Reshaping the tumor microenvironment of cold soft-tissue sarcomas with oncolytic viral therapy: a phase 2 trial of intratumoral JX-594 combined with avelumab and low-dose cyclophosphamide.

Most soft-tissue sarcomas (STS) exhibit an immunosuppressive tumor microenvironment (TME), leading to resistance against immune checkpoint inhibitors (ICIs) and limited therapeutic response. Preclinical data suggest that oncolytic viral therapy can remodel the TME, facilitating T cell accumulation and enhancing the immunogenicity of these tumors.We conducted the METROMAJX, a phase II clinical trial, to investigate the combination of JX-594, an oncolytic vaccinia virus engineered for selective tumor cell replication, with metronomic cyclophosphamide and the PD-L1 inhibitor avelumab in patients with advanced, 'cold' STS, characterized by an absence of tertiary lymphoid structures. The trial employed a two-stage Simon design. JX-594 was administered intratumorally at a dose of 1.109 pfu every 2 weeks for up to 4 intra-tumoral administrations. Cyclophosphamide was given orally at 50 mg twice daily in a week-on, week-off schedule, and avelumab was administered at 10 mg/kg biweekly. The primary endpoint was the 6-month non-progression rate.Fifteen patients were enrolled, with the most frequent toxicities being grade 1 fatigue and fever. Fourteen patients were assessable for efficacy analysis. At 6 months, only one patient remained progression-free, indicating that the trial did not meet the first stage endpoint of Simon's design. Analysis of sequential tissue biopsies and plasma samples revealed an increase in CD8 density and upregulation of immune-related protein biomarkers, including CXCL10.Intra-tumoral administration of JX-594 in combination with cyclophosphamide and avelumab is safe and capable of modulating the TME in cold STS. However, the limited efficacy observed warrants further research to define the therapeutic potential of oncolytic viruses, particularly in relation to specific histological subtypes of STS.

CD95/Fas and metastatic disease: What does not kill you makes you stronger.

CD95 (also known as Fas) is the prototype of death receptors; however, evidence suggests that this receptor mainly implements non-apoptotic signaling pathways such as NF-κB, MAPK, and PI3K that are involved in cell migration, differentiation, survival, and cytokine secretion. At least two different forms of CD95 L exist. The multi-aggregated transmembrane ligand (m-CD95 L) is cleaved by metalloproteases to release a homotrimeric soluble ligand (s-CD95 L). Unlike m-CD95 L, the interaction between s-CD95 L and its receptor CD95 fails to trigger apoptosis, but instead promotes calcium-dependent cell migration, which contributes to the accumulation of inflammatory Th17 cells in damaged organs of lupus patients and favors cancer cell invasiveness. Novel inhibitors targeting the pro-inflammatory roles of CD95/CD95 L may provide attractive therapeutic options for patients with chronic inflammatory disorders or cancer. This review discusses the roles of the CD95/CD95 L pair in cell migration and metastasis.

Disrupting the CD95-PLCγ1 interaction prevents Th17-driven inflammation.

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.

Probing the side chain tolerance for inhibitors of the CD95/PLCγ1 interaction.

Proceeding our effort to study protein-protein interaction between the death receptor CD95 and phospholipase PLCγ1, we present in the current work chameleon-like traits of peptidomimetic inhibitors. Minute analysis of the interaction suggests that most of the binding energy relies on van der Waals contacts rather than more specific features, such as hydrogen bonds or salt bridges. The two most important positions of the peptoid for its interaction with PLCγ1 (Arg184 and Arg187) were modified to test this hypothesis. While Arg184 proves to be exchangeable for Trp, with no alteration in affinity, the nature of the amino acid replacing Arg187 is more dependent on its positive charge. However, affinity can be partially recovered by increasing van der Waals interactions. Overall, this study shows that for both positions, a subtle balance exists between hydrophobicity, surface contacts and affinity for CD95/PLCγ1, and provides information for the generation of new therapeutic compounds toward this druggable target.

Synthesis of peptidomimetics and chemo-biological tools for CD95/PLCγ1 interaction analysis.

The death receptor CD95 (also known as Fas) induces apoptosis through protein/protein association and the formation of the death-inducing signaling complex. On the other hand, in certain biological conditions, this receptor recruits different proteins and triggers the formation of another complex designated motility-inducing signaling complex, which promotes cell migration and inflammation. This pathway relies on a short sequence of CD95, called calcium-inducing domain (CID), which interacts with the phospholipase PLCγ1. To better understand how CID/PLCγ1 interaction occurs, we synthesized different α-AA peptides mimicking CID. Some of these peptidomimetics are as potent as the natural peptide to disrupt the CID/PLCγ1 interaction and cell migration, and showed improved pharmacokinetic properties. We also generated biotinyl- and palmitoyl-labelled peptidomimetics, useful chemico-biological tools to further explore the pro-inflammatory signal of CD95, which plays an important role in the pathogenesis of lupus and other autoimmune diseases.

MEK1/2 overactivation can promote growth arrest by mediating ERK1/2-dependent phosphorylation of p70S6K.

The extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway has been involved in the positive and negative regulation of cell proliferation. Upon mitogen stimulation, ERK1/ERK2 activation is necessary for G1- to S-phase progression whereas when hyperactived, this pathway could elicit cell cycle arrest. The mechanisms involved are not fully elucidated but a kinase-independent function of ERK1/2 has been evidenced in the MAPK-induced growth arrest. Here, we show that p70S6K, a central regulator of protein biosynthesis, is essential for the cell cycle arrest induced by overactivation of ERK1/2. Indeed, whereas MEK1 silencing inhibits cell cycle progression, we demonstrate that active mutant form of MEK1 or MEK2 triggers a G1 phase arrest by stimulating an activation of p70S6K by ERK1/2 kinases. Silencing of ERK1/2 activity by shRNA efficiently suppresses p70S6K phosphorylation on Thr421/Ser424 and S6 phosphorylation on Ser240/244 as well as p21 expression, but these effects can be partially reversed by the expression of kinase-dead mutant form of ERK1 or ERK2. In addition, we demonstrate that the kinase p70S6K modulates neither the p21 gene transcription nor the stability of the protein but enhances the translation of the p21 mRNA. In conclusion, our data emphasizes the importance of the translational regulation of p21 by the MEK1/2-ERK1/2-p70S6K pathway to negatively control the cell cycle progression.

Deciphering the correlation between metabolic activity through 18F-FDG-PET/CT and immune landscape in soft-tissue sarcomas: an insight from the NEOSARCOMICS study.

Metabolic elevation in soft-tissue sarcomas (STS), as documented with 18F-Fluorodeoxyglucose positron emission tomography (18F-FDG-PET/CT) has been linked with cell proliferation, higher grade, and lower survivals. However, the recent diagnostic innovations (CINSARC gene-expression signature and tertiary lymphoid structure [TLS]) and therapeutic innovations (immune checkpoint inhibitors [ICIs]) for STS patients underscore the need to re-assess the role of 18F-FDG-PET/CT. Thus, in this correspondence, our objective was to investigate the correlations between STS metabolism as assessed by nuclear imaging, and the immune landscape as estimated by transcriptomics analysis, immunohistochemistry panels, and TLS assessment. Based on a prospective cohort of 85 adult patients with high-grade STS recruited in the NEOSARCOMICS trial (NCT02789384), we identified 3 metabolic groups according to 18F-FDG-PET/CT metrics (metabolic-low [60%], -intermediate [15.3%] and high [24.7%]). We found that T-cells CD8 pathway was significantly enriched in metabolic-high STS. Conversely, several pathways involved in antitumor immune response, cell differentiation and cell cycle, were downregulated in extreme metabolic-low STS. Next, multiplex immunofluorescence showed that densities of CD8+, CD14+, CD45+, CD68+, and c-MAF cells were significantly higher in the metabolic-high group compared to the metabolic-low group. Lastly, no association was found between metabolic group and TLS status. Overall, these results suggest that (i) rapidly proliferating and metabolically active STS can instigate a more robust immune response, thereby attracting immune cells such as T cells and macrophages, and (ii) metabolic activity and TLS could independently influence immune responses.

TROP2 Is Associated with Primary Resistance to Immune Checkpoint Inhibition in Patients with Advanced Non-Small Cell Lung Cancer.

PURPOSE: Mechanisms of primary resistance to inhibitors of the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling axis in non-small cell lung cancer (NSCLC) are still poorly understood. While some studies suggest the involvement of trophoblast cell surface antigen 2 (TROP2) in modulating tumor cell resistance to therapeutic drugs, its specific role in the context of PD-1/PD-L1 axis blockade is not definitively established. EXPERIMENTAL DESIGN: We performed high-throughput analysis of transcriptomic data from 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy in two large randomized clinical trials. To confirm our results at the protein level, we complemented this transcriptional approach by performing a multiplex immunofluorescence analysis of tumor tissue samples as well as a proteomic profiling of plasma. RESULTS: We observed a significant association of TROP2 overexpression with worse progression-free survival and overall survival on PD-L1 blockade, independent of other prognostic factors. Importantly, we found increased TROP2 expression to be predictive of survival in patients treated with atezolizumab but not chemotherapy. TROP2 overexpression was associated with decreased T-cell infiltration. We confirmed these results at the proteomic level both on tumor tissue and in plasma. CONCLUSIONS: Our results suggest an important contribution of TROP2 expression to the primary resistance to PD-L1 blockade in NSCLC. TROP2-biomarker-based strategy may be relevant in selecting patients with NSCLC who are more likely to benefit from a combination of immunotherapy and an anti-TROP2 agent.

MAPK signaling in cisplatin-induced death: predominant role of ERK1 over ERK2 in human hepatocellular carcinoma cells.

Hepatocellular carcinoma treatment by arterial infusion of cis-diamminedichloroplatinum-II (cisplatin) exhibits certain therapeutic efficacy. However, optimizations are required and the mechanisms underlying cisplatin proapoptotic effect remain unclear. The mitogen-activated protein kinase (MAPK) pathway plays a key role in cell response to cisplatin and the functional specificity of the isoform MAPK/ERK kinase 1 and 2 (MEK1/2) and ERK1/2 could influence this response. The individual contribution of each kinase on cisplatin-induced death was thus analyzed after a transient or stable specific inhibition by RNA interference in the human hepatocellular carcinoma cells Huh-7 or in knockout mice. We demonstrated here that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas MEK1 and MEK2 acted in a redundant manner. Indeed, at clinically relevant concentrations of cisplatin, ERK1 silencing alone was sufficient to protect cells from cisplatin-induced death both in vitro, in Huh-7 cells and ERK1(-/-) hepatocytes, and in vivo, in ERK1-deficient mice. Moreover, we showed that ERK1 activity correlated with the induction level of the proapoptotic BH3-only protein Noxa, a critical mediator of cisplatin toxicity. On the contrary, ERK2 inhibition upregulated ERK1 activity, favored Noxa induction and sensitized hepatocarcinoma cells to cisplatin. Our results point to a crucial role of ERK1 in cisplatin-induced proapoptotic signal and lead us to propose that ERK2-specific targeting could improve the efficacy of cisplatin therapy by increasing ERK1 prodeath functions.

Antitumor Effects of PRMT5 Inhibition in Sarcomas.

Patients with advanced soft-tissue sarcomas (STS) have few therapeutic options. Protein arginine methyltransferase 5 (PRMT5), an anticancer target, has been extensively investigated in recent years in epithelial tumors. To date, no data related to the biological role of PRMT5 inhibition and its potential effect as a treatment in STS have been reported.To investigate the therapeutic potential of PRMT5 targeting in STS, we first evaluated the prognostic value of PRMT5 expression in two different cohorts of patients with STS. We then used the potent and selective GSK3326595 (GSK595) compound to investigate the antitumor effect of the pharmacologic inhibition of PRMT5 in vitro via MTT, apoptosis, cell cycle, clonogenicity, and proliferation assays. In vivo studies were performed with two animal models to evaluate the effects of GSK595 on tumor growth. The mechanisms of action were investigated by RNA sequencing, metabolic pathway analysis, Western blotting, and glucose uptake/lactate production assays.High PRMT5 gene expression levels were significantly associated with worsened metastasis-free survival of patients with STS. GSK595 decreased the global symmetric dimethylarginine level, the proliferation rate and clonogenicity of STS cell lines in vitro and tumor growth in vivo. Moreover, PRMT5 inhibition regulated aerobic glycolysis through downregulation of key enzymes of glycolysis as well as glucose uptake and lactate production.The current study demonstrated that PRMT5 regulates STS cell metabolism and thus represents a potential therapeutic target for STS. Additional studies in diverse sarcoma subtypes will be essential to confirm and expand upon these findings. SIGNIFICANCE: STSs have limited therapeutic options. We show here the poor prognostic value of high PRMT5 expression in STS. Moreover, we demonstrate that the pharmacologic inhibition of PRMT5 has significant antitumor activity through the downregulation of glycolysis. Our findings support the clinical investigation of PRMT5 inhibition in STSs.

Upregulation of Indoleamine 2,3-Dioxygenase 1 in Tumor Cells and Tertiary Lymphoid Structures is a Hallmark of Inflamed Non-Small Cell Lung Cancer.

PURPOSE: Overexpression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) has been reported in several tumor types, including non-small cell lung cancer (NSCLC), and has been shown to promote tumor-immune evasion and inhibit T-cell activation through increased tryptophan degradation and the production of several immunosuppressive metabolites collectively known as kynurenines. However, it remains unclear whether IDO1 expression by tumor cells is detrimental specifically in the context of programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis blockade. EXPERIMENTAL DESIGN: We analyzed the transcriptome of 891 NSCLC tumor samples from patients enrolled in two large randomized clinical trials investigating the safety and activity of atezolizumab, a humanized IgG1 mAb that targets PD-L1, versus docetaxel in patients with advanced NSCLC. We complemented these transcriptomics results at the protein level by using multiplex immunofluorescence and at the functional level with in vitro experiments. RESULTS: The increased expression of the tryptophan-catabolizing enzyme IDO1 was significantly associated with improved objective response, progression-free survival, and overall survival in patients treated with PD-L1 inhibitors, but not in those treated with chemotherapy. Strikingly, inflamed tumors had higher levels of IDO1, and IDO1 was also expressed in tertiary lymphoid structures (TLS) by mature follicular dendritic cells. L-kynurenine impaired the differentiation of antibody-producing B cells induced by follicular helper T (Tfh)/B-cell interactions, a hallmark process within TLS. CONCLUSIONS: IDO1 pathway in NSCLC is driven by the immune system rather than by tumor cells. Targeting IDO1 in combination with anti-PD-1/PD-L1 might be beneficial only in patients with inflamed tumors and particularly in those bearing TLS.

The complexity of ERK1 and ERK2 MAPKs in multiple hepatocyte fate responses.

Recent reports suggest that extracellular signal-regulated kinase (ERK1) and ERK2 mitogen-activated protein kinases (MAPK) may direct specific biological functions under certain contexts. In this study, we investigated the role of early and sustained epidermal growth factor (EGF) stimulation on long-term hepatocyte differentiation and the possible role of ERK1 and ERK2 in this process. We demonstrate a long-term survival and an elevated level of differentiation up to 3 weeks. The differentiation state of hepatocytes is supported by sustained expression of aldolase B, albumin, and the detoxifying enzymes CYP1A2, 2B2, and 3A23. Similarly to freshly isolated cells, cultured hepatocytes also retain the ability to respond to 3-methylcholanthrene (3MC) and phenobarbital (PB), two known CYP inducers. In addition, we show evidence that continuous MAPK/ERK kinase (MEK) inhibition enhances the level of differentiation. Using RNA interference approaches against ERK1 and ERK2, we demonstrate that this effect requires both ERK1 and ERK2 activity, whereas the specific ERK1 knockdown promotes cell survival and the specific ERK2 knockdown regulates cell proliferation. In conclusion, we demonstrate that early and sustained EGF stimulation greatly extends long-term hepatocyte survival and differentiation, and that inhibition of the ERK1/2 MAPK pathway potentiates these pro-survival/pro-differentiation phenotypes. We clearly attest that specific ERK1 and ERK2 MAPKs determine hepatocyte survival and proliferation, respectively, whereas dual inhibition is required to stabilize a highly differentiated state.

Targeting Tryptophan Catabolism in Cancer Immunotherapy Era: Challenges and Perspectives.

Metabolism of tryptophan (Trp), an essential amino acid, represent a major metabolic pathway that both promotes tumor cell intrinsic malignant properties as well as restricts antitumour immunity, thus emerging as a drug development target for cancer immunotherapy. Three cytosolic enzymes, namely indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO2), catalyzes the first-rate limiting step of the degradation of Trp to kynurenine (Kyn) and modulates immunity toward immunosuppression mainly through the aryl hydrocarbon receptor (AhR) activation in numerous types of cancer. By restoring antitumor immune responses and synergizing with other immunotherapies, the encouraging preclinical data of IDO1 inhibitors has dramatically failed to translate into clinical success when combined with immune checkpoints inhibitors, reigniting the debate of combinatorial approach. In this review, we i) provide comprehensive evidences on immunomodulatory role of the Trp catabolism metabolites that highlight this pathway as relevant target in immuno-oncology, ii)ii) discuss underwhelming results from clinical trials investigating efficacy of IDO1 inhibitors and underlying mechanisms that might have contributed to this failure, and finally, iii) discuss the current state-of-art surrounding alternative approaches of innovative antitumor immunotherapies that target molecules of Trp catabolism as well as challenges and perspectives in the era of immunotherapy.

Phase 2 trial of intravenous oncolytic virus JX-594 combined with low-dose cyclophosphamide in patients with advanced breast cancer.

Breast cancer is one the most common cause of cancer death in women worldwide. We report here the first phase II study investigating a virus genetically engineered for tumor-selective replication in patients with breast cancer. Ten patients were treated with a combination of low-dose oral cyclophosphamide and intra-venous JX-594, a thymidine kinase gene-inactivated oncolytic vaccinia virus engineered for the expression of transgenes encoding human granulocyte-macrophage colony-stimulating factor (GM-CSF) and ß-galactosidase. Best response as per RECIST criteria was stable disease for 2 patients and progressive disease for 8 patients. Median progression-free and overall survival were 1.6 months (95% CI: [1.1-1.9]) and 14.4 months (95% CI: [2.0 - NA]) respectively. High throughput analysis of sequential plasma samples revealed an upregulation of protein biomarkers reflecting immune induction such as IFN gamma. Whether the combination of JX-594 with an immune checkpoint inhibitor is associated with meaningful clinical activity is therefore worth to investigate.

Randomized phase 2 trial of intravenous oncolytic virus JX-594 combined with low-dose cyclophosphamide in patients with advanced soft-tissue sarcoma.

JX-594 is an oncolytic vaccinia virus genetically modified to replicate selectively in tumor cells. Metronomic chemotherapy has shown preclinical synergy with oncolytic viruses. We report here the results of the METROMAJX which is a randomized phase II clinical trial investigating the combination of JX-594 combined with metronomic cyclophosphamide (arm 1) or metronomic cyclophosphamide (arm 2) in patients with advanced STS. A two-stage Simon design was used. JX-594 was administered intra-venously at the dose 1.109 every 2 weeks for the first 3 injections and then every 3 weeks. Cyclophosphamide was given orally at the dose of 50 mg BID 1 week on 1 week off. The primary endpoint was the 6-month non progression rate. 20 patients were included (arm 1:15, arm 2:5). The two most frequent toxicities were grade 1 fatigue and fever and grade 2 fatigue and grade 2 lymphopenia in arms 1 and 2, respectively. In arm 1, 12 patients were assessable for the efficacy analysis. None of them were progression-free at 6 months indicating that the first stage of the Simon's design was not satisfied. One patient out 4 assessable for efficacy was progression-free at 6 months in arm 2. High throughput analysis of sequential plasma samples revealed an upregulation of protein biomarkers reflecting immune induction such as CXCL10 and soluble CD8 antigen in arm 1. Systemic treatment with JX-594 is safe in patients with advanced STS. Further investigations are needed to improve immune response to oncolytic viruses and define their therapeutic potential in patients with STS.

Spatial transcriptomics of macrophage infiltration in non-small cell lung cancer reveals determinants of sensitivity and resistance to anti-PD1/PD-L1 antibodies.

BACKGROUND: Tumor-associated macrophages (TAMs) having immunosuppressive properties are one of the most abundant immune cells in the tumor microenvironment (TME). Preclinical studies have highlighted the potential role of TAMs in resistance to immune checkpoint blockers (ICBs). Here, we investigated the predictive value of TAM infiltration in patients with non-small cell lung cancer (NSCLC) treated with ICBs and characterized their transcriptomic profiles. METHODS: Tumor samples were collected from 152 patients with NSCLC before ICB treatment onset. After immunohistochemical staining and image analysis, the correlation between CD163+ cell infiltration and survival was analyzed. Spatial transcriptomic analyses were performed using the NanoString GeoMx Immune Pathways assay to compare the gene expression profile of tumors with high or low levels of CD163+ cell infiltration and to identify determinants of response to ICBs in tumors with high CD163+ infiltration. RESULTS: Low intratumoral CD163+ cell infiltration was associated with longer progression-free survival (PFS; HR 0.61, 95% CI 0.40 to 0.94, p=0.023) and overall survival (OS; HR 0.48, 95% CI 0.28 to 0.80, p=0.004) under ICB treatment. Spatial transcriptomic profiles of 16 tumors revealed the upregulation of ITGAM, CD27, and CCL5 in tumors with high CD163+ cell infiltration. Moreover, in tumors with high macrophage infiltration, the upregulation of genes associated with the interferon-γ signaling pathway and the M1 phenotype was associated with better responses under immunotherapy. Surprisingly, we found also a significantly higher expression of CSF1R in the tumors of responders. Analysis of three independent data sets confirmed that high CSF1R expression was associated with an increased durable clinical benefit rate (47% vs 6%, p=0.004), PFS (median 10.89 months vs 1.67 months, p=0.001), and OS (median 23.11 months vs 2.66 months, p<0.001) under ICB treatment. CONCLUSIONS: Enrichment of TAMs in the TME of NSCLC is associated with resistance to immunotherapy regardless of the programmed death ligand 1 status and is driven by upregulation of CD27, ITGAM, and CCL5 gene expression within the tumor compartment. Our transcriptomic analyses identify new potential targets to alter TAM recruitment/polarization and highlight the complexity of the CSF1R pathway, which may not be a suitable target to improve ICB efficacy.

Pembrolizumab combined with low-dose cyclophosphamide and intra-tumoral injection of the toll-like receptor 4 agonist G100 in patients with advanced pretreated soft tissue sarcoma: results from the PEMBROSARC basket study.

Soft tissue sarcomas (STS) are heterogeneous mesenchymal tumors with limited therapeutic options in the advanced setting. Immune checkpoint inhibitors have been shown to have significant clinical activity in inflamed STS which are characterized by the presence of tertiary lymphoid structures (TLS). New strategies are needed to sensitize TLS-negative STS to immunotherapy. Engagement of the toll-Like Receptor 4 (TLR4) signal pathway contributes to the development of a favorable tumor microenvironment in solid tumors. G100 is a highly potent toll-like receptor 4 (TLR4) agonist. We hypothesized that intra-tumoral G100 would induce a robust local and potentially systemic anti-tumor immune response in the microenvironment of TLS-negative sarcoma, leading to improved response to PD1 inhibition. Twenty metastatic STS patients who had a superficial injectable lesion were treated with 50 mg of cyclophosphamide (CP) orally twice daily (1 week on and 1 week off), 200 mg of pembrolizumab intravenously on day 8 of a planned 21-day cycle and G100 20 µg one weekly intra-tumoral injection for at least 6 weeks and for a maximum of 12 weeks (1st injection one week before CP administration, ie. Day -7). Biopsies and blood were collected pre and post treatment. Of the 17 patients assessable for efficacy analysis, 2 were progression-free at 6 months, and the 6-month non-progression rate was 11.8% (95% CI: 1.5-36.4), indicating that the first endpoint of the study was not reached. In 8 patients, there was an increase in T-cell infiltration into tumor after treatment. The ratio CD8/Fox-P3 + CD4 on treatment decreased in 11 cases out of 14 suggesting a predominant induction of Treg. Soluble PDL1 levels at baseline were also with adverse outcome. G100 appears to modulate the tumor microenvironment with significant infiltration of T cells. However, clinical activity in combination with PD1 inhibition was limited and no clear correlation was observed between tumor shrinkage and increased inflammation. TLR4 stimulation might have both antitumor and pro-tumor consequences.Trial registration: This study was registered with ClinicalTrial.gov, number NCT02406781.

Regorafenib-avelumab combination in patients with biliary tract cancer (REGOMUNE): a single-arm, open-label, phase II trial.

BACKGROUND: Regorafenib has shown substantial clinical activity in patients with advanced biliary tract cancers (BTCs). Preclinical data suggested that this drug modulates antitumour immunity and is synergistic with immune checkpoint inhibition. PATIENTS AND METHODS: This is a single-arm, multicentric phase II trial. Regorafenib was given 3 weeks/4, 160 mg quaque die (once a day) (QD); avelumab 10 mg/kg IV was given every two weeks, beginning at C1D15 until progression or unacceptable toxicity. The primary end-point was the confirmed objective response rate under treatment, as per Response Evaluation Criteria in Solid Tumours 1.1. The secondary end-points included the following: 1-year non-progression rate; progression-free survival (PFS) and overall survival; safety and biomarkers studies performed on sequential tumour samples obtained at baseline and at cycle 2 day 1. RESULTS: Thirty-four patients were enrolled in four centres. Twenty-nine patients were assessable for efficacy after central radiological review. The best response was partial response for four patients (13.8%), stable disease for 11 patients (37.9%) and progressive disease for 14 patients (48.3%). The median PFS and overall survival were 2.5 months (95% confidence interval [CI] [1.9-5.5]) and 11.9 months (95%CI [6.2-NA]) respectively. The most common grade 3 or 4 clinical adverse events related to treatment were hypertension (17.6%), fatigue (14.7%) and maculopapular rash (11.8%). High baseline levels of programmed cell death ligand 1 and of indoleamine 2, 3-dioxygénase expression were associated with improved outcomes. CONCLUSIONS: Regorafenib combined with avelumab has antitumour activity in a subset of heavily pretreated biliary tract cancer population. Further investigations are needed in patients selected based on tumour microenvironment features. CLINICAL TRIAL REGISTRATION: NCT03475953.

Signaling by the tyrosine kinase Yes promotes liver cancer development.

Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage and have few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or a dominant oncogene that can be targeted pharmacologically, unlike in other cancer types. Here, we report the identification of a previously uncharacterized oncogenic signaling pathway in HCC that is mediated by the tyrosine kinase Yes. Using genetic and pharmacological interventions in cellular and mouse models of HCC, we showed that Yes activity was necessary for HCC cell proliferation. Transgenic expression of activated Yes in mouse hepatocytes was sufficient to induce liver tumorigenesis. Yes phosphorylated the transcriptional coactivators YAP and TAZ (YAP/TAZ), promoting their nuclear accumulation and transcriptional activity in HCC cells and liver tumors. We also showed that YAP/TAZ were effectors of the Yes-dependent oncogenic transformation of hepatocytes. Src family kinase activation correlated with the tyrosine phosphorylation and nuclear localization of YAP in human HCC and was associated with increased tumor burden in mice. Specifically, high Yes activity predicted shorter overall survival in patients with HCC. Thus, our findings identify Yes as a potential therapeutic target in HCC.

Trabectedin plus Durvalumab in Patients with Advanced Pretreated Soft Tissue Sarcoma and Ovarian Carcinoma (TRAMUNE): An Open-Label, Multicenter Phase Ib Study.

PURPOSE: Trabectedin has shown preclinical synergy with immune checkpoint inhibitors in preclinical models. PATIENTS AND METHODS: TRAMUNE is a phase Ib study investigating the combination of trabectedin with durvalumab through a dose escalation phase and two expansion cohorts, soft tissue sarcoma (STS) and ovarian carcinoma. Trabectedin was given at three dose levels (1 mg/m2, 1.2 mg/m2, and 1.5 mg/m2) on day 1, in combination with durvalumab, 1,120 mg on day 2, every 3 weeks. The primary endpoints were the recommended phase II dose (RP2D) of trabectedin combined with durvalumab and the objective response rate (ORR) as per RECIST 1.1. The secondary endpoints included safety, 6-month progression-free rate (PFR), progression-free survival (PFS), overall survival, and biomarker analyses. RESULTS: A total of 40 patients were included (dose escalation, n = 9; STS cohort, n = 16; ovarian carcinoma cohort, n = 15, 80% platinum resistant/refractory). The most frequent toxicities were grade 1-2 fatigue, nausea, neutropenia, and alanine/aspartate aminotransferase increase. One patient experienced a dose-limiting toxicity at dose level 2. Trabectedin at 1.2 mg/m2 was selected as the RP2D. In the STS cohort, 43% of patients experienced tumor shrinkage, the ORR was 7% [95% confidence interval (CI), 0.2-33.9], and the 6-month PFR was 28.6% (95% CI, 8.4-58.1). In the ovarian carcinoma cohort, 43% of patients experienced tumor shrinkage, the ORR was 21.4% (95% CI, 4.7-50.8), and the 6-month PFR was 42.9% (95% CI, 17.7-71.1). Baseline levels of programmed death-ligand 1 expression and CD8-positive T-cell infiltrates were associated with PFS in patients with ovarian carcinoma. CONCLUSIONS: Combining trabectedin and durvalumab is manageable. Promising activity is observed in patients with platinum-refractory ovarian carcinoma. See related commentary by Digklia et al., p. 1745.