cancer, suggesting these markers may enrich for tumor-reactive CD4+ T cells in many cancers.ConclusionsThese results identify a subset of tumor infiltrating conventional CD4+ T cells in melanoma that are enriched for reactivity to tumor antigens and exist in multiple phenotypic states. Correlations of the presence of these cells with the frequency and phenotype of other immune cells suggest roles for these tumor antigen-specific CD4+ T cells in providing CD8+ T cell help, driving recruitment and maturation of B cells, and activating macrophages. Isolating such cells based on their unique phenotype and utilizing them for adoptive therapy could alter the tumor microenvironment for therapeutic benefit.Ethics ApprovalAll Patient samples in this study were obtained from patients who signed informed consent in a study approved by the institutional review board of the Fred Hutchinson Cancer Research Center (protocol #2643).

Gastrointestinal stromal tumor (GIST) is the most common sarcoma, often resulting from a KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutation. The lineage transcription factor ETV1 is expressed similarly in GISTs regardless of malignant potential. Although the related transcription factor ETV4 has been associated with metastasis and tumor progression in other cancers, its role in GIST is unknown. In this study, we found that ETV4 levels were high in a subset of human GISTs and correlated with high mitotic rate. Through Gene Set Enrichment Analysis in selected human GISTs, we identified a relationship between ETV4 levels and β-catenin signaling, especially in advanced GISTs. GIST specimens with high ETV4 levels overexpressed cell cycle regulating genes and had aberrant activation of the canonical Wnt pathway. In human GIST cell lines, ETV4 RNA interference suppressed cell cycle genes and Wnt/β-catenin signaling. ETV4 knockdown also reduced tumor cell proliferation, invasion, and tumor growth in vivo. Conversely, ETV4 overexpression increased cyclin D1 expression and Wnt/β-catenin signaling. Moreover, we determined that ETV4 knockdown destabilized nuclear β-catenin and increased its degradation via COP1, an E3 ligase involved in both ETV4 and β-catenin turnover. Aberrant accumulation of ETV4 and nuclear β-catenin was found in patient derived xenografts created from metastatic GISTs that became resistant to tyrosine kinase inhibitors. Collectively, our findings highlight the significance of ETV4 expression in GIST and identify ETV4 as a biomarker in human GISTs.

Background: Multiple factors, including dense stroma and high infiltration of suppressive immune cells make pancreatic ductal adenocarcinoma (PDA) challenging to treat with immunotherapy. The presence of intra-tumoral T cells positively correlates with improved survival for PDA patients. Gamma chain cytokines (GCCs) can augment T cell mediated anti-tumor immunity and various GCC agonist drugs have been applied in clinical trials for cancer. Besides evaluating the effect of GCC derived agonists using recovered blood or biopsies of treated patients, it is challenging to mechanistically study the effect of GCCs on tumor-resident immune cells in human samples. Methods: Here we apply organotypic slice culture of surgically resected tumor tissues to study the effect of GCCs on intra-tumoral immune cells. We treated slices of PDAs with GCCs (IL-2, IL-7, IL-15 and IL-21) for 6 days. Immune cells, especially T cells emigrated from the tumor slices into the culture supernatant. On day 6 we fixed intact tumor slices for multiplex immunohistochemistry (mIHC) and analyzed emigrated cells using flow cytometry. Results: IL-15 and IL-7 potently induced T cell proliferation. The effect of IL-7 and IL-15 on T cell proliferation was higher for T cells that expressed markers of antigen experience such as CD39 and PD1 (Table 1). While IL-15 had the strongest effect on tumor-derived T cells, IL-15 and IL-7 were similar in their effect on T cells in the blood of the same patient, suggesting that IL-15 is especially effective in inducing the proliferation of tumor-infiltrating T cells. The effect of IL-15 on enhanced T cell proliferation was also seen within tumor slices, as revealed by mIHC. Conclusion: Our data suggest that IL-15 is the most potent GCC at inducing in situ expansion of tumor-resident T cells, including T cells that show signs of antigen experience. Table 1.IL-15 induces in situ T cell proliferation in PDA.AssayGroupsUntreatedIL-2IL-7IL-15IL-21ParameterFlow Cytometry%Ki67+CD8+cells of live cells0.090.040.360.595.001.3514.331.200.310.14%CD39+CD103+of CD8+0.130.150.110.040.290.140.620.180.130.08%PD1+of CD8+25.107.2223.233.1030.484.7446.483.5037.402.32mIHC%CD8+Ki67+ of all cells0.16 0.04(n=2)0.83 (n=1)0.550.38(n=2)2.200.04(n=2)0.490.41(n=2)n=4 slices for each group for flow cytometry. Data are from one experiment*. MeanSD is shown. Two doses (10-fold difference) for each cytokine were tested and results only for the low dose are shown.*Another experiment with groups IL-2 and IL-2+IL-15 was performed and had similar results for the effect of IL-15 Citation Format: Karan Kohli, Shihong Zhang, Xiuyun Jiang, Cynthia Hsu, Arezou Abbasi, Teresa S. Kim, Venu G. Pillarisetty. IL-15 is the most potent of tested gamma chain cytokines at inducing in situ proliferation of T cells in human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 607.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy. Experimental Design: Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization. Results: mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis. Conclusions: Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA. See related commentary by Medina and Miller, p. 3747

In the original article Kaitlin C. McLoughlin’s name is spelled incorrectly. It is correct as reflected here.

Importance Melanoma is epidemiologically linked to UV exposure, particularly childhood sunburn. Public health campaigns are increasing sun-protective behavior in the United States, but the effect on melanoma incidence is unknown. Objective To examine the incidence of melanoma in the United States and whether any age-specific differences are present. Design, Setting, and Participants Observational, population-based registry data were extracted on July 3, 2018, from the combined National Program of Cancer Registries–Surveillance Epidemiology and End Results United States Cancer Statistics database for 2001-2015. Deidentified data for 988 103 cases of invasive melanoma, withInternational Classification of Diseases for Oncologyhistologic categorization codes 8720 to 8790, were used for analysis. Data analysis was performed from July 1, 2018, to March 1, 2019. Main Outcomes and Measures The annual rates of melanoma in pediatric, adolescent, young adult, and adult age groups were determined. Analyses were stratified by sex, and incidence rates were age-adjusted to the 2000 US standard population. Annual percentage change (APC) in incidence rate was calculated over the most recent decade for which data were available (2006-2015) using the weighted least squares method. Results In 2015, 83 362 cases of invasive melanoma were reported in the United States, including 67 in children younger than 10 years, 251 in adolescents (10-19 years), and 1973 in young adults (20-29 years). Between 2006 and 2015, the overall incidence rate increased from 200.1 to 229.1 cases per million person-years. In adults aged 40 years or older, melanoma rates increased by an APC of 1.8% in both men (95% CI, 1.4%-2.1%) and women (95% CI, 1.4%-2.2%). In contrast, clinically and statistically significant decreases were seen in melanoma incidence for adolescents and young adults. Specifically, incidence rates decreased by an APC of −4.4% for male adolescents (95% CI, −1.7% to −7.0%), −5.4% for female adolescents (95% CI, −3.3% to −7.4%), −3.7% for male young adults (95% CI, −2.5% to −4.8%), and −3.6% for female young adults (95% CI, −2.8% to −4.5%). Data on skin pigmentation and sun protection history were unavailable; similar trends were observed with data limited to non-Hispanic whites. Young adult women appeared to have twice the risk of melanoma as young adult men. Conclusions and Relevance The incidence of invasive melanoma in the United States appeared to decrease in adolescents and young adults from 2006 to 2015, and this finding contrasted with increases in older populations. These incidence trends suggest that public health efforts may be favorably influencing melanoma incidence in the United States.

BACKGROUND. Gastric adenocarcinoma is a heterogenous disease that results from complex interactions between environmental and genetic factors, which may contribute to the disparate outcomes observed between different patient populations. This study aimed to determine whether genomic differences exist in a diverse population of patients by evaluating tumor mutational profiles stratified by race. METHODS. All patients with gastric adenocarcinoma between 2012 and 2016 who underwent targeted next-generation sequencing of cancer genes by the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets platform were identified. Patient race was categorized as Asian, African American, Hispanic, or Caucasian. Fisher’s exact test was used to examine differences in mutation rates between racial designations for the most common mutations identified. The p values in this study were adjusted using the false discovery rate method. RESULTS. The study investigated 595 mutations in 119 patients. The DNA alterations identified included missense mutations (66%), frame-shift deletions (13%), and nonsense mutations (9%). Silent mutations were excluded. The most frequently mutated genes were ARID1A, CDH1, ERBB3, KRAS, PIK3CA, and TP53. Of these, TP53 was the most frequently mutated gene, affecting 50% of patients. The proportion of patients with TP53 mutations differed significantly between races (p = 0.012). The findings showed TP53 mutations for 89% (16/18) of the African American patients, 56% (10/18) of the Asian patients, 43% (9/21) of the Hispanic patients, and 40% (25/62) of the Caucasian patients. CONCLUSIONS. Significantly higher rates of TP53 mutations were identified among the African American patients with gastric adenocarcinoma. This is the first study to evaluate tumor genomic differences in a diverse population of patients with gastric adenocarcinoma.

Incidentally discovered pancreatic cystic lesions are increasingly common, affecting up to 10% to 15% of patients undergoing cross-sectional imaging. Although some pancreatic cystic neoplasms harbor invasive malignancy or the potential to progress over time, a majority are benign and can be observed safely. Accurate diagnosis is key to appropriate management. Diagnosis requires a multidisciplinary and multimodal approach. This review discusses each type of pancreatic cystic neoplasm and the current data on diagnosis and treatment.

The current era of gastric surgery is marked by low morbidity and mortality rates, innovative strategies to approach resections with a minimally invasive fashion or hyperthermic intraperitoneal chemotherapy (HIPEC), as well as improved understanding of the biology of sporadic and hereditary stromal, neuroendocrine, and epithelial malignancies. In 2017, the Society for Surgery of the Alimentary Tract convened a State-of-the-Art Conference on Current Surgical Management of Gastric Tumors with both international experts and emerging leaders in the field of gastric surgery. Martin D. McCarter, MD of the University of Colorado discussed the current management of gastric gastrointestinal stromal tumors (GIST). Kaitlyn J. Kelly, MD of the University of California, San Diego discussed the management of gastric carcinoid tumors. Jeffrey A. Norton of Stanford University discussed recent advances in the management of gastric adenocarcinoma including a focus on hereditary diffuse gastric cancer (HDGC). Joseph Kim, MD of Stony Brook University discussed a systematic approach to minimally invasive gastrectomy for cancer. Joyce Wong, MD of Pennsylvania State University discussed the role for cytoreductive surgery (CRS) and HIPEC for gastric adenocarcinoma. This review provides gastrointestinal surgeons with a concise update on the current surgical management of gastric tumors.

Gastrointestinal stromal tumor (GIST) is the most common type of sarcoma and usually harbors either a KIT or PDGFRA mutation. However, the molecular basis for tumor malignancy is not well defined. Although the Wnt/β-catenin signaling pathway is important in a variety of cancers, its role in GIST is uncertain. Through analysis of nearly 150 human GIST specimens, we found that some human GISTs expressed β-catenin and contained active, dephosphorylated nuclear β-catenin. Furthermore, advanced human GISTs expressed reduced levels of the Wnt antagonist DKK4. Accordingly, in human GIST T1 cells, Wnt stimulation increased β-catenin–mediated transcriptional activity in a reporter assay as well as transcription of the downstream target genes Axin2 and CCND1. In contrast, DKK4 overexpression in GIST T1 cells reduced Wnt/β-catenin signaling. In addition, we showed that nuclear β-catenin stability was partially regulated by the E3 ligase COP1, as demonstrated with coimmunoprecipitation and COP1 knockdown. Three molecular inhibitors of the Wnt/β-catenin pathway demonstrated antitumor efficacy in various GIST models, both in vitro and in vivo. Notably, the tankyrase inhibitor G007-LK alone had substantial activity against tumors of genetically engineered KitV558Δ/+ mice, and the effect was increased by the addition of the Kit inhibitor imatinib mesylate. Collectively, our findings demonstrate that Wnt/β-catenin signaling is a novel therapeutic target for selected untreated or imatinib-resistant GISTs. Mol Cancer Ther; 16(9); 1954–66. ©2017 AACR.