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Sinonasal intestinal-type adenocarcinoma (sITAC) is histomorphologically indistinguishable from colorectal adenocarcinoma (CRC) leading to diagnostic challenges. Metastases from CRCs to the sinonasal tract have been reported. The aim of the study was to identify a biomarker making it possible to distinguish between sITAC and metastases of colorectal origin. Formalin-fixated paraffin-embedded (FFPE) tissue from 20 consecutive patients with sITAC treated at Rigshospitalet, Denmark from 2005 to 2017, 20 patients with CRC, and second patients with both sinonasal and colorectal carcinomas were included, and RNA-sequencing was performed on all samples. Moreover, a series of 26 samples from metastasizing CRC were included (in-house data). 3139 differentially expressed genes were identified, of these several were deemed as possible biomarkers, including CSDE1, for which immunohistochemical staining was performed. sITAC and CRC differ in genomic expression. CSDE1, previously found upregulated in CRC, was significantly differentially expressed. Using immunohistochemical staining, no sITACs displayed strong and diffuse staining for CSDE1, which represents a potential marker to use in distinguishing sITAC from a metastasis of colorectal origin. This knowledge could improve the diagnostic process and hopefully the outcome in patients with this rare tumor.

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Purpose:We evaluated the clinical benefit of tumor molecular profiling to select treatment in the phase I setting.Experimental Design:Patients with advanced solid cancers and exhausted treatment options referred to a phase I unit were included in a prospective, single-center, single-arm open-label study (NCT02290522). Tumor biopsies were obtained for comprehensive genomic analysis including whole-exome sequencing and RNA sequencing. When possible, patients were treated with regimen matched to the genomic profile. Primary endpoint was progression-free survival (PFS).Results:From May 2013 to January 2017, a total of 591 patients were enrolled, with 500 patients undergoing biopsy. Genomic profiles were obtained in 460 patients and a potential actionable target was identified in 352 (70%) of 500 biopsied patients. A total of 101 patients (20%) received matched treatment based on either gene mutations or RNA expression levels of targets available in early clinical trials or off-label treatment. Objective response according to RECIST1.1 was observed in 15 of 101 patients (0% complete response, 15% partial response), with a median PFS of 12 weeks (95% confidence interval, 9.9–14.4).Conclusions:Our study supports the feasibility of genomic profiling to select patients in the phase I setting and suggests that genomic matching can be beneficial for a minor subset of patients with no other treatment options. Randomized studies may validate this assumption.See related commentary by Ratain, p. 1136

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Potentiel actionable targets divided in cancer subtypes

BACKGROUND: Mutational analysis of circulating tumor DNA (ctDNA) can potentially be used for early detection of recurrence after resection for hepatocellular carcinoma (HCC). Mutations from tumor may be identified in plasma as an early sign of recurrence. We conducted a pilot study investigating if somatic mutations could be detected in plasma in patients undergoing liver resection for HCC and in patients with advanced non-resectable HCC.METHODS AND RESULTS: We prospectively included patients undergoing curative liver resection for HCC. Tumor tissue was investigated with whole exome sequencing and preoperative blood samples were evaluated for ctDNA using targeted next-generation sequencing (NGS) with TruSight Oncology 500 including 523 cancer-associated genes. Subsequently, the method was evaluated in patients with advanced HCC. We included eight patients curatively resected for HCC, where tumor tissue mutations were identified in seven patients. However, only in one patient tumor specific mutations were found in the preoperative blood sample. In all three patients with advanced HCC, tumor mutations were detected in the blood.CONCLUSIONS: In patients with resectable HCC, ctDNA could not be reliably detected using the applied targeted NGS method. In contrast, ctDNA was detected in all patients with advanced HCC. Small tumors, tumor heterogeneity and limited sequencing coverage may explain the lack of detectable ctDNA.

Background Psychological distress may be present among patients who are considering enrollment in phase 1 cancer trials, as they have advanced cancer and no documented treatment options remain. However, the prevalence of psychological distress has not been previously investigated in larger cohorts. In complex phase 1 cancer trials, it is important to ensure adequate understanding of the study premises, such as the undocumented effects and the risk of adverse events. Materials and methods In a prospective study, patients completed questionnaires at two time points. We investigated psychological distress, measured as stress, anxiety, and depression, among patients at their first visit to the phase 1 unit (N = 229). Further, we investigated the understanding of trial information among patients who were enrolled in a phase 1 cancer trial (N = 57). Results We enrolled 75% of 307 eligible patients. We found a lower mean score of stress in our population compared to population norms, while the mean scores of anxiety and depression were higher. A total of 9% showed moderate to severe symptoms of anxiety and 11% showed moderate to severe symptoms of depression, which indicates higher levels than cancer patients in general. A total of 46 (81% of enrolled patients) completed questionnaires on trial information and consent. The understanding of the information on phase 1 cancer trials in these patients was slightly lower than the level reported for cancer trials in general. Some aspects relating to purpose, benefit, and additional risks were understood by fewer than half of the patients. Conclusion Our results suggest that distress is not as prevalent in the population of patients referred to phase 1 cancer trials as in the general cancer population. Although patients' understanding of trial information was reasonable, some aspects of complex phase 1 cancer trials were not easily understood by enrolled patients.

Background Immune checkpoint inhibitors (ICIs) have revolutionized the cancer therapeutic landscape and our perception of interactions between the immune system and tumor cells. Despite remarkable progress, disease relapse and primary resistance are not uncommon. Understanding the biological processes that tumor-infiltrating lymphocytes (TILs) undergo during ICI, how this affects the tumor microenvironment (TME) and, ultimately, clinical outcome is, therefore, necessary to further improve treatment efficacy. Aim In the current study, we sought to characterize TILs from patients with metastatic solid tumors undergoing ICI correlating flowcytometric findings with clinical outcome. Methods In total, 20 patients with 10 different metastatic solid tumors treated with ICIs targeting programmed-cell death-1 (PD-1)/PD-L1 axis were included in this study. The phenotype of T cells deriving from biopsies obtained prior to treatment initiation and on-treatment was investigaded. Analyses were focused on T cells’ degree of differentiation and activity and how they correlate with transcriptomic changes in the TME. Results Data indicate that patients benefitting from ICIs accumulate CD8+central memory T cells. TILs developed an effector-like phenotype over time, which was also associated with a cytolytic gene signature. In terms of modulation of T-cell responses, we observed that high expression of checkpoint molecules pre-treatment (i.e., PD-1, lymphocyte activation gene-3 [LAG-3], B and T-lymphocyte attenuator [BTLA] and T-cell immunoglobulin and mucin domain containing-3 [TIM-3]) was associated with similar gene signature and correlated to treatment benefit. Increasing expression of LAG-3 and BTLA in the CD8 compartment and their co-expression with PD-1 during treatment were, however, a common feature for patients who failed to respond to ICIs. Conclusions Besides identifying immune profiles suggestive of response to ICI, our results provide a more nuanced picture regarding expression of checkpoint molecules that goes beyond T-cell anergy.

BackgroundThe selective TRK inhibitor larotrectinib was approved for paediatric and adult patients with advanced TRK fusion-positive solid tumours based on a primary analysis set of 55 patients. The aim of our analysis was to explore the efficacy and long-term safety of larotrectinib in a larger population of patients with TRK fusion-positive solid tumours.MethodsPatients were enrolled and treated in a phase 1 adult, a phase 1/2 paediatric, or a phase 2 adolescent and adult trial. Some eligibility criteria differed between these studies. For this pooled analysis, eligible patients were aged 1 month or older, with a locally advanced or metastatic non-CNS primary, TRK fusion-positive solid tumour, who had received standard therapy previously if available. This analysis set includes the 55 patients on which approval of larotrectinib was based. Larotrectinib was administered orally (capsule or liquid formulation), on a continuous 28-day schedule, to adults mostly at a dose of 100 mg twice daily, and to paediatric patients mostly at a dose of 100 mg/m2 (maximum of 100 mg) twice daily. The primary endpoint was objective response as assessed by local investigators in an intention-to-treat analysis. Contributing trials are registered with ClinicalTrials.gov, NCT02122913 (active not recruiting), NCT02637687 (recruiting), and NCT02576431 (recruiting).FindingsBetween May 1, 2014, and Feb 19, 2019, 159 patients with TRK fusion-positive cancer were enrolled and treated with larotrectinib. Ages ranged from less than 1 month to 84 years. The proportion of patients with an objective response according to investigator assessment was 121 (79%, 95% CI 72-85) of 153 evaluable patients, with 24 (16%) having complete responses. In a safety population of 260 patients treated regardless of TRK fusion status, the most common grade 3 or 4 larotrectinib-related adverse events were increased alanine aminotransferase (eight [3%] of 260 patients), anaemia (six, 2%), and decreased neutrophil count (five [2%]). The most common larotrectinib-related serious adverse events were increased alanine aminotransferase (two [

PURPOSE CheckMate 032 is an open-label, multicohort study that includes patients with unresectable locally advanced or metastatic urothelial carcinoma (mUC) treated with nivolumab 3 mg/kg monotherapy every 2 weeks (NIVO3), nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses followed by nivolumab monotherapy 3 mg/kg every 2 weeks (NIVO3+IPI1), or nivolumab 1 mg/kg plus ipilimumab 3 mg/kg every 3 weeks for four doses followed by nivolumab monotherapy 3 mg/kg every 2 weeks (NIVO1+IPI3). We report on the expanded NIVO1+IPI3 cohort and extended follow-up for the NIVO3 and NIVO3+IPI1 cohorts. METHODS Patients with platinum-pretreated mUC were enrolled in this phase I/II multicenter study to receive NIVO3, NIVO3+IPI1, or NIVO1+IPI3 until disease progression or unacceptable toxicity. Primary end point was investigator-assessed objective response rate per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, including duration of response. RESULTS Seventy-eight patients were treated with NIVO3 (minimum follow-up, 37.7 months), 104 with NIVO3+IPI1 (minimum follow-up, 38.8 months), and 92 with NIVO1+IPI3 (minimum follow-up, 7.9 months). Objective response rate was 25.6%, 26.9%, and 38.0% in the NIVO3, NIVO3+IPI1, and NIVO1+IPI3 arms, respectively. Median duration of response was more than 22 months in all arms. Grade 3 or 4 treatment-related adverse events occurred in 21 (26.9%), 32 (30.8%), and 36 (39.1%) patients treated with NIVO3, NIVO3+IPI1, and NIVO1+IPI3, respectively. Grade 5 treatment-related pneumonitis occurred in one patient each in the NIVO3 and NIVO3+IPI1 arms. CONCLUSION With longer follow-up, NIVO3 demonstrated sustained antitumor activity alone and in combination with ipilimumab. NIVO1+IPI3 provided the greatest antitumor activity of all regimens, with a manageable safety profile. This result not only supports additional study of NIVO1+IPI3 in mUC, but demonstrates the potential benefit of immunotherapy combinations in this disease.

Selecting patients for early clinical trials is a challenging process and clinicians lack sufficient tools to predict overall survival (OS). Circulating cell-free DNA (cfDNA) has recently been shown to be a promising prognostic biomarker. The aim of this study was to investigate whether baseline cfDNA measurement could improve the prognostic information of the Royal Marsden Hospital (RMH) score. Solid tumour patients referred for phase I trials were included in the Copenhagen Personalized Oncology (CoPPO) programme. Baseline characteristics were collected prospectively, including the RMH prognostic score, Eastern Cooperative Oncology Group (ECOG) performance status and concentration of cfDNA per millilitre plasma. Cox proportional hazards model was used to assess the prognostic value of baseline variables. Plasma cfDNA concentration was quantifiable in 302 patients out of a total of 419 included in the study period of 2 years and 5 months. The RMH score was confirmed to be associated with OS. Cell-free DNA was shown to be an independent prognostic marker of OS and improved the risk model, including RMH, performance status and age. Furthermore, both plasma cfDNA concentration and RMH score were associated with treatment allocation (p

Despite encouraging results with immune checkpoint inhibition (ICI), a large fraction of cancer patients still does not achieve clinical benefit. Finding predictive markers in the complexity of the tumor microenvironment is a challenging task and often requires invasive procedures. In our study, we looked for putative variables related to treatment benefit among immune cells in peripheral blood across different tumor types treated with ICIs. For that, we included 33 patients with different solid tumors referred to our clinical unit for ICI. Peripheral blood mononuclear cells were isolated at baseline, 6 and 20 weeks after treatment start. Characterization of immune cells was carried out by multi-color flow cytometry. Response to treatment was assessed radiologically by RECIST 1.1. Clinical outcome correlated with a shift towards an effector-like T cell phenotype, PD-1 expression by CD8+T cells, low levels of myeloid-derived suppressor cells and classical monocytes. Dendritic cells seemed also to play a role in terms of survival. From these findings, we hypothesized that patients responding to ICI had already at baseline an immune profile, here called ‘favorable immune periphery’, providing a higher chance of benefitting from ICI. We elaborated an index comprising cell types mentioned above. This signature correlated positively with the likelihood of benefiting from the treatment and ultimately with longer survival. Our study illustrates that patients responding to ICI seem to have a pre-existing immune profile in peripheral blood that favors good outcome. Exploring this signature can help to identify patients likely to achieve benefit from ICI.

Purpose: The purpose of this study was to identify mechanisms of resistance to BRAF targeted therapy using proteomics together with genomics and transcriptomics in patients with BRAFV600E mutated solid tumors. Experimental procedures: A total of nine patients with BRAFV600E mutated advanced solid tumors (5 with colorectal cancer, 2 with neuroendocrine carcinoma, 1 with cholangiocarcinoma and 1 with breast cancer) treated with BRAF targeted therapy (BRAF inhibitor in combination with either MEK inhibitor and/or EGRF antibody) as part of the Copenhagen Prospective Personalized Oncology study, were included in this study. Tumor biopsies at baseline and at disease progression were analyzed with whole exome/genome sequencing (WES/WGS), transcriptomics (RNA sequencing) and proteomics. Genomic variants were analyzed together with changes in protein expression. Three filtering steps were used to identify potential resistance mechanisms from the proteomics measurements. Proteins were filtered for 1) proteins with a high shift in abundance between baseline and progression 2) proteins with known associations to the patient’s primary cancer based on text-mining and 3) proteins in pathways where BRAF is also involved. Results: Alterations conferring resistance were identified in 2 out of 9 patients when comparing data from WES/WGS and RNA sequencing at baseline and at disease progression (one patient with PTBP2-BRAF fusion and one with NRAS mutation). Genomic or transcriptomic alterations conferring resistance were not detected in the remaining 7 patients. Notably, there was not a single protein shared between all 9 patients after our filtering, but when comparing overlap on pathway level, six pathways related to RAF and MAPK signaling were affected in all 9 patients. Particularly, paradoxical activation of RAF signaling seems to be an interesting candidate to explain resistance, where formation of RAF dimer structures happens as a response to treatment with BRAF inhibitors, selectively inhibiting BRAF monomer structures (i.e. BRAFV600E). Conclusion: With a multiomic approach using proteomics together with genomics and transcriptomics, potential mechanisms of resistance were detected in all patients at disease progression, where resistance to BRAF targeted therapies had occurred. Six mechanisms of resistance were shared independently of diagnosis and BRAF inhibitor combination regimes. The potential of integrating proteomics with genomics and transcriptomics is promising and may potentially guide therapy for future patients with treatment resistant BRAFV600E mutated solid tumors. Citation Format: Martina Eriksen, Annelaura Bach Nielsen, Filip Mundt, Josephine Kerzel Duel, Matthias Mann, Ulrik Lassen, Christina Westmose Yde, Camilla Qvortrup, Martin Højgaard, Iben Spanggaard, Kristoffer Staal Rohrberg. Multiomics detect potential mechanisms of resistance to BRAF targeted therapy in patients with BRAFV600E mutated 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 3254.

Gene rearrangements involving the neurotrophic receptor kinase genes NTRK1, NTRK2, and NTRK3 (referred to as TRK, encoding TRKA, TRKB, and TRKC, respectively) result in highly oncogenic fusions. TRK fusions are rare, with a prevalence of < 1% in solid tumors. Detection of TRK fusions can be based on fluorescence in-situ hybridization (FISH), immunohistochemistry (IHC), and next-generation sequencing (NGS), where RNA sequencing is the most sensitive method. Inhibition of TRK fusions with highly selective small-molecule TRK inhibitors (TRKi) such as entrectinib and larotrectinib, results in profound responses in most cancer patients, regardless of cancer histology. Even response in CNS metastases is relatively common. Although responses are often durable, many patients develop resistance to TRKi due to mutations in one of the TRK genes, or due to genetic alterations conferring activation of alternative oncogenic signaling pathways. Second-generation TRKi have been developed, which can overcome some of the TRK resistance mutations. TRKi are well tolerated, with most common adverse events being related to on-target/off-tumor inhibition of TRKs.

Purpose: BAY1436032, an inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1), was active against multiple IDH1-R132X solid tumors in preclinical models. This first-in-human study was designed to determine the safety and pharmacokinetics of BAY1436032, and to evaluate its potential pharmacodynamics and antitumor effects. Patients and Methods: The study comprised of dose escalation and dose expansion cohorts. BAY1436032 tablets were orally administered twice daily on a continuous basis in subjects with mIDH1 solid tumors. Results: In dose escalation, 29 subjects with various tumor types were administered BAY1436032 across five doses (150–1,500 mg twice daily). BAY1432032 exhibited a relatively short half-life. Most evaluable subjects experienced target inhibition as indicated by a median maximal reduction of plasma R-2-hydroxyglutarate levels of 76%. BAY1436032 was well tolerated and an MTD was not identified. A dose of 1,500 mg twice daily was selected for dose expansion, where 52 subjects were treated in cohorts representing four different tumor types [lower grade glioma (LGG), glioblastoma, intrahepatic cholangiocarcinoma, and a basket cohort of other tumor types]. The best clinical outcomes were in subjects with LGG (n = 35), with an objective response rate of 11% (one complete response and three partial responses) and stable disease in 43%. As of August 2020, four of these subjects were in treatment for >2 years and still ongoing. Objective responses were observed only in LGG. Conclusions: BAY1436032 was well tolerated and showed evidence of target inhibition and durable objective responses in a small subset of subjects with LGG.

Background Selicrelumab is a human IgG2 agonistic anti-CD40 monoclonal antibody. Binding of the antibody to CD40 expressed on antigen-presenting cells results in T- cell priming and T-cell dependent anti-tumor activity. In response to T-cell activation, tumor cells express programmed-death ligand 1 (PD-L1) that can suppress effector T-cells. Atezolizumab interrupts this feedback loop by blocking PD-L1, thereby supporting the combination with selicrelumab. Methods This phase Ib open-label, multicenter, dose escalation (DE)/expansion clinical study (NCT02304393) investigated safety, pharmacokinetic (PK), pharmacodynamics (PD) and efficacy of selicrelumab in combination with atezolizumab in unselected patients with advanced/metastatic solid tumors, not amenable to standard therapy. In DE cohorts, a single dose of selicrelumab was given, either by intravenous (IV) infusion at a 16 mg fixed dose or subcutaneously (SC) at a range from 1 to 64 mg/dose. In dose-expansion cohorts (small bowel and colorectal cancer, head and neck squamous cell carcinoma [HNSCC] and non-small cell lung carcinoma), patients received multiple doses of selicrelumab SC at a dose of 16 mg. In all treatment cohorts, patients received atezolizumab at a fixed dose of 1200 mg IV Q3W. Results In this study, 140 patients were treated. This included 95 patients in DE cohorts (6 patients in the IV cohort, 89 patients in the SC cohorts) and 45 patients in dose-expansion cohorts. In the IV cohort, infusion related reaction was the most frequent treatment-related adverse event (TRAE; 50%), while Grade ≥ 3 TRAE occurred in 1 patient (16.7%). In this cohort one dose-limiting toxicity (DLT) was reported (Grade 3 pancytopenia). In the SC cohorts, the most frequent TRAE was injection site reaction (ISR; 92%). Four DLTs were reported in four patients: three Grade 3 ISR and one Grade 3 transaminase increase. Grade ≥ 3 TRAE were reported in 22 patients (16.4%). Anti-tumor activity was observed across cohorts receiving SC selicrelumab (dose range 1 to 36 mg). Eight of 80 evaluable patients in DE cohorts experienced objective responses (9% ORR). In the dose-expansion HNSCC cohort, three of 16 evaluable patients responded (15.8% ORR). There were no objective responses in the IV cohort. Treatment with selicrelumab resulted in significant peripheral B-cell depletion and activation and CD8+ T cell proliferation. Conclusions Treatment with selicrelumab in combination with atezolizumab was well tolerated in patients with advanced solid tumors. Signals of clinical and PD activity were observed. However, efficacy of the combination in this unselected population was limited, when compared to monotherapy efficacy of atezolizumab. Trial Registration NCT02304393 Ethics Approval This study was approved by the local IRB at each participating study site.

Background ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody developed as a first in class tumor-localizing CTLA-4 antibody for improved efficacy and reduced toxicity. Methods The study (NCT03782467) is a first-in-human dose escalation study followed by an expansion part. In the dose escalation patients with refractory solid malignancies are enrolled and the expansion part will enroll patients with cutaneous or mucosal malignant melanoma. Patients receive ATOR-1015 intravenously Q2W as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. Intra-patient dose escalation is allowed. The primary objective is to assess the safety and tolerability of ATOR-1015. Secondary objectives include pharmacokinetics, immunogenicity, pharmacodynamics, and clinical efficacy as assessed by iRECIST. Pharmacodynamic analyses include serum cytokines, immunophenotyping of peripheral blood mononuclear cells. Tumor biopsies before and after ATOR-1015 will be analyzed. Results As of June 26, 2020, 23 patients have been exposed to ATOR-1015. The median age of the patients is 54 years (range 40–72). Patients have received a median of 5 prior lines of therapy (range 1–16). Most common cancer type is colorectal cancer.Dose levels from 0.043 mg to 600 mg have been evaluated and declared safe. Dose escalation is ongoing, and currently two patients have been enrolled at 750 mg dose level. The median time on study was 8.4 weeks (range 0.1–34.3). Five patients are on study and 18 patients have discontinued. Reasons for discontinuation included clinical deterioration (n=10), disease progression (n=5), death due to disease progression (n=2), and investigator’s decision (n=1). Twelve of the 23 patients experienced a drug-related adverse event (AE). Two patients experienced a grade 3 drug-related AE, for all other patients AEs were grade 1 or 2. Infusion-related reactions (IRR) were reported in nine patients. Predominant symptoms of the IRR were chills, rash and pain. Potentially immune-related AEs grade 1 were reported in three patients: one patient had rash, one vitiligo, and one exanthema and eczema. No dose-limiting toxicities have occurred. Best response is stable disease. Pharmacokinetic data show dose proportional kinetics up to 600 mg. Preliminary biomarker analysis shows pharmacodynamic activity of ATOR-1015. Conclusions ATOR-1015 has been safe and well-tolerated up to 600 mg. Currently 750 mg is under evaluation. Best response is stable disease. Following the dose escalation phase, an expansion cohort for patients with advanced malignant melanoma will be initiated. Ethics Approval The study is approved by the Ethic Boards in Sweden and Denmark.

Background RO7122290 (RO) is a bispecific antibody-like fusion protein that simultaneously targets FAP, abundantly expressed by cancer-associated fibroblasts in most solid tumors, and 4-1BB, transiently expressed on activated T cells. Pre-clinical experiments revealed strong intra-tumoral CD8+ T cells infiltration in FAP-positive tumors, cytokines induction and significant anti-tumor activity mediated by RO (signal 2 of T cell activation), upon TCR/CD3 engagement (signal 1) or in combination with atezolizumab (ATZ). In this first-in-human study, the pharmacodynamic (PD) effects of RO were assessed, both as single agent (SA) (Part A) and in combination with ATZ (Part B). Methods Pts with advanced and/or metastatic solid tumors were eligible for this ongoing Phase 1/1b trial (EUDRACT 2017-003961-83). RO was administered intravenously, weekly (QW) at escalating dose levels (DLs). In Part A, 62 pts were treated at 13 DLs of RO, dose range 5–2000 mg. In Part B, 39 pts were treated at 8 DLs of RO, dose range 45–2000 mg, with ATZ 1200 mg Q3W. Secondary biomarker objective was characterization of PD effects in tumor tissue and blood. The endpoints were change from baseline in intra-tumoral density (cell/mm2) and proliferation (Ki67) of CD8+ T cells measured by immunohistochemistry (IHC), and change in activation (4-1BB) and proliferation (Ki67) of peripheral CD8+ T cells measured by flow cytometry. Exploratory objectives were characterization of PD effects in plasma/serum and measurement of intra-tumoral immune activation. The endpoints were change in peripheral cytokines (TNF-alfa, IFN-gamma, IL-6) and soluble(s) factors (sCD25, s4-1BB) concentration measured by ELISA, and intra-tumoral changes in gene expression measured by RNAseq. Results In the periphery, we observed transient expression of 4-1BB on CD4+ and CD8+ T cells, along with secretion of s4-1BB and inflammatory cytokines, suggesting 4-1BB targeting and potent T cell activation. The concomitant induction of proliferating T cells indicated the potential association to priming and formation of tumor-reactive T cells. In the tumor, we detected increased CD8+ T cells infiltration and proliferation, in both Parts. Proliferating CD8+ T cells increased in both tumor nests and surrounding stroma, with a preferential accumulation in the latter. RNAseq analysis revealed induction of 4-1BB, PD-1 and IFN-gamma, indicating intra-tumoral T cells activation in Part B. Conclusions PD activity was consistent with the postulated MoA, confirming RO to be a potent tumor-targeted 4-1BB agonist in the clinical setting. Our observations suggest that RO can synergize with endogenous T cell receptor stimulation, both as SA and in combination with ATZ.

Metastatic cancer spread is responsible for most cancer-related deaths. To colonize a new organ, invading cells adapt to, and remodel, the local extracellular matrix (ECM), a network of proteins and proteoglycans underpinning all tissues, and a critical regulator of homeostasis and disease. However, there is a major lack in tools to study cancer cell behavior within native 3D ECM. Here, an in-house designed bioreactor, where mouse organ ECM scaffolds are perfused and populated with cells that are challenged to colonize it, is presented. Using a specialized bioreactor chamber, it is possible to monitor cell behavior microscopically (e.g., proliferation, migration) within the organ scaffold. Cancer cells in this system recapitulate cell signaling observed in vivo and remodel complex native ECM. Moreover, the bioreactors are compatible with co-culturing cell types of different genetic origin comprising the normal and tumor microenvironment. This degree of experimental flexibility in an organ-specific and 3D context, opens new possibilities to study cell–cell and cell–ECM interplay and to model diseases in a controllable organ-specific system ex vivo.

Background Agonistic 4-1BB monoclonal antibodies were preclinically validated as promising cancer immunotherapies, both as monotherapy and as potentiators of the activity of PD-(L)1–blocking agents. However, toxicity and a narrow therapeutic window have hampered their clinical development. DuoBody-PD­-L1×4-1BB, a first-in-class, bispecific, next-generation checkpoint immunotherapy, was designed to overcome these limitations by activating T cells through conditional 4-1BB costimulation, while simultaneously blocking the PD-L1 axis. We present preliminary data from the ongoing, first-in-human, open-label, phase I/IIa trial of DuoBody-PD-L1×4-1BB in advanced solid tumors (NCT03917381). Methods During dose escalation, patients with metastatic or unresectable solid tumors not eligible for standard therapy received flat-dose DuoBody-PD-L1×4-1BB (25–1200 mg) intravenously every 3 weeks until disease progression or unacceptable toxicity. Primary endpoints were dose-limiting toxicities (DLTs) and adverse events (AEs). Secondary endpoints included pharmacokinetic parameters and antitumor activity (RECIST 1.1). Pharmacodynamic biomarkers and antitumor activity (iRECIST) were assessed as exploratory endpoints. Results As of June 22, 2020, 61 patients were enrolled (median age: 59 years). The most common cancer types were colorectal (19.7%), ovarian (14.8%), pancreatic (9.8%), and NSCLC (9.8%). Patients had previously received a median (range) of 3 (1–11) treatments; 44.2% had prior anti-PD-(L)1 immunotherapy. Patients received a median (range) of 4 (1–15) treatment cycles; Cmax was observed shortly after the end of infusion (mean T½: 2.3–10.3 days). Maximum tolerated dose was not reached; 6 patients experienced DLTs. The most common (=10%) treatment-related AEs (all grades; grades 3–4) were transaminase elevation (24.6%; 9.8%), hypothyroidism (16.4%; 1.6%), and fatigue (13.1%; 1.6%). Treatment-related grade-3 transaminase elevations decreased upon corticosteroid administration; no treatment-related bilirubin increases or grade-4 transaminase elevations occurred. Disease control, including stable disease at first assessment and partial responses in triple-negative breast cancer, ovarian cancer, and immune checkpoint inhibitor (ICI)–pretreated NSCLC, occurred in 40/61 patients (65.6%). Pharmacologic activity, as measured by modulation of adaptive immunity mediators, was observed across a broad range of dose levels. Peripheral proliferating (Ki67+) CD8+ effector memory T cells and serum interferon-gamma levels showed maximum induction relative to baseline (p=0.01) 8 days following treatment. Conclusions DuoBody-PD-L1×4-1BB demonstrated biologic activity and a manageable safety profile. Encouraging early clinical activity across different dose levels was observed in a heavily pretreated population with advanced solid tumors, including those resistant to prior immunotherapy or typically less sensitive to ICIs. Expansion cohorts of patients for whom DuoBody-PD-L1×4-1BB treatment could be relevant and biologically sound have started enrollment. Updated data will be presented. Acknowledgements The authors thank Manish Gupta, Lei Pang, and Thomas Breuer at Genmab A/S; Alice Bexon, Alexander Muik, and Friederike Gieseke at BioNTech SE; and Zuzana Jirakova (formerly at BioNTech SE) for their valuable contributions. This trial was funded by Genmab A/S and BioNTech SE. Trial Registration ClinicalTrials. gov; trial number: NCT03917381 Ethics Approval This trial is undertaken following full approval of the final protocol, amendments, informed consent form, applicable recruiting materials, and subject compensation programs by the Independent Ethics Committee/Institutional Review Board. Consent Written informed consent, in accordance with principles that originated in the Declaration of Helsinki 2013, current ICH guidelines including ICH-GCP E6(R2), applicable regulatory requirements, and sponsor policy, was provided by the patients.

Genomic screening of cancer patients for predisposing variants is traditionally based on age at onset, family history and type of cancer. Whereas the clinical guidelines have proven efficient in identifying families exhibiting classical attributes of hereditary cancer, the frequency of patients with alternative presentations is unclear. We identified and characterized germline variants in 636 patients with advanced solid cancer using whole exome sequencing. Pathogenic and likely pathogenic germline variants among 168 genes associated with hereditary cancer were considered. These variants were identified in 17.8% of the patients and within a wide range of cancer types. In particular, patients with mesothelioma, ovarian cancer, cervical cancer, urothelial cancer, and cancer of unknown primary origin displayed high frequencies of pathogenic variants. Variants were predominantly found in DNA-repair pathways and about half were within genes involved in homologous recombination repair. Twenty-two BRCA1 and BRCA2 germline variants were identified in 12 different cancer types, of which 10 (45%) were not previously identified in these patients based on the current clinical guidelines. Loss of heterozygosity and somatic second hits were identified in several of the affected genes, supporting possible causality for cancer development. A potential treatment target based on the pathogenic germline variant could be suggested in 25 patients (4%). The study demonstrates a high frequency of pathogenic germline variants in the homologous recombination pathway in patients with advanced solid cancers. We infer that genetic screening in this group of patients may reveal high-risk families and identify patients with potential PARP inhibitor sensitive tumors.

ATOR-1015 is evaluated in this first-in-human phase 1 study for safety and tolerability, as well as pharmacokinetics, immunogenicity and clinical efficacy (NCT03782467). ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody developed as a next generation CTLA-4 antibody with enhanced immune activation and tumor-directed activity for improved clinical efficacy and reduced toxicity. The primary objective of the study is to determine the maximum tolerated dose (MTD) or the recommended phase 2 dose (RP2D) in patients with advanced solid malignancies. Safety and tolerability of ATOR-1015 are assessed by adverse events (AEs), vital signs, ECG, laboratory evaluations and physical examinations. Secondary objectives include pharmacokinetics (PK), immunogenicity and clinical efficacy. Clinical efficacy is assessed with CT scans using Response Evaluation Criteria in Solid Tumors (iRECIST) for immune-based therapeutics. The study is designed with single patient cohorts for doses less than 100 mg. Thereafter, the study follows a modified 3+3 design. Intra-patient dose escalation is allowed. After the dose escalation, there is an option for expansion at or below the MTD. A total of up to 53 patients will be enrolled in the study. ATOR-1015 is administered intravenously biweekly as a single agent. Patients will receive ATOR-1015 treatment until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. The first patient was dosed in March 2019. Sixteen patients have been enrolled, one screen failure and 15 exposed to ATOR-1015. The following dose levels have been evaluated; 0.043 mg; 0.137 mg; 0.438 mg; 1.4 mg; 4.4 mg; 14 mg; 42 mg and 100 mg. Dose escalation is ongoing, and the 200 mg dose level is under evaluation. The following cancer types have been included; colorectal cancer (n=9), uveal melanoma (n=2), pancreatic cancer (n=2), ovarian cancer (n=2), and cholangiocarcinoma (n=1). The median age of the patients is 52 years (range 40-72). Patients received a median of 6 prior lines of therapy (range 3-16). The median time on study was 7.4 weeks (range 0.1-32.1). Six patients are on study and nine patients have discontinued treatment. Reasons for discontinuation include clinical deterioration (n=6), death due to disease progression (n=2) and confirmed disease progression (n=1). Six patients out of 15 experienced drug-related AEs that were all grade 2 or less. Four patients had infusion-related reactions (IRR), and one of those four also had abdominal pain, one patient had vitiligo, and one patient had rash. The IRR symptoms were predominantly rash. No Dose-Limiting Toxicities have occurred. Preliminary PK data show dose-proportional kinetics up to 100 mg. Conclusion: The dosing of ATOR-1015 has been safe and well-tolerated up to 100 mg. Dose escalation continues and the current dose level is 200 mg. Citation Format: Jeffrey Yachnin, Gustav J. Ullenhag, Ana Carneiro, Dorte L. Nielsen, Kristoffer Staal Rohrberg, Anne Månsson Kvarnhammar, Erika Bågeman, Camilla S. Wennersten, Charlotte Astrid Russell. A first-in-human phase 1 study in patients with advanced and/or refractory solid malignancies to evaluate the safety of ATOR-1015, a CTLA-4 x OX40 bispecific antibody [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 CT145.

3061 Background: ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody developed to be a next generation CTLA-4 antibody with enhanced immune activation and tumor-directed activity for improved efficacy and reduced toxicity. Methods: The primary objective of the study is to determine the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) in patients with advanced solid malignancies. Safety and tolerability of ATOR-1015 are assessed by adverse events (AEs), vital signs, ECG, laboratory evaluations and physical examinations. Secondary objectives include pharmacokinetics (PK), immunogenicity and clinical efficacy. Clinical efficacy is assessed using Response Evaluation Criteria in Solid Tumors for immune-based therapeutics (iRECIST). The study is designed with single patient cohorts for doses below 100 mg followed by a modified 3+3 design (NCT03782467). Intra-patient dose escalation is allowed. ATOR-1015 is administered intravenously every other week as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. Results: From March 2019 to February 2020, 15 patients have been exposed to ATOR-1015. The median age of the patients is 52 years (range 40-72). The following cancer types have been included: colorectal cancer (n=8), uveal melanoma (n=2), pancreatic cancer (n=2), ovarian cancer (n=2), and cholangiocarcinoma (n=1). Patients received a median of 6 prior lines of therapy (range 3-16). Dose levels from 0.043 mg to 200 mg have been evaluated and declared safe. Dose escalation is ongoing, and 400 mg is under evaluation. The median time on study was 8 weeks (range 2.1-34.3). Four patients are on study and eleven patients have discontinued treatment . Reasons for discontinuation include clinical deterioration (n=7), death due to disease progression (n=2), confirmed disease progression (n=1) and investigator´s decision (n=1). Six of the 15 patients experienced drug-related AEs which were grade 2 or less. Infusion-related reactions (IRR) were reported in four patients. One of those four also had abdominal pain and mediastinal burning sensation. The IRR symptoms were predominantly rash. One patient had vitiligo, and one had rash. No dose-limiting toxicities have occurred. Preliminary PK data show dose-proportional kinetics up to 200 mg. Conclusions: The dosing of ATOR-1015 has been safe and well-tolerated up to 200 mg. Dose escalation continues and the current dose level under evaluation is 400 mg. Clinical trial information: NCT03782467 .

// Lise Barlebo Ahlborn 1, 2 , Ida Viller Tuxen 1 , Florent Mouliere 3 , Savvas Kinalis 2 , Ane Y. Schmidt 2 , Kristoffer Staal Rohrberg 1 , Eric Santoni-Rugiu 4 , Finn Cilius Nielsen 2 , Ulrik Lassen 1 , Christina Westmose Yde 2 , Olga Oestrup 2 and Morten Mau-Sorensen 1 1 The Phase I Unit, Department of Oncology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark 2 Center for Genomic Medicine, Rigshospitalet, Copenhagen University, Copenhagen, Denmark 3 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom 4 Department of Pathology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark Correspondence to: Lise Barlebo Ahlborn, email: lise.barlebo.ahlborn@regionh.dk Keywords: BRAF inhibitor; circulating tumor DNA; mutant allele fraction; early phase study; solid cancer Received: June 04, 2018 Accepted: July 29, 2018 Published: August 24, 2018 ABSTRACT Purpose: We evaluated longitudinal tracking of BRAF V600E in circulating cell-free DNA (cfDNA) as a marker of treatment response to BRAF inhibitor (BRAFi) combination therapies in non-melanoma solid tumors included in the Copenhagen Prospective Personalized Oncology (CoPPO) program. Experimental design: Patients with BRAF V600E-mutated tumors were treated with combination therapies including BRAFi. Quantification of mutant cfDNA from plasma was determined and correlated to clinical outcomes. Exome sequencing was performed to identify possible resistance mutations. Results: Twenty-three patients had BRAF-mutated tumors out of 455 patients included in CoPPO and 17 started BRAFi combination (EGFRi/MEKi) therapy. Tumor responses were achieved in 8 out of 16 evaluable patients and the median overall- and progression-free survival (OS and PFS) was 15 and 4.8 months, respectively. Longitudinal measurements of BRAF V600E-mutant cfDNA indicated disease progression prior to radiological evaluation and a reduction in the mutant fraction of more than 50% after 4 and 12 weeks of therapy was associated with a significantly longer PFS (p=0.003 and p=0.029) and OS (p=0.029 and p=0.017). Furthermore, the baseline mutant fraction and total level of cfDNA positively correlated with tumor burden (p=0.026 and p=0.024). Finally, analysis of cfDNA at progression revealed novel mutations potentially affecting the MAPK pathway. Conclusion: BRAFi combination therapies showed a response rate of 50% in BRAF V600E-mutated non-melanoma tumors. The fraction of BRAF-mutant cfDNA represent a sensitive indicator for clinical outcomes with plasma collected at week 4 and 12 as crucial time points for monitoring response and disease progression.

Purpose: We evaluated the clinical benefit of tumor molecular profiling to select treatment in the phase I setting. Experimental Design: Patients with advanced solid cancers and exhausted treatment options referred to a phase I unit were included in a prospective, single-center, single-arm open-label study (NCT02290522). Tumor biopsies were obtained for comprehensive genomic analysis including whole-exome sequencing and RNA sequencing. When possible, patients were treated with regimen matched to the genomic profile. Primary endpoint was progression-free survival (PFS). Results: From May 2013 to January 2017, a total of 591 patients were enrolled, with 500 patients undergoing biopsy. Genomic profiles were obtained in 460 patients and a potential actionable target was identified in 352 (70%) of 500 biopsied patients. A total of 101 patients (20%) received matched treatment based on either gene mutations or RNA expression levels of targets available in early clinical trials or off-label treatment. Objective response according to RECIST1.1 was observed in 15 of 101 patients (0% complete response, 15% partial response), with a median PFS of 12 weeks (95% confidence interval, 9.9–14.4). Conclusions: Our study supports the feasibility of genomic profiling to select patients in the phase I setting and suggests that genomic matching can be beneficial for a minor subset of patients with no other treatment options. Randomized studies may validate this assumption. See related commentary by Ratain, p. 1136

Background MET gene amplification/mutation is implicated in oncogenesis of many tumor types. Sym015 is a recombinant antibody (Ab) mixture containing 2 humanized Abs binding non-overlapping epitopes on MET which has superior preclinical activity compared to other MET Abs. Here we present interim results from the First in Human study of Sym015. Methods In part 1 dose escalation, Sym015 was evaluated on every 2-week (q2w) schedule at 6/12/18/24 mg/kg doses in late line advanced solid tumor patients (pts). Based on PK/PD assessment, 18 mg/kg loading dose followed by 12 mg/kg q2w was selected as RP2D. The on-going Part 2 Dose expansion enrolls pts with MET amplification (METAmp >5 MET copies by Next Generation Sequencing (NGS) or FISH MET/CEP7 ratio >2.2 updated to ≥ 3.0) and/or MET exon 14 deletion (METEx14Δ) positive tumors. Central FISH confirmation of tumor MET status and longitudinal circulating tumor (ct)DNA profiling is performed. Results By April 2019, 51 pts (median age 61 yrs) have been treated, 12 pts in Part 1 and 39 pts in Part 2, the majority are non small cell lung cancer (NSCLC; 14 pts; 6 METAmp,7 METEx14Δ, 1 both) and gastric cancer (GC; 12 METAmp pts). Treatment-related adverse events (TRAE) occurred overall in 41,2%; TRAE ≥ gr3 in 9,8% (edema, colitis, septic shock, hypoalbuminemia, hypophosphatemia, increased amylase). No pts discontinued or died due to TRAE. The most common TRAE of ≥ 5% were fatigue 15.7%, peripheral edema 7,8%, nausea, decreased appetite, pruritus and abdominal pain reported by 5.9% of pts each. The table shows preliminary efficacy in NSCLC patients. Sym015 PK was slightly non-linear, with a dose-dependent t½ of 7-10 days for the first dose. 83% METAmp concordance (10/12 samples) between tumor and blood was observed. Table . 1490PD NSCLC Pt MET Status (Local Assessment) Best Response (Best % Change From Baseline) Previous MET TKI therapy Duration of Response (weeks) 1 METAmp NGS 6.8 Copies PR (-53%) No 80 2 METAmp NGS >5 Copies SD (-29%) No 9, Ongoing 3 METAmp NGS >5 Copies SD (+3%) No 9, Ongoing 4 METEx14Δ SD (-28%) No 7, Ongoing 5 METAmp FISH MET/ CEP7:4.9 SD (+6%) No 11 6 METEx14Δ SD (-4%) Yes 8 7 METEx14Δ SD (-21%) Yes 15 8 METEx14Δ SD (-20%) Yes 8 9 METAmp SISH MET/ CEP7: 15.2 Pending No Ongoing 10 METEx14Δ Pending Yes Ongoing 11 METEx14Δ + METAmp Pending Pending Ongoing 12 METEx14Δ Pending No Ongoing 13 METEx14Δ Pending No Ongoing 14 METEx14Δ Not Evaluable Yes Not Evaluable Conclusions Sym015 was well tolerated with early evidence of antitumor activity. Predictive biomarker analysis is underway. The study continues to enroll NSCLC patients with METAmp and/or METEx14Δ. MET screening in blood seems feasible and may be included in future trials. Clinical trial identification NCT02648724. Legal entity responsible for the study Symphogen A/S. Funding Symphogen A/S. Disclosure D.R. Camidge: Advisory / Consultancy: 2019: Takeda, CBT Pharmaceuticals, Daiichi-Sankyo (ILD adjudication committee), G1 Therapeutics (DSMB), Bio-Thera (DSMB), Blueprint; Advisory / Consultancy: 2018: AstraZeneca, Takeda, Arrys/Kyn, Regeneron, Hengrui, G1 Therapeutics (DSMB), Daiichi Sankyo (ILD adjudication committee), Hansoh (SRC), Bio-Thera (DSMB), Ribon, BMS, Blueprint, Roche/Genentech, Inivata; Advisory / Consultancy: 2017: Genoptix, G1 Therapeutics (DSMB), Mersana Therapeutics, Roche/Genentech, Takeda, Ignyta, Daiichi Sankyo (ILD adjudication committee), Hansoh SRC, Bio-Thera DSMB, Lycera, Revolution Med; Research grant / Funding (self), Research grant / Funding (institution): 2017: Takeda Investigator-initiated Trial; Research grant / Funding (self), Research grant / Funding (institution), Company Sponsored Trials at Institution: 2018/9: AbbVie, AstraZeneca, BMS, Hansoh, Lycera, MedImmune, Merck, Pfizer, Phosplatin, Roche/Genentech, Seattle Genetics, Symphogen, Takeda. F. Janku: Research grant / Funding (self): Novartis, Genentech, BioMed Valley Discoveries, Astellas, Agios, Plexxikon, Deciphera, Piqur, Symphogen, Bristol-Myers Squibb, Asana, Upsher-Smith Laboratories; Advisory / Consultancy: Guardant Health, IFM Therapeutics, Synlogic, Deciphera; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Trovagene; Advisory / Consultancy: Immunomet. D.V. Catenacci: Honoraria (self), Advisory / Consultancy: Merck, BMS, Lilly, Astellas, Gritstone, Taiho, Five Prime, Genentech Roche, Foundation Medicine, Guardant Health, Tempus. H.C. Chung: Honoraria (self): Merck-Serono, Lilly, Foundation Medicine; Advisory / Consultancy: Taiho, Celltrion, MSD, Lilly,Quintiles, BMS, Merck-Serono; Research grant / Funding (institution): Lilly, GSK, MSD, Merck-Serono, BMS-ONO, Taiho. A. Dowlati: Advisory / Consultancy: Seattle genetics, Takeda, AbbVie; Research grant / Funding (institution): Takeda, Taiho, Roche, EMD Serono, Bayer, Tesaro, Regeneron, Amgen, Mirati, BMS. K.S. Rohrberg: Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Research grant / Funding (institution): Loxo Oncology, Orion Pharma, Pfizer, PUMA, Cantargia, Genmab, Novartis, Incyte, Bayer, AstraZeneca, Alligator, Merck, BMS; Travel / Accommodation / Expenses: Sanofi; Research grant / Funding (institution): Symphogen. T.T. Poulsen: Full / Part-time employment: Symphogen A/S. H. Rudbaek: Full / Part-time employment: Symphogen A/S. L. Alifrangis: Full / Part-time employment: Symphogen A/S. R.P. Dalal: Full / Part-time employment: Symphogen A/S. All other authors have declared no conflicts of interest.

Background: Immunomodulation has improved anti-tumor activity. However, this treatment modality is only effective in a proportion of patients. Hence, analyzing changes in the tumor microenvironment is essential for understanding the variety of response patterns seen among patients undergoing the treatment. It also provides clinicians with valuable knowledge of possible new therapeutic targets. Aim: Assessment of the repertoire of T-cells (tumor-infiltrating lymphocytes -TIL) in the tumor microenvironment during check-point inhibition in patients with metastatic solid tumors. Material and methods: 35 patients with different metastatic solid tumors were included in the study at our Unit for Experimental Treatment at Rigshospitalet, Denmark. Patients were given treatment with check-point inhibitors targeting PD-1/PD-L1/CTLA-4 either in monotherapy or combined with other agents. Fresh tumor biopsies were taken at two time-points, namely, at baseline and on-treatment (50 days interval). T-cells were expanded in vitro with high doses of IL2 (6,000IU/mL IL2), harvested and cryopreserved for later use. Samples were analyzed using a multicolor flow cytometry panel. Response to the treatment was assessed radiologically using to RECIST criteria 1.1 and patients were divided in responder and non-responders. Results: Expansion was successful in seventeen patients and five other follow-up samples are still in culture at the moment. The current data analysis was run based on the seventeen cryopreserved samples. Among responders, we observed that baseline samples showed a statistically significant high percentage of effector CD8+(CD45RO+CCR7-) and CD8+naïve T cells(CD62L+), with p value 0.034 and 0.009, respectively. The presence of a high proportion of naïve CD8+Tcells in tumor microenvironment of responders could be explained by the fact that there is a high proportion of T cells surviving and becoming memory T cells and eventually effector T cells. No statistical difference was seen at the baseline expression of PD-1, either in the CD8 (p 0.66) or in the CD4 compartment (p 0.88). For follow-up samples, CD4+ effector memory T cells seemed to be more present among responders (p 0.009). Interestingly, NK-cells expressing CD56 (p 0.046) and co-expressing CD137 (p 0.005), known as an activation marker, appeared to be more present in samples from the responder group. CD4+T cells deriving form follow-up samples were also co-stained for LAG-3 revealing that responders had a low percentage of TILs expressing both markers (p 0.016). Paired-sample test will be run as the expansion phase for the remaining 5 samples still in culture is over. Conclusion: The preliminary findings of our study suggest that response achieved during check-point inhibition is reliant on the subset of T cells present in the tumor microenvironment and on how its composition changes over time. Citation Format: Vinicius Araujo Barbosa de Lima, Morten Hansen, Kristoffer Staal Rohrberg, Iben Spangaard, Inge Marie Svane, Ulrik Lassen. Exploring T cell- repertoire in the tumor microenvironment during check-point inhibition in patients with metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2810.

TPS2653 Background: ATOR-1015 is a human bispecific IgG1 antibody targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and the tumor necrosis factor receptor superfamily member 4, OX40 (also known as CD134). Both in vitro and in vivo, ATOR-1015 induces activation of cytotoxic T cells and depletion of regulatory T cells (1). In syngeneic tumor models, using human OX40 transgenic mice cross-reacting with both targets, ATOR-1015 is demonstrated to localize to the tumor. Further, the effects of ATOR-1015 are shown to occur in the tumor area and not in the spleen (1). Treatment with ATOR-1015 also reduces tumor growth and improves survival in several tumor models in mice, including bladder, colon and pancreatic cancer (1). The non-clinical safety profile and the pharmacokinetics were established in cynomolgus monkeys and the data were used for the dosing schedule. Methods: This is a multicenter, open-label, dose escalation study enrolling patients with advanced and/or refractory solid malignancies (NCT03782467). The primary objective of the study is to determine the maximum tolerated dose (MTD) or the recommended phase 2 dose (RP2D) and to establish the safety profile of ATOR-1015. ATOR-1015 is administered intravenously biweekly as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. The study will start with single patient cohorts until grade 2 toxicities are observed, thereafter the study follows a modified 3+3 design. At the MTD/RP2D, or a lower dose, up to 20 patients are planned for additional safety and efficacy evaluation. Study enrollment was initiated in January 2019. A total of up to 53 patients are estimated to be enrolled in the study. (1) Månsson Kvarnhammar et al. Journal for ImmunoTherapy of Cancer 2018; 6(Suppl 1):115. Abstract P683. Clinical trial information: NCT03782467.

The epidermal growth factor receptor (EGFR) and angiogenesis are well established targets in anti-cancer therapy. Several targeted anti-cancer therapies are in clinical trials in pancreatic and gastroesophageal (GEJ) cancer. However, many patients do not respond to these targeted therapies and there is therefore an increasing need for biomarkers for selection of patients to these therapies. We investigated the expression of EGFR, vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2) in tumour tissue by immunohistochemistry, and soluble EGFR (sEGFR), soluble VEGFR-2 (sVEGFR-2), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), plasminogen activator inhibitor 1 (PAI-1), and different forms of the urokinase plasminogen activator receptor (uPAR): uPAR (I), uPAR (I-III), and uPAR (I-III)+(II-III) in plasma by quantitative immunoassays in 14 patients with pancreatic and GEJ cancer. We found expression in tumour tissue and the plasma levels to be similar to those found in patients with other tumour types. No correlation was found between the blood levels of soluble receptors and the corresponding tumour tissue levels. We conclude that these markers are present in pancreatic and GEJ cancer patients, and could be investigated further as predictive biomarkers in such patients treated with EGFR or angiogenesis targeted therapies.

Introduction. Gemcitabine based regimens have been widely used in patients with advanced cholangiocarcinoma (CC), but no standard therapy exists. In this study we aimed to find the maximally tolerated dose (MTD) of a two-week schedule of fixed dose rate (FDR) gemcitabine (G), oxaliplatin (O) and capecitabine (C), and evaluate the safety and efficacy of this regimen in patients with advanced cholangiocarcinoma (CC). Methods. In the Phase I part of the study a dose-escalation schedule of FDR G, O and C, administered every two weeks, was performed in patients with solid tumours and no other treatments or advanced CC. In the Phase II part response rate, toxicity, progression-free survival (PFS) and overall survival was evaluated in patients with newly diagnosed advanced CC. Results. Thirty-six patients entered the Phase I part and G 1 000 mg/m(2) day 1 and 15, O 60 mg/m(2) day 1 and 15, and C 1 000 mg/m(2) BID day 1-7 and day 15-21 were established as MTD. In the Phase II part, 41 patients with advanced CC were included. Overall response rate was 34% and 51% had stable disease, resulting in a clinical benefit rate of 85%. Grade III and IV adverse events were rare. Median survival was 12.5 months (95% CI 9.2-15.9) and median progression-free survival (PFS) was 6.9 months (95% CI 5.1-8.6). Conclusions. This outpatient regimen was very feasible with significant activity and a favourable safety profile. Further studies will explore this combination with addition of newer targeted agents.

e15155Background: High-dose (HD) IL-2 is approved for patients with melanoma and renal cell carcinoma but is associated with significant toxicity, given only in an inpatient setting. RO6874281 is a...

// Lise B. Ahlborn 1, 2 , Kristoffer S. Rohrberg 1 , Migle Gabrielaite 2 , Ida V. Tuxen 1 , Christina W. Yde 2 , Iben Spanggaard 1 , Eric Santoni-Rugiu 3 , Finn C. Nielsen 2 , Ulrik Lassen 1 , Morten Mau-Sorensen 1, * , Olga Ostrup 2, * 1 The Phase I Unit, Department of Oncology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark 2 Center for Genomic Medicine, Rigshospitalet, Copenhagen University, Copenhagen, Denmark 3 Department of Pathology, Rigshospitalet, Copenhagen University, Copenhagen, Denmark * These authors contributed equally to this work Correspondence to: Lise B. Ahlborn, email: lise.barlebo.ahlborn@regionh.dk Keywords: cfDNA; liquid biopsy; genomic profile; biopsy; WES Received: October 24, 2014 Accepted: January 17, 2019 Published: February 15, 2019 ABSTRACT Purpose: Access to genomic tumor material is required to select patients for targeted therapies. However, tissue biopsies are not always feasible and therefore circulating cell-free DNA (cfDNA) has emerged as an alternative. Here we investigate the utility of cfDNA for genomic tumor profiling in the phase I setting. Study design: Peripheral blood was collected from patients with advanced solid cancers eligible for phase I treatment. Patients failing the initial tissue biopsy due to inaccessible lesions or insufficient tumor cellularity (

Background. Patients with upper gastrointestinal cancers have a poor prognosis and only few treatment options. The epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) are valid targets in many solid tumours, and they have synergistic effects in preclinical studies. Methods. In this multi-center phase II trial patients with chemoresistant, metastatic upper gastrointestinal cancer were treated with erlotinib (150 mg daily) and bevacizumab (10 mg/kg every two weeks). Primary endpoint was overall response rate (ORR). Secondary endpoints were progression free survival (PFS), overall survival (OS), toxicity and biomarker correlates. Plasma samples were analysed for EGFR and angiogenesis related markers using quantitative immunoassays. Results. One hundred and two patients were enrolled in the trial between June 2006 and October 2007. The most common toxicities were skin reaction, diarrhoea, and fatigue. ORR was 6%, median PFS was 2.2 months, and OS 4.3 months. Low concentration of urokinase plasminogen activator receptor (uPAR) domain I was correlated to longer PFS and OS. Discussion. The combination of erlotinib and bevacizumab is well tolerated, however, with low clinical activity in patients with chemoresistant UGI cancer. Some patients do benefit from the therapy, and uPAR forms are potential biomarkers in these patients.

Malignancies in the upper gastrointestinal (UGI) tract are amongst the most aggressive cancers and only few treatment options exist. We have recently analysed data from a phase II trial where patients with UGI cancers were treated with erlotinib and bevacizumab. The combination therapy could not be recommended in an unselected population of patients with chemo-refractory UGI cancer. However, a subpopulation of patients did benefit from the therapy. In this prospectively planned biomarker study we investigated vascular endothelial growth factor A (VEGF-A), VEGF receptor 2 (VEGFR-2) and epidermal growth factor receptor (EGFR) by immunohistochemistry and KRAS mutation status detected by PCR as potential predictors of effect of therapy. High VEGF-A expression was correlated to longer overall survival (HR: 0.8, 95%CI: 0.7-0.9) and high VEGFR-2 expression to shorter progression free survival (HR: 1.4, 95%CI: 1.0-1.9). EGFR expression and KRAS mutation status were not correlated to response or survival. We conclude that VEGF-A and VEGFR-2 could potentially be predictive markers in patients with UGI cancers treated with erlotinib and bevacizumab.

35 Background: In colorectal cancer (CRC) several independent blood-borne biomarkers have been proposed as prognostic and/or predictive markers. However only CEA is at present recommended as a serological CRC biomarker. We have identified 6 other biomarkers and the aim of this study is to see if a combination of markers improves the prognostic and/or predictive value. Methods: Two-hundred and twenty-eight patients with CRC have been included in this study, all with TNM stages I-III. Overall survival (OS) was chosen as the primary endpoint with 93 events and the minimum follow-up was 47 months. Seven biomarkers measured in plasma or serum were analysed: CEA, TIMP-1, the 3 soluble uPAR forms suPAR(I-III), suPAR(I-III)+(II-III) and uPAR(I), PAI-1 and YKL-40. Multivariable analyses of OS were done using regression analysis and results presented by 3 and 5 year OS rates with area under the receiver operating characteristic curve (AUC). All biomarker levels were analysed on the log scale (base 2). Results: High levels of each of the included biomarker were significantly associated in a multivariable analysis (adjusted for age, gender, TNM stage, tumor localization, adjuvant chemotherapy,interaction between adjuvant therapy and biomarker) to poor prognosis in patients not receiving adjuvant chemotherapy. The results are shown for those patients (see Table). The uPAR forms were the only biomarkers significantly associated to OS in patients receiving adjuvant chemotherapy. Combining the markers resulted in an enhanced prediction of 3 and 5 year OS. For TNM stage II patients not receiving adjuvant therapy, the AUC was 0.765 for 3 year OS and 0.745 for 5 year OS. The corresponding AUC’s for TNM stage III were 0.859 and 0.861. Conclusions: The presented combination of blood biomarkers are shown to predict OS with higher precision than any single marker for patients not receiving adjuvant chemotherapy and may provide useful information for use in the management of patients with CRC. [Table: see text]