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Phase 2 study of ipilimumab, nivolumab, and tocilizumab combined with stereotactic body radiotherapy in patients with refractory pancreatic cancer (TRIPLE-R)

Open AccessPublished:December 31, 2022DOI:https://doi.org/10.1016/j.ejca.2022.11.035

      Highlights

      • Ipilimumab/nivolumab/tocilizumab/radiation was evaluated in pancreatic cancer.
      • No clinically meaningful efficacy was observed.
      • No unanticipated safety signals were registered.
      • No changes in the tumour-infiltrating lymphocytes were induced.
      • The serum IL-6 level prior to treatment was associated with survival.

      Abstract

      Background

      Interleukin-6 blockade and radiation combined with immunotherapy may modulate the tumour microenvironment to overcome immune resistance. We assessed the efficacy of ipilimumab, nivolumab, and tocilizumab combined with stereotactic body radiotherapy (SBRT) in patients with refractory pancreatic cancer (PC).

      Methods

      Patients with PC who had progressive disease (PD) or intolerance to gemcitabine- or fluorouracil-containing regimens were enrolled in Part A of the two-part, single-centre, phase 2 study (NCT04258150). SBRT with 15 Gy was administered on day one of the first cycle. Ipilimumab was administered (1 mg/kg every 6 weeks) for a maximum of two infusions. Nivolumab (6 mg/kg) and tocilizumab (8 mg/kg) were given every four weeks until the PD or unacceptable toxicity, or for up to one year. The primary end-point was the objective response rate, with a threshold of 15%.

      Results

      Twenty-six patients were enrolled and treated between April 17, 2020, and January 25, 2021. The median follow-up time at the time of data cutoff (February 7, 2022) was 4.9 months (interquartile range 2.1–7.7). No responses were observed. Five patients (19%; 95% confidence intervals [CI], 7–39) achieved a stable disease. The median progression-free survival was 1.6 months (95% CI 1.4–1.7), and the median overall survival was 5.3 months (95% CI 2.3–8.0). Overall, 19 (73%) experienced adverse events related to the treatment including two (8%) with grade 3 or higher events.

      Conclusion

      The combination of ipilimumab, nivolumab, tocilizumab, and SBRT in patients with PC did not meet the prespecified criteria for expansion for full accrual.

      Keywords

      1. Introduction

      Pancreatic cancer (PC) is the fourth leading cause of cancer-related death in the United States [
      • Siegel R.L.
      • et al.
      Cancer statistics.
      ]. Survival rates are low, currently approaching 11% at 5 years post-diagnosis, and they have not improved over the last 50 years [
      • Siegel R.L.
      • et al.
      Cancer statistics.
      ]. PC is highly resistant to conventional treatments, and nearly all the patients develop metastases and die. Since the incidence of PC is increasing continuously, while the treatment response rates remain low, the lack of effective therapy options is a cause for concern and highlights the need for further research in this area.
      The use of single-agent or combined immunotherapy with immune checkpoint inhibitors (ICIs) targeting the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed death-ligand 1 (PD-L1) have shown repeated durable responses in patients with various types of cancer [
      • Ribas A.
      • Wolchok J.D.
      Cancer immunotherapy using checkpoint blockade.
      ]. However, these successes have not been repeated in PC [
      • Brahmer J.R.
      • et al.
      Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.
      ,
      • Royal R.E.
      • et al.
      Phase 2 trial of single agent ipilimumab (anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma.
      ,
      • O'Reilly E.M.
      • et al.
      Durvalumab with or without tremelimumab for patients with metastatic pancreatic ductal adenocarcinoma: a phase 2 randomized clinical trial.
      ]. The most recent data have suggested that one of the factors that is considered to be the primary mediator of this resistance in PC is a highly immunosuppressive tumour microenvironment (TME), supported partly by the activation of the interleukin (IL)-6 pathway [
      • Bear A.S.
      • Vonderheide R.H.
      • O'Hara M.H.
      Challenges and opportunities for pancreatic cancer immunotherapy.
      ]. The immune-modulating cytokine IL-6 promotes the inflammation cascade and other key pathways associated with immune resistance within the TME [
      • Lee J.W.
      • et al.
      Hepatocytes direct the formation of a pro-metastatic niche in the liver.
      ,
      • Flint T.R.
      • et al.
      Tumor-induced IL-6 reprograms host metabolism to suppress anti-tumor immunity.
      ]. Murine models have demonstrated that the blockade of the IL-6-regulated signalling pathways may inhibit liver metastasis, immunomodulate the suppressive TME, and increase the intratumoural effector T lymphocytes [
      • Lee J.W.
      • et al.
      Hepatocytes direct the formation of a pro-metastatic niche in the liver.
      ,
      • Eriksson E.
      • et al.
      IL-6 signaling blockade during CD40-mediated immune activation favors antitumor factors by reducing TGF-beta, collagen type I, and PD-L1/PD-1.
      ,
      • Mace T.A.
      • et al.
      IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer.
      ]. Furthermore, IL-6 inhibition has been observed to suspend the immunosuppressive effects and to enhance the efficacy of anti-PD-1/PD-L1 checkpoint inhibitor therapy in preclinical models [
      • Mace T.A.
      • et al.
      IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer.
      ,
      • Tsukamoto H.
      • et al.
      Combined blockade of IL6 and PD-1/PD-L1 signaling abrogates mutual regulation of their immunosuppressive effects in the tumor microenvironment.
      ,
      • Liu H.
      • Shen J.
      • Lu K.
      IL-6 and PD-L1 blockade combination inhibits hepatocellular carcinoma cancer development in mouse model.
      ,
      • Bent E.H.
      • et al.
      Microenvironmental IL-6 inhibits anti-cancer immune responses generated by cytotoxic chemotherapy.
      ]. IL-6 is a negative prognostic biomarker in many cancers including PC and is associated with a worse outcome [
      • Kjaergaard A.D.
      • et al.
      Inflammatory biomarker score identifies patients with six-fold increased risk of one-year mortality after pancreatic cancer.
      ]. Radiation combined with immunotherapy has been recommended in order to modulate the immune TME, thereby overcoming immune resistance [
      • Azad A.
      • et al.
      PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy.
      ,
      • Twyman-Saint Victor C.
      • et al.
      Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer.
      ]. Furthermore, in a recent phase 2 study where nivolumab, ipilimumab, and stereotactic body radiotherapy (SBRT) with 15 Gy were administered to patients with PC, the lower levels of serum IL-6 that were observed after treatment have been associated with improved clinical outcomes [
      • Chen I.M.
      • et al.
      Randomized phase II study of nivolumab with or without ipilimumab combined with stereotactic body radiotherapy for refractory metastatic pancreatic cancer (CheckPAC).
      ]. Therefore, in order to address the clinical impact of IL-6 inhibition together with immunotherapy plus radiotherapy, we initiated a phase 2 trial of ipilimumab, nivolumab, tocilizumab (an anti-IL-6 receptor monoclonal antibody), and SBRT in patients with advanced PC. Herein, we reported on the safety and efficacy of this combination.

      2. Methods

      2.1 Study design and participants

      This phase 2, single-centre, open-label study was conducted at Herlev University Hospital in Denmark (Appendix 1, p 11). This two-part study is expected to consist of Part A, the Lead–in and a possible Part B. Based on the predictive probability of the responses after 15 patients completed two post-baseline tumour assessments, a total of up to 30 patients with PC were expected to be enrolled in Part A, the Lead–in cohort. Part B was planned as either a non-randomised expansion or a randomised controlled study depending on responses from Part A. The study followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guidelines (Appendix 1, pp 4–5). The protocol, amendments, and informed consent forms were approved by the Ethics Committee of the Capital Region of Denmark before the start of the study (H-19087729). Informed consent was obtained from each participant before inclusion in the study.
      Eligible patients were ≥18 years of age with histologically or cytologically confirmed locally advanced or metastatic PC and who had progressed during or after at least one line of systemic chemotherapy (gemcitabine or fluorouracil-based regimens). All of the patients had an Eastern Cooperative Oncology Group performance status (ECOG PS) score of 0 or 1 and adequate organ and haematologic functions. The key exclusion criteria were the presence of an active autoimmune disease and the requirement for >10 mg/day or equivalent of prednisone. Concomitantly, patients were included in the Danish BIOPAC study “Biomarkers in Patients with Pancreatic Cancer” (NCT03311776).

      2.2 Procedures

      The patients received a single-fraction SBRT with 15 Gy for a single primary or metastatic lesion administered on day 1 of the first cycle. The organs at risk were prioritised over volume coverage during the SBRT planning. Ipilimumab (1 mg/kg) was administered intravenously on day 1 and repeated once after 6 weeks. Nivolumab (6 mg/kg) and tocilizumab (8 mg/kg) were given intravenously on day 1 and every 4 weeks for up to 1 year or until there was confirmed disease progression, unacceptable toxicity, withdrawal of consent, or clear clinical deterioration according to the investigator's judgement. Patients with disease progression continued receiving nivolumab and tocilizumab if they did not experience clinical deterioration and were deriving clinical benefits from treatment. Patients were offered one reinduction course, if disease progression was noted during the follow-up, and if the patient had not received other anticancer treatment and had not met the criteria for discontinuation.
      Safety assessments were performed from the start of the study to 100 days after the last nivolumab dose, in accordance with the National Cancer Institute Common Terminology Criteria, version 5.0. The target and non-target lesions were assessed according to the Response Evaluation Criteria in Solid Tumors (version 1.1; RECIST v1.1) at baseline [
      • Eisenhauer E.A.
      • et al.
      New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).
      ]. The patients underwent tumour assessments using cross-sectional imaging every 8 weeks for the first 48 weeks, relative to the date of the first infusion, and then every 12 weeks.

      2.3 Blood and tumour sample processing

      Blood samples were obtained at baseline, 4 weeks, and every 8 weeks after treatment initiation until PD. The cytokines IL-6, IL-8, and IL-17 were analysed using the R&D Systems assay (Abingdon, UK) and the C-reactive protein (CRP) levels were measured as part of routine analyses. All the assays were performed in accordance with the manufacturer's instructions.
      When feasible, the ultrasound-guided core needle tumour biopsies were collected prior to (baseline) and 14–21 days after treatment initiation (on-treatment). The tumour biopsies were obtained from 13 of the patients who were enrolled in the clinical study (13 baseline and eight on-treatment biopsies) and processed immediately after collection (Appendix 1, p 6). The TILs were isolated from freshly processed uncultured tumour digests (FTDs) (Appendix 1, p 3). The expanded TILs that were successfully obtained from 20 of 21 biopsies were established in vitro from minced tumour fragments. The phenotype of the uncultured TILs was determined using flow cytometry with a broad panel of fluorochrome-conjugated monoclonal antibodies: CD56-BV605, CD16-PE-AF700, CD3-BV786, CD4-BV510, CD8-APC-R700, HLA-DR-PerCP-Cy5·5, CD28-APC, CD27-BV711, CD38-PE-Cy5, CD57-FITC, PD1-PE-CF594, CD39-BV421, CD103-PE, CD45RA-BV650, and CCR7-PE-Cy7 (Appendix 1, p 7). Due to the limited number of CD3+ cells detected in the FTDs (Appendix 1, p 6), the phenotypic characterisation of the uncultured TILs could only be performed on six paired baseline and on-treatment samples with sufficient uncultured TILs.
      The establishment of autologous tumour cell lines was more laborious than expected, resulting in 17 established adherent cell cultures (ACCs, 11 baseline and six on-treatment biopsies) (Appendix 1, p 6). Tumour-specific TIL activation after the co-culture with autologous ACCs was defined as the percentage of live CD3+CD4+ or CD3+CD8+ TILs that stained positive for the activation marker CD137, minus the control (TIL alone).

      2.4 Outcomes

      The primary end-point of this study was the objective response rate (ORR, defined as the proportion of patients with a complete response or partial response) on the basis of the investigator-assessed RECIST v1.1. The key secondary end-points were safety, based on the assessment of adverse events (AEs) and serious AEs, duration of response (DoR, defined as the time from the date of first documented response until date of documented progression or death in the absence of disease progression), disease control rate at 16 weeks (DCR16, defined as confirmed complete or partial response or stable disease (SD) for ≥16 weeks), progression-free survival (PFS), and overall survival (OS). The exploratory analyses included the assessment of biomarkers (including TILs, tumour-specific TIL activation, cytokines, and CRP) that were hypothesised to show a potential correlation with clinical activity.

      2.5 Statistical analysis

      The clinical cut-off date was February 7, 2022, for Part A. Once the 15 patients completed two post-baseline tumour assessments (at weeks eight and 16), the predictive probability was calculated to assess the possibility of observation in at least five out of 30 responses (>15% ORR, and/or at least 15/30 DCR at 16 weeks (≥50% DCR16). If the predictive probability fell below 10% for both end-points, enrolment would have been stopped. However, recruitment continued, and the predictive probability was evaluated. Expansion to Part B was based on responses from Part A. A safety analysis was performed on the as-treated population, defined as all the enrolled patients who received one or more doses of ipilimumab, nivolumab, and tocilizumab. The ORR was assessed in the response-evaluable population, defined as patients who received at least one dose of study treatment and had one or more post-baseline scans or experienced clinical disease progression or death. The ORR, DCR, DCR16, and the accompanying 95% confidence intervals (CIs) were estimated using the exact binomial method. The DoR was estimated using the Kaplan–Meier method. The statistical efficacy analyses were performed using R software (version 3.6.1). To characterise the TILs and the tumour-specific TIL activation, statistical tests were conducted using a paired Wilcoxon matched-pair signed-rank test. The baseline and on-treatment cytokines and CRP were analysed, with blinded clinical data, correlated with the survival using the Kaplan–Meier method, and stratified using the median values for the cytokines and a cut-off of 10 mg/L for CRP. P was set than 0.05. GraphPad Prism version 9.2.0 was used to perform the statistical analyses and generate graphs.

      3. Results

      3.1 Patients

      Between April 17, 2020, and January 25, 2021, 33 patients with PC were screened for eligibility, of whom 26 were enrolled and assigned to ipilimumab, nivolumab, tocilizumab, and SBRT with 15 Gy (Fig. 1). The median age of the study population was 62 years (interquartile range [IQR] 54–71) (Table 1). Fourteen (54%) patients had an ECOG PS of zero and 12 (46%) had an ECOG PS of 1.24 (92%) of patients had ductal adenocarcinoma, one patient had mucinous carcinoma, and one had unspecified carcinoma. All patients had metastatic disease by the time of inclusion. Liver metastases were registered in 20 (77%) patients, and 16 (62%) had a weight loss of ≥5%. The median time from diagnosis of advanced disease to inclusion was 12.9 months (IQR 10.3–18.1). Twenty-one (81%) patients were pre-treated heavily with ≥2 therapies prior to inclusion. All the patients had received previous gemcitabine-based chemotherapy, and 20 (77%) fluorouracil-based therapy (Appendix 1, pp 8–9). The resection of primary tumour was performed in seven (27%) patients combined with adjuvant or neoadjuvant chemotherapy in four and three patients, respectively. All the patients had mismatch repair-proficient tumours, as determined by the immunohistochemical staining of MLH1, PMS2, MSH2, and MSH6.
      Fig. 1
      Fig. 1Consort diagram. Abbreviations: AE, adverse event; PD, progressive disease.
      Table 1Baseline patient and demographic and disease characteristics.
      Characteristic(n = 26)
      Median age, years (IQR)62 (54–71)
      Sex, No. (%)
       Male16 (62)
      ECOG PS, No. (%)
       014 (54)
       112 (46)
      Weight loss, No. (%)
       <5%6 (23)
       ≥5%16 (62)
      Number of metastatic sites, No. (%)
       18 (31)
       28 (31)
       ≥310 (38)
      Sites of metastasis, No. (%)
       Liver20 (77)
       Lung9 (35)
       Peritoneum3 (12)
      Previous Whipple procedure, No. (%)
       Yes7 (27)
      Biliary stent, No. (%)
       Yes2 (8)
      Thromboembolic event, No. (%)
       Yes11 (42)
       CA19–9, kU/L, median (IQR)4425 (157–52800)
       NLR, median (IQR)3.6 (3.0–4.8)
       Albumin, g/L, median (IQR)37 (35–40)
       CRP, mg/L, median (IQR)13.5 (4–25)
      mGPS, No. (%)
       012 (46)
       19 (35)
       25 (19)
      Number of prior therapy lines for advanced disease, No. (%)
       15 (19)
       ≥221 (81)
      Type of prior therapy, No. (%)
       Gemcitabine-based26 (100)
       Fluorouracil-based20 (77)
       Immunotherapy2 (8)
      BOR to last prior therapy, No (%)
       Partial response5 (19)
       Stable disease12 (46)
       Progressive disease8 (31)
       Not evaluable1 (4)
      Median time from diagnosis of advanced disease to inclusion, months (IQR)12.9 (10.3–18.1)
      MMR-proficient tumours, n (%)26 (100)
      NOTE: Data are number (%) unless otherwise indicated. Columns can add up to greater than 100% because some patients are listed in more than one group.
      Abbreviations: BOR, best overall response; CA19-9, carbohydrate antigen 19-9; CRP, C-reactive protein; ECOG PS, Eastern Cooperative Oncology Group Performance Status; IQR, interquartile range; mGPS, modified Glasgow Prognostic Score; MMR, mismatch repair; NLR, Neutrophil-to-Lymphocyte Ratio.

      3.2 Efficacy

      At the time of the data cut-off, the median follow-up time after the first dose was 4.9 months (IQR 2.1–7.7). All the patients showed disease progression, and 25 patients died. According to RECIST v1.1, there were no responders; the percentage change in the target lesion size and the best overall response are summarised in Fig. 2A. Five patients achieved SD, resulting in a DCR of 19% (95% CI, 7–39), and the DCR16 was 4% (95% CI 0–20) (Appendix 1, p 10). The trial did not meet the pre-specified criteria for expansion to full accrual. The median PFS was 1.6 months (95% CI 1.4–1.7) (Fig. 3A). The 3- and 6-months PFS rate were 15% (95% CI, 6–38) and 4% (95% CI, 1–26), respectively. The median OS was 4.9 months (95% CI 2.3–8.0) (Fig. 3B). At 6 months and 1 year, the rates of surviving patients were 42% (95% CI 27–66) and 4% (95% CI 1–26), respectively.
      Fig. 2
      Fig. 2Efficacy data based on radiologic response per RECIST v1.1 in evaluable patients (A). Best change in sum of diameters for target lesion is presented. Each bar represents one patient coloured according to the best overall response (∗ Overall PD due to progression of non-target lesions or new lesions). Efficacy outcomes in all analysed patients (B). The length of the bar represents time on study. Responses in patients without radiologic assessment were based on clinical assessment. Abbreviations: BOR, best overall response; PD, progressive disease; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease.
      Fig. 3
      Fig. 3Kaplan–Meier plot of PFS (A) and OS (B). Abbreviations: CI, confidence interval; OS, overall survival; PFS, progression-free survival.

      3.3 Safety

      All participants received SBRT with 15 Gy. A median of two doses (range 1–7) of nivolumab and tocilizumab, and a median of one dose (range 1–2) of ipilimumab were administered. Toxicity was evaluable for toxicity (Table 2). The most common treatment-related adverse events (TRAEs) (all grades, all cycles) experienced by patients were hyperthyroidism (35%), rash (27%), pruritus (23%), nausea (15%), mucositis oral (15%), arthralgia (15%), hypothyroidism (12%), increased alanine aminotransferase (12%), and increased aspartate aminotransferase (12%). Grade 3 or 4 AEs included nausea (4%) and increased aspartate aminotransferase levels (4%). Three patients discontinued treatment owing to AEs, of which two were related to treatment. One patient developed gastrointestinal bleeding, and gastroscopic examination revealed extensive tumour bleeding, which eventually resulted in death. No other grade 5 AEs were observed.
      Table 2Summary of treatment related AEs occurred in at least 5% of all treated patients.
      (n = 26)
      Any gradeGrades 3–4
      Any AEs25 (96)13 (50)
      TRAEs19 (73)2 (8)
      Fatigue2 (8)0
      Anorexia2 (8)0
      Nausea4 (15)1 (4)
      Vomiting2 (8)0
      Diarrhoea2 (8)0
      Mucositis oral4 (15)0
      Adrenal insufficiency2 (8)0
      Hypothyroidism3 (12)0
      Hyperthyroidism9 (35)0
      Arthralgia4 (15)0
      Increased ALT3 (12)0
      Increased AST3 (12)1 (4)
      Blood bilirubin increased2 (8)0
      Pruritus6 (23)0
      Rash maculo-papular7 (27)0
      NOTE: Data are n (%) unless otherwise indicated.
      Abbreviations: AEs, adverse events; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TRAEs, treatment related AEs.

      3.4 TIL phenotype characterisation and activation status during immunotherapy

      To monitor the effects of immunotherapy on the proportion, differentiation, potential tumour reactivity, and dysfunctional status of intratumoural T cells, we first analysed FTDs in paired biopsies obtained at baseline and during treatment. No significant differences in the proportion of TILs or distribution of CD4+ and CD8+ TIL subsets were observed (Appendix 1, p 12). In addition, we observed no changes in the expression levels of CD57, CD27, CD28, HLA-DR, CD38, CD39 + CD103 (Appendix 1, p 12), CD45RA, and CCR7 (data not shown). However, we also observed a reduction in PD-1 expression upon treatment that was likely related to competition between nivolumab and the flow cytometry PD-1 antibody used in these analyses [
      • Fung K.L.
      • et al.
      Steric effects of therapeutic anti-PD-1 drugs, pembrolizumab and nivolumab, that interfere with binding of diagnostic anti-PD-1 antibodies.
      ].
      Finally, we examined the tumour reactivity of the expanded TILs by measuring the upregulation of CD137 after the co-culture with autologous ACCs. This was tested in an autologous matched fashion against both the baseline and on-treatment ACCs, with 17 available ACCs. However, we did not observe an upregulation of CD137 in any of the TILs (data not shown), thereby failing to provide any evidence of tumour reactivity.

      3.5 Cytokines and CRP analyses

      Lower baseline serum concentrations of IL-6 were associated with a longer median OS (below versus above median values), 6.9 months (95% CI 5.0–non-reached) versus 2.2 months (95% CI 1.6–non-reached), Appendix 1, p 13). The normal CRP prior treatment tended to be associated with better survival (Appendix 1, p 13). However, no markers that were measured after treatment were associated with survival (Appendix 1, pp 14–15). The serum IL-6 levels increased rapidly after treatment and more profoundly (up to 24 × ) 8 weeks after the first tocilizumab administration (Appendix 1, p 16). Suppression of the CRP was observed, while IL-8 and IL-17 did not change significantly after treatment at the 4- and 8-week time points (Appendix 1, p 16).

      4. Discussion

      The therapeutic options for patients with chemorefractory PC are limited. We hypothesised that the combination of ipilimumab, nivolumab, tocilizumab, and SBRT with 15 Gy would demonstrate clinical activity in patients with PC who had progressed on at least one line of chemotherapy. The threshold for efficacy was not met, as no responses in terms of RECIST v1.1 were observed. To our knowledge, this study was the first phase 2 trial to evaluate an IL-6 blockade combined with the use of ICI and radiation in patients with refractory PC.
      The combination of ipilimumab, nivolumab, tocilizumab, and SBRT with 15 Gy was well tolerated and the overall safety was consistent with outcomes observed with ipilimumab, nivolumab, and SBRT with 15 Gy in patients with resistant PC (CheckPAC study) [
      • Chen I.M.
      • et al.
      Randomized phase II study of nivolumab with or without ipilimumab combined with stereotactic body radiotherapy for refractory metastatic pancreatic cancer (CheckPAC).
      ]. However, the treatment exposure in the present trial was low, and only 11 of the 26 patients (42%) had at least two infusions of each investigational drug, that is, received treatment for more than 6 weeks. Most of the patients discontinued treatment because of PD. The most common TRAE were hyperthyroidism and rash (mostly grade 1 or 2). Notably, the most frequently observed grade 3–4 TRAE that we observed in the CheckPAC trial, such as diarrhoea, fatigue, adrenal insufficiency, colitis, arthralgia, myalgia, and increased serum aspartate aminotransferase levels, were not registered or occurred at a low prevalence in our study. These observations supported the notion that the addition of an IL-6 blockade has the potential to reduce immune-related AEs in ICI-treated patients [
      • Stroud C.R.
      • et al.
      Tocilizumab for the management of immune mediated adverse events secondary to PD-1 blockade.
      ,
      • Holmstrøm R.B.
      • et al.
      Results from an intervention study investigating IL-6 blockade with tocilizumab for treatment of immune checkpoint inhibitor induced colitis and arthritis.
      ,
      • Dimitriou F.
      • et al.
      Interleukin-6 blockade for prophylaxis and management of immune-related adverse events in cancer immunotherapy.
      ].
      The presence of an immunosuppressive TME and the paucity of T cells in PC tumours are believed to be responsible for resistance to most single-agent immunotherapeutic approaches [
      • Bear A.S.
      • Vonderheide R.H.
      • O'Hara M.H.
      Challenges and opportunities for pancreatic cancer immunotherapy.
      ]. A combination of multimodal immune-modulating strategies aimed at enhancing the immune response and neutralising immunosuppressive elements within the TME has been recommended to overcome immune resistance. In our recently published CheckPAC study, we found that the combination of nivolumab and ipilimumab plus SBRT resulted in a 37.2% clinical benefit rate in patients with refractory metastatic PC [
      • Chen I.M.
      • et al.
      Randomized phase II study of nivolumab with or without ipilimumab combined with stereotactic body radiotherapy for refractory metastatic pancreatic cancer (CheckPAC).
      ]. Decreased serum IL-6 levels during treatment were associated with better OS. This was consistent with published data from ICI-treated melanoma patients, demonstrating that increased levels of IL-6 were associated with shorter survival [
      • Laino A.S.
      • et al.
      Serum interleukin-6 and C-reactive protein are associated with survival in melanoma patients receiving immune checkpoint inhibition.
      ]. In a phase 2 study aimed at investigating the value of ipilimumab, nivolumab, and tocilizumab inhibition in patients with melanoma, 14 RECIST responses in 20 patients (70% ORR) were observed at the initial assessment after the first stage with a 6-month median follow-up [
      • Weber J.S.
      • et al.
      Phase II trial of ipilimumab, nivolumab and tocilizumab for unresectable metastatic melanoma.
      ]. Additionally, reduced immune-related AEs were observed in 41 patients evaluable for toxicity.
      Despite compelling preclinical rationale and biomarker data, the addition of tocilizumab to ICI and radiation in the present study did not show any clinically relevant benefit in patients with PC. In contrast to the responses observed in the CheckPAC study, that used the same indication and inclusion criteria, the therapeutic role of IL-6 blockade in patients with PC can be questioned. In the present study, the serum IL-6 level prior to treatment was associated with survival, reflecting the disease burden. However, we have not tested tumour expression of IL-6 and/or IL-6 receptor, which levels can influence susceptibility to tocilizumab-mediated death [
      • Hong C.
      • et al.
      cGAS-STING drives the IL-6-dependent survival of chromosomally instable cancers.
      ]. The serum IL-6 levels increased markedly after tocilizumab administration. We hypothesised that increases in serum IL-6 levels occurring after IL-6 signalling inhibition by tocilizumab may have pathologic significance, and whether IL-6 signalling was completely inhibited by the tocilizumab that was given every 4 weeks in our study, remains to be fully understood. Furthermore, IL-6 is a multipotent cytokine with a wide range of biological activities, which in turn, aside from pro-tumour functions may also exert antitumour effects. However, the potential impact of IL-6 on the generation of antitumour immune responses within a specific TME, including in PC, remains unclear [
      • Fisher D.T.
      • Appenheimer M.M.
      • Evans S.S.
      The two faces of IL-6 in the tumor microenvironment.
      ].
      Our translational analyses failed to observe any significant change in the TIL proportion or phenotype ex vivo that was induced by immunotherapy. PC is a cold tumour with a low TIL density compared to melanoma, and overall, we recovered a very low number of CD3+ cells within the FTDs [
      • Blando J.
      • et al.
      Comparison of immune infiltrates in melanoma and pancreatic cancer highlights VISTA as a potential target in pancreatic cancer.
      ,
      • Ino Y.
      • et al.
      Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer.
      ]. Therefore, it remains unclear whether the absence of treatment-induced changes was due to the poor immunological response to immunotherapy and, subsequently, the lack of significant T cell infiltration in the tumours, or other technical issues, such as the low number of cells recovered and the small patient cohort, failing to provide sufficient statistical power. Despite the decrease in PD-1 surface expression on TILs during treatment, it was likely due to competition between nivolumab and the flow cytometry PD-1 antibody [
      • Fung K.L.
      • et al.
      Steric effects of therapeutic anti-PD-1 drugs, pembrolizumab and nivolumab, that interfere with binding of diagnostic anti-PD-1 antibodies.
      ]. These results confirmed indirectly that immunotherapy treatment affected TILs either by reaching the TME directly or by inducing changes in the TME indirectly (e.g., clonal repopulation of TILs from nivolumab-bound peripheral T cells). Our experiments failed to provide any evidence of tumour reactivity; however, our assay could not confirm whether this was due to a genuine absence of tumour-specific TILs in the TME or a lack of naturally presented tumour antigens in our autologous ACCs due to the prevalence of other cell types such as fibroblasts.
      The study had some limitations. First, only 26 patients received the experimental treatment, and no responses were observed. This prevented the assessment of biomarkers that correlate with clinical activity. Second, the median time from diagnosis to inclusion of the current cohort does not reflect general population. Third, this was a single-arm study, which did not allow for direct comparisons with other therapeutic options. However, most of the patients included in our study received ≥2 prior therapy lines for metastatic disease, and for these patients, no established therapeutic options exist other than enrolment in a clinical trial.
      In conclusion, we did not observe efficacy with the combination of ipilimumab, nivolumab, tocilizumab, and SBRT with 15 Gy in patients with refractory PC; however, the treatment had a manageable safety profile. Studies to understand the immunosuppressive TME of PC and its resistance mechanisms are recommended.

      Data sharing

      Appendix 2 presents the study protocol. The trial protocol did not include a data-sharing plan; therefore, data from the trial will not be shared publicly, as data sharing was not included when ethical approval was requested. The anonymised participant data will be made available when the trial is complete upon requests directed to the corresponding author. The proposals will be reviewed and approved by the sponsor, investigator, and collaborators based on scientific merit. After approval of a proposal, data can be shared through a secure online platform after signing a data-access agreement.

      Funding

      This study was supported by grants from the Danish Cancer Society (grant no. R268-A15344) and Jørgen Holm and Elisa f. Hansens Mindelegat (Grant No. 20105).

      Conflict of interest statement

      The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
      IMC reports institutional research grants from Roche, Bristol Myers Squibb, Celgene, Genis, and Varian Medical Systems; advisory board fees from Amgen; and travel and accommodation expenses from Roche, Bristol Myers Squibb, Celgene, and Bayer. PG reports institutional research grants from Roche, Bristol Myers Squibb, Pfizer, MSD, and Merck and travel and accommodation expenses from Novartis, Bristol Myers Squibb, and Pfizer. IMS reports institutional research grants from Adaptimmune, Enara Bio, Evaxion, Lytix Biopharma, TILT Biotherapeutics, Bristol Myers Squibb, Novartis, and Roche; advisory fees from Bristol Myers Squibb, Novartis, and Pierre Fabre; consulting honoraria from Bristol Myers Squibb, MSD, Novartis, Pierre Fabre, Roche, and IO Biotech; honoraria for lectures from Bristol Myers Squibb, MSD, Novartis, Pierre Fabre, and Roche; stock and ownership interests in IO Biotech. JSJ reports institutional research grants from Roche, Bristol Myers Squibb, and Celgene and travel and accommodation expenses from Roche, Bristol Myers Squibb, Celgene, and Bayer. DN reports institutional research grants from Eli Lilly, Merck, MSD, Genmab, and Boehringer Ingelheim. MD reports honoraria for lectures from Roche (past two years), access to research data from Bristol Myers Squibb, and from Genentech, an advisory role for Achille Therapeutics.
      All other authors declare no competing interests.

      Clinical trial information

      NCT04258150 (TRIPLE-R) and EudraCT No. 2019-004438-40.

      Role of the funding source

      The funders played no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data that was used in the study and had final responsibility for the decision to submit for publication.

      Author statement

      IMC, JSJ, and DN: Conceptualisation, Methodology. IMC, MD, CAC, AWPJ, AD, EH, AT, TL, EEW, PG, IMS, JSJ, and DN: Investigation and Project administration. IMC, KM, and ST: Formal analysis, Visualisation and Data curation. IMC: Supervision and Writing – Original draft preparation. All the authors: Reviewing and editing.

      Acknowledgements

      The authors would like to thank the patients, their families, caregivers, and all participating clinical teams for making this study possible. We are very grateful to the Department of Oncology, Herlev & Gentofte Hospital, for their support. Many thanks to the biomedical laboratory scientists, Charlotte Falk and Vibeke H. Holm for their assistance with blood sampling. We thank MSc Christina Grønhøj for her assistance with tissue sampling. The nurses Marianne Christiansen, Karen C. Petersen, Anne B. Christiansen, Merete Facius, and Charlotte Føge Vebner are acknowledged for their contribution in terms of the inclusion of patients, data registration, and sample collection. We thank Editage for English language editing. We thank the physicists, Dorte Klitgaard and Karen Andersen, for their support with stereotactic body radiotherapy plans.

      Appendix A. Supplementary data

      The following are the supplementary data to this article:

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