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Local recurrence following resection of retroperitoneal liposarcoma (RLPS) is common. Well-differentiated (WD) and dedifferentiated (DD) RLPS are distinct entities with differing outcomes. A few reports suggest that WDLPS can recur as DDLPS and that DDLPS can recur as WDLPS. This study evaluates whether this change in differentiation from the primary tumor to the first local recurrence impacts long-term outcomes. Retrospective review from 22 sarcoma centers identified consecutive patients who underwent resection for a first locally recurrent RLPS from January 2002 to December 2011. Outcomes measured included overall survival, local recurrence, and distant metastasis. A total of 421 RPLS patients were identified. Of the 230 patients with primary DDLPS, 34 (15%) presented WDLPS upon recurrence (DD → WD); and of the 191 patients with primary WDLPS, 54 (28%) presented DDLPS upon recurrence (WD → DD). The 6-year overall survival probabilities (95% CI) for DD → DD, DD → WD, WD → WD, and WD → DD were 40% (32–48%), 73% (58–92%), 76% (68–85%), and 56% (43–73%) (p < 0.001), respectively. The 6-year second local recurrence incidence was 66% (59–73%), 63% (48–82%), 66% (57–76%), and 77% (66–90%), respectively. The 6-year distant metastasis incidence was 13% (9–19%), 3% (0.4–22%), 5% (2–11%), and 4% (1–16%), respectively. On multivariable analysis, DD → WD was associated with improved overall survival when compared with DD → DD (p < 0.001). Moreover, WD → DD was associated with a higher risk of LR (p = 0.025) A change in RLPS differentiation from primary tumor to first local recurrence appears to impact survival. These findings may be useful in counseling patients on their prognosis and subsequent management.

Background Robotic hepatectomy (RH) is increasingly utilized for minor and major liver resections. The IWATE criteria were developed to classify minimally invasive liver resections by difficulty. The objective of this study was to apply the IWATE criteria in RH and to describe perioperative and oncologic outcomes of RH over the last decade at our institution. Methods Perioperative and oncologic outcomes of patients who underwent RH between 2011 and 2019 were retrospectively collected. The difficulty level of each operation was assessed using the IWATE criteria, and outcomes were compared at each level. Univariate linear regression was performed to characterize the relationship between IWATE criteria and perioperative outcomes (OR time, EBL, and LOS), and a multivariable model was also developed to address potential confounding by patient characteristics (age, sex, BMI, prior abdominal surgery, ASA class, and simultaneous non-hepatectomy operation). Results Two hundred and twenty-five RH were performed. Median IWATE criteria for RH were 6 (IQR 5–9), with low, intermediate, advanced, and expert resections accounting for 23% (n = 51), 34% (n = 77), 32% (n = 72), and 11% (n = 25) of resections, respectively. The majority of resections were parenchymal-sparing approaches, including anatomic segmentectomies and non-anatomic partial resections. 30-day complication rate was 14%, conversion to open surgery occurred in 9 patients (4%), and there were no deaths within 30 days postoperatively. In the univariate linear regression analysis, IWATE criteria were positively associated with OR time, EBL, and LOS. In the multivariable model, IWATE criteria were independently associated with greater OR time, EBL, and LOS. Two-year overall survival for hepatocellular carcinoma and intrahepatic cholangiocarcinoma was 94% and 50%, respectively. Conclusion In conclusion, the IWATE criteria are associated with surgical outcomes after RH. This series highlights the utility of RH for difficult hepatic resections, particularly parenchymal-sparing resections in the posterosuperior sector, extending the indication of minimally invasive hepatectomy in experienced hands and potentially offering select patients an alternative to open hepatectomy or other less definitive liver-directed treatment options.

This study aimed to evaluate perioperative morbidity after surgery for first locally recurrent (LR1) retroperitoneal sarcoma (RPS). Data concerning the safety of resecting recurrent RPS are lacking. Data were collected on all patients undergoing resection of RPS-LR1 at 22 Trans-Atlantic Australasian Retroperitoneal Sarcoma Working Group (TARPSWG) centers from 2002 to 2011. Uni- and multivariable logistic models were fitted to study the association between major (Clavien-Dindo grade ≥ 3) complications and patient/surgery characteristics as well as outcome. The resected organ score, a method of standardizing the number of organs resected, as previously described by the TARPSWG, was used. The 681 patients in this study had a median age of 59 years, and 51.8% were female. The most common histologic subtype was de-differentiated liposarcoma (43%), the median resected organ score was 1, and 83.3% of the patients achieved an R0 or R1 resection. Major complications occurred for 16% of the patients, and the 90-day mortality rate was 0.4%. In the multivariable analysis, a transfusion requirement was found to be a significant predictor of major complications (p

Single agent checkpoint inhibitor therapy has not been effective for most gastrointestinal solid tumors, but combination therapy with drugs targeting additional immunosuppressive pathways is being attempted. One such pathway, the CXCL12-CXCR4/CXCR7 chemokine axis, has attracted attention due to its effects on tumor cell survival and metastasis as well as immune cell migration. CXCL12 is a small protein that functions in normal hematopoietic stem cell homing in addition to repair of damaged tissue. Binding of CXCL12 to CXCR4 leads to activation of G protein signaling kinases such as P13K/mTOR and MEK/ERK while binding to CXCR7 leads to β-arrestin mediated signaling. While some gastric and colorectal carcinoma cells have been shown to make CXCL12, the primary source in pancreatic cancer and peritoneal metastases is cancer-associated fibroblasts. Binding of CXCL12 to CXCR4 and CXCR7 on tumor cells leads to anti-apoptotic signaling through Bcl-2 and survivin upregulation, as well as promotion of the epithelial-to-mesechymal transition through the Rho-ROCK pathway and alterations in cell adhesion molecules. High levels of CXCL12 seen in the bone marrow, liver, and spleen could partially explain why these are popular sites of metastases for many tumors. CXCL12 is a chemoattractant for lymphocytes at lower levels, but becomes chemorepellant at higher levels; it is unclear exactly what gradient exists in the tumor microenvironment and how this influences tumor-infiltrating lymphocytes. AMD3100 (Plerixafor or Mozobil) is a small molecule CXCR4 antagonist and is the most frequently used drug targeting the CXCL12-CXCR4/CXCR7 axis in clinical trials for gastrointestinal solid tumors currently. Other small molecules and monoclonal antibodies against CXCR4 are being trialed. Further understanding of the CXCL12- CXCR4/CXCR7 chemokine axis in the tumor microenvironment will allow more effective targeting of this pathway in combination immunotherapy.

Purpose: This clinical trial combined pembrolizumab and vorinostat in recurrent/metastatic squamous cell carcinomas of the head and neck (HN), and salivary gland cancer (SGC). Patients and Methods: Patients with progressing incurable HN and SGC, Eastern Cooperative Oncology Group (ECOG) ≤1, no prior immunotherapy, RECIST1.1 measurable disease, and normal organ function were eligible. Pembrolizumab 200 mg was given intravenous every 21 days, and vorinostat 400 mg given orally 5 days on and 2 days off during each 21-day cycle. Primary endpoints were safety and objective response rates. Results: From November 2015 to August 2017, 25 patients with HN and 25 SGC were enrolled. Median age was 61 (range, 33–86) years, 39 (78%) were male, 21 (62%) were never smokers, and 27 (54%) had ECOG 0. In HN, 13 (52%) were p16+ oropharynx. Most common SGC histologies were adenoid cystic 12 (48%), acinic cell 3 (12%), and mucoepidermoid 3 (12%). Adverse events (AEs) in all patients were: 27 (54%) with grade ≥ 1 and 18 (36%) with grade ≥ 3. The most common AEs in all patients were renal insufficiency in seven, (14%), fatigue in six, (12%), and nausea in three (6%). There were three (12%) deaths on study. Responses in HN were complete response (CR) 0, partial response (PR) eight (32%), and stable disease (SD) five (20%). Efficacy in SGCs was CR 0, PR four (16%) in one lymphoepithelioma-like carcinoma, two acinic cell, one adenoid cystic, and SD 14 (56%). In the HN group, median follow-up (mFUP) was 12.6 months, median overall survival (mOS) was 12.6 months, and median progression-free survival (mPFS) was 4.5 months. In SGC, mFUP was 13.1 months, mOS was 14.0 months, and mPFS was 6.9 months. Conclusions: This combination demonstrated activity in HN, with fewer responses in SGC. Toxicities were higher than reported with pembrolizumab alone.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Neuroendocrine and Adrenal Gland Tumors focus on the diagnosis, treatment, and management of patients with neuroendocrine tumors (NETs), adrenal tumors, pheochromocytomas, paragangliomas, and multiple endocrine neoplasia. NETs are generally subclassified by site of origin, stage, and histologic characteristics. Appropriate diagnosis and treatment of NETs often involves collaboration between specialists in multiple disciplines, using specific biochemical, radiologic, and surgical methods. Specialists include pathologists, endocrinologists, radiologists (including nuclear medicine specialists), and medical, radiation, and surgical oncologists. These guidelines discuss the diagnosis and management of both sporadic and hereditary neuroendocrine and adrenal tumors and are intended to assist with clinical decision-making. This article is focused on the 2021 NCCN Guidelines principles of genetic risk assessment and counseling and recommendations for well-differentiated grade 3 NETs, poorly differentiated neuroendocrine carcinomas, adrenal tumors, pheochromocytomas, and paragangliomas.

Although intra-abdominal accessory spleens are commonly found in 10-30% of the general population, epidermoid cyst within an intrapancreatic accessory spleen (ECIPAS) is an extremely rare entity and is often misdiagnosed preoperatively as a cystic malignancy. We present the case of a 51-year-old man who was referred to our clinic because of an incidentally found pancreatic tail cystic mass. Due to CA 19-9 level over 2000 and high suspicion of malignancy, the patient underwent distal pancreatectomy with splenectomy. Histopathological analysis revealed a squamous epithelial lining with splenic parenchyma within the cyst wall, which was consistent with ECIPAS. The patient was discharged on postoperative day 4 without any complication. As this disease may mimic malignancy with no characteristic features in preoperative imaging, it should be considered in the differential diagnosis of pancreatic cystic lesions.

Interferon-γ (IFNγ) has been studied as a cancer treatment with limited evidence of clinical benefit. However, it could play a role in cancer immunotherapy combination treatments. Despite high expression of immunogenic cancer–testis antigens, synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) have a cold tumor microenvironment (TME), with few infiltrating T cells and low expression of major histocompatibility complex class I (MHC-I). We hypothesized that IFNγ treatment could drive inflammation in a cold TME, facilitating further immunotherapy. We conducted a phase 0 clinical trial treating 8 SS or MRCL patients with weekly systemic IFNγ. We performed pre- and posttreatment biopsies. IFNγ changed the SS and MRCL TME, inducing tumor-surface MHC-I expression and significant T-cell infiltration (P < 0.05). Gene-expression analysis suggested increased tumor antigen presentation and less exhausted phenotypes of the tumor-infiltrating T cells. Newly emergent antigen-specific humoral and/or T-cell responses were found in 3 of 7 evaluable patients. However, increased expression of PD-L1 was observed on tumor-infiltrating myeloid cells and in some cases tumor cells. These findings suggest that systemic IFNγ used to convert SS and MRCL into “hot” tumors will work in concert with anti–PD-1 therapy to provide patient benefit.

BACKGROUND In this series from the Transatlantic Australasian Retroperitoneal Sarcoma Working Group (TARPSWG), the authors examined longitudinal outcomes of patients with a second recurrence of retroperitoneal sarcoma (RPS) after complete resection of a first local recurrence (LR). METHODS Data from patients undergoing resection of a first LR from January 2002 to December 2011were collected from 22 sarcoma centers. The primary outcome was overall survival (OS) after second recurrence. RESULTS Second recurrences occurred in 400 of 567 patients (70.5%) after an R0/R1 resection of a first locally recurrent RPS. Patterns of disease recurrence were LR in 323 patients (80.75%), distant metastases (DM) in 55 patients (13.75%), and both LR and DM in 22 patients (5.5%). The main subtype among the LR group was liposarcoma (77%), whereas DM mainly were leiomyosarcomas (43.6%). In patients with a second LR only, a total of 200 patients underwent re-resection (61.9%). The 5-year OS rate varied significantly based on the pattern of failure (P

Summary Metastatic colorectal cancer (CRC) is a major cause of cancer-related death, and incidence is rising in younger populations (younger than 50 years). Current chemotherapies can achieve response rates above 50%, but immunotherapies have limited value for patients with microsatellite-stable (MSS) cancers. The present study investigates the impact of chemotherapy on the tumor immune microenvironment. We treat human liver metastases slices with 5-fluorouracil (5-FU) plus either irinotecan or oxaliplatin, then perform single-cell transcriptome analyses. Results from eight cases reveal two cellular subtypes with divergent responses to chemotherapy. Susceptible tumors are characterized by a stemness signature, an activated interferon pathway, and suppression of PD-1 ligands in response to 5-FU+irinotecan. Conversely, immune checkpoint TIM-3 ligands are maintained or upregulated by chemotherapy in CRC with an enterocyte-like signature, and combining chemotherapy with TIM-3 blockade leads to synergistic tumor killing. Our analyses highlight chemomodulation of the immune microenvironment and provide a framework for combined chemo-immunotherapies., Graphical Abstract, Highlights CRLM slice culture can assess immune response to chemotherapy Single-cell analysis identifies cancer subtypes with differing response to chemotherapy 5-FU+irinotecan modulates interferon and PD-L1 pathways in stem-like CRLM Combining chemotherapy with TIM-3 blockade is synergistic in enterocyte-like CRLM, The response of colorectal liver metastases (CRLM) to chemotherapy is analyzed in 3D organotypic tumor slices by single-cell RNA-seq. Jabbari et al. find two subtypes (stem-like and enterocyte-like) of CRLM that express different immune checkpoint ligands and respond differently to chemotherapy. The study highlights the chemomodulation of the tumor immune microenvironment that can be targeted therapeutically.

Background The tumor microenvironment (TME) is critical to every aspect of cancer biology. Organotypic tumor slice cultures (TSCs) preserve the original TME and have demonstrated utility in predicting drug sensitivity, but the association between clinicopathologic parameters and in vitro TSC behavior has not been well-defined. Methods One hundred and eight fresh tumor specimens from liver resections at a tertiary academic center were procured and precisely cut with a Vibratome to create 250 μm × 6 mm slices. These fixed-dimension TSCs were grown on polytetrafluoroethylene inserts, and their metabolic activities were determined by a colorimetric assay. Correlation between baseline activities and clinicopathologic parameters was assessed. Tissue CEA mRNA expression was determined by RNAseq. Results By standardizing the dimensions of a slice, we found that adjacent tumor slices have equivalent metabolic activities, while those derived from different tumors exhibit >30-fold range in baseline MTS absorbances, which correlated significantly with the percentage of tumor necrosis based on histologic assessment. Extending this to individual cancers, we were able to detect intra-tumoral heterogeneity over a span of a few millimeters, which reflects differences in tumor cell density and Ki-67 positivity. For colorectal cancers, tissue CEA expression based on RNAseq of tumor slices was found to correlate with clinical response to chemotherapies. Conclusions We report a standardized method to assess and compare human cancer growth ex vivo across a wide spectrum of tumor samples. TSC reflects the state of tumor behavior and heterogeneity, thus providing a simple approach to study of human cancers with an intact TME.

Author(s): Kohli, Karan; Yao, Lu; Nowicki, Theodore Scott; Zhang, Shihong; Black, Ralph Graeme; Schroeder, Brett A; Farrar, Erik A; Cao, Jianhong; Sloan, Heather; Stief, Dawn; Cranmer, Lee D; Wagner, Michael J; Hawkins, Douglas S; Pillarisetty, Venu G; Ribas, Antoni; Campbell, Jean; Pierce, Robert H; Kim, Edward Y; Jones, Robin L; Riddell, Stanley R; Yee, Cassian; Pollack, Seth M | Abstract: BackgroundSynovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression.MethodWe performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15.ResultsFour patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers.ConclusionsETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

Current clinical treatment regimens, including many emergent immune strategies (e.g., checkpoint inhibitors) have done little to affect the devastating course of pancreatic ductal adenocarcinoma (PDA). Clinical trials for PDA often employ multi-modal treatment, and have started to incorporate stromal-targeted therapies, which have shown promising results in early reports. Focused ultrasound (FUS) is one such therapy that is uniquely equipped to address local and systemic limitations of conventional cancer therapies as well as emergent immune therapies for PDA. FUS methods can non-invasively generate mechanical and/or thermal effects that capitalize on the unique oncogenomic/proteomic signature of a tumor. Potential benefits of FUS therapy for PDA include: (1) emulsification of targeted tumor into undenatured antigens in situ, increasing dendritic cell maturation, and increasing intra-tumoral CD8+/ T regulatory cell ratio and CD8+ T cell activity; (2) reduction in intra-tumoral hypoxic stress; (3) modulation of tumor cell membrane protein localization to enhance immunogenicity; (4) modulation of the local cytokine milieu toward a Th1-type inflammatory profile; (5) up-regulation of local chemoattractants; (6) remodeling the tumor stroma; (7) localized delivery of exogenously packaged immune-stimulating antigens, genes and therapeutic drugs. While not all of these results have been studied in experimental PDA models to date, the principles garnered from other solid tumor and disease models have direct relevance to the design of optimal FUS protocols for PDA. In this review, we address the pertinent limitations in current and emergent immune therapies that can be improved with FUS therapy for PDA.

BACKGROUND Patients with metastatic sarcomas have poor outcomes and although the disease may be amenable to immunotherapies, information regarding the immunologic profiles of soft tissue sarcoma (STS) subtypes is limited. METHODS The authors identified patients with the common STS subtypes: leiomyosarcoma, undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), well-differentiated/dedifferentiated liposarcoma, and myxoid/round cell liposarcoma. Gene expression, immunohistochemistry for programmed cell death protein (PD-1) and programmed death-ligand 1 (PD-L1), and T-cell receptor Vβ gene sequencing were performed on formalin-fixed, paraffin-embedded tumors from 81 patients. Differences in liposarcoma subsets also were evaluated. RESULTS UPS and leiomyosarcoma had high expression levels of genes related to antigen presentation and T-cell infiltration. UPS were found to have higher levels of PD-L1 (P≤.001) and PD-1 (P≤.05) on immunohistochemistry and had the highest T-cell infiltration based on T-cell receptor sequencing, significantly more than SS, which had the lowest (P≤.05). T-cell infiltrates in UPS also were more oligoclonal compared with SS and liposarcoma (P≤.05). A model adjusted for STS histologic subtype found that for all sarcomas, T-cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression levels (P≤.01). CONCLUSIONS In the current study, the authors provide the most detailed overview of the immune microenvironment in sarcoma subtypes to date. UPS, which is a more highly mutated STS subtype, provokes a substantial immune response, suggesting that it may be well suited to treatment with immune checkpoint inhibitors. The SS and liposarcoma subsets are less mutated but do express immunogenic self-antigens, and therefore strategies to improve antigen presentation and T-cell infiltration may allow for successful immunotherapy in patients with these diagnoses. Cancer 2017;123:3291-304. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Purpose: The role of surgery for first relapse locally recurrent retroperitoneal sarcoma (RPS-LR1) is uncertain. We report outcomes of the largest RPS-LR1 series and propose a new prognostic nomogram. Experimental Design: Patients with consecutive RPS-LR1 without distant metastases who underwent resection at 22 centers (2002–2011) were included. Endpoints were disease-free and overall survival (DFS, OS) and crude-cumulative-incidence (CCI) of local/distant recurrence from second surgery. Nomograms predicting DFS and OS from second surgery were developed and validated (calibration plots); discrimination was assessed (Harrell C index). Results: Of 684 patients identified, full prognostic variable data were available for 602. Initial surgery for primary RPS was performed at our institutions in 188 patients (31%) and elsewhere in 414 (69%). At a median follow-up of 119 months [Interquartile range (IQR), 80–169] from initial surgery and 75 months (IQR 50–105) from second surgery, 6-year DFS and OS were 19.2% [95% confidence interval (CI), 16.0–23.0%] and 54.1% (95% CI, 49.8–58.8%), respectively. Recurrence patterns and survival probability were histology-specific, with liposarcoma subtypes having the highest 6-year CCI of second local recurrence (LR, 60.2%–70.9%) and leiomyosarcoma (LMS) having higher 6-year CCI of distant metastasis (DM, 36.3%). Nomograms included age at second surgery, multifocality, grade, completeness of second surgery, histology, chemotherapy/radiotherapy at first surgery, and number of organs resected at first surgery. OS and DFS nomograms showed good calibration and discriminative ability (C index 0.70 and 0.67, respectively). Conclusions: We developed nomograms to predict DFS and OS for patients undergoing RPS-LR1 resection. Nomograms provide individualized, disease-relevant estimations of survival for RPS-LR1 patients and assist in clinical decisions.

Robotic technology is being utilized in multiple hepatobiliary procedures, including hepatic resections. The benefits of minimally invasive surgical approaches have been well documented; however, there is some concern that robotic liver surgery may be prohibitively costly and therefore should be limited on this basis. A single-institution, retrospective cohort study was performed of robotic and open liver resections performed for benign and malignant pathologies. Clinical and cost outcomes were analyzed using adjusted generalized linear regression models. Clinical and cost data for 71 robotic (RH) and 88 open (OH) hepatectomies were analyzed. Operative time was significantly longer in the RH group (303 vs. 253 min; p = 0.004). Length of stay was more than 2 days shorter in the RH group (4.2 vs. 6.5 days; p

Background Interleukin-10 (IL-10) is an immunosuppressive cytokine that inhibits innate and adaptive antitumor immune responses in the tumor microenvironment (TME). Antibody neutralization of IL-10 in organotypic tumor slice cultures (TSC) in colorectal cancer liver metastases (CRCLM) leads to cancer cell death through. To target IL-10 blockade to the TME, we genetically engineered macrophages (GEM) to produce an IL-10 neutralizing antibody which we hypothesized would reactivate tumor infiltrating immune cells and increase cancer cell death in TSC. Methods We created a lentiviral construct to transduce GEMs with a publicly available neutralizing IL-10 antibody sequence (BT-063). CD14+ monocytes were isolated from healthy donor peripheral blood mononuclear cells and differentiated into macrophages, which were transduced with lentivirus to create control (cGEMs) and anti-IL-10 GEMs (αIL10 GEMs). To test the efficacy of αIL10 GEMs, punch biopsies (6 mm) were obtained from resected CRCLM, sliced to 250 um thickness with a vibratome and placed in individual culture wells. Slices were treated in triplicates with either 20ug/mL of human monoclonal αIL10 (αIL10), 1e5 cGEMs or αIL10 GEMs + 20ug/mL αIL10. GEMs and tumor cells were visualized with a Leica SP8X confocal microscope and apoptosis was measured with SR-FLICA fluorescent poly-caspase assay kit. Supernatant interferon-gamma (IFN-γ) levels were measured with Bioplex immunoassay and IL-10 neutralizing antibody was quantified with an enzyme-linked immunosorbent assay. Results Allogenic GEMs were transduced with BT-063-T2A-CD19t at 31% efficiency. cGEMs and αIL10 GEMs persisted in tumor slice culture for at least 6 days. αIL10 GEM-treated slice cultures contained 6.6ng/mL and 6.5ng/mL of αIL10 antibody after 3 and 6 days, respectively. At day 6 in culture, cancer cell apoptosis was increased in the αIL10 treated groups with 7%, 15%, 25%, 31% and 39% of cells were apoptotic in the no treatment control, cGEM, αIL10, cGEM + αIL10 and αIL10 GEM groups, respectively (One-way ANOVA, p = 0.0004). Elevation in IFN-γ was observed on day 6 in slices treated cGEMs (7.2ng/mL+4.3) and αIL10 GEMs (9.2ng/mL+5) compared to isotype control. Conclusion GEMs transduced with lentivirus encoding IL-10 neutralizing antibody maintain its production and are associated with increased cancer cell apoptosis. Citation Format: Kevin P. Labadie, Shannon A. Kreuser, Katherine J. Brempelis, Xiuyun Jiang, Sara K. Daniel, Courtney A. Crane, Venu G. Pillarisetty. Neutralization of interleukin-10 by genetically engineered macrophages increase cancer cell death in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5545.

732 Background: 5-7% of PC pts exhibit deleterious germline mutations (MUT) in HR tumor suppressor genes BRCA1 and BRCA2. BROCA-HR is a targeted capture and massively parallel sequencing assay designed to detect all mutation classes including gene rearrangements, copy number variations, and gene aberrations within the Fanconi Anemia-BRCA HR, non-homologous end joining (NHEJ) DNA repair, and DNA mismatch repair pathways. BROCA-HR has been successfully used in breast and ovarian cancer pts for overall prognosis and prediction of response to platinum-based therapies. While BRCA1/2 MUT may confer survival advantage for PC pts if treated with platinum-chemotherapy, the survival impact of HRD is less well defined. Methods: We retrospectively identified 100 consecutive pts who underwent surgical resection for suspected PC at University of Washington Medical Center between 1999 and 2008. Formalin-fixed paraffin embedded resected tumors were sequenced using BROCA-HR. HRD was grouped based on the following deleterious genetic mutations: 1) BRCA1, BRCA2; 2) core HRD: BARD1, BRIP1, RAD51C, RAD51D, PALB2, CDK12, NBN; 3) non-core HRD: ATM, ATR, ATRX, BAP1, BLM, CHEK1/2, ERCC, FANC A/C/D2/E /F/G/L, MRE11, RAD50/51/51B, RIF1, SLX4; 4) HR proficient. Overall survival (OS) was measured from diagnosis until death or last follow-up. Results: 95 pts had histologically confirmed PC, and 81 pts had adequate tumor DNA for analysis. Six pts (7%) had BRCA1/2 MUT (n = 5), or BRCA1 methylation (n = 1), 1 pt (1%) had non-BRCA core HRD ( PALB2 MUT), 7 pts (9%) had non-core HRD: ERCC (2), CHEK2 (2), ATR, RAD51D, and FANCA MUT (1 each). Median OS was: all pts 1.93 yrs (95% C.I. 1.53, 2.16), BRCA1/2 pts 3.09 yrs (95% CI 0.41, 12.21), all core HRD pts 1.21 yrs (95% CI 0.41, 12.21), all core and non-core HRD pts 1.89 yrs (95% CI 0.57, 4.96), HR proficient pts 1.93 yrs (95% CI 1.51, 2.15). There were no OS differences between pts with HRD vs those HR proficient. Conclusions: HRD is common (17%) but does not affect OS for pts with resected PC. Prospective clinical trials should test neo/adjuvant therapies including platinum chemotherapy and PARP inhibitors for pts with HRD.

Extra-adrenal paragangliomas are rare tumors arising from the chromaffin cells of the autonomic nervous system. Retroperitoneal paragangliomas may present as a pancreatic mass. We present a case of a 61-year-old woman with an incidentally found pancreatic mass (7.2 × 6.5 cm) in the CT scan. EUS- guided FNA result was compatible with pancreatic neuroendocrine tumor. Patient underwent pancreaticoduodenectomy and histopathologic assessment revealed the mass was an extra-adrenal paraganglioma. Preoperative diagnosis of pancreatic paragangliomas can be challenging due to imaging and histopathologic similarities with pancreatic neuroendocrine tumors.

Uterine leiomyosarcoma is a rare and aggressive malignancy with poor overall prognosis. There have been few reports of metastatic leiomyosarcoma in the gallbladder. We report a case of a 41-year-old female who underwent total abdominal hysterectomy due to presumed uterine fibroids. The postoperative pathology revealed high-grade pleomorphic leiomyosarcoma, with involvement of the uterine serosal surface. She subsequently underwent exploratory laparotomy, followed by pelvic radiation and chemotherapy. Since initial management she has developed metastatic disease and has been under treatment and surveillance for 11 years. She has undergone multiple surgical procedures and numerous lines of systemic therapy for metastatic leiomyosarcoma, including cholecystectomy for a metastatic lesion in the gallbladder. There have been no previous reports of metastatic leiomyosarcoma in the gallbladder. Despite extensive metastatic disease this patient has had prolonged survival with multi-modality management.

The NCCN Guidelines for Neuroendocrine and Adrenal Tumors provide recommendations for the management of adult patients with neuroendocrine tumors (NETs), adrenal gland tumors, pheochromocytomas, and paragangliomas. Management of NETs relies heavily on the site of the primary NET. These NCCN Guidelines Insights summarize the management options and the 2018 updates to the guidelines for locoregional advanced disease, and/or distant metastasis originating from gastrointestinal tract, bronchopulmonary, and thymus primary NETs.

IntroductionPancreaticoduodenectomy is a complex surgical procedure associated with high morbidity and prolonged length of stay. Enhanced recovery after surgery principles have reduced complications rate and length of stay for multiple types of operations. We hypothesized that implementation of a standardized perioperative care pathway would facilitate safe discharge by five days after pancreaticoduodenectomy.MethodsWe performed a retrospective cohort study of patients undergoing pancreaticoduodenectomy 18 months prior to and 18 months following implementation of a perioperative care pathway at a quaternary center performing high volume pancreatic surgery.ResultsA total of 145 patients underwent pancreaticoduodenectomy (mean age 63 ± 10 years, 52% female), 81 before and 64 following pathway implementation, and the groups were similar in terms of preoperative comorbidities. The percentage of patients discharged within 5 days of surgery increased from 36% to 64% following pathway implementation (p = 0.001), with no observed differences in post-operative serious adverse events (p = 0.34), pancreatic fistula grade B or C (p = 0.28 and p = 0.27 respectively), or delayed gastric emptying (p = 0.46). Multivariate regression analysis showed length of stay ≤5 days three times more likely after pathway implementation. Rates of readmission within 30 days (20% pre- vs. 22% post-pathway (p = 0.75)) and 90 days (27% pre- vs. 36% post-pathway (p = 0.27)) were unchanged after pathway implementation, and were no different between patients discharged before or after day 5 at both 30 days (19% ≤5 days vs. 23% ≥ 6 days (p = 0.68)) and 90 days (32% ≤5 days vs. 30% ≥ 6 days (p = 0.81)).ConclusionsStandardizing perioperative care via enhanced recovery protocols for patients undergoing pancreaticoduodenectomy facilitates safe discharge by post-operative day five.

INTRODUCTION: Retroperitoneal sarcoma (RPS) is a rare disease accounting for 0.1%–0.2% of all malignancies. Management of RPS is complex and requires multidisciplinary, tailored treatment strategies at all stages, but especially in the context of metastatic or multifocal recurrent disease. Due to the rarity and heterogeneity of this family of diseases, the literature to guide management is limited. METHODS: The Trans-Atlantic Retroperitoneal Sarcoma Working Group (TARPSWG) is an international collaboration of sarcoma experts from all disciplines convened in an effort to overcome these limitations. The TARPSWG has compiled the available evidence surrounding metastatic and multifocally recurrent RPS along with expert opinion in an iterative process to generate a consensus document regarding the complex management of this disease. The objective of this document is to guide sarcoma specialists from all disciplines in the diagnosis and treatment of multifocal recurrent or metastatic RPS. RESULTS: All aspects of patient assessment, diagnostic processes, local and systemic treatments, and palliation are reviewed in this document, and consensus recommendations provided accordingly. Recommendations were guided by available evidence, in conjunction with expert opinion where evidence was lacking. CONCLUSIONS: This consensus document combines the available literature regarding the management of multifocally recurrent or metastastic RPS with the practical expertise of high-volume sarcoma centers from multiple countries. It is designed as a tool for decision making in the complex multidisciplinary management of this condition and is expected to standardize management across centers, thereby ensuring that patients receive the highest quality care.

Neuroendocrine tumors (NETs) comprise a broad family of tumors that may or may not be associated with symptoms attributable to hormonal hypersecretion. The NCCN Clinical Practice Guidelines in Oncology for Neuroendocrine Tumors discuss the diagnosis and management of both sporadic and hereditary NETs. This selection from the guidelines focuses on sporadic NETs of the pancreas, gastrointestinal tract, lung, and thymus.

Retroperitoneal sarcomas (RPS) are rare malignancies, comprising just 10-15 % of all soft-tissue sarcomas. These are challenging tumors to treat, with surgical resection being the only modality capable of providing a cure. This study analyzed the management and survival of patients resected at a large academic institution.A retrospective study of all patients with primary localized RPS referred to the University of Washington between January 2000 and January 2013 was performed. Univariate and multivariate Cox regression models were used to analyze progression-free survival (PFS) and overall survival (OS) by patient, tumor, and treatment variables.The study identified 132 patients. Median follow-up was 31.8 months. Median PFS was 33 months, and median OS was 111 months. Sixty patients (45.5 %) underwent a margin-negative resection (R0), 59 (44.7 %) had a microscopic margin-positive resection (R1), and 7 (5.3 %) had a macroscopic margin-positive resection (R2). Forty (30.3 %) patients received preoperative radiation, 28 (21.2 %) received neoadjuvant chemotherapy, and 7 (5.3 %) received both. Tumor grade and microscopic margin status emerged as statistically significant predictors for both PFS and OS. Tumor size was also found to correlate with PFS. No significant difference in OS or PFS was observed for histologic subtype, neoadjuvant chemotherapy, or neoadjuvant radiation.Complete surgical resection should remain the mainstay of management for RPS, with emphasis on achieving negative microscopic margins. Neither neoadjuvant chemotherapy nor radiation was shown to significantly improve survival, and their unclear role in the management of RPS requires evaluation in a prospective setting.

Introduction: Although pancreatic neuroendocrine tumors (PNET) are less common and less deadly than pancreatic adenocarcinoma, they remain a considerable source of morbidity and mortality. The cases increasingly discovered due to advanced cross sectional imaging and the majority of PNETs are malignant when diagnosed. Immunotherapy for PNET has not yet been successfully demonstrated, although we and others have previously shown that they often contain robust T cell infiltrates. We hypothesized that combined immunotherapy could reactivate endogenous antitumor activity in organotypic tumor slice cultures of human PNET. Methods: Human PNET tumors were collected from the operating room and 250μm slices were cut using a vibratome and place on 0.4μm pore size membrane inserts (6 tumors). Slice culture survival testing was conducted using immunohistochemistry (IHC), the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, or live immunofluorescence imaging. To test the combining immunotherapeutic effects, slices were treated with a PD-1 blocking monoclonal antibody or isotype control, with or without the CXCR4 inhibitor, AMD3100. For time-lapse live imaging, slices were stained with fluorescently conjugated antibodies for CD8 and EpCAM, as well as a reagent that binds to activated caspase 3/7 enzymes to indicate induction of apoptosis. The live slice was first imaged alone to serve as a non-treated control, then anti-PD1 antibody and AMD3100 were applied, and the slice imaged immediately afterward to monitor response after treatment. Results: We confirmed that cultured slices maintain their baseline morphology and architecture over 9 days in culture. The MTT assay showed stable metabolic activity over the same period. The demonstration of the live and dynamic microenvironment was performed first via live multicolor IF including PNET cells (EpCAM+) and immune cells (both CD45+ and CD4+ cells) within the microenvironment. IHC demonstrated 10% or 12% increased of cleaved-Caspase-3+ cells with combined PD-1 and CXCR4 blockade, as compared to control, after 2 days or 7 days in culture. To confirm the role of cytotoxic T cells, live stained slices were imaged before and after addition of PD-1 blockade and AMD3100. We demonstrated that caspase activation was increased in EpCAM+ cells proximal to CD8+ T cells immediately following combination drug treatment. Furthermore, CD8+ cells proximity to EPCAM+ cells ( Conclusion: Our study demonstrates that combination PD-1 and CXCR4 blockade enhances CD8+ T cell migration and antitumor activity in human PNET tumor slice cultures. Citation Format: Xiuyun Jiang, Kevin Sullivan, Kevin Labadie, Sara K. Daniel, David Seo, Arezou Abbasi, Teresa Kim, Raymond Yeung, Venu Pillarisetty. Combination immunotherapy with PD-1 and CXCR4 blockade activates antitumor immunity against pancreatic neuroendocrine tumors [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 3196.

Background: Colorectal cancer (CRC) is the 4th most common cancer in the US, and the liver is the most common site of metastatic disease. Immune checkpoint inhibitor therapy has not been successful in achieving a clinical response in most patients with CRC liver metastases (CRCLM). The liver is known to induce tolerance to foreign antigens as a result of immunosuppressive cytokines including IL-10. We hypothesized that blockade of IL-10 signaling in CRCLM would potentiate tumor infiltrating lymphocyte (TIL)-mediated tumor cell death. Methods: We performed single-cell RNA sequencing (scRNAseq) of CRCLM using the 10x platform to evaluate for expression of IL-10 or IL-10 receptor (IL-10R) RNA within the tumor (n=8). To confirm if the IL-10R protein was present within the tumor microenvironment (TME), we also performed immunohistochemistry (IHC) (n=3). In order to study the functional effects of IL-10 blockade, we utilized a tumor slice culture (TSC) model, which allows for the study of cancers with their intact TME including immune cells. For TSCs, cores (6 mm diameter) were taken from freshly resected sterile human CRCLM and cut to 250 µm thick slices using a vibratome (n=3). Duplicate slices were treated with either IgG control or anti-IL-10 monoclonal antibodies and cultured for up to 6 days. To evaluate for histological evidence of necrosis and cell apoptosis within the tumor slice, we stained slides with either hematoxylin and eosin (H&E) or cleaved-Caspase-3 (CC3). To gain insight into the activation state of TIL after treatment, we measured levels of cytokines within the culture supernatants. Results: We found by scRNAseq that that IL-10 was expressed by a subset of tumor-associated macrophages, and IL-10R was expressed by both CD4+ and CD8+ T cells as well as macrophages. We confirmed that IL-10R protein was present within the CRCLM TME by IHC, and IL-10R expression was distributed throughout the stroma in non-tumor cells. In TSC treated with anti-IL-10 antibody, CC3+ cells were found to be 82.8% of total cells, compared to 36.1% of control (p = 1 x 10-6) at day 6. These findings were consistent across all human tumor samples treated with IL-10 blockade versus control at all time points examined. Furthermore, IL-10 blockade led to histologic evidence of generalized necrosis compared to an intact TME seen in the control group. Analysis of cytokines released into the media confirmed that IL-10 was present in controls, but absent in slices blocked with anti-IL-10 antibody. We also found increased levels of granzyme B, IL-2, GM-CSF, and IL-18, as well as a reduction in the immune checkpoint receptor TIM3, after one day of IL-10 blockade in culture. Conclusion: Treatment of human CRCLM TSCs with anti-IL-10 antibody leads to a marked increase in immune-mediated cell death within the tumor. Our data suggest that IL-10 serves as a critical regulator of anti-tumor immunity in the CRCLM TME and may serve as an important immunotherapeutic target. Citation Format: Kevin M. Sullivan, Xiuyun Jiang, Yongwoo David Seo, Heidi L. Kenerson, Xiaowei Yan, Chris Lausted, Changting Meng, Neda Jabbari, Kevin P. Labadie, Sara K. Daniel, Qiang Tian, Teresa S. Kim, Raymond S. Yeung, Venu G. Pillarisetty. IL-10 blockade reactivates antitumor immunity in human colorectal cancer liver metastases [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 4489.

Despite recent advancements in multimodal therapy, pancreatic ductal adenocarcinoma (PDA) continues to have a dismal prognosis. In the era of burgeoning immune therapies against previously difficult-to-treat malignancies, there has been growing interest in activating the immune system against PDA; however, unlike in other cancers such as melanoma and lymphoma, immunotherapy has not yielded many clinically significant results. To harness these mechanisms for therapeutic use, an in-depth understanding of T-cell programming in the immune microenvironment of PDA must be achieved. The outcome of T-cell programming against pathogens or cancer depends on the uptake and presentation of foreign antigens by dendritic cells and macrophages to T cells, and the expression of various co-stimulatory molecules and cytokines. Subsequent immune responses are kept in check via regulatory mechanisms such as immune checkpoints (for example, programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4)), as well as other immunosuppressive cell types such as regulatory T cells (Treg) and M2 macrophages. PDA presents a challenge from the perspective of immune therapy because of many immunosuppressive mechanisms at play in its microenvironment. The tumor itself produces IL-10 and transforming growth factor beta (TGF-β) that downregulate T-cell activation as well as the activity of antigen-presenting cells. At the same time, PDA also appears to recruit more regulatory elements into its milieu; higher infiltration of Treg, for instance, has been associated with poorer prognosis in PDA patients. M2 macrophages and myeloid-derived suppressive cells are also highly prevalent in the tumor microenvironment. T cells in PDA have high expression of PD-1, whereas the tumor has high expression of PD-L1, which likely inhibits activation of tumor antigen-specific T cells. Many of these immunosuppressive mechanisms have been targeted as potential immune therapies of PDA. Immune checkpoint inhibitors, which target PD-1 and CTLA-4, have been shown to be effective in other cancers such as melanoma; however, they have not demonstrated outcome benefits in PDA so far. Other novel investigational approaches under study currently include inhibiting the homing of immunosuppressive cell types to the tumor milieu, as well as vaccines designed to boost the adaptive response to PDA antigens. As our understanding of the nuanced and complex interactions of the immune microenvironment expands, more targeted approaches can be taken toward achieving therapeutic success in immune therapy against PDA.

BackgroundTumor-infiltrating lymphocyte (TIL) counts in colorectal cancer liver metastases (CRCLM) predict survival following resection. While CD4 and CD8 T cells have been correlated with outcome following CRCLM resection, the role of regulatory T cells (Treg) is not well defined.MethodsTIL in 188 patients who underwent CRCLM resection between 1998 and 2000 were analyzed by immunohistochemistry using tissue microarrays. Correlation between TIL composition and outcome was determined while controlling for established prognostic factors. Total T cells (CD3), helper T cells (CD4), cytotoxic T cells (CD8), and Treg (FoxP3) were analyzed.ResultsMedian follow-up time was 40 months for all patients and 95 months for survivors. Overall survival (OS) at 5 and 10 years was 40 and 25%, respectively. The CD4 T cell count correlated with OS (p = .02) and recurrence-free survival (p = .04). A high number of CD8 T cells relative to total T cells (CD8:CD3 ratio) predicted longer OS times (p = .05). Analysis of Treg revealed that high FoxP3:CD4 (p = .03) and FoxP3:CD8 (p = .05) ratios were independent predictors of shorter OS. Patients with a high clinical risk score (CRS) were more likely to have a high number of intratumoral Treg, and patients ≥65 years old had a less robust CRCLM T cell infiltration.ConclusionsA high number of Treg relative to CD4 or CD8 T cells predicted poor outcome, suggesting an immunosuppressive role for FoxP3 + TIL. The intratumoral immune response was an independent predictor of outcome in patients with colorectal liver metastases.

With the widespread adoption of molecular profiling in clinical oncology practice, many physicians are faced with making therapeutic decisions based upon isolated genomic alterations. For example, epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are effective in EGFR-mutant non-small cell lung cancers (NSCLC) while anti-EGFR monoclonal antibodies are ineffective in Ras-mutant colorectal cancers. The matching of mutations with drugs aimed at their respective gene products represents the current state of “precision” oncology. Despite the great expectations of this approach, only a fraction of cancers responds to ‘targeted’ interventions, and many early responders will ultimately develop resistance to these agents. The underwhelming success of mutation-driven therapies across all cancer types is not due to an inability to detect genetic changes in tumors; rather a deficit in functional insight into the genomic alterations that give rise to each cancer. The Achilles heel of precision oncology thus remains the lack of a robust functional understanding of an individual cancer genome that then allows prediction of the best therapy and resultant outcome for that patient. Current practice focuses on one ‘actionable’ mutation at a time, while solid cancers typically possess many mutations that involve different cellular sub-populations within a tumor. No method or platform currently exists to guide the interpretation of these complex data, nor to accurately predict response to treatment. This problem is particularly germane to primary liver cancers (PLC), for which only a handful of targeted therapies have been introduced. Here, we will review strategies aimed at overcoming some of these challenges in precision oncology, using liver cancer as an example.

Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) cells homogeneously express high levels of NY-ESO-1 and other cancer/testis antigens, which makes these sarcoma types good candidates for T cell immunotherapy. However, these tumors demonstrate a “cold” microenvironment, with low cell surface expression of human leukocyte antigen (HLA) molecules and few infiltrating T cells. Interferon gamma (IFNγ) has been used both as a single agent and in combination with other immunotherapies in cancers with “hot”, highly inflamed tumor microenvironments such as melanoma. To date, little work has explored its impact on immunologically quiet tumors. In order to increase the expression of HLA on the tumor cell surface and improve the T cell recognition and cytotoxicity, we initiated a pilot “window of opportunity” clinical trial, 6 SS/MRCL patients were treated with subcutaneous systemic interferon gamma (IFNγ) weekly for 2 or 4 weeks at a dose of 100 mcg/m2. The primary objective of the study was to determine if HLA expression increased on post-treatment biopsies in comparison to pre-treatment biopsies. The treatment was relatively well-tolerated, patients experienced flu-like symptoms with no severe adverse effects. Patients' sera, PBMCs and tumor biopsies were collected pre- and post-IFNγ treatment for further analysis. Several cytokines including interleukin-16, microphage migration inhibitory factor and CXCL-10 etc. changed considerably in patients' periphery. Transcriptome profiling on the PBMCs showed significant changes in some immune regulatory genes in T cells and monocytes. Flow cytometry analysis of the tumor biopsies showed significant increase of both class I and class II HLA on the tumor surface in all treated patients. Tumor infiltrating immune cell frequencies increased by 3-10 times, of which, the majority were T cells. Functional assay of expanded tumor infiltrating lymphocytes (TIL) showed an increased and broadened tumor-associated peptide recognition capability. However, in all evaluable tumors there was an increase in PD-L1 either on tumor or on infiltrating macrophages. In conclusion, weekly IFNγ treatment is well tolerated in SS/MRCL patients and it can significantly enhance the expression of HLA on tumor surface, and increase T cell infiltration; however, increased PD-L1 expression on tumor cells and infiltrating immune cells may help tumors evade T cell elimination. Strategies combining PD-1 blockade and IFNγ should be explored. Citation Format: Shihong Zhang, Sara Cooper, Bailey Donahue, Venu G. Pillarisetty, Robin L. Jones, Stanley R. Riddell, Brian A. Van Tine, Seth M. Pollack. Interferon gamma induced transformation of the cold tumor microenvironment in patients with NY-ESO-1 expressing sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 616.

6025Background: Epigenetic modification is increasingly recognized as a mechanism for tumor immune evasion. This clinical trial explored the activity of P with the HDAC inhibitor V in HN and SGC. M...

71 Background: Soft tissue sarcomas (STS) are heterogeneous tumors which are morbid and lethal. G100 is under investigation in multiple clinical trials and contains a potent TLR4 agonist (oil-in-water emulsion of glucopyranosyl lipid A) that has been tested as vaccine adjuvant. We hypothesized IT G100 would induce robust local and potentially systemic anti-tumor immune response, leading to improved outcomes. Methods: 15 metastatic STS patients with superficial lesions were treated with weekly IT G100 for 8-12 wks; 12 patients received radiation (RT) for 2 wks to start, while 3 received IT G100 for 6 wks prior to RT. Biopsies and PBMC were collected pre and post treatment, and flow cytometry was performed on biopsies. TIL and PBMC were analyzed with TCR deep sequencing. PBMC were analyzed by single cell multiplex cytokine profiling. Results: No grade ≥ 3 toxicity was observed, and local tumor control was achieved in all evaluable tumors (14/14). Treated tumors tracked post-trial (mean 156 days) had persistent local control with 1 CR (7%), 1 PR (7%), and 11 SD (79%). In 3 patients with long term follow up, treated lesions remained controlled vs index lesions (-53% vs +31% at mean 235 days, p = 0.002). In all tumors after G100 alone, T cell infiltration increased. In P14, CD3 live cells in tumor rose from < 1% to 62%. PBMC clonality increased in 8/14 tested including P06, who had 4× increase in clonality and CR in the injected lesion; PBMC and TIL TCR overlap increased from 13.4% to 21.5%. P13 had a 2.3× rise in TIL clonality; the top clone (a CD4 T cell) expanded from 0.1% to 38% and expressed more TNFα than the rest (p < 0.0001). Single cell cytokine analysis of PBMC showed 7/13 (54%) increased in polyfunctionality (producing > 2 cytokines) in CD4 T cells; no consistent increase was seen in CD8 T cells. TNFα levels in pre-treatment monocytes correlated with PFS (R2= 0.5, p = 0.02). Conclusions: IT G100 is a viable agent for local control of metastatic STS lesions. With or without RT, G100 appears to cause CD4 T cell mediated local and systemic response. Combination of G100 with other immunomodulators could induce clinically significant systemic responses, as seen in follicular NHL treated with G100. Clinical trial information: NCT02180698.

The aggressive nature of medullary thyroid cancer (MTC) is evidenced by its propensity to present early with lymph node (LN) metastases. The clinical significance of sporadic MTC ≤1 cm (micro-MTC) is not clearly defined. We performed a retrospective review of the clinical, laboratory, and pathologic data for all patients treated or followed at our institution for sporadic micro-MTC from 1987 through 2008. A total of 18 patients met the criteria for inclusion in our study. All tumors were unifocal and C-cell hyperplasia distant from the tumors was uniformly absent. Fourteen (78%) patients underwent total thyroidectomy, and the remaining four (22%) patients with tumors ≤0.5 cm had lobectomy with isthmusectomy alone. Four (22%) patients were found to have LN metastases. However, none of the nine patients with tumors

While tumor infiltrating lymphocytes (TIL) have been shown to independently predict survival in primary colorectal cancer, the prognostic implications of TIL in resectable colorectal cancer liver metastases (CRCLM) have not been previously defined. This study examines the correlation between TIL numbers and survival following hepatic resection. We studied patients who survived ≤2 or ≥10 years following CRCLM resection. Immunohistochemistry was performed on tissue microarrays (TMAs) to determine the number of T cells within CRCLM. Correlation between TIL frequency and ≤2 or ≥10 year survival was determined while controlling for established prognostic factors. Of 162 patients, 104 survived ≤2 years and 58 survived ≥10 years. Independent correlates of 10-year survival following CRCLM resection included a high number of CD8 T cells, a low number of CD4 T cells, and a clinical risk score of ≤2 (P

Intraductal papillary mucinous neoplasm (IPMN) is a mucin-producing epithelial neoplasm that carries a risk of progression to invasive pancreatic ductal adenocarcinoma. Lynch syndrome is an autosomal dominant condition caused by germline mutations in mismatch repair genes such as MSH2 that lead to microsatellite instability and increased risk of tumor formation. Although families with Lynch syndrome have an increased risk of pancreatic cancer, a clear connection between Lynch syndrome and IPMN has not been drawn. We present a report of a 58 year-old Caucasian woman with multiple cancers and a germline mutation of MSH2 consistent with Lynch syndrome. A screening abdominal computed tomography scan revealed a dilated main pancreatic duct and cystic ductular structure in the uncinate process that were consistent with IPMN of the main pancreatic duct on excision. Immunohistochemistry and polymerase chain reaction of the patient's pancreas specimen did not reveal microsatellite instability or mismatch repair gene loss of expression or function. Our findings may be explained by the fact that loss of mismatch repair function and microsatellite instability is a late event in neoplastic transformation. Given the relative rarity of main duct IPMN, its appearance in the setting of somatic MSH2 mutation suggests that IPMN may fit into the constellation of Lynch syndrome related malignancies.

Introduction Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease that is rarely cured. Given the recent successes with immunotherapy for other malignancies and the fact that PDA is heavily infiltrated by effector T cells, we postulate that accurate modeling of the PDA immune microenvironment would allow us to unveil mechanisms of immunosuppression that could be overcome for therapeutic benefit. Methods PDA tumors were collected from the operating room and transported in cold media to the laboratory, where 250μm slices were cut using a vibratome and placed on 0.4μm pore size membrane inserts pre-coated with collagen gel and culture. Proliferation was measured using MTT assay and weight-adjusted optical density. Slices were fixed in 4% PFA for imaging after live immunofluorescence (IF) staining or for subsequent immunohistochemistry (IHC) prior to imaging. For proteomic analysis, slices were digested with trypsin and analyzed by mass spectrometry. To assess for immune cell migration into the tumor slices, isolated autologous splenocytes were stained with CFSE and added to the slice culture and imaged using IF after 6 days. Results Using light microscopy, we confirmed that cultured slices maintain their baseline morphology, architecture and surface area over 9 days in culture. The MTT assay showed stable growth over the same period; IHC for Ki-67 and cleaved Caspase-3 corroborated a similar pattern of proliferation and apoptosis after 6 days culture when compared to day 1. T cells (CD3+, CD8+ and FOXP3+), macrophages (CD68+, CD163+, and HLA-DR+), and stromal myofibroblasts (αSMA+) were confirmed to be present through day 6 using IHC. Quantitative proteomic analysis of over 3000 proteins showed that only 2-3% of proteins showed significant changes of abundance greater than 2 fold after 6 days in culture. However, when slice were challenged with cytotoxic drugs (staurosporine and cyclohexamide), there was a time- and dose-dependent response in proliferation and apoptosis compared with controls. The demonstration of the live and dynamic microenvironment was performed first via live multicolor IF including PDA cells (EpCAM+), fibroblasts (fibronectin+) and immune cells (both CD11b+ and CD8+ cells) within the microenvironment. Furthermore, co-culture of CFSE-labeled splenocytes on top of slices for 6 days. Z-stacked live confocal imaging confirmed their migration from the surface into the slice microenvironment. Conclusion Our study demonstrates that the PDA slice culture maintains original tumor microenvironment, and overall proteomic production and dynamic function response in vitro. The slice culture model thus represents a novel platform to evaluate both pharmaceutical and cell-based immunotherapies for mechanisms of action and potential synergism, effects on specific cell types of the microenvironment, and tumor-specific immunogenic potential. Citation Format: Xiuyun Jiang, David Seo, Jae Chang, Eslam Nigjeh, Sheng Pan, Florencia Jalikis, Venu Pillarisetty. Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment [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 3692. doi:10.1158/1538-7445.AM2017-3692

Introduction: Soft tissue sarcomas (STS) are heterogeneous mesenchymal tumors which are both morbid and lethal. G100 is a stable oil-in-water emulsion of glucopyranosyl lipid adjuvant, a highly potent toll-like receptor 4 (TLR4) agonist, which has been utilized for intratumoral (IT) injections and as vaccine adjuvants without significant toxicity. We hypothesized that IT G100 would induce a robust local and potentially systemic anti-tumor immune response in the STS microenvironment, leading to improved outcomes. Methods: 15 metastatic STS patients who had a superficial injectable lesion were treated with weekly IT G100 for 8-12 weeks; 12 patients received concurrent radiation for 2 weeks at the start, while 3 got IT G100 alone for 6 weeks prior to radiation. Biopsies and blood were collected pre and post treatment, and flow cytometry was performed on fresh tumor samples. T-cell receptor (TCR) deep sequencing of tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) was performed on 7 patients. RECIST v1.1 and the Common Terminology Criteria for Adverse Events were used to monitor clinical outcomes. Results: Patients had a median of 3 (0~5) prior lines of therapy and mean tumor size of 5.6cm (1~20cm). No grade 3 or higher treatment-related toxicity was observed, and local tumor control was achieved in 93% (14/15). 6 (40%) had stable disease after treatment, and 1 (P06) had complete regression of injected tumor. This tumor had a high percentage of infiltrating pre-treatment immune cells (12% CD45+ on flow cytometry versus 2.7% for all other tumors). TCR sequencing showed that the increase in clonality of PBMC after treatment was greater in P06 (389%) compared to 6 other patients (mean 34%). There was also higher overlap in TCR sequence between TIL versus PBMC after treatment (13% versus 22%), suggesting systemic expansion of tumor-specific T-cells. In 7 patients evaluable for tumor-associated macrophages (tumors with >1000 CD45+CD11b+ cells), 71% had a shift from an M2 to M1 phenotype. In all patients who received G100 alone, there was an increase in T-cell infiltration into tumor after treatment. In one patient (P14), the proportion of CD3+ live cells in tumor went from Conclusion: IT G100 provides a potentially viable agent for local control of metastatic STS. With or without radiation, G100 appears to shift the tumor microenvironment into a more inflammatory state with significant infiltration of T cells. The increase in clonality in PBMC and TIL, as well as increased overlap of tumor-associated versus peripheral TCR sequences, suggest induction of a tumor-specific response. Combination of G100 with other immunomodulators may further enhance the adaptive anti-tumor response. Citation Format: Yongwoo D. Seo, Edward Y. Kim, Ernest U. Conrad, Ryan B. O'Malley, Sara Cooper, Bailey Donahue, Lee D. Cranmer, Hailing Lu, Frank Hsu, Elizabeth T. Loggers, Taylor Hain, Darin J. Davidson, Lynn Bonham, Venu G. Pillarisetty, Gabrielle M. Kane, Stanley R. Riddell, Robin L. Jones, Seth M. Pollack. Intratumoral injection of the toll-like receptor 4 agonist G100 induces a T-cell response in the soft tissue sarcoma microenvironment [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 2947. doi:10.1158/1538-7445.AM2017-2947

23 Background: The success of immunotherapy has raised new issues regarding the selection of patients, design of combination strategies, and sequencing of various regimens. Sarcomas have poor outcomes in the metastatic setting but may be amenable to immune therapies. However, we currently have limited knowledge of the immunologic profiles of different soft tissue sarcoma (STS) subtypes. Methods: We identified patients with the relatively common STS subtypes: leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS) and liposarcoma. Formalin fixed paraffin embedded (FFPE) tumor samples from 81 patients underwent gene expression analysis, immunohistochemistry for PD-1 and PD-L1, and sequencing of the T cell receptor Vβ region. Differences in liposarcoma subsets were also evaluated. Results: UPS and LMS had high expression levels of genes related to antigen presentation and T cell infiltration. UPS had higher levels of PD-L1 (p ≤ 0.001) and PD-1 (p ≤ 0.05) on IHC. UPS also had the highest T cell infiltration based on TCR sequencing, significantly more than SS, which had the lowest (p ≤ 0.05). UPS and LMS both had higher clonality compared with SS and liposarcoma (p ≤ 0.05). A model adjusted for STS histologic subtype found that for all sarcoma T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 staining levels (p ≤ 0.01). Conclusions: In a model adjusted for sarcoma histologic subtypes, T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression, consistent with the emerging view of tumor immunity that highly inflamed tumors acquire inhibitory ligands to evade tumor-specific T cells. UPS, which is a more highly mutated STS subtype, provokes a strong immune response evidenced by multiple inflammatory features suggesting that it may be well-suited to checkpoint inhibitor based approaches. SS and liposarcoma subsets are less highly mutated but do express immunogenic self-antigens therefore strategies to improve antigen presentation and T cell infiltration may be valuable for allowing immunotherapeutic success in these tumor types.

Objective To determine if the density of FOXP3+ lymphocytes in primary tumors and lymph nodes in pancreatic cancer correlates with the presence of lymph node metastases. Methods FOXP3+ lymphocyte density in primary pancreatic cancer tissue and draining lymph nodes was measured using immunohistochemistry. We analyzed the clinical and pathological aspects associated with the accumulation of FOXP3+ lymphocytes in pancreatic cancer. We also analyzed the correlation of density of FOXP3+ lymphocytes in lymph nodes with the nodal status and distance from the primary tumor. Results FOXP3+ lymphocyte density in pancreatic cancer was significantly higher than in paratumoral pancreatic tissue. The density of FOXP3+ lymphocytes in local tumor tissue correlated significantly with the histological grade and overall lymph node status. Furthermore, FOXP3+ lymphocyte density was significantly higher in positive lymph nodes than in negative ones, while it had no correlation with the distance of the lymph node from the primary tumor. Conclusion FOXP3+ lymphocyte density in primary tumor tissue in patients with pancreatic cancer correlates with lymph node metastasis. Lymph nodes containing metastases having higher FOXP3+ lymphocyte densities than do negative lymph nodes.

Tumor infiltrating lymphocytes (TIL) have been demonstrated to predict oncologic outcomes following resection of primary intrahepatic neoplasms and metastatic liver tumors. Despite strong immunosuppressive factors within the intrahepatic space, TIL are frequently demonstrated in liver tumors. The presence of TIL within liver tumors provides evidence of a host immune response that may be protective, but often is rendered ineffective by tumor induced immune dysfunction. In this review, we discuss techniques involved in studying TIL and subsets of TIL commonly identified. We emphasize the unique nature of the intrahepatic milieu that promotes immunosuppression, and how liver TIL and TIL ratios can be used as indicators of prognosis. Several types of primary and metastatic liver tumors are considered to highlight the similarities and important differences in TIL responses, which likely reflect how intrahepatic immunity is influenced by tumor biology. The studies we discuss indicate that tumor infiltration by suppressor cells and expression of immunoinhibitory molecules by TIL limits the anti-tumor immune function of effector T cells. Most patients fail to mount an adequate immune response to liver tumors, which provides compelling rationale for clinical study of immunotherapy for intrahepatic neoplasms.

Objective The immune response to pancreatic ductal adenocarcinoma (PDA) may play a role in defining its uniquely aggressive biology; therefore, we sought to clearly define the adaptive immune infiltrate in PDA. Design We used immunohistochemistry and flow cytometry to characterize the immune infiltrate in human PDA and compared our findings to the patients’ peripheral blood. Results In contrast to the myeloid cell predominant infiltrate seen in murine models, T cells comprised the majority of the hematopoietic cell component of the tumor stroma in human PDA. Most intratumoral CD8+ T cells exhibited an antigen-experienced effector memory cell phenotype and were capable of producing IFN-γ. CD4+ regulatory T cells (Treg) and IL-17 producing T helper cells were significantly more prevalent in tumor than in blood. Consistent with the association with reduced survival in previous studies, we observed higher frequencies of both myeloid cells and Treg in poorly differentiated tumors. The majority of intratumoral T cells expressed the co-inhibitory receptor programmed death-1 (PD-1), suggesting one potential mechanism through which PDA may evade antitumor immunity. Successful multimodal neoadjuvant therapy altered the immunoregulatory balance and was associated with reduced infiltration of both myeloid cells and Treg. Conclusion Our data show that human PDA contains a complex mixture of inflammatory and regulatory immune cells, and that neoadjuvant therapy attenuates the infiltration of intratumoral cells associated with immunosuppression and worsened survival.