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Background: Oncogenic alterations (gene amplification, mutation, and fusion/rearrangements) of fibroblast growth factor receptor 2 (FGFR2) are rare and occur at varying frequencies across solid tumor types and have become critical therapeutic targets in drug development. First generation FGFR pan-kinase inhibitors non-selectively inhibit FGFR1-4 and are associated with dose limiting toxicities and narrow therapeutic windows. Off-isoform toxicity (FGFR1-hyperphosphatemia; FGFR4-diarrhea) and on-target acquired resistance have led to limited efficacy, which is primarily seen in FGFR2-fusion+ intrahepatic cholangiocarcinoma. Therapies that selectively target FGFR2 remain an unmet need in advanced breast cancer as well as other solid tumor types. RLY-4008 is a novel, oral FGFR2 inhibitor designed to overcome the limitations of pan-FGFR inhibitors (FGFRi) by potently and selectively targeting primary oncogenic FGFR2 alterations and acquired resistance mutations. RLY-4008 is > 200-fold selective over FGFR1, > 80- and > 5000-fold selective over FGFR3 and FGFR4 respectively. Here we describe a phase 1/2 study to investigate the safety and antitumor activity in advanced FGFR2 altered cancers, including breast cancer. Methods: ReFocus is a phase 1/2 open label global study evaluating the safety and efficacy of RLY-4008 (NCT04526106) in adult patients with advanced unresectable and/or metastatic cancers harboring an FGFR2 alteration. Key eligibility criteria include: documented FGFR2 alteration in blood or tissue per local assessment, ECOG performance status of 0-1, disease that is refractory or not adequately responding to standard therapy, has no available standard therapy, or patient is intolerant of, or declined standard therapy (including pan-FGFRi), and measurable or evaluable disease per RECIST 1.1. FGFR2 alteration will be confirmed retrospectively by central laboratory assessment. Part 1 dose escalation employed the Bayesian Optimal Interval (BOIN) design to determine the MTD/RP2D of RLY-4008. Part 2 dose expansion is presently enrolling patients at the RP2D of RLY-4008 and includes 5 cohorts comprised of patients with: 1. FGFR2 fusion+ cholangiocarcinoma previously treated with an FGFRi; 2. FGFR2 fusion+ cholangiocarcinoma not previously treated with an FGFRi; 3. FGFR2 fusion+ solid tumors; 4. FGFR2 mutation+ solid tumors and 5. FGFR2 amplified solid tumors. Solid tumors in cohorts 3, 4 and 5 will have a focus in breast cancer. The primary endpoint is objective response rate (ORR); key secondary endpoints include: duration of response, safety and tolerability, correlation of FGFR2 genotype by central tissue assessment with antitumor response, characterization of PK profile, and quality of life. US enrollment began September 2020 and has expanded into Europe and Asia. Clinical trial information: NCT04526106. Citation Format: Suneel Kamath, David Tai, Irene Moreno, Hani Babiker, Zhaohui Jin, Changhoon Yoo, Fabien Ricard, Kai Yu Jen, Jim Coward, Jia Liu, Frans Opdam, Michael Millward, Mariano Ponz-Sarvise, Jeffrey Yachnin, Richard Kim, Joon Oh Park, Vivek Subbiah, Alison M. Schram. ReFocus: A Phase 1/2 Study of the Highly Selective FGFR2 Inhibitor, RLY-4008, in Patients with Advanced Solid Tumors Including Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-24-01.

Experimental treatments (A) and tumor size follow-up until sacrifice (B) of tumors used to prepare cell suspensions for figures 3, 5 and supplementary figure 4. (C) Shows individual tumor lesion weights used to calculate lymphocytes/gram densities.

FACS analyses on cell suspensions (n=4) derived from non-irradiated MC38-derived tumors

Myeloid cell content (density per gram) in cell suspension from non-irradiated tumors as those in figure 5. Macrophages are defined as CD45+ CD11b+ F4/80+; Dendritic cells (DC) as CD45+CD11b+CD11c+; myeloid-derived suppressor cells (MDSC) as CD45+CD11b+Ly6C+ or CD45+CD11b+Ly6G+ to estimate monocytic (M-MDSC) and granulocytic (G-MDSC) subtypes.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti–PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGFβ expression or activation increases in irradiated tissues, we tested whether TGFβ blockade may further enhance abscopal effects in conjunction with the anti–PD-1 plus anti-CD137 mAb combination. Indeed, TGFβ blockade with 1D11, a TGFβ-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGFβ in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGFβ hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGFβ blockade in combination with radioimmunotherapy results in greater efficacy.

Assessment of spontaneous lung metastases of 4T1 tumors in the indicated treatment groups corresponding to mice treated as in figure 2A. Metastases were detected and quantitated -number of lesions in the surface of the lung counted upon inspection under a magnification lens (left) and by real time RT-PCR for tumor specific gp70 RNA (right)-.

Suppl. Figure 3. A. Immunoblot of LAMC2 upon Lamc2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines. B. Quantification of organoid number upon LAMC2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines.C. Quantification of organoid area (AU) in same cell lines as in B. Error bars correspond to s.d. D. Immunoblot of LAMC2 upon LAMC2 knockdown in HPAFII cell line. E. Quantification of organoid number and organoid area (AU) in HPAFII cell line expressing a TET-inducible shRNA against LAMC2 (bottom) or a control shRNA (top) cultured in 3D. F. Representative images of the subcutaneous tumors from PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. G. Representative images of the subcutaneous tumors from HPAFII cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. H and I. Representative IHC of LAMC2 expression in subcutaneous tumors from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice J. Representative images of the tumors derived from intrapancreatic injection of PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. K. Representative images of the subcutaneous tumors from PM12167 mouse cells expressing a shRNA against Lamc2 or a control shRNA at sacrifice. L and M. Representative images of the subcutaneous (sc) tumors from PATU8902 and HPAFII expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. ShRNA expression induced after tumor establishment at Day 8. N and O. Representative IHC of LAMC2 expression in subcutaneous tumors (Tx) from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA, shRNA expression was induced at day 8 of tumor establishment. P. Quantification of Ki67 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx. Q. Quantification of cleaved caspase3 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx.

This file includes the Suppl. Methods, Table (1) and References

Purpose:The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.Experimental Design:LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.Results:LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.Conclusions:LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Supplementary Table 4

An Id1 gene signature is dependent of KEAP1-related genes and a FOSL1 network.

Clinical implication of Id1 expression in LUAD patients.

Supplementary tables and figure's headings

Supplementary Table 1

Purpose:KRAS is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells.Experimental Design:We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities.Results:Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies.Conclusions:Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both in vitro and in vivo.

Purpose:Pancreatic ductal adenocarcinoma (PDAC) is largely unresponsive to checkpoint inhibitors. Blockade of the CXCR4/CXCL12 axis increases intratumoral trafficking of activated T cells while restraining immunosuppressive elements. This study evaluates dual blockade of CXCR4 and PD1 with chemotherapy in PDAC.Patients and Methods:Multicenter, single-arm, phase II study to evaluate the safety and efficacy of motixafortide and pembrolizumab combined with chemotherapy in patients with de novo metastatic PDAC and disease progression on front-line gemcitabine-based therapy (NCT02826486). Subjects received a priming phase of motixafortide daily on days 1–5, followed by repeated cycles of motixafortide twice a week; pembrolizumab every 3 weeks; and nanoliposomal irinotecan, fluorouracil, and leucovorin every 2 weeks (NAPOLI-1 regimen). The primary objective was objective response rate (ORR). Secondary objectives included overall survival (OS), progression-free survival (PFS), disease control rate (DCR), safety, and tolerability.Results:A total of 43 patients were enrolled. The ORR according to RECISTv1.1 was 21.1% with confirmed ORR of 13.2%. The DCR was 63.2% with median duration of clinical benefit of 5.7 months. In the intention-to-treat population, median PFS was 3.8 months and median OS was 6.6 months. The triple combination was safe and well tolerated, with toxicity comparable with the NAPOLI-1 regimen. Notably, the incidence of grade 3 or higher neutropenia and infection was 7%, lower than expected for this chemotherapy regimen.Conclusions:Triple combination of motixafortide, pembrolizumab, and chemotherapy was safe and well tolerated, and showed signs of efficacy in a population with poor prognosis and aggressive disease.

Supplementary Legends

Supplementary Figure 2

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2–M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD.Significance:These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

A shRNA-resistant Id1 cDNA rescues Id1 loss.

Supplementary Figure 1

Supplementary Figures 1-3

Supplementary Table 2

Regulation of kinases by Id1 expression

Supplementary Table 3

Supplementary Figure 3

Purpose:The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.Experimental Design:LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.Results:LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.Conclusions:LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Liver cancer including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) is the third leading cause of cancer-related deaths worldwide. HCC arises from hepatocyte or hepatic stem cells, while iCCA originates from biliary epithelial cells, and the respective biological context are very different. Despite screening programs, the diagnosis of liver cancer is in most cases made when curative treatments such as surgery or ablation are not possible. In 2020, after a decade of using only tyrosine kinase inhibitors (TKI), a combination of an immune-check point inhibitor (ICI) and a VEGF antagonist proved superior to a TKI as first line therapy of advanced HCC. In 2022, the addition of an ICI to standard chemotherapy demonstrated an improvement of patient survival in iCCA. Moreover, ICI offer an unprecedented rate of durable responses to HCC and iCCA patients. Nevertheless, still two thirds of patients do not respond to ICI-based combinations, and research efforts are focused on deciphering the mechanisms of immune evasion of these lethal cancers. Reliable predictive and prognostic biomarkers are still lacking, but the molecular phenotyping of the tumor microenvironment is currently providing potential candidates for patient stratification. In this review, we will summarize the current knowledge on the immune biology of the liver, the discovery of cell-intrinsic and immune cell-mediated mechanisms of immune evasion by means of high-resolution single cell data, the main targets of current immunotherapy approaches, and the recent milestones in immunotherapy of HCC and iCCA.

Checkpoint inhibitors are being added to standard-of-care chemotherapy in multiple clinical trials. Success has been reported in non–small and small cell lung carcinomas and urothelial, head and neck, gastric, and esophageal cancers, and promising results are already available in triple-negative breast and pancreatic malignancies. The potential mechanisms of synergy include immunogenic tumor cell death, antiangiogenesis, selective depletion of myeloid immunosuppressive cells, and lymphopenia, which reduces regulatory T cells and makes room for proliferation of effector T cells. However, chemotherapy regimens have not been optimized for such combinations, perhaps explaining some recent clinical trial disappointments. Approaches to make the most of chemoimmunotherapy include neoadjuvant and adjuvant schemes.Significance: Immunotherapy of cancer based on PD-1/PD-L1 blockade has prompted a revolution in cancer clinical management. Evidence in phase III clinical trials already supports combinations of immunotherapy with standard-of-care chemotherapy for a number of malignant diseases. This review focuses on such evidence and provides an overview of the potential synergistic mechanisms of action and the opportunities to optimize chemoimmunotherapy regimens.

Background Adoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation. Methods PD-1− and PD-1+ CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation. Results Tumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1+ fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137+ cells within the PD-1+CD8+ TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products. Conclusion We have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1+ TILs.

Programmed cell death 1 (PD-1) inhibitors have limited effect in pancreatic ductal adenocarcinoma (PDAC), underscoring the need to co-target alternative pathways. CXC chemokine receptor 4 (CXCR4) blockade promotes T cell tumor infiltration and is synergistic with anti-PD-1 therapy in PDAC mouse models. We conducted a phase IIa, open-label, two-cohort study to assess the safety, efficacy and immunobiological effects of the CXCR4 antagonist BL-8040 (motixafortide) with pembrolizumab and chemotherapy in metastatic PDAC (NCT02826486). The primary outcome was objective response rate (ORR). Secondary outcomes were overall survival (OS), disease control rate (DCR) and safety. In cohort 1, 37 patients with chemotherapy-resistant disease received BL-8040 and pembrolizumab. The DCR was 34.5% in the evaluable population (modified intention to treat, mITT; N = 29), including nine patients (31%) with stable disease and one patient (3.4%) with partial response. Median OS (mOS) was 3.3 months in the ITT population. Notably, in patients receiving study drugs as second-line therapy, the mOS was 7.5 months. BL-8040 increased CD8+ effector T cell tumor infiltration, decreased myeloid-derived suppressor cells (MDSCs) and further decreased circulating regulatory T cells. In cohort 2, 22 patients received BL-8040 and pembrolizumab with chemotherapy, with an ORR, DCR and median duration of response of 32%, 77% and 7.8 months, respectively. These data suggest that combined CXCR4 and PD-1 blockade may expand the benefit of chemotherapy in PDAC and warrants confirmation in subsequent randomized trials. Results from the phase IIa COMBAT trial combining CXCR4 and PD-1 inhibition in patients with metastatic cancer show encouraging clinical responses in association with enhanced antitumor immune activation.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

Background & Aims: Cholangiocarcinoma (CCA) is a rare and heterogeneous biliary cancer, whose incidence and related mortality is increasing. This study investigates the clinical course of CCA and subtypes (intrahepatic [iCCA], perihilar [pCCA], and distal [dCCA]) in a pan-European cohort. Methods: The ENSCCA Registry is a multicenter observational study. Patients were included if they had a histologically proven diagnosis of CCA between 2010-2019. Demographic, histomorphological, biochemical, and clinical studies were performed. Results: Overall, 2,234 patients were enrolled (male/female=1.29). iCCA (n = 1,243) was associated with overweight/ obesity and chronic liver diseases involving cirrhosis and/or viral hepatitis; pCCA (n = 592) with primary sclerosing cholangitis; and dCCA (n = 399) with choledocholithiasis. At diagnosis, 42.2% of patients had local disease, 29.4% locally advanced disease (LAD), and 28.4% metastatic disease (MD). Serum CEA and CA199 showed low diagnostic sensitivity, but their concomitant elevation was associated with increased risk of presenting with LAD (odds ratio 2.16; 95% CI 1.43-3.27) or MD (odds ratio 5.88; 95% CI 3.69-9.25). Patients undergoing resection (50.3%) had the best outcomes, particularly with negative-resection margin (R0) (median overall survival [mOS] = 45.1 months); however, margin involvement (R1) (hazard ratio 1.92; 95% CI 1.53-2.41; mOS = 24.7 months) and lymph node invasion (hazard ratio 2.13; 95% CI 1.55-2.94; mOS = 23.3 months) compromised prognosis. Among patients with unresectable disease (49.6%), the mOS was 10.6 months for those receiving active palliative therapies, mostly chemotherapy (26.2%), and 4.0 months for those receiving best supportive care (20.6%). iCCAs were associated with worse outcomes than p/dCCAs. ECOG performance status, MD and CA19-9 were independent prognostic factors. Conclusion: CCA is frequently diagnosed at an advanced stage, a proportion of patients fail to receive cancer-specific therapies, and prognosis remains dismal. Identification of preventable risk factors and implementation of surveillance in high-risk populations are required to decrease cancer-related mortality. Lay summary: This is, to date, the largest international (pan-European: 26 hospitals and 11 countries) observational study, in which the course of cholangiocarcinoma has been investigated, comparing the 3 subtypes based on the latest International Classification of Diseases 11th Edition (ICD-11) (i.e., intrahepatic [2C12], perihilar [2C18], or distal [2C15] affected bile ducts), which come into effect in 2022. General and tumor-type specific features at diagnosis, risk factors, biomarker accuracy, as well as patient management and outcomes, are presented and compared, outlining the current clinical state of cholangiocarcinoma in Europe. The ENSCCA Registry is competitively funded by the European Association for the Study of the Liver (EASL; Registry grant awards 2016 and 2019) and Incyte Pharma (grant award 2020 to JMB). JMB also received grants from Spanish Carlos III Health Institute (ISCIII) [FIS PI18/01075,PI21/00922 and Miguel Servet Program CPII19/00008), CIBERehd (ISCIII), Department of Health of the Basque Country (2017111010), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD), "Fundacion Cientifica de la Asociacion Espanola Contra el Cancer" (AECC Scientific Foundation) and the European Union's Horizon 2020 Research and Innovation Program [grant number 825510, ESCALON: to JMB, AL, AV and JWV]. AL also received funding from The Christie Charity. AF received grant support from ISCIII (PI18/00542). CB is recipient of the Lord Kelvin Adam Smith Leadership fellowship at University of Glasgow. ALl was funded by "Fondazione AIRC per la Ricerca sul Cancro" (IG2019 project number 23408). RIRM received grant support from ISCIII (PI20/00189), co-funded ERDF/ESF, "A way to make Europe"/"Investing in your future" and "Centro Internacional sobre el Envejecimiento" (0348_CIE_6_E), Spain. JJGM received grant support from ISCIII (PI19/00819), CIBERehd (ISCIII), "Fundacio la Marato de TV3" (201916-31) and Junta de Castilla y Leon (SA074P20), Spain. NBJ received grant support from Cancer Research UK (grant number A25813), and is a recipient of the Lord Kelvin Adam Smith Leadership fellowship at the University of Glasgow.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti–PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGFβ expression or activation increases in irradiated tissues, we tested whether TGFβ blockade may further enhance abscopal effects in conjunction with the anti–PD-1 plus anti-CD137 mAb combination. Indeed, TGFβ blockade with 1D11, a TGFβ-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGFβ in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGFβ hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGFβ blockade in combination with radioimmunotherapy results in greater efficacy.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2–M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. Significance: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

Background: AAC-HPV consists of autologous RBCs engineered using Cell Squeeze® technology to deliver synthetic long peptides (SLP) of HPV16 E6, E7 and the adjuvant, poly I:C. After intravenous (IV) administration, the AACs are designed to be phagocytosed by endogenous antigen presenting cells (APCs) and the E6 and E7 antigens presented on the APC’s major histocompatibility complexes (MHCs). The concurrent delivery of antigen and adjuvant to the APCs is designed to elicit a potent antitumor T cell response to drive HPV16+ tumor killing. Preclinical experiments demonstrated that post IV administration of mouse AAC-HPV in a HPV16 E7-expressing TC-1 mouse model, E7 specific CD8+ T cells are recruited to the tumor, inhibiting tumor growth, prolonging survival, and forming immunological memory. Murine AAC-HPV treatment was also shown to be synergistic with cisplatin combinations. In vitro studies with human volunteer derived SQZ-AAC-HPV demonstrated to be endocytosed by dendritic cells and drive IFN-gamma production by T cell clones specific to human E7. The Phase 1/2 study’s biomarker program investigates whether pharmacodynamic effects observed in nonclinical studies correlate with potential clinical benefit. Immunogenic and pharmacodynamic endpoints include quantification and characterization of TILs and tumor microenvironment, changes in numbers and functions of circulating blood cells. In addition, cytokine responses and cell-free HPV16 DNA will be measured. Methods: ENVOY-001 (NCT04892043) is open for enrollment to HLA A*02+ patients with HPV16+ recurrent, locally advanced, or metastatic solid tumors and includes escalation cohorts for monotherapy and in combination with anti-PD-1 and anti-CTLA-4 checkpoint inhibitors. The study is divided in two parts, the first will assess the safety in monotherapy dose-escalation following a Bayesian Optimal Interval approach. The second part of the study will assess the safety of SQZ-AAC-HPV when combined with nivolumab 360 mg q3w and/or ipilimumab 3 mg/kg q3w x4 or 1 mg/kg q6w when combined with nivolumab. The cycle length is 3 weeks, and patients will receive SQZ-AAC-HPV for up to 1 year or until available autologous drug product is exhausted. Eligible patients including but not limited to anal, cervical and head and neck tumors will undergo a 200 mL whole blood collection at the study site. The manufacturing process takes less than 24 hours to produce multiple cryopreserve patient doses and this therapy does not necessitate pre-conditioning. The vein-to-vein time for the 1st administration is approximately one week. Patients must have a lesion that can be biopsied with acceptable clinical risk and agree to have a fresh biopsy at Screening and on study. A Data Monitoring Committee is in place. No formal statistical hypothesis testing will be performed. Results: N/A Conclusions: N/A Citation Format: Victoria Villaflor, Rajwanth Veluswamy, Elena Garralda, Richard Maziarz, Emese Zsiros, Anthony Shields, Mariano Ponz-Sarvise, Martijn Lolkema, Mehdi Brahmi, Julia Jennings, Nathan Miselis, Lindsay Moore, Katarina Blagovic, Rui-Ru Ji, Scott Loughhead, Ricardo Zwirtes, Sandip Patel. ENVOY-001: A phase 1, multicenter, open-label study of SQZ-AAC-HPV as monotherapy and in combination with immune checkpoint inhibitors in HLA-A*02+ patients with HPV16+ recurrent, locally advanced, or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT241.

Cytokines are soluble proteins that mediate cell-to-cell communication. Based on the discovery of the potent anti-tumour activities of several pro-inflammatory cytokines in animal models, clinical research led to the approval of recombinant interferon-alpha and interleukin-2 for the treatment of several malignancies, even if efficacy was only modest. These early milestones in immunotherapy have been followed by the recent addition to clinical practice of antibodies that inhibit immune checkpoints, as well as chimeric antigen receptor T cells. A renewed interest in the anti-tumour properties of cytokines has led to an exponential increase in the number of clinical trials that explore the safety and efficacy of cytokine-based drugs, not only as single agents, but also in combination with other immunomodulatory drugs. These second-generation drugs under clinical development include known molecules with novel mechanisms of action, new targets, and fusion proteins that increase half-life and target cytokine activity to the tumour microenvironment or to the desired effector immune cells. In addition, the detrimental activity of immunosuppressive cytokines can be blocked by antagonistic antibodies, small molecules, cytokine traps or siRNAs. In this review, we provide an overview of the novel trends in the cytokine immunotherapy field that are yielding therapeutic agents for clinical trials.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is largely unresponsive to checkpoint inhibitors. Blockade of the CXCR4/CXCL12 axis increases intratumoral trafficking of activated T cells while restraining immunosuppressive elements. This study evaluates dual blockade of CXCR4 and PD1 with chemotherapy in PDAC. Patients and Methods: Multicenter, single-arm, phase II study to evaluate the safety and efficacy of motixafortide and pembrolizumab combined with chemotherapy in patients with de novo metastatic PDAC and disease progression on front-line gemcitabine-based therapy (NCT02826486). Subjects received a priming phase of motixafortide daily on days 1–5, followed by repeated cycles of motixafortide twice a week; pembrolizumab every 3 weeks; and nanoliposomal irinotecan, fluorouracil, and leucovorin every 2 weeks (NAPOLI-1 regimen). The primary objective was objective response rate (ORR). Secondary objectives included overall survival (OS), progression-free survival (PFS), disease control rate (DCR), safety, and tolerability. Results: A total of 43 patients were enrolled. The ORR according to RECISTv1.1 was 21.1% with confirmed ORR of 13.2%. The DCR was 63.2% with median duration of clinical benefit of 5.7 months. In the intention-to-treat population, median PFS was 3.8 months and median OS was 6.6 months. The triple combination was safe and well tolerated, with toxicity comparable with the NAPOLI-1 regimen. Notably, the incidence of grade 3 or higher neutropenia and infection was 7%, lower than expected for this chemotherapy regimen. Conclusions: Triple combination of motixafortide, pembrolizumab, and chemotherapy was safe and well tolerated, and showed signs of efficacy in a population with poor prognosis and aggressive disease.

[EN] Background & Aims: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. Methods: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Followup RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. Results: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. Conclusions: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. Lay summary: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention. A.V. was supported by ADA of the University of Navarra, Spain, O.E. by FSE; MINECO; FJCI-2017-34233, Spain, R.E. by a donation from Mauge Burgos de la Iglesia’s family, Spain, and P. Olaizola by the Basque Government (PRE_2016_1_0269), Basque Country, Spain. M.J.P. was funded by ISCIII [FIS PI14; 00399, PI17; 00022] cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; Spanish Ministry of Economy and Competitiveness (MINECO: “Ramón y Cajal” Program RYC-2015-17755), Spain. M.A.A was funded by La Caixa Foundation, HEPACARE project, Spain, ISCIII FIS PI16/01126 cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain, and “Fundación Científica de la Asociación Española Contra el Cáncer’’ (AECC Scientific Foundation) Rare Cancers 2017, Spain. J.M.B. was funded by the Spanish Carlos III Health Institute (ISCIII) (FIS PI15; 01132, PI18; 01075 and Miguel Servet Program CON14; 00129 and CPII19; 00008), Spain, co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; “Euskadi RIS3” (2019222054) and BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15; CA; 016; BD), Basque Country, Spain; “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientific Foundation) Rare Cancers 2017, Spain. S.V. was supported by FEDER; MINECO (SAF2017-89944-R), Spain, by the Government of Navarra-Health Research Department (58; 2018), Navarra, Spain, by La Caixa and Caja Navarra Foundation-CIMA agreement, Spain. None of the funding sources were involved in the decision to submit the article for publication. This article is based upon work from COST Action CA18122 European Cholangiocarcinoma Network, supported by COST (European Cooperation in Science and Technology). COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks (www.cost.eu).

Protocol tittle: Study of BO-112 with pembrolizumab for colorectal or gastric/GEJ cancer with liver metastasis.Background: Gastric (GC), oesophagogastric junction (EGJC) and colorectal tumors (CRC) with microsatellite stability (MSS) are poorly responders to PD(L)-1 inhibition, due to low CD8+ T cell infiltration and PD-L1 expression. Therefore, modulation of the tumor microenvironment (TME) could increase disease control. In these tumors the presence of liver lesions is difficult to control because of the hepatic immune tolerance. Control of the hepatic disease could increase survival.BO-112 is a non-coding dsRNA agonist of TLR3, MDA5 and RIG-I which activates the innate and adaptative immune response, inducing apoptosis and immunogenic cell death, with a local and a systemic effect. A prior Phase I trial showed that the combination of pembrolizumab and intratumoral (IT) BO-112 has a good safety profile and encouraging activity. Study design: This is a single arm phase IIa trial to assess the efficacy of intrahepatic IT BO-112 and iv pembrolizumab in 3W cycles in patients with MSS CRC, GC or EGJ tumors. Up to 69 patients with at least one liver lesion and 2 prior lines for CRC and 1 for GC/EGJC will be enrolled. Primary endpoints: ORR and related TEAEs ≥ grade 3. Secondary endpoints: ORR (iRECIST), PFS, 6-months OS and safety. Exploratory endpoint: Changes in TME (PD-L1 expression and CD8+ T cell infiltration) from baseline to C2D1.Results: A total of 11 CRC and 7 GC/EGJC patients were enrolled since July 2020. This abstract focuses on the CRC patients´ biomarker expression. These patients had low PD-L1 CPS (combined positive score) expression and CD8+ T cell infiltration with a median value of 5 and 4%, respectively, in the TME at baseline. Following two IT BO-112 injections, an important increase in PD-L1 expression and CD8+ T cell infiltration was observed in 5 and 7 patients, respectively. PD-L1 CPS median value increased to 37.9 while CD8+ T cell infiltration median values raised to 12.9%.Conclusions: A meaningful increase in PD-L1 expression and CD8+ T cell infiltration was achieved in MSS CRC patients after one cycle of BO-112-pembrolizumab. Table 1.Baseline characteristics and TME changesBaseline characteristicsCRC N=11Demographics-Sex-Male-Female-Age (mean, range)7 (63.6%)4 (36.4%)55 (38-74)Location of primary tumor-Right colon-Left colon2 (36.4%)7 (63.6%)Presence of extrahepatic disease-No-YesLocation of the extrahepatic disease-Lungs-Lymph nodes-Ohers (bone, adrenal gland, peritoneum, pancreas)2 (18.2%)9 (81.8%)6 (54.5%)5 (45.5%)1 (9.1%)Molecular characteristics:-MMR status-MSS-KRAS-Wild type-Mutant -PD-L1-CPS Citation Format: Mariano Ponz-Sarvisé, Elena Élez, Andrés Muñoz, Marc Van den Eynde, Antonio Gaetano Rampoldi, Alain Hendlisz, Hans Prenen, Mercedes Rodríguez, María Teresa Cano, Marya Chaney, Helena Escuin, Vanesa Pons, Marisol Quintero, Andrés Cervantes. Phase IIa open-label clinical study of intratumoural administration of BO-112 in combination with pembrolizumab in subjects with liver metastasis from colorectal cancer or gastric/gastro-oesophageal junction cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT192.

Background RO7122290 (RO) is a potent next generation 4-1BB agonist simultaneously targeting fibroblast activation protein (FAP). FAP is highly expressed on cancer-associated fibroblasts in many solid tumors, while expression in healthy tissue is low. 4 1BB stimulation via FAP mediated cross linking augments the cytotoxicity, proliferation, and longevity of immune effector cells in FAP-positive tumors. RO was labelled with Zirconium-89 (89Zr) to visualize FAP-dependent tumor accumulation and assess the biodistribution in patients (pts) after successful proof of principle of 89Zr-based PET in preclinical pilot studies. Methods Pts with advanced and/or metastatic solid tumors were eligible for this sub-study of an ongoing Phase 1/1b trial (EUDRACT 2017-003961-83). RO was administered intravenously once at a total dose of 5, 45, 200 or 500 mg consisting of RO (cold) + 5 mg 89Zr-RO (hot, 37.0 [± 3.4] MBq).Up to three 89Zr-PET scans were performed over a period of nine days followed by a single tumor biopsy before pts could switch to Part A (single agent) and subsequently to Part B (combination with atezolizumab) of the main study to continue treatment. Tracer uptake was calculated as peak standardized uptake value (SUVPeak) of the lesion with the highest uptake. Results 14 pts were exposed to a total dose of 5, 45, 200 (all n=3) or 500 mg (n=5); median age was 60 years (range 26–74) with seven male and female pts each. Primary tumor sites included colon (n=4), lung (n=3), thymus (n=2), anus (n=1), breast (n=1), bile duct (n=1), pleura (n=1), and uvea (n=1). In healthy tissues, uptake of 89Zr-RO was predominantly observed in the liver and spleen across all doses and in non-malignant lymph nodes in 2/3 patients at 5 mg. Consistent with the target expression, tracer uptake was detected in FAP-positive scarring tissue and tissues with ongoing remodeling. Intra-tumoral accumulation of 89Zr-RO was observed at all doses. 96 hours p.i., SUVPeak values were 12.1 [± 4.1], 10.0 [± 4.9], and 7.0 [± 3.7] for the 5, 45 and 200 mg cohorts, respectively, while SUVPeak for the 500 mg cohort was even lower at 4.9 [± 0.5]. Conclusions 89Zr-PET confirmed tumor-specific uptake and expected biodistribution pattern of 89Zr-RO. The decrease in SUV with increasing doses suggest that more cold antibodies saturated FAP binding sites in the tumor. 89Zr-PET results supported together with clinical PK, PD, safety and response data the selection of the recommended phase 2 dose and schedule of RO in combination with atezolizumab.