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Hepatopancreaticobiliary Resection Arginine Immunomodulation (PRIMe) trial: protocol for a randomised phase II trial of the impact of perioperative immunomodulation on immune function following resection for hepatopancreaticobiliary malignancy
  1. Ramy Behman1,
  2. Rebecca C Auer2,3,
  3. Lev Bubis1,
  4. Grace Xu4,
  5. Natalie G Coburn1,4,5,
  6. Guillaume Martel6,7,
  7. Julie Hallet1,4,5,
  8. Fady Balaa6,
  9. Calvin Law1,4,5,
  10. Kimberly A Bertens6,
  11. Jad Abou Khalil6,
  12. Paul Jack Karanicolas1,4,5
  1. 1Surgery, University of Toronto, Toronto, Ontario, Canada
  2. 2Cancer Research Program, The Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
  3. 3Surgery, University of Ottawa, Ottawa, Ontario, Canada
  4. 4Evaluative Clinical Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada
  5. 5Surgery, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada
  6. 6Surgery, The Ottawa Hospital, Ottawa, Ontario, Canada
  7. 7Clinical Epidemiology Program, The Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
  1. Correspondence to Dr Paul Jack Karanicolas; paul.karanicolas{at}sunnybrook.ca

Abstract

Introduction Surgical stress results in immune dysfunction, predisposing patients to infections in the postoperative period and potentially increasing the risk of cancer recurrence. Perioperative immunonutrition with arginine-enhanced diets has been found to potentially improve short-term and cancer outcomes. This study seeks to measure the impact of perioperative immunomodulation on biomarkers of the immune response and perioperative outcomes following hepatopancreaticobiliary surgery.

Methods and analysis This is a 1:1:1 randomised, controlled and blinded superiority trial of 45 patients. Baseline and perioperative variables were collected to evaluate immune function, clinical outcomes and feasibility outcomes. The primary outcome is a reduction in natural killer cell killing as measured on postoperative day 1 compared with baseline between the control and experimental cohorts.

Ethics and dissemination This trial has been approved by the research ethics boards at participating sites and Health Canada (parent control number: 223646). Results will be distributed widely through local and international meetings, presentation, publication and ClinicalTrials.gov (identifier: NCT04549662). Any modifications to the protocol will be communicated via publications and ClinicalTrials.gov.

Trial registration number ClinicalTrials.gov identifier: NCT04549662.

  • IMMUNOLOGY
  • NUTRITION & DIETETICS
  • Hepatobiliary tumours
  • Hepatobiliary surgery
  • Pancreatic surgery
http://creativecommons.org/licenses/by-nc/4.0/

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Strengths and limitations of this study

  • This is the first randomised, blinded and controlled study investigating the impact of immunonutrition on immune cell function in patients having hepatopancreaticobiliary resection for cancer.

  • Extensive data collection allows for analysis of immune function, clinical and feasibility outcomes.

  • The broad eligibility criteria allow for better generalisation of nutritional supplement immunomodulation in hepatopancreaticobiliary resections.

  • Lack of standardisation in care, anaesthesia and surgical technique may yield variability.

Introduction

Background

Surgical resection remains the only curative intent option for patients with hepatopancreaticobiliary (HPB) malignancies. Although perioperative morbidity and mortality have been declining due to advancements, there are still numerous complications following pancreas or liver resections. Common complications include infections, intra-abdominal haemorrhage, coagulation disorders, pancreatic leaks, bile leakage and liver failure.1–5 Furthermore, HPB malignancies have a high likelihood of cancer recurrence and ultimately cancer-related mortality following resection.6–12

Surgical stress results in immune dysfunction, predisposing patients to infections in the postoperative period.13 Surgical stress results in the immediate release of myeloid derived suppressor cells (MDSC) from the bone marrow. MDSC express high levels of the enzyme arginase and rapidly metabolise arginine.14 The resulting arginine deficiency results in impaired T-lymphocytes, which are essential for adequate wound healing. In addition to T-cell function, the proliferative capacity, cytotoxicity and cytokine production (interferon-ɣ) of natural killer (NK) cells is greatly impaired in the absence of arginine.

There is strong preclinical rationale to support the concept that surgery facilitates cancer recurrences and metastases secondary to postoperative immune suppression and that reversing this effect can improve cancer outcomes. Experiments using a validated animal model of surgical stress and metastases demonstrated that surgery is associated with increased metastases secondary to impaired NK cell killing. The key mediators of this effect are MDSC, which upregulate arginase (ARG1) expression and are significantly better at suppressing NK cell killing following surgical stress.15 These preclinical studies provide a link between perioperative arginine supplementation and reduced metastases and recurrence. Furthermore, omega-3 fatty acids attenuate the activation of MDSCs arising from surgically-inducted trauma. This mechanism protects against the metabolism of arginine by lowering the upregulation of ARG1. Therefore, these studies suggest that perioperative supplementation with arginine and omega-3 fatty acids may help counter the expected immune suppression that arises from surgery itself.16 17

The only published study to evaluate the ability of perioperative arginine supplementation to improve cancer survival demonstrated a significant improvement in disease-specific survival which included 32 malnourished patients with head and neck cancer.18

A perioperative nutritional formulation, enhanced in arginine, has been shown in a number of randomised studies to reduce infectious postoperative complications and hospital length of stay (LOS) following major surgery.19 In a meta-analysis including 35 studies (1280 total patients) examining perioperative effects of supplementation with arginine, nucleotides and omega-3-fatty acids, there was a statistically significant reduction in overall infectious complications (pooled risk ratio (RR)=0.59) and reduced overall LOS (weighted mean difference of 2.4 days) when compared with a control nutritional supplement.19 Furthermore, several studies have demonstrated a reduction in the incidence of anastomotic leak following procedures for head and neck malignancies.20–22 To date, there are no studies specifically evaluating the effect of perioperative immunonutrition with arginine on immune cell function in patients undergoing HPB resection for cancer.

Trial objectives

  1. To measure the impact of perioperative immunomodulation with arginine±omega-3 fatty acids on biomarkers of the immune response following HPB surgery.

  2. To determine the impact of perioperative immunomodulation on perioperative outcomes following resection of an HPB malignancy.

  3. To evaluate the feasibility to enrol, randomise and follow patients undergoing resection for HPB malignancy in anticipation of a future phase III trial.

Overview of trial design

This is a 1:1:1 randomised, controlled and blinded superiority trial to evaluate the impact of perioperative immunosupplementation on NK-cell function following resection of HPB malignancies. Two variations of immunosupplementation will be compared with the control nutritional supplement. Participants were recruited from two sites: (1) Sunnybrook Health Sciences Centre (SHSC) in Toronto, Ontario, Canada; and (2) The Ottawa Hospital in Ottawa, Ontario, Canada. The first participant was randomised on 16 September 2021 and the last participant was randomised on 20 October 2023. Please refer to figure 1 for the schedule of events.

Figure 1

Schedule of events. HPB, hepatopancreaticobiliary.

Methods

Immunosupplementation intervention

A blinded, pre-packaged powder manufactured and provided in kind by Enhanced Medical Nutrition under the trade name ‘INergy’, which contains supraphysiological arginine (Active A) will be provided for study participants in experimental group A. In addition, participants in experimental group A will receive a lipid bolus containing omega-3 fatty acids. Active A will also be provided for study participants in experimental group B with a placebo oil that does not contain omega-3 fatty acids. The control will consist of a pre-packaged powder similar to the experimental drug. This will be provided by the same manufacturer as the experimental formula (Enhanced Medical Nutrition). This formulation will only consist of whey protein and will not contain arginine (Active B). Participants in the comparator arm will also receive a teaspoon (tsp) of placebo oil which does not contain omega-3 fatty acids.

Participants will mix the powder (Active A or Active B) with 250 mL of water and drink the formula three times per day for 5 days. Each drink will be followed by consuming one tsp of the lipid bolus or placebo oil. This will be done for 5 days prior to the operation and up to 5 days after the operation, beginning as soon as they are able to tolerate fluids orally. If a participant is unable to tolerate oral intake after starting the supplements postoperatively, the intervention will be discontinued permanently.

Outside of investigational product intake and blood sample collection, preoperative and postoperative care will be left to the discretion of the healthcare providers as per their baseline standards of care. Please refer to table 1 for the schedule of study assessments.

Table 1

Schedule of study assessments

Primary outcome

The primary outcome is the reduction in NK cell killing as measured on postoperative day (POD) 1 compared with baseline between the control and experimental cohorts. A flow cytometry-based killing assay will be used to specifically measure NK cell cytotoxicity by using fluorescent cell dyes. This will distinguish between live or dead effector cells (the participant peripheral blood mononuclear cells (PBMCs) or the control NK92 cell line) and target cells (the K562 erythroleukaemia cell line). Arginine supplementation will be considered effective at reducing postoperative NK cell dysfunction if there is a 50% or greater improvement in postoperative suppression of NK cell cytotoxicity (reduction of 27% from baseline).

Secondary outcomes

  • Secondary immune function outcomes:

    • Flow cytometric analysis of different immune cell subsets (example panel: CD3, CD14, CD56, CD16, CD4, CD8) to compare their proportions at all time points.

    • Flow cytometric characterisation of NK cell activating and inhibitory receptors (example panel: CD3, CD56, CD16, CD226, NKG2D, CD96, CD69, TIGIT) to assess whether they are affected by immunosupplementation.

    • Systemic blood levels of amino acids will be measured via liquid chromatography-mass spectrometry protein card analysis.

  • Secondary clinical outcomes (exploratory)

    • Incidence of pancreatic fistula (in a subgroup of patients having pancreatic surgery) using the Pancreatic fistula of International Study Group on Pancreatic Fistula (ISGPF) Grade B and C (ie, clinically significant).

    • Incidence of liver insufficiency (in a subgroup of patients having liver resection) using the posthepatectomy liver failure of International Study Group of Liver Surgery (ISGLS) Grade B and C (ie, clinically significant).

    • Postoperative wound complication and surgical site infection.

    • LOS (number of days from POD 0 to discharge).

    • 90-day postoperative complications (Clavien-Dindo Grades 3–5).

    • 90-day postoperative readmissions and LOS.

    • 90-day postoperative mortality.

Feasibility outcomes

Feasibility data for phase III trial—before embarking on a national definitive randomised controlled trial (RCT), this trial has six specific feasibility objectives:

  1. To determine the proportion of participants screened that meet the eligibility criteria for a definitive trial.

  2. To calculate the proportion of eligible participants who consent to participate in this trial.

  3. To assess our ability to randomise participants and adherence to the group allocations.

  4. To examine our ability to collect complete perioperative data and samples for biochemical testing.

  5. To assess adherence with perioperative immunosupplementation as per the protocol.

  6. To estimate the resource requirements for potential participating sites to conduct the full RCT.

Criteria for subject eligibility

Since this is a preliminary exploratory study, we elected to include patients with a range of upper gastrointestinal malignancies undergoing major cancer surgery. It is possible, although unlikely, that the impact of arginine and omega-3-fatty acids differ in patients based on underlying cancer or procedure. Although this study lacks the power to examine this fully, future larger studies may explore this possibility with subgroup analyses. Further, the stratified randomisation based on the type of surgery should balance this potential confounder between groups.

A small minority of participants may be found to have unresectable malignancies intraoperatively, at which point the resection will not be performed. Alternatively, a small minority of participants may be found to not have malignancy on histopathology. Some outcome measures may not be relevant to these two subsets of participants.

Inclusion criteria

Patients aged 18 or older at the time of a diagnosis of resectable presumed liver, pancreas and/or bile duct malignancy (ie, liver metastasis, hepatocellular carcinoma (HCC), cholangiocarcinoma, pancreatic or periampullary adenocarcinoma, neuroendocrine tumours) are eligible for the trial. Patients must also have an anticipated stay of at least 3 days postoperatively, and be able to tolerate oral intake and comply with protocol requirements at the time of consent.

Exclusion criteria

Patients will be excluded if any of the following criteria are met:

  1. Documented significant immunodeficiency due to underlying illness (eg, HIV/AIDS) and/or medication (eg, systemic corticosteroids, azathioprine, ciclosporin A). Subjects may be on physiological doses of replacement prednisone or equivalent doses of corticosteroid (<7.5 mg daily).

  2. Subjects with resting hypotension (blood pressure <90/50 at rest).

  3. History of autoimmune disease, such as but not restricted to, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, scleroderma or multiple sclerosis.

  4. Serious, active, intercurrent chronic or acute illness or other active illness considered by the investigator as an unwarranted high risk for an investigational product.

  5. Active infection of any site and/or active herpes requiring ongoing treatment.

  6. Known pregnancy or nursing mothers.

  7. Allergy to the nutritional supplement or comparator including milk, fish and/or shellfish allergies.

  8. Religious or other objections to consuming fish or shellfish.

  9. Severe asthma is defined as asthma not controlled with inhaled corticosteroids and additional controllers or by oral corticosteroid treatment (arginine can cause allergic response or make swelling in airways worse).

  10. Known inherited guanidinoacetate methyltransferase deficiency (due to an inability to convert arginine to creatine).

  11. Known current liver cirrhosis.

  12. Documented myocardial infarction or life-threatening arrhythmia.

  13. Known current cardiac failure or coronary artery disease causing unstable angina.

Sample size and sample size justification

Immunonutrition will be considered effective at reducing postoperative NK cell dysfunction if there is a 50% or greater improvement in postoperative suppression of NK cell cytotoxicity (reduction of 27% from baseline) as measured on POD 1 as compared with baseline. Previous studies in patients undergoing resection for solid tumours have demonstrated a reduction in NK cell cytotoxicity of 54% (0.54, SD 0.22) on POD 1 as compared with baseline when measured in a standard ex vivo 51Cr release assay (the gold standard in measuring NK-cell function).23–26 Given this baseline data, a sample size of 15 participants per group provides greater than 90% power to detect a 50% improvement in the experimental cohort at a significance level (alpha) of 0.05. If this study demonstrates adequate feasibility and a signal of efficacy, a phase III trial with a larger sample size will be explored.

Recruitment

Participants will be enrolled by their operating surgeons preoperatively with the assistance of delegated study personnel at the SHSC and the Ottawa Hospital. Delegated study personnel will obtain written informed consent from the participant or substitute decision-maker prior to any intervention. The informed consent form has been included as an online supplemental file 1 for reference.

Randomisation

We will conduct 1:1:1 randomisation using a computer-based randomisation programme designed by the team statistician. Randomisation will be stratified by site and by type of surgery (liver/bile duct vs pancreas).

Blinding (masking)

Participants, healthcare providers, outcome adjudicators and data collectors will not be aware of group allocation. The three experimental group products will be pre-packaged identically and distributed by blinded study personnel. Allocation codes will be maintained centrally and distributed to investigators only following data analysis. Only if there is a compelling medical or safety reason to do so, and only with approval from the medical monitor, will unblinding occur. The unblinding procedure will be standardised and will be detailed in trial operational documents.

Patient and public involvement

None.

Data collection

Pretreatment evaluation

Pretreatment evaluation will confirm that eligibility criteria are met. This includes the patient’s demographics, anticipated procedure, brief medical history, baseline laboratory values and research-specific blood samples.

Perioperative evaluation

Participants who are deemed ineligible or withdraw after starting the immunomodulation intervention are to permanently discontinue the intervention. No further study tests will be done, but safety data will still be collected (ie, complications, readmissions). Preoperative intake adherence, surgical information (American Society of Anaesthesiologists (ASA) score, blood loss, tissue(s) resected, etc), postoperative intake, research specific blood samples and corresponding laboratory values will be collected perioperatively. Intake adherence is recorded through patient diaries and hospital staff when applicable.

POD 30 evaluation (±7 days)

Blood samples to evaluate blood cells and serum markers for evidence of immune function.

POD 90 (±14 days) evaluation

Readmissions, complications including pancreatic leak (using ISGPF) and liver insufficiency (using ISGLS), final pathology and date of death if applicable will be collected at the POD 90 time point. This follow-up may be performed by phone or medical record.

Toxicities/side effects

Previous studies have demonstrated no side effects associated with ingestion of this type of product. We do not anticipate any toxicities associated with the trial intervention. Adverse events will be monitored up to 5 days after immunomodulation intervention.

Statistical methods

Primary biological endpoint—criteria for the efficacy of immune modulation

Immunonutrition will be considered effective at reducing postoperative NK cell dysfunction if there is a 50% or greater improvement in postoperative suppression of NK cell cytotoxicity (reduction of 27% from baseline) as measured on POD 1 compared with baseline.

Postoperative immune function (including NK-cell function as well as other biochemical markers of immune function) will be compared with preoperative baseline values using paired t-tests or Wilcoxon signed-rank tests as appropriate.

Clinical secondary outcomes including incidence of pancreatic fistula, liver insufficiency, wound complications, surgical site infections and mortality will all be recorded in a binary fashion. Univariate and multivariable regression analyses will be performed to assess the impact of the intervention on these clinical outcomes.

Participants who do not undergo resection of all visible disease (eg, due to more extensive disease identified intraoperatively) will be excluded from the primary analysis, but will still be included in the analysis for safety and toxicity.

Additional analyses

Subgroup analysis will be performed by histological subtype of malignancy, tumour stage and type of procedure performed if there is enough data available pertaining to the subgroup.

Secondary analysis will be performed per-protocol for the explanatory purpose of assessing the impact of adherence to the intervention on outcomes.

Monitoring

Site monitoring plan

The delegated monitor will evaluate study processes and documentation based on the approved protocol/amendment(s), Part C, Division 5 of the Food and Drug Regulations, the International Conference on Harmonisation, E6: Good Clinical Practice guidelines and institutional policies.

Auditing and inspecting

The investigator will provide direct access to source data/documents for the purposes of study-related monitoring, audits and inspections by the research ethics board (REB), the sponsor and applicable regulatory bodies. The investigator will permit the review of all study-related documents and will ensure access to applicable study-related facilities.

Subject privacy

Participant consent will be conducted by delegated study personnel. The eligible participant will be given as much time as they need in order to have all of their questions answered to their satisfaction and to fully understand the trial before deciding to participate. The study personnel will inform the participant of any trial-related deadlines that may impact their choice to participate.

Data confidentiality

Participant source documents will be de-identified and kept in locked file cabinets if hard copies are applicable. An online database will be used to enter data into electronic case report forms which will be password-protected and accessible via the internet.

Ethics statements

Patient consent for publication

Acknowledgments

The authors would like to thank the Centre for Clinical Trial Support (CCTS) staff for their data management and multicenter coordination (Sonya Mergler, Shirley Xu). Study data are collected and managed using Research Electronic Data Capture (REDCap

™) by CCTS (Kathryn Mangoff, Michael Lau, Suzanne Chung). REDCap™ is a secure, password protected, web-based application designed to support data capture for research studies (www.projectredcap.org).The authors would also like to thank their research staff at SHSC and The Ottawa Hospital who play a vital role in the day-to-day management of this trial. Most importantly, we would like to thank all past, present and future study participants for their contribution to this research and putting their trust in us. CCTS at the SHSC is responsible for the site training, start-up and activation, essential document management, supply management, database development, and data management. Study data will be entered by each site and maintained on REDCap.The qualified investigator for each site may appoint co-investigators to assist in trial oversight and execution. Each site will also onboard at least one research staff to assist in the daily coordination and data collection of the trial. A task delegation log will be completed at each site prior to activation including the investigators and research staff. Appropriate clinical and trial specific training documentation will also be collected.

References

Supplementary materials

  • Supplementary Data

    This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

Footnotes

  • X @ChamoGui

  • Contributors RB and LB participated in the design of the study. RCA participated in the design of the study and is leading the laboratory analysis. GX assisted in protocol write-up and data collection. NGC, GM, JH, FB, CL, KAB and JAK participated in the coordination and data collection for the study. PJK spearheaded the design, coordination and data collection for the study.

  • Funding This work was supported by Enhanced Medical Nutrition, the Odette Cancer Centre (OCC) Clinical Research Grant Competition, the Sunnybrook AFP Association through the Innovation Fund of the Alternative Funding Plan from the Academic Health Sciences Centres of Ontario (Project Code: SHS-17-003) and the Sunnybrook Health Sciences Centre. Funders were/are not involved in the design, coordination, data collection or publication of the study.

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.