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Despite progress toward equity within our broad social context, the domains of gender as a social, cultural, and structural variable continue to exert influence on the delivery of oncology care. Although there have been vast advances in our understanding of the biological underpinnings of cancer and significant improvements in clinical care, disparities in cancer care for all women—including cisgender, transgender, and gender diverse women—persist. Similarly, despite inclusion within the oncology physician workforce, women and gender minorities, particularly those with additional identities under-represented in medicine, still face structural barriers to clinical and academic productivity and career success. In this article, we define and discuss how structural sexism influences both the equitable care of patients with cancer and the oncology workforce and explore the overlapping challenges in both realms. Solutions toward creating environments where patients with cancer of any gender receive optimal care and all physicians can thrive are put forward.

Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.

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Cholangiocarcinoma (CCA) is a highly morbid cancer with rising incidence and few approved standard therapies. The main anatomical sites of cholangiocarcinoma are intra-hepatic (iCCA), perihilar, an...

TPS817 Background: Metastatic disease remains a consistent challenge for patients with gastrointestinal (GI) malignancies. Multiple immune checkpoint inhibitors (ICI) have been FDA approved for several GI indications. Still, patients will progress on these therapies, either immediately or after initial response. Palliative radiation therapy (RT) is an effective treatment that can be delivered to symptomatic metastases. It also has been reported to lead to an abscopal effect, which is an induction of response in a distant tumor. This is thought to result from increased tumor immunogenicity, tumor microenvironment modulation, and immune cell recruitment. Thus, palliative RT could not only relieve symptoms, but also potentially reinvigorate the immune response, prolong ICI efficacy, and delay next-line systemic therapy that carries risks of unknown efficacy and tolerability. Ongoing studies seek to elucidate prognostic and predictive biomarkers of the abscopal effect. However, cohorts of patients with GI malignancies are limited, so there remains a need for research in combining RT and immunotherapy in the context of metastatic GI cancer. ARM-GI aims to study the radiation-augmented immune response in patients with metastatic GI cancers progressing on immunotherapy. Methods: ARM-GI is a phase 2, single arm study. Key eligibility criteria include confirmed metastatic GI malignancy, progressive disease per RECIST v1.1 on any ICI, and at least 2 metastases, one of which can be safely left unirradiated. The target prescription dose is 30 Gy in 5 fractions to symptomatic sites through intensity modulated RT or stereotactic body RT. Patients will continue the same immunotherapy after RT and be evaluated per RECIST v1.1 longitudinally. The primary endpoint is overall response rate (ORR) per RECIST v1.1. Secondary endpoints include ORR per iRECIST, progression free survival, overall survival, local control in radiated lesion(s), effect of distant radiation on unirradiated target lesions, incidence of new metastatic lesions, safety by CTCAE v5.0, and time to new systemic therapy. The study will enroll 28 patients, with recruitment ongoing. Serial peripheral blood samples are collected for exploratory studies that will analyze for association with disease response and quantification of changes in circulating immune cell subsets after RT compared to baseline. Clinical trial information: NCT04221893 .

450 Background: Neoadjuvant chemoradiation (CRT) followed by surgical resection is standard of care for patients with locally advanced esophageal/gastroesophageal junction (E/GEJ) cancer. A pathologic complete response (pCR) at surgery is associated with improved survival outcomes. Sotigalimab (sotiga) is a potent CD40 agonist mAb capable of inducing and expanding anti-tumor immune responses. A recently reported non-randomized phase II clinical trial of sotiga combined with CRT in E/GEJ cancer patients showed promising pCR rates that compared favorably to historical data1. Here, deep immune profiling was performed on a subset of patients to gain insight into the mechanism of sotiga. Methods: In the initial safety cohort (n=6) of this phase II E/GEJ clinical trial, tumor and blood samples were obtained pre-treatment and again following a single lead-in dose of sotiga, prior to initiation of CRT. Immune modulation in the tumor microenvironment and blood of patients was examined using high dimensional techniques, including mass cytometry, multiplexed ion beam imaging, and single cell RNA sequencing. Results: Sotiga administration dramatically re-models the tumor microenvironment, inducing immune infiltration and shifting the immune composition predominantly toward an inflammatory phenotype. At baseline, E/GEJ tumors were infiltrated with myeloid cells and T cells, the majority of which were Tregs. Sotiga increased antigen-presenting cell (APC) infiltration in tumors and activated dendritic cells as demonstrated by expression of MHCII and CD86. Sotiga treatment also induced infiltration of activated cytotoxic T cells and decreased the proportion of Tregs. There was also evidence of T cell activation in peripheral blood with increased expression of 4-1BB, CTLA-4, ICOS and PD-L1. Therapeutic responses to sotiga were associated with distinct T cell signatures both at baseline and on-treatment. At baseline, pCR patients (representing 4 of the 6 pts (67%) in this cohort) had a higher ratio of memory CD4+ T cells to Tregs which further increased following sotiga treatment, whereas patients who did not have a pCR had a higher proportion of myeloid cells at baseline and did not show a decrease in Treg cells after sotiga treatment. Conclusions: Sotiga induced dramatic changes in the tumor microenvironment including increased frequency of activated T cells and APCs, and decreased frequency of Tregs. A distinct signature of T cell infiltration in baseline tumor biopsies was observed in patients who achieved a pCR versus those who did not, potentially identifying patients that may benefit from this novel treatment strategy.

TPS630 Background: Hepatocellular Carcinoma (HCC) is among the most common types and leading causes of cancer death globally, and the incidence is on the rise. For patients with advanced hepatocellular carcinoma (HCC), immune checkpoint inhibitors (ICI) in combination with anti-angiogenic agents or other ICI improve survival compared with tyrosine kinase inhibition. Though ICI-based regimens can achieve prolonged responses in some patients and prolong overall survival, only a subset of patients achieve durable responses. There is an urgent need for strategies to overcome primary and acquired resistance. Many of these patients have symptomatic sites of disease which could benefit from local palliative radiotherapy. Radiotherapy also has the potential to elicit an abscopal immune response when combined with ICI. We hypothesize that combining Radiation Treatment (RT) with Durvalumab or the combination of Durvalumab + Tremelimumab (combination D+T in STRIDE regimen showed OS improvement in HIMALAYA trial in 1st line HCC) will achieve a meaningful response rate in advanced HCC patients after progression on other ICI-based therapies. Methods: This is an open label, single center, two-arm, non-comparative phase II trial. Key eligibility criteria are; histologically-diagnosed HCC with progression during or after prior PD-(L)1 checkpoint inhibitor immunotherapy and at least 1 RECIST 1.1-measurable tumor present which has not received RT or other local therapy prior to enrollment, ECOG 0-1, and Child Pugh score of A, B7, or B8. Patients are assigned sequentially to either single-agent durvalumab initiated starting 3 to 10 days after completing RT (25 Gy in 5 fractions to 1-5 sites of disease), at fixed dose of 1500 mg IV every 28 days, or a single fixed dose of tremelimumab of 300 mg IV administered on Day 1, initiated within 3-10 days of completing RT, in combination with durvalumab at fixed dose of 1500 mg IV every 28 days; for up to 24 months or until confirmed radiographic progression. The primary end point is overall response rate (ORR) and secondary endpoints include proportion of participants with adverse events, overall survival, progression free survival and duration of response. Exploratory endpoints include profiling of peripheral blood mononuclear cell immune cells and plasma biomarkers. We plan to accrue 30 patients to this study, 15 to each Arm, to evaluate ORR which would allow us to detect at least a 20% ORR, which would be a clinically meaningful improvement in efficacy in this population with otherwise poor treatment options assuming a statistical power of 87% and a directional 5% statistical significance level based upon an exact binomial test. The trial is open and enrolling. Clinical trial information: NCT04430452 .

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide and confers a poor prognosis. Beyond standard systemic therapy with multikinase inhibitors, recent studies demonstrate the potential for robust and durable responses from immune checkpoint inhibition in subsets of HCC patients across disease etiologies. The majority of HCC arises in the context of chronic inflammation and from within a fibrotic liver, with many cases associated with hepatitis virus infections, toxins, and fatty liver disease. Many patients also have concomitant cirrhosis which is associated with both local and systemic immune deficiency. Furthermore, the liver is an immunologic organ in itself, which may enhance or suppress the immune response to cancer arising within it. Here, we explore the immunobiology of the liver from its native state to chronic inflammation, fibrosis, cirrhosis and then to cancer, and summarize how this unique microenvironment may affect the response to immunotherapy.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n = 9) and matched healthy donors (n = 8). Following anti-PD-1 treatment, CD14

Summary:In this issue, Kamata-Sakurai and colleagues describe an agonist antibody to CD137 (4-1BB) that takes on an active conformation in environments with high ATP concentrations, characteristic of tumors. This represents a novel advancement in developing immunotherapies that can be administered systemically, but act locally to induce antitumor immune responses without the usual attendant toxicities.See related article by Kamata-Sakurai et al., p. 158.

Background/aim The majority of colorectal cancer (CRC) cases, which are microsatellite stable (MSS) and do not harbor mismatch repair deficiency/microsatellite instability, are resistant to immunotherapy. Identification of patients with exceptional responses in MSS CRC and predictive biomarkers is an unmet need that needs to be addressed. Case report We report three cases of MSS CRC with durable clinical benefit from immunotherapy with anti-PD-1 checkpoint inhibitors. Two cases bear a POLE P286R mutation, which has been associated with lack of immunotherapy response in MSS CRC. Two cases bear alterations in Ataxia-Telangiectasia Mutated (ATM) which may contribute to observed responses, including interaction with a co-administered intratumoral stimulator of interferon genes (STING) pathway agonist in one patient. Conclusion Novel DNA damage repair alterations, including mutations in ATM, can provide insight into additional mechanisms by which genomic alterations can sensitize MSS CRC to diverse immunotherapies.

Background Nivolumab demonstrated durable responses and safety in patients with hepatocellular carcinoma (HCC) with Child-Pugh class A cirrhosis in the CheckMate 040 trial, with rates of hepatotoxicity that were similar to those of non-HCC populations. To the authors' knowledge, the safety and efficacy of nivolumab has not been established in patients with Child-Pugh class B (CPB) cirrhosis, a population with limited therapeutic options and a poor prognosis. Methods The authors conducted a retrospective case series of patients with advanced HCC and CPB cirrhosis who were treated with nivolumab and enrolled in the University of California at San Francisco Hepatobiliary Tissue Bank and Registry. Safety endpoints included rates of grade ≥3 adverse events (AEs) (graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.03]) and serious AEs, immune-related AEs (irAE), steroid requirement, and discontinuation. Efficacy endpoints included time on treatment, the objective response rate according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, overall survival, and progression-free survival. Results A total of 18 patients were included, with 72% of them (13 of 18 patients) previously treated with sorafenib. The majority of patients (94%; 17 of 18 patients) experienced a grade ≥3 AE, with treatment-related grade ≥3 AEs reported in 28% of patients (5 of 18 patients). irAEs were reported to occur in approximately 50% of patients (9 of 18 patients), and 28% (5 of 18 patients) required steroids. Treatment-related AEs required discontinuation in 4 patients (22%). The median time on treatment was 2.3 months (95% CI, 1.9 months to upper bound not estimable). The objective response rate was 17% (3 of 18 patients), including 2 partial responses and 1 complete response. The median overall survival from the time of nivolumab initiation was 5.9 months (95% CI, 3 months to upper bound not estimable), with a median progression-free survival of 1.6 months (95% CI, 1.4-3.5 months). Conclusions Patients with CPB HCC experienced high rates of AEs, although the frequency of irAEs was similar to that of patients with Child-Pugh class A HCC in the CheckMate 040 trial. A subset of patients experienced prolonged tumor responses. Nivolumab warrants further study in patients with CPB HCC.

3565 Background: MSS mCRC rarely responds to pembrolizumab monotherapy, but capecitabine and bevacizumab may induce immune-stimulatory effects. This study evaluates the safety, tolerability and preliminary efficacy of pembrolizumab in combination with capecitabine and bevacizumab in MSS mCRC. Methods: Single-center, phase 2 trial with safety lead-in to confirm the recommended phase 2 dose (RP2D) for capecitabine and expansion cohorts (NCT03396926). Key eligibility: MSS mCRC with stable disease (SD) or progressive disease (PD) on prior fluoropyrimidine-based therapy. Treatment: Capecitabine 1000 mg/m2 PO BID D1-14 Q21 days (confirmed RP2D) plus pembrolizumab 200 mg IV D1 Q21 days and bevacizumab 7.5 mg/kg IV D1 Q21 days. Endpoints: Primary: Objective response rate (ORR) by RECIST 1.1. Key secondary: Safety, duration of response (DOR), progression-free survival (PFS), overall survival (OS). Results: From 04/2018-10/2021, 44 patients (pts) were enrolled. Overall: Median age 53 years (range 28-79); female 50%; Caucasian 61%. Liver metastases at enrollment 80%. Prior therapies: median prior lines of therapy 2 (range 1-5); PD on fluoropyrimidine-containing regimens 91%; prior exposure to bevacizumab 86%. Complete toxicity data are available for 36 off-treatment pts. Grade ≥ 3 treatment-related (tr)AEs occurred in 10 (28%) pts, including grade 3 immune-related AEs in 4 (11%) pts. All-cause serious (s)AEs occurred in 13 (36%) pts and trSAEs in 5 (14%) pts. (tr)AEs leading to dose interruptions, reductions, or delays occurred in 21 (58%) pts, most commonly palmar-plantar erythrodysesthesia syndrome in 17 (47%) pts. Disposition: of 44 pts enrolled, 35 were removed for PD and 1 was removed for treatment noncompliance; 8 treatment ongoing. ORR in 40 evaluable pts was 5% (95% CI: 0.6,16.9). Best response by RECIST 1.1: partial response (PR) in 2 (5%); SD in 26 (65%); PD in 12 (30%). 2 responders: DOR 12 and 15 months, both with liver metastases. Median follow up was 7 months (range 1-45), with median PFS 4.3 months (95% CI: 3.9, 6.1), PFS at 6 months 31.1% (95% CI: 19.2%, 50.4%), and median OS 9.6 months (95% CI: 6.2, 13). Median time on treatment was 5 months (range 1-26). Single cell RNA sequencing on a subset of paired pre- and on-treatment biopsies demonstrated changes in the frequency of dendritic cells. Conclusions: The combination of pembrolizumab with capecitabine and bevacizumab was found to be tolerable with an expected toxicity profile in MSS mCRC pts. The ORR of 5% did not meet the prespecified target of ≥ 15%, however nearly a third of pts had PFS > 6 months. Immune profiling of tumor biopsies and peripheral blood is ongoing. Clinical trial information: NCT03396926.

Pancreatic cancer has demonstrated low response rates to immunotherapy, due in part to late diagnosis, inherent immune resistance, and desmoplastic stroma. Signals in the tumor microenvironment that suppress effector immune cells include the physical barrier of the stroma and suppressive pro-oncogenic molecules secreted by immune myeloid cells and non-immune cells such as fibroblasts. Different strategies to overcome these barriers including vaccines, targeting suppressive myeloid cells, depleting stroma, and T cell therapy have been used thus far with mixed success. The outlook for pancreatic cancer includes rationale combinations of different treatment modalities and identification of biomarkers of response.

Women are under-represented at the highest levels of leadership in health care, so many institutions have started forming "women in medicine" affinity groups. In this The Clinical Teacher's Toolbox, we review the history of women's professional peer-to-peer networking groups in health care, describe the rationale for establishing a women's group, discuss the goals and common content covered by successful women's groups, share best practices on forming and sustaining women's groups, and describe common pitfalls to avoid. When forming a women's group, identifying the group's vision, mission, and primary aim statements are important, and early meetings should deliberately establish a tone of inclusion. We acknowledge that the term "women's groups" implies that gender identity is binary - in reality, these groups are for all who want to combat gender inequities in health care. While early stages of women's groups typically focus on community-building, peer networking, and inviting guest speakers to speak about relevant topics, successful groups often ultimately pivot to advocacy, internal capacity-building, evaluation, and dissemination. To sustain and maintain the group, succession planning, regular opportunities for evaluation, and deliberate planning are essential. Although usual principles of successful small group creation apply, this article outlines unique considerations for how women's groups can advance gender equity.

Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.

BackgroundAdvanced biliary cancers (ABC) have a poor prognosis and low rates of response to immune checkpoint inhibition (CPI), with overall response rates ranging from 3–13%.1–3 Although suppressive myeloid cells have been proposed as a mechanism of resistance to immunotherapy in general, their relationship to response to CPI is unknown.MethodsWe used multiplexed simultaneous single cell RNA sequencing and cell surface proteomics (CITE-seq) to profile circulating immune cells in ABC patients receiving anti-PD-1 at longitudinal timepoints pre-immunotherapy and on treatment, as well as from healthy donors. We also performed single cell RNA sequencing on resected biliary tumors.ResultsWe identified a novel population of circulating cancer-enriched myeloid cells (CEM) characterized by chemokines and extracellular matrix digestion-related gene expression, which were present pre-treatment. Anti-PD-1 treatment drove the CEMs into two diverging states that were associated with response or resistance to treatment. CEM induced in non-responders constituted over 40% of the circulating myeloid cells and expressed immunosuppressive programs, including the upregulation of suppressive cytokines and chemokines. The frequency of these myeloid cells were correlated with the abundance of SOCS3-expressing CD4+ T cells. These SOCS3+CD4+ T cells also colocalized with tumor-infiltrating myeloid cells that share CEM gene expression signatures in the biliary cancer microenvironment. Moreover, CEM can directly induce SOCS3-expressing T cells, which despite their naïve phenotype are functionally unresponsive. Finally, expression signatures of CEM and of SOCS3+CD4+ T cells are associated with worse survival in a larger cohort of ABC patients.ConclusionsThese results demonstrate the capacity of CEM to induce T cell paralysis as an alternate mode of tumor-mediated immunosuppression. A deeper understanding of immune cell biology in ABC provides insights for developing novel therapeutics that can overcome immunotherapy resistance in biliary cancer as well as other tumor types.Trial RegistrationNCT02703714ReferencesUeno M, et al. Nivolumab alone or in combination with cisplatin plus gemcitabine in Japanese patients with unresectable or recurrent biliary tract cancer: a non-randomised, multicentre, open-label, phase 1 study. Lancet Gastroenterol Hepatol 2019;4:611–621.Piha-Paul SA, et al. Efficacy and safety of pembrolizumab for the treatment of advanced biliary cancer: results from the KEYNOTE-158 and KEYNOTE-028 studies. Int J Cancer 2020.Kim RD, et al. A Phase 2 Multi-institutional study of nivolumab for patients with advanced refractory biliary tract cancer. JAMA Oncol 2020;6:888–894.Ethics ApprovalInformed consent was obtained from all patients for participation in the listed trial and for use of blood and tumor samples in research studies.

Background Although gemcitabine plus platinum chemotherapy is the established first-line regimen for advanced biliary cancer (ABC), there is no standard second-line therapy. This study evaluated current practice and outcomes for second-line chemotherapy in patients with ABC across 3 US academic medical centers. Methods Institutional registries were reviewed to identify patients who had received second-line chemotherapy for ABC from April 2010 to March 2015 along with their demographics, diagnoses and staging, treatment histories, and clinical outcomes. Overall survival from the initiation of second-line chemotherapy (OS2) was estimated with Kaplan-Meier methods. Results This study identified 198 patients with cholangiocarcinoma (intrahepatic [61.1%] or extrahepatic [14.1%]) or gallbladder carcinoma (24.8%); 52% received at least 3 lines of systemic chemotherapy. The median OS2 was 11 months (95% confidence interval [CI], 8.8-13.1 months). The median OS2 for patients with intrahepatic cholangiocarcinoma was 13.4 months (95% CI, 10.7-17.8 months), which was longer than that for patients with extrahepatic cholangiocarcinoma (6.8 months; 95% CI, 5-10.6 months) or gallbladder carcinoma (9.4 months; 95% CI, 7.2-12.3 months; P = .018). The median time to second-line treatment failure was 2.2 months (95% CI, 1.8-2.7 months), and it was similar across tumor locations (P = .60). Conclusions In this large cohort of patients with ABC treated across 3 academic medical centers after the failure of first-line chemotherapy, the time to treatment failure on standard therapies was short, although the median OS2 was longer than has been reported previously, and more than half of the patients received additional lines of treatment. This multicenter collaboration represents the largest cohort studied to date of second-line chemotherapy for ABC and provides a contemporary benchmark for future clinical trials.

Background: Patients with advanced biliary cancer (ABC) have low rates of response to immune checkpoint inhibition (CPI) monotherapy. We conducted a phase II clinical trial studying combination immunotherapy with GM-CSF and the anti-PD-1 antibody, pembrolizumab (NCT02703714), with preliminary objective response rate of 19%, higher than the reported response rate of 5.8% for pembrolizumab monotherapy in ABC. Mechanisms of response to CPI in ABC are unknown, and to our knowledge, there have not been studies of the immune response in this patient population at the single cell level. Methods: Cryopreserved serial peripheral blood mononuclear cells (PBMC) samples from n=27 ABC patients treated with pembrolizumab plus GM-CSF were assessed with single cell RNA sequencing combined with CITE-Seq, the staining of cells with >90 oligo-tagged antibodies. This allows for the simultaneous identification of protein and RNA expression on an individual cell basis. We used high-throughput multiplexing in which PBMC from different patients could be deconvolved from other mixed samples using individual specific SNPs (“demuxlet”). >100 PBMC samples, including pre-treatment and 1 week, 3 weeks, and 6 weeks post-treatment, were studied. Nine of 27 patients were characterized as responders, while 18 were non-responders. We also performed CITE-Seq on PBMC from age/gender-matched healthy controls (n=9). Results: Unbiased clustering revealed all major immune populations were present in PBMC from both ABC and controls, though the immune compositions differed, most strikingly within the myeloid compartment. A distinct population of CXCL5-expressing “cancer-associated myeloid cells”, distinct from classical and non-classical monocytes and dendritic cells, was found only within cancer patients. We also analyzed the effects of treatment on immune sub-populations within responders and non-responders and found that following anti-PD-1 monotherapy, there was an increased frequency and absolute number of non-classical monocytes in responders. Conversely, the addition of GM-CSF to anti-PD-1 therapy decreased the frequency of circulating non-classical monocytes, though there was a robust induction of pro-inflammatory genes following treatment with GM-CSF in responders. Regardless of responder status, GM-CSF also dramatically induced monocyte-derived dendritic cells within 1 week of administration. Conclusions: The compositions of the myeloid compartment differed between ABC patients and healthy individuals, with a circulating cancer-associated myeloid population identified in ABC patients. Circulating non-classical monocytes increased following anti-PD-1 monotherapy in all patients, but declined after combination with GM-CSF in responders, suggesting that anti-PD-1-induced myeloid populations may be associated with response to anti-PD1. Pre-existing and induced myeloid cell states may represent a mechanism of response to anti-PD-1 and GM-CSF. Citation Format: Bridget P. Keenan, Elizabeth E. McCarthy, Arielle Ilano, David S. Lee, Yang Sun, Brenna Sheldon, Alexander Cheung, Chun Jimmie Ye, Robin Kate Kelley, Lawrence Fong. Identification of circulating myeloid cells induced in advanced biliary cancer patients responding to anti-PD1 through combined single cell RNA sequencing and protein expression analysis [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 5512.

Background: Advanced biliary cancers (ABC) including cholangiocarcinoma and gallbladder adenocarcinoma are rising in incidence with limited standard treatment options. While checkpoint inhibition (CPI) achieves durable tumor responses in subsets of patients across many malignancies, less than 10% of ABC patients respond to single agent PD-1-targeted therapy. Combination immunotherapy aims to overcome pre-existing and adaptive resistance to immunotherapy. GM-CSF is a cytokine that activates and matures antigen-presenting cells, suggesting the potential to enhance immune responses. The exact mechanisms of action of this cytokine have not been defined in cancer patients. The combination of GM-CSF and anti-CTLA-4 CPI demonstrated safety along with inducing clinical responses in melanoma and prostate cancer. We conducted a phase II trial of the novel combination of GM-CSF and pembrolizumab (Pembro) in patients with ABC which has resulted in durable clinical responses in greater proportion than previously reported with anti-PD-1 monotherapy. Methods: We assessed peripheral blood mononuclear cells (PBMC) from patients on Pembro monotherapy and combined Pembro/GM-CSF by mass cytometry (CyTOF) and T cell receptor (TCR) sequencing. We explored for the differences between clinical responders versus non-responders. Results: We find that the addition of GM-CSF to Pembro leads to a higher frequency of myeloid cells overall; however, certain sub-populations of classical monocytes and conventional dendritic cells decreased in peripheral blood following upfront Pembro followed by GM-CSF. GM-CSF did not seem to change the phenotypes or relative frequencies of circulating T cell subsets. We also find that clinical responders possess specific circulating populations of classical monocyte and conventional dendritic cells prior to treatment. Responders also had higher percentages of CD8+ T cells expressing CD39 following Pembro treatment compared to non-responders. Combination therapy with Pembro/GM-CSF led to different effects on the T cell repertoire compared to Pembro alone. Conclusions: The addition of GM-CSF to Pembro leads to dynamic shifts in myeloid cell subsets in the peripheral blood of ABC patients treated with immunotherapy, whereas Pembro alone led to changes in the activation states of T cell subsets. The TCR repertoire shifts reflect distinct mechanisms of action for Pembro monotherapy versus Pembro/GM-CSF. Future studies will explore the mechanisms driving the increased response rate seen with combination immunotherapy in comparison to Pembro alone in ABC patients, both through the study of peripheral immune responses as well as via immune-profiling of tumors from sequential biopsies of patients on Pembro monotherapy versus combined Pembro/GM-CSF. Citation Format: Bridget P. Keenan, Whitney Tamaki, Eric Liu, Brandon Chen, Alexander Cheung, John D. Gordan, Brenna Sheldon, Li Zhang, Robin K. Kelley, Lawrence Fong. Single cell immune profiling of patients with advanced biliary cancers treated with combination checkpoint inhibition and GM-CSF reveals diverse T cell and myeloid cell mechanisms of action [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 4063.

TPS464 Background: The antiangiogenic tyrosine kinase inhibitor (TKI) SOR prolongs survival in advanced HCC, but responses occur in fewer than 5% of patients (pts) and median progression-free survival (PFS) is less than 6 months. Immune checkpoint inhibition (CPI) with the PD-1 inhibitor, NIVO, achieved an overall response rate (ORR) of approximately 20% in sorafenib-naïve pts enrolled on CheckMate 040 trial though most pts had progression. In preclinical studies, TKI can inhibit regulatory T cells and myeloid derived suppressor cells, immune cell subsets which may contribute to CPI resistance. CD8+ T cells in sorafenib-resistant tumors are characterized by PD-1 expression, providing rationale for a combined approach. The combination of TKI or the anti-angiogenic bevacizumab with CPI improves anti-tumor activity in HCC mouse models and in preliminary clinical studies. This study will examine the safety, maximum tolerated dose (MTD), and ORR of the combination of SOR plus NIVO in advanced HCC pts, along with correlative analyses of tumor and circulating immune cells. Methods: Eligible pts must have Child-Pugh A liver function and advanced HCC, without prior systemic therapy and measurable by RECIST 1.1. In Part 1 (3+3 dose escalation), SOR dose will be 400 mg QD or BID plus NIVO 240 mg IV Q2 weeks. In Part 2, Arm 1, pts will start NIVO Cycle 1, Day 1 (C1D1), with addition of SOR at MTD on C1D15; in Part 2, Arm 2, SOR is given at MTD on C1D1 with addition of NIVO on C1D15. Primary endpoints are MTD of SOR (Part 1) and ORR by RECIST 1.1 with H0 7.5% vs. H1 25% (Part 2). For expected sample size of 24 evaluable pts in Part 2, the power is 83% with 1-sided alpha 5% to determine ORR >25% by Chi-square tests. Secondary endpoints are safety, duration of response, PFS, and overall survival. Exploratory endpoints include peripheral and tumor immune cell profiling, PD-L1 expression, and alpha-fetoprotein (AFP) response. An interim safety analysis will be performed after 50% enrollment in Part 2. Optional paired biopsies in Part 2 will allow for investigation of the tumor microenvironment on SOR, NIVO, and combination. Clinical trial information: NCT03439891.

The fourth AACR Special Conference “Tumor Immunology: Basic and Clinical Advances” was held in Miami, FL in December 2012. The overall objective of this meeting was to discuss emerging concepts in cancer immunology and immunotherapy. The key findings that emerged from this meeting included: (i) multiple immune checkpoints should be inhibited to increase effective T-cell therapy, (ii) successful adoptive T-cell therapy will rely on obtaining the proper T-cell phenotype, (iii) chimeric antigen receptors have shown promise in treating some B-cell malignancies, and (iv) multiple pathways of inflammation within the tumor microenvironment are immunotherapy targets. Cancer Immunol Res; 1(1); 16–23. ©2013 AACR.

Recent advances in immunotherapy have demonstrated that single agent vaccines can be effective when given as primary prevention before exposure to the causative agent, and partially effective in some patients with existing cancer. However, as tumors develop and progress, tumor-induced immune suppression and tolerance present the greatest barrier to therapeutic success. Preneoplastic disease represents an important opportunity to intervene with tumor antigen–targeted vaccines before these mechanisms of immune evasion outpace efforts by the immune system to destroy precancerous cells. However, as we discuss in this review, emerging evidence suggests that procarcinogenic inflammatory changes occur early in cancer development, in both patients and mouse models of cancer progression. Defining early inhibitory signals within tumor microenvironments will yield insights that can eventually be used in the clinic to target these events and deliver treatments that can be used in addition to cancer vaccines to prevent premalignant and early invasive cancers.

4087Background: The efficacy of immune checkpoint inhibition (CPI) has not been established in ABC. The combination of CPI plus the myeloid cytokine GM-CSF was safe with prolonged overall survival ...

386 Background: The efficacy of immune checkpoint inhibition (CPI) has not been established in ABC. GM-CSF modulates immune effector cells and has demonstrated safety and improved survival (OS) in combination with ipilimumab in melanoma. This phase 2 trial aims to evaluate the efficacy and safety of PEM in combination with GM-CSF in ABC. Methods: Design: Simon’s 2-stage. Key eligibility: ABC with progression/intolerance on ≥ 1 standard therapy, no prior CPI, bilirubin ≤1.5xULN. Treatment: PEM 200 mg IV Q21 days plus 2 cycles of GM-CSF 250 µg SC D1-14 Q21 days in cycles 2 and 3 (Stage 1 Safety Cohort) or in cycles 1 and 2 (Stage 2). Endpoints: 1◦: Progression-free survival at 6 months (PFS6) with H0 25% vs. H1 50%. Key 2◦: Safety, overall response rate (ORR) and duration (DOR), OS, PD-L1 expression. Exploratory: PBMC and tumor immune cell profiles, tumor genotype, microsatellite (in)stability (MSI or MSS). Results: Accrual has completed with 27 patients (pts) enrolled 5/2016-6/2017: F/M 13/14; median age 61 (range 37-77); intrahepatic 19 (70%), extrahepatic 7 (26%), mixed 1 (4%) cholangiocarcinoma; stage IVA/B 85%, II/III 15%; median prior therapies 2 (range 1-6). Adverse events (AE): Related grade(Gr) ≥3 AE occurred in 4/27 (15%) pts including immune-related (ir)AE of Gr4 diabetes mellitus and Gr3 thrombocytopenia in 1 pt each. Gr≤2 irAE in ≥5% were: arthralgia (33%), dry eye/mouth (15%), hyperthyroid/thyroiditis (15%), hypothyroid (15%), neuropathy (11%), rash (11%), and adrenal insufficiency (7%). Steroids were required in 3/27 (11%) pts. Disposition: 19 pts removed for PD, 1 for Gr2 irAE; 7 pts remain active on treatment. Median time on treatment: 6 cycles (range 2-22+). Best response by RECIST 1.1: Partial response (PR) in 5/24 (21%) evaluable pts (1 MSI, 4 MSS); minor regression and ≥50% CA 19-9 decline in 2 additional MSS pts for 11+ and 16+ months. PBMC analyses show changes in expression of activating and inhibitory markers including PD-1 on various immune cell populations. Conclusions: PEM plus induction GM-CSF is safe and tolerable in ABC. Durable radiographic and tumor marker responses including MSS pts warrant further study. PFS6, OS, and correlative analyses are ongoing. Clinical trial information: NCT02703714.

In mammals, rapid mRNA turnover directed by AU-rich elements (AREs) is mediated by selective association of cellular ARE-binding proteins. These trans-acting factors display overlapping RNA substrate specificities and may act to either stabilize or destabilize targeted transcripts; however, the mechanistic features of AREs that promote preferential binding of one trans-factor over another are not well understood. Here, we describe a hairpin-like structure adopted by the ARE from tumor necrosis factor alpha (TNFalpha) mRNA that modulates its affinity for selected ARE-binding proteins. In particular, association of the mRNA-destabilizing factor p37(AUF1) was strongly inhibited by adoption of the higher order ARE structure, whereas binding of the inducible heat shock protein Hsp70 was less severely compromised. By contrast, association of the mRNA-stabilizing protein HuR was only minimally affected by changes in ARE folding. Consistent with the inverse relationship between p37(AUF1) binding affinity and the stability of ARE folding, mutations that stabilized the ARE hairpin also inhibited its ability to direct rapid mRNA turnover in transfected cells. Finally, phylogenetic analyses and structural modeling indicate that TNFalpha mRNA sequences flanking the ARE are highly conserved and may stabilize the hairpin fold in vivo. Taken together, these data suggest that local higher order structures involving AREs may function as potent regulators of mRNA turnover in mammalian cells by modulating trans-factor binding selectivity.

Background & Aims Premalignant lesions and early stage tumors contain immunosuppressive microenvironments that create barriers for cancer vaccines. Kras G12D/+ ;Trp53 R172H/+ ;Pdx-1-Cre (KPC) mice, which express an activated form of Kras in pancreatic tissues, develop pancreatic intraepithelial neoplasms (PanIN) that progress to pancreatic ductal adenocarcinoma (PDA). We used these mice to study immune suppression in PDA. Methods We immunized KPC and Kras G12D/+ ; Pdx-1-Cre mice with attenuated intracellular Listeria monocytogenes (which induces CD4 + and CD8 + T-cell immunity) engineered to express Kras G12D (LM-Kras). The vaccine was given alone or in sequence with an anti-CD25 antibody (PC61) and cyclophosphamide to deplete T-regulatory (Treg) cells. Survival times were measured; pancreatic and spleen tissues were collected and analyzed by histologic, flow cytometry, and immunohistochemical analyses. Results Interferon γ–mediated, CD8 + T-cell responses were observed in KPC and Kras G12D/+ ; Pdx-1-Cre mice given LM-Kras, but not in unvaccinated mice. Administration of LM-Kras to KPC mice 4–6 weeks old (with early stage PanINs), depleted of Treg cells, significantly prolonged survival and reduced PanIN progression (median survival, 265 days), compared with unvaccinated mice (median survival, 150 days; P = .002), mice given only LM-Kras (median survival, 150 days; P = .050), and unvaccinated mice depleted of Treg cells (median survival, 170 days; P = .048). In 8- to 12-week-old mice (with late-stage PanINs), LM-Kras, alone or in combination with Treg cell depletion, did not increase survival time or slow PanIN progression. The combination of LM-Kras and Treg cell depletion reduced numbers of Foxp3 + CD4 + T cells in pancreatic lymph nodes, increased numbers of CD4 + T cells that secrete interleukin 17 and interferon γ, and caused CD11b + Gr1 + cells in the pancreas to acquire an immunostimulatory phenotype. Conclusions Immunization of KPC mice with Listeria monocytogenes engineered to express Kras G12D , along with depletion of Treg cells, reduces progression of early stage, but not late-stage, PanINs. This approach increases infiltration of the lesion with inflammatory cells. It might be possible to design immunotherapies against premalignant pancreatic lesions to slow or prevent progression to PDA.

Cancer vaccines have shown success in curing tumors in pre-clinical models. Accumulating evidence also supports their ability to induce immune responses in patients. In many cases, these responses correlate with improved clinical outcomes. However, cancer vaccines have not yet demonstrated their true potential in clinical trials. This is likely due to the difficulty in mounting a significant antitumor response in patients with advanced disease because of preexisting tolerance mechanisms that are actively turning off immune recognition in cancer patients. This review will examine the recent progress being made in the design and implementation of whole cell cancer vaccines, one vaccine approach that simultaneously targets multiple tumor antigens to activate the immune response. These vaccines have been shown to induce antigen specific T cell responses. Pre-clinical studies evaluating these vaccines given in sequence with other agents and cancer treatment modalities support the use of immunomodulating doses of chemotherapy and radiation, as well as immune modulating pathway targeted monoclonal antibodies, to enhance the efficacy of cancer vaccines. Based on emerging pre-clinical data, clinical trials are currently exploring the use of combinatorial immune based therapies for the treatment of cancer.

Bevacizumab, a humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), is used in the treatment of several cancers, macular degeneration, and proliferative diabetic retinopathy as a means of limiting angiogenesis, a hallmark feature of these diseases. Nonspecific skin eruption has been reported in some patients receiving bevacizumab, and recently, bevacizumab-associated acneiform eruption was reported in the treatment of colon cancer (systemic administration) and macular degeneration (intravitreal administration).1-3 The eruption in these patients was strikingly similar to that characterized in patients treated with epidermal growth factor receptor (EGFR) inhibitors.1-4 We report a case of bevacizumab-induced acneiform eruption in a patient with glioblastoma multiforme.

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR., link_to_OA_fulltext

Human pancreatic ductal adenocarcinoma (PDA) remains a fatal disease. Biologically, it is naturally resistant to therapy and characterized by an intense desmoplastic reaction to the tumor. Genetically-engineered mouse models of spontaneously developing PDA tumors mimic the multistep progression from pre-cancer to invasive cancer in humans, and are ideal for studying the tumor microenvironment. We investigated the cellular and molecular signals underlying pancreatic intra-epithelial neoplasm (PanIN) progression, and examined the changes in these signals, PanIN progression, and survival following immunotherapy. The KrasG12D/+; Trp53R172H/+; Pdx-1-Cre mouse model of pancreatic ductal adenocarcinoma combines a Kras dominant active mutation with a p53 loss of function mutation, specifically expressed in the pancreas under the control of the Pdx-1 promoter. Mice begin developing PanINs at several weeks old, progressing to PDA and accompanied by the characteristic desmoplastic reaction seen in human PDA. We evaluated the effects of an attenuated Listeria monocytogenes expressing mutated Kras (LM-Kras) vaccine capable of inducing a Kras-specific T cell responses together with regulatory T cell (Treg)-depleting therapy (Cyclophosphamide and anti-CD25 monoclonal antibody). Combination therapy given to mice with early stage PanINs increases survival and delays PanIN progression, whereas intervention in older mice or with single agent immunotherapy fails to alter the course of tumor progression. Combination treatment administered during early PanINs correlated with enhanced CD8+ T cell infiltration and activation. In addition, combination treatment induced a Th1/Th17 cytokine expression pattern in CD4+ T cells and decreased infiltration by CD4+Foxp3+ Tregs. Local, rather than systemic, changes in T cell activation correlated with delay in PanIN progression. Immunohistochemistry revealed granulocytes, myeloid cells, and macrophages infiltrating untreated PanINs and PDA. Analysis of upregulated genes in laser capture micro-dissected specimens comparing advanced PanIN stages and invasive PDA included those associated with recruitment of MDSCs, induction of a suppressive phenotype in these cells, and mediators secreted by cells with a M2 or N2 phenotype. We are further investigating the effect of combination immunotherapy on the granulocyte and myeloid populations with the hypothesis that these signals will be altered with treatment. Thus, vaccination targeting an early genetic event in PDA can slow progression from early PanINs to invasive cancer when Tregs are also altered. Additional studies are ongoing to determine whether PanIN progression can be delayed when vaccination is administered at a time of advanced PanIN development if additional suppressive populations of immune cells within the tumor microenvironment are also inhibited. This abstract is also presented as Poster A20. Citation Format: Bridget Keenan, Yvonne Saenger, Michel Kafrouni, Ashley Leubner, Peter Lauer, Anirban Maitra, Andrew Gunderson, Lisa Coussens, Todd Armstrong, Elizabeth Jaffee. Prevention of pancreatic intra-epithelial neoplasm progression by a Listeria monocytogenes vaccine targeting mutated Kras, an early genetic event in pancreatic tumor development. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR6.