TRITICC is an interventional, prospective, open-label, non-randomised, exploratory, multicentre, single-arm phase IIA clinical trial organized and sponsored by the University of Dusseldorf. Eligible patients with histologically or cytologically confirmed, non-resectable, locally advanced or metastatic cholangiocarcinoma or gall bladder carcinoma will receive a second line combination chemotherapy with FTD/TPI and irinotecan in cycles of 14 days. In total 28 patients (including 3 calculated dropouts and invalid cases) after failure of a gemcitabine based first-line therapy will be enrolled at up to 6 centers. Screening is expected to be necessary in 40 patients.

Study duration

The recruitment of patients for the inclusion of the study was started in Q3 2021. The recruitment period will last 18 months. Patients will be treated until radiological progression. In average this is estimated being about 4 months. A follow up is planned every 3 months up to 6 months. Last patient last visit is anticipated for August 2023. Based on these assumptions, duration will be in total 40 months including 12 months evaluation and clinical study report.

Trial population

Patients with cholangiocarcinoma or gall bladder carcinoma after progressing on a gemcitabine based first-line therapy and fulfilling the other inclusion criteria will be eligible to participate in this clinical trial.

Inclusion criteria 1.

Written informed consent incl. participation in translational research and any locally-required authorization (EU Data Privacy Directive in the EU) prior to performing any protocol-related procedures, including screening evaluations

2.

Age ≥ 18 years at time of study entry

3.

Histologically or cytologically confirmed, non-resectable, locally advanced or metastatic cholangiocarcinoma or gall bladder carcinoma

4.

Measurable or assessable disease according to RECIST

5.

Documented disease progression after prior gemcitabine or gemcitabine containing therapy. Examples of permitted therapies include, but are not limited to: a) Single agent gemcitabine); b) Any gemcitabine-based regimen, with or without maintenance gemcitabine

6.

ECOG performance status 0–1

7.

Ability to take medications orally

8.

Adequate blood count, liver-enzymes, and renal function:

9.

ANC > 1,500 cells/μL without the use of hematopoietic growth factors; and Platelet count ≥ 100 × 109/L (> 100,000 per mm3) and Hemoglobin > 9 g/dL (blood transfusions are permitted for patients with hemoglobin levels below 9 g/dL)

10.

Serum total bilirubin ≤ 1.5 × upper normal limit (ULN) (biliary drainage is allowed for biliary obstruction; elevated bilirubin should be caused by obstruction not impaired liver function as assessed by albumin and INR values):

11.

Albumin levels ≥ 3.0 g/dL

12.

Patients not receiving therapeutic anticoagulation must have an INR < 1.5 ULN and PTT < 1.5 ULN within 7 days prior to inclusion. The use of full dose anticoagulants is allowed as long as the INR or PTT is within therapeutic limits (according to the medical standard in the institution) and the patient has been on a stable dose for anticoagulants for at least three weeks at the time of inclusion.

13.

AST (SGOT) and ALT (SGPT) ≤ 5 × institutional upper limit of normal

14.

Serum Creatinine ≤ 1.5 × ULN and a calculated glomerular filtration rate ≥ 30 mL per minute Adequate renal and bone marrow function

15.

In case of liver cirrhosis: Child–Pugh A

16.

Women of childbearing potential must have a negative pregnancy test and must agree to adequate birth control if conception is possible. Males must agree to adequate birth control

Exclusion criteria 1.

Age < 18 years

2.

CNS metastases

3.

Active, uncontrolled infection

4.

Additional malignancy within the past 2 years (except adequately treated in-situ carcinoma of the cervix or non-melanoma skin cancer)

5.

Clinically significant gastrointestinal disorders including bleeding, inflammation, occlusion, or diarrhea > grade 1

6.

Any condition or comorbidity that, in the opinion of the investigator, would interfere with evaluation of study treatment or interpretation of patient safety or study results

7.

Known hypersensitivity to FTD/TPI or Irinotecan or their components

8.

Medication that is known to interfere with any of the agents applied in the trial

9.

Pregnancy or lactating female

10.

Prior partial or total gastrectomy

11.

Previous radio- or radiochemotherapy, previous transarterial chemoembolisation (TACE), radiofrequencyablation (RFA) or selective intraarterial radiotherapy (SIRT) within 3 months prior to inclusion (except radiation for bone metastases)

12.

Patients who might be dependent on the sponsor, site or the investigator

13.

Patients who have been incarcerated or involuntarily institutionalized by court order or by the authorities § 40 Abs. 1 S. 3 Nr. 4 AMG.

14.

Patients who are unable to consent because they do not understand the nature, significance and implications of the clinical trial and therefore cannot form a rational intention in the light of the facts [§ 40 Abs. 1 S. 3 Nr. 3a AMG].

15.

Use of other investigational treatment within 5 half-lives of enrollment is prohibited.

Gender and age selection

Adults of all genders equal or above 18 years are eligible for this clinical trial.

Tumor imaging

For tumor assessment according to RECIST version 1.1 whenever possible, MRI should be chosen as examination method. However, conventional CT is permitted as alternative. The examination method once selected is to be retained for all examinations within the context of the study. Chest CT and additional skeletal scintigraphy /X-ray images of all affected bones in case of suspected bone metastases and additional cranial MRI or CT (MRI preferred whenever possible) in case of suspected CNS metastases are to be implemented. Additional imaging may be required in symptomatic patients if clinically indicated.

Treatment regimen

Dosage and schedule were selected according to the results of Phase I study of FTD/TPI Plus Irinotecan and Bevacizumab in Advanced Gastrointestinal Tumors (Varghese AM et al.) [20].

FTD/TPI will be administered at a dose of 25 mg/m2 / dose twice daily on days 1–5 followed by a 9-days recovery period from day 6 trough day 14 of each 14-days treatment cycle.

The dosage is calculated according to body surface area (BSA) as displayed in Table 2. The dosage must not exceed 60 mg/dose. Table 2 also displays the necessary number of 15 mg and 20 mg tablets Lonsurf® to be taken. If any doses are missed, these doses must not be made up by the patient.

Table 2 Calculation of dosing for FTD/TPI in mg according to BSA used in the TRITICC trial

Irinotecan will be administered at the same time as FTD/TPI on day 1 of each cycle at a dose of 180 mg/m2 / dose.

Treatment will be administered until radiological progress or inacceptable toxicity.

Dose modifications and dose delays

Toxicity is classified according to NCI-CTCAE version 5.0; the dose modifications are made based on the grading given in the latter. The treatment must not be modified if adverse reactions occur which, according to the investigator's estimation, do not result in severe or life-threatening consequences (e.g. alopecia). Should several different toxicities appear simultaneously, the respective greatest dose reduction decrement should be applied. If an adverse reaction is unequivocally attributable to only one of the cytostatic agents the dosages of the other substances do not need to be modified. A further dose escalation is not permitted.

In case permanent discontinuation or treatment interruption of more than two cycles of one of the substances is required due to toxicity, study treatment has to be terminated. The further treatment will be at the treating physician's discretion.

Dose modification of FTD/TPI

A maximum of 1 dose reduction level (20 mg/m2 BSA BID) is permitted for FTD/TPI. Table 3 displays the dose in mg to be taken twice daily calculated per m2 BSA. (Tables 4 and 5).

Table 3 Calculation of dose reduction for FTD/TPI in mg according to BSA used in the TRITICC trialTable 4 Dose interruption and resumption criteria for haematological toxicities related to myelosuppression [21]Table 5 Recommended dose modifications for FTF/TPI in case of haematological and non-haematological adverse reactions [21]

Dose escalation of FTD/TPI is not permitted after it has been reduced.

Dose modification of irinotecan

Table 6 displays recommended dose modifications for Irinotecan in case of haematological or non-haematological adverse reactions.

Table 6 Recommended dose interruption/ modification criteriaConcomitant medication

All medication, which is considered necessary for the patient’s welfare, and which is not expected to interfere with the evaluation of FTD/TPI, may be given at the discretion of the investigator. All concomitant medications must be recorded in the patient’s source documentation, as well as in the appropriate pages of the eCRF. Subjects should receive full supportive care, including transfusions of blood and blood products, anti-infective drugs, and antiemetics when appropriate.

All patients should be closely monitored for side effects of all concomitant medications regardless of the path of elimination.

Antiemetics

During the treatment with FTD/TPI and Irinotecan, owing a moderate emetic potential, an antiemetic treatment should be administered according to center-specific protocols using, among others, dexamethasone and 5-HT3 antagonists. In case of insufficient effect, aprepitant (Emend®) may be considered. Appropriate prophylactic antiemesis is recommended.

Anticoagulants

The use of full dose anticoagulants is allowed as long as the INR or PTT is within therapeutic limits (according to the medical standards in the institution) and the patient has been on a stable dose for anticoagulants for at least two weeks at the time of registration. During treatment close monitoring of INR for oral anticoagulants is recommended.

Growth factors

Hematopoietic growth factors (i.e., G-CSF) may be used according to institutional guidelines to treat febrile neutropenia, but should not be used as primary or secondary prophylaxis. Growth factors must be discontinued at least 48 h prior to initiation of the next treatment of chemotherapy.

Prohibited concomitant medication

The following medications/treatments are not allowed during the study participation, unless the patient is in the follow-up phase and has permanently discontinued any study medication:

Any investigational medicinal product or experimental therapy or participation in another clinical trial. Any other anti-tumor treatment including chemotherapy, targeted therapy, antibodies, any antihormonal tumor treatment, and immunotherapy other than FTD/TPI and Irinotecan.

Current data in gastric cancer indicate a potential worse survival for patients receiving fluoropyrimidine-derivates with the concomitant use of protone-pump inhibitors, which should be avoided if possible. For details of contraindications, Investigators should refer to the respective SmPC. Caution is required when using medicinal products that are human thymidine kinase substrates, e.g., zidovudine. Such medicinal products, if used concomitantly with FTD/TPI, may compete with the effector, trifluridine, for activation via thymidine kinases. Therefore, when using antiviral medicinal products that are human thymidine kinase substrates, monitor for possible decreased efficacy of the antiviral medicinal product, and consider switching to an alternative antiviral medicinal product that is not a human thymidine kinase substrate, such as lamivudine, and abacavir. It is unknown whether FTD/TPI may reduce the effectiveness of hormonal contraceptives. Therefore, women using hormonal contraceptive must also use a barrier contraceptive method.

Statistical considerations

The primary endpoint, time till progression-free survival, will be analyzed using a non-parametric survival estimate. To this end the method of Turnbull will be used to compute the survival curve [22]. The variance of the survival will be estimated using the Greenwood formula. The log transformation will be used for the calculation of confidence intervals.

An exploratory analysis of secondary outcomes will be performed. Overall survival will be analyzed using the Kaplan–Meier estimate of survival. For the secondary outcomes, objective response and occurrence of adverse events the proportion of patients with the outcome and confidence intervals using the Wilson method will be calculated [23].

The statistical analysis will be carried out by the Coordination Center for Clinical Trials Düsseldorf (KKSD).

Sample size determination

Sample size consideration for this study is based on the precision of the survival estimate for the outcome progression-free survival. Based on previous results with the combination of Irinotecan and 5-FU therapy in patients with biliary tract cancers we assume a median survival time of 3 months. Assuming an exponential failure time with a median survival of 3 months (hazard rate: 0.23 per month) and a censoring rate of 0.04 per month, a total sample size of 28 patients (including 3 calculated drop outs and invalid cases) results in an expected standard error of 0.09 for the 3 months survival.

Assessment of severity/intensity of adverse events

The severity of any (S)AE's occurring will be assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE), version 5.0.

Personnel of the trial center that is involved with the study must have access to a copy of the NCI-CTCAE version 5.0.

AE's are to be documented with the following details:

Indication of the diagnosis or syndrome, duration of the AE, severity, assessment of whether an AE is serious or not (if an AE is to be classified as a SAE, additional reporting as SAE is required), assessment of the causal relationship with the study medication and the outcome of the AE's.

In this study, all adverse events occurring from the time of signed informed consent by the patient until 30 days after the last administration of the study medication are to be documented as adverse events.

A serious adverse event (SAE) or serious adverse drug reaction (SADR) is any AE that fulfils at least one of the following criteria: results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, is a congenital anomaly/birth defect or constitutes an important medical event.

Important medical events are defined as those occurrences that may not be immediately life threatening or result in death, hospitalization, or disability, but may jeopardize the subject or require medical or surgical intervention to prevent one of the other outcomes listed above. Medical and scientific judgment should be exercised in deciding whether such an AE should be considered serious. SAE’s must be reported immediately within 24 h knowledge of the event.

Translational research

It is intended to collect plasma and serum samples from all patients, following separate declaration of consent. Samples are to be taken at baseline, at each restaging visit and at the end of the treatment visit. After centrifugation, the serum and plasma have to be stored at -80° Celsius for further analyses. The aim is to measure concentrations of inflammatory cytokines using FACS bead technology in the serum of patients. This panel will include analysis of Osteopontin, suPAR, APRIL, TWEAK, TRAIL, CCL1, 2, 5, and CXCR1, 2, 5, 9, which all have been demonstrated being regulated in the serum of patients with gastrointestinal cancers. Moreover concentrations of circulating miRNAs and lncRNAs will be measured in these samples. The ultimate aim of these retrospective analyses is to study the predictive value with regard to response, survival periods and development of resistance.

Liquid biopsies will be taken to perform translational research on cell-free DNA in blood, following separate declaration of consent. cfDNA exome sequencing will be performed using a panel of 28 mutations (e.g. IDH1, IDH2, SMAD4). These mutations will be recapitulated in patients´ serum by using cfDNA exome sequencing and correlated with clinical response, survival and development of resistance.

Stool analyses will be collected at baseline, at each restaging visit and at the end of the study treatment, following separate declaration of consent. In order to analyze the gut macrobiota we will perform different molecular-genetic examinations. First stool DNA will be prepared and bacterial gene sequences coding for 16S RNA will be quantified. As a screening examination a quantitative PCR using group-specific primer against variable parts of bacterial 16S RNA will be used. Next we will use a defined panel of 15 primer-pairs against the most prominent species of the gut microbiota to analyze rifaximin dependent alterations. Moreover it is planned to apply the highly innovative high-throughput sequencing of bacterial 16S RNA for complete and unbiased analysis of the gut microbiota.

QoL assessment

Quality of life analysis will be performed by means of the EORTC QLQ-C30 [24,25,26,27,28] and the EQ-5D-5L- questionnaires [29]. The EORTC QLQ-C30 was developed as an instrument to measure patient-reported health-related quality of life. The current version 3.0 of the EORTC QLQ-C30 consists of 30 questions. It incorporates nine multi-item scales: five functional scales (physical, role, cognitive, emotional, and social); three symptom scales (fatigue, pain, and nausea/vomiting); and a global health and quality-of-life scale. Several single-item symptom measures are also included (dypnoea, insomnia, decreased appetite, constipation, diarrhea and financial difficulties due to physical condition/medical treatment).

EQ-5D is a standardized measure of health status developed by the EuroQol Group. The EQ-5D-5L essentially consists of the EQ-5D descriptive system and the EQ visual analogue scale (EQ VAS). The EQ-5D-5L descriptive system comprises the following 5 dimensions: mobility, self-care, usual activities, pain/discomfort and anxiety/depression. Each dimension has 5 levels: no problems, slight problems, moderate problems, severe problems, and extreme problems. The respondent is asked to indicate his/her health state by ticking (or placing a cross) in the box against the most appropriate statement in each of the 5 dimensions. This decision results in a 1-digit number expressing the level selected for that dimension. The digits for 5 dimensions can be combined in a 5-digit number describing the respondent’s health state. It should be noted that the numerals 1–5 have no arithmetic properties and should not be used as a cardinal score [29].

The EQ VAS records the respondent’s self-rated health on a 20 cm vertical, visual analogue scale (VAS) where the endpoints are labelled ‘the best health you can imagine’ and ‘the worst health you can imagine’. This information can be used as a quantitative measure of health outcome as judged by the individual respondents. Respondents are asked to simply ‘mark an X on the scale to indicate how your health is TODAY’ and then to ‘write the number you marked on the scale in the box below [29].

Results from the EQ-5D-5L descriptive system will be presented as health profile and index values. EQ VAS scores will be presented as mean values with standard deviation.

The EORTC QLQ-C30 and EQ-5D-5L questionnaires as paper version will be handed out to and completed by the patient, independently of the study personnel, on Day 1 of the first cycle visit prior to other study measures. Moreover the questionnaires will be handed out to and completed by the patient before each following cycle. The investigator should explain to the patient how to complete the questionnaires but neither he/she nor the research staff can assist the patient with their completion. The patient should complete the questionnaires by him/herself in a quiet place.

EORTC QLQ-C30 and EQ-5D-5L questionnaires filled in by the patient at the trial center have to be filed together with the patient’s medical file until the end of study visit in the trial center.

Ethical considerations and trial registration

The protocol V03F from 09.08.2021 was developed and approved by the sponsor. The study will be performed in accordance with the ethical principles that have their origin in the Declaration of Helsinki and the ICH Good Clinical Practice guidelines. The local Ethics Committee of the University of Dusseldorf and the local ethic committees of the participating centers throughout Germany approved the study.

Any change in the study protocol and/or informed consent form will be presented to the named Ethics Committee. They have to be approved by the Ethics Committee before implementation (except for changes in logistics and administration or when necessary to eliminate immediate hazards). The sponsor, the competent authorities and the ethics committee may stop the clinical trial or participation of a clinical trial site in the clinical trial for medical, safety, regulatory, administrative or other reasons consistent with ICH-GCP, the respective European Union’s and national legislation. The study is registered at the ‘European Union Drug Regulating Authorities Clinical Trials’ (EudraCT 2018–002936-26) and clinicalstrials.gov (NCT04059562).