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9 clinical monitoring

Clinical site monitoring is conducted to ensure that the rights of the participants are protected, that the study is implemented per the approved protocol, Good Clinical Practice and standard operating procedures, and that the quality and integrity of study data and data collection methods are maintained. Monitoring for this study will be performed by NCI CCR Office of Sponsor and Regulatory Oversight (OSRO) Sponsor and Regulatory Oversight Support (SROS) Services contractor. Clinical site monitoring activities will be based on OSRO standards, FDA Guidance E6(R2) Good Clinical Practice: Integrated Addendum to ICH E6(R1) March 2018, and applicable regulatory requirements. Details of clinical site monitoring will be documented in a Clinical Monitoring Plan (CMP) developed by OSRO. CMPs will be protocol-specific, risk-based and tailored to address human subject protections and integrity of the study data. OSRO will determine the intensity and frequency of monitoring based on several factors, including study type, phase, risk, complexity, expected enrollment rate, and any unique attributes of the study and the site. The Sponsor will conduct a periodic review of the CMP to confirm the plan's continued appropriateness. A change to the protocol, significant or pervasive non-compliance with GCP, or the protocol may trigger CMP updates. OSRO SROS Monitoring visits and related activities will be conducted throughout the life cycle of each protocol. The first activity is before the study starts to conduct a Site Assessment Visit (SAV) (as warranted), followed by a Site Initiation Visit (SIV), Interim Monitoring Visit(s) (IMVs), and a study Close-Out Visit (COV). Some monitoring activities may be performed remotely, while others will take place at the study site(s). Monitoring visit reports will describe visit activities, observations, findings of protocol non-adherence and associated action items or follow-up required for resolution of findings. Monitoring reports will be distributed to the study PI, NCI CCR QA, coordinating center (if applicable) and the OSRO regulatory file. The site Monitor will inform the study team of any deviations observed during monitoring visits. If unresolved, the Monitor will request that the site Staff enter the deviations in the CCR Protocol Deviation Tracking System (PDTS) for deviation reporting to the Sponsor and as applicable per institutional and IRB guidance. 10 STATISTICAL CONSIDERATIONS
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10.1 statistical hypothesis

10.1.1 Primary Objective To determine whether the 5-month PFS of the trametinib plus hydroxychloroquine (HCQ)
combination in subjects with refractory bile tract carcinoma (BTC) with KRAS mutation exceeds 25%.
10.1.2 Secondary Objectives  
To determine the safety, tolerability and feasibility of the combination with trametinib plus HCQ in refractory BTC with KRAS mutation.  
To evaluate the response rate and overall survival the combination with trametinib plus HCQ in patients with refractory BTC with KRAS mutation.
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10.2 sample size determination

The primary objective of this study is to determine in a preliminary, limited size trial whether trametinib in combination with HCQ is associated with a 5-month progression free probability, which exceeds 25% when used to treat patients with cholangiocarcinoma. Based on our analysis of data from previous trials of patients with similar eligibility requirements the median progression free survival in BTC is 2.5 months[8]. This translates to 25% without progressive disease at 5 months following an exponential failure model. Based on these results, it would be useful to demonstrate whether trametinib in combination with hydroxychloroquine is able to be associated with a median PFS of 5 months, which would correspond to 50% of patients having stable disease at 5 months. The trial will be conducted using a two-stage optimal design [52]. In order to attempt to determine if the agent offers any improvement, using alpha=0.10 and beta =0.10 as acceptable error probabilities, the trial will target 50% as the desirable proportion of patients who are still without progression at the five-month evaluation (p1=0.50), and will be considered inadequate if only a fraction consistent with 20% are without progression by the same evaluation time (p0=0.20). Initially, 10 patients will be enrolled and evaluated for progression. Enrollment will be temporarily halted after the 10th patient has been accrued, unless we know that 3 patients have passed the 5month point without progression. If 3 or more of the first 10 patients enrolled have not progressed at the 5-month evaluation, then accrual will continue until a total of 17 patients have been entered. If, among the first 10 patients accrued, 0 to 2 are able to be progression-free at the 5-month evaluation, then no further patients will be enrolled after such a determination has been made. If 6 or more of the total cohort of 17 patients have been found to be progression-free at 5 months, then this will indicate an adequate progression free probability to justify further consideration of trametinib in combination with hydroxychloroquine in this population of patients. On the other hand, if 3 to 5 of 17 are progression-free at 5 months, this will be considered insufficient. Under the null hypothesis (20% progression free at 5 months), the probability of being able to stop accrual after 10 patients have been evaluated at 5 months is 68%. In addition to evaluation of the proportion of patients that are progression-free at 5 months, the progression-free survival for patients will also be analyzed via a Kaplan-Meier curve. This curve will be compared informally to other published results in similar patients. As well, the overall response rate will be reported, and the overall survival will be reported using a Kaplan-Meier curve. It is anticipated that up to 10 patients per year will be able to enroll onto this protocol. Thus, it is expected that accrual of up to 17 total patients can be completed in approximately 2 years. In order to allow for a small number of unevaluable patients and screen failures, the accrual ceiling will be set at 30 patients. To evaluate the response rate, proportion of patients obtaining CR and PR per RECIST 1.1 criteria of all evaluable patients will be analyzed.  
To address safety, tolerability and feasibility of trametinib in combination with hydroxychloroquine, adverse events will be tabulated by grade according to CTCAE and analyzed and reported descriptively. The fraction of patients who experience a response will be reported along with confidence intervals.
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10.3 population for analysis

Any patient who receives at least one dose of both agents will be included in the efficacy evaluations. Any patients who receive at least one dose of either agent will be included in the safety evaluations.
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10.4 statistical analysis

10.4.1 General approach The fraction of patients who do not have progressive disease by 5 months will be reported. 10.4.2 Analysis of the Primary Endpoints The fraction of patients who are able to not have progressive disease at 5 months will be reported along with a 95% confidence interval. A Kaplan-Meier curve of PFS will also be created and reported along with the 5-month PFS probability and its 95% confidence interval. 10.4.3 Analysis of the Secondary Endpoint The safety of the treatment will be monitored, and any toxicities identified will be reported by type and grade. The feasibility and tolerability will be assessed and reported descriptively. In each cohort, the fraction of patients who experience a response (PR + CR) will be reported along with 80% and 95% two-sided confidence intervals. Overall survival will be calculated from the on-study date using the Kaplan-Meier method. 10.4.4 Safety analysis The safety of the treatment will be monitored, and any toxicities identified will be reported by type and grade. 10.4.5 Baseline Descriptive Statistics Baseline demographic characteristics will be reported. 10.4.6 Planned Interim Analyses There are no planned interim analyses for efficacy. 10.4.7 Sub-group analyses None are planned. 10.4.8 Tabulations of individual participant data None. 10.4.9 Exploratory Analyses The following objectives will result in descriptive or comparative analyses when adequate data exist to perform them:  
To evaluate the bioactivity of the combination of trametinib plus HCQ against the MEK1/2 and autophagy pathways by immunohistochemistry.  
To measure changes in immune parameters in the peripheral blood and tumors of patients with refractory KRAS mutation BTC treated with the combination of trametinib plus HCQ.  
To explore mechanism of primary and secondary resistance to the combination of trametinib plus HCQ in patients with refractory KRAS mutation BTC.  
To evaluate the relationship between the RR, OS and immune parameters, in patients with refractory KRAS mutation BTC treated with the combination of trametinib plus HCQ.  
Any exploratory evaluations which generate quantitative measures will be done using descriptive statistics including confidence intervals when appropriate. Any statistical tests performed for evaluation of exploratory objectives will be done without formal adjustment for multiple comparisons, but in the context of the number of tests performed.
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11 collaborative agreements 11.1 cooperative

CRADA for this protocol is currently being negotiated between Thoracic & GI Malignancy Branch, NCI, NIH and Novartis, the manufacturer of trametinib.
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12 human subjects protections 12.1

No individual who meets the criteria for eligibility will be excluded from participation based on their race, ethnicity, gender, or socioeconomic status. Particular attention will be made to acquire a broad and diversified population.
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12.2 participation of children

Children (younger than 18 years) will not be included in this protocol due to the limited data on this combination in children and the different biology of childhood malignancy.
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12.3 participation of subjects unable

Adults unable to consent are excluded from enrolling in the protocol. However, it is possible that subjects enrolled in the protocol may permanently lose the capacity to consent for themselves during the course of this study. If a subject loses the capacity to consent during the course of the study for reasons unrelated to protocol participation and is receiving direct benefit from the study therapy in the opinion of the PI, the subject may be retained on the study therapy with the permission of the alternate decision maker. If the permanent loss of capacity is due to toxicity from the study therapy or disease progression, the participant will be removed from study therapy as required per protocol, but may be retained on study to be followed for safety reasons and/or overall survival as described in the Study Calendar 3.4. The follow-up procedures involve non-invasive, low risk studies, with the exception of scanning. Scanning during follow-up will be performed only in participants who are taken of treatment for reasons other than disease progression and only within 5 months of study initiation. These scans are clinically indicated. The benefit of a safety follow up would outweigh the risks of such procedures. All subjects will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another  
person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Please see section **12.5** for the consent procedure.
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12.4 risk/benefit assessment for all

12.4.1 Known Potential Risks The primary risk to patients participating in this research study is from the toxicity of study drugs. All care will be taken to minimize study treatment side effects, but they can be unpredictable in nature and severity. 12.4.1.1 Risk of Biopsy All care will be taken to minimize risks that may be incurred by tumor sampling. However, there are procedure-related risks (such as bleeding, infection, visceral injury, hematoma and death) that will be explained fully during informed consent. However, it may be noted that we have not experienced a single bleeding event in approximately 250 patients who have undergone liver biopsy procedures recently for other standard of care or research testing. The biopsies will be performed in interventional radiology. 12.4.1.2 Risks of Exposure to Ionizing Radiation The study will involve radiation from the following sources:  
Up to 7 CT scans per year for disease assessment 2 CT scans for the collection of 2 mandatory biopsies
Subjects in this study may be exposed to approximately 9.3 rem per year. This amount is more than would be expected from everyday background radiation. Being exposed to excess radiation can increase the risk of cancer. The risk of getting cancer from the radiation exposure in this study is 0.9 out of 100 (0.9%) and of getting a fatal cancer is 0.5 out of 100 (0.5%). 12.4.1.3 Risks of CT Scans In addition to the radiation risks discussed above, CT scans may include the risks of an allergic reaction to the contrast. Participants might experience hives, itching, headache, difficulty breathing, increased heartrate and swelling. 12.4.1.4 Risks of sedation Biopsies will be done under sedation. Potential side effects of sedation include headache, nausea and drowsiness. These side effects usually go away quickly. 12.4.1.5 Risks of ophthalmologic exam Risks of ophthalmologic exam include discomfort, blurry vision and light sensitivity. 12.4.1.6 Risks of Blood Collection Risks of blood draws include pain and bruising in the area where the needle is placed, lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood cell count (anemia) can develop. Up to the 170 ml of blood may be collected at any visit, but no more than 550 ml per 8 weeks period.  
12.4.1.7 Risks of EKG Risks include some minor skin irritation from the electrodes. 12.4.1.8 Risks of Echocardiogram Risks include mild discomfort during the test. 12.4.1.9 Other Risks Risks include the possible occurrence of any of a range of side effects which are listed in the Consent Document or this protocol document. Frequent monitoring for adverse effects will help to minimize the risks associated with administration of the study agents.
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12.4.1.10 risk of losing data

This includes the risk that data obtained during this study, including data related to genotype, DNA sequencing or risk for disease tendency or trait can be released to members of the public, insurers, employers, or law enforcement agencies. Although there are no plans to release results to the patients, family members or health care providers, this risk will be included in the informed consent document. 12.4.1.11 Risk of receiving unwanted information Anxiety and stress may arise as a result of the anticipation that unwanted information regarding disease related DNA sequencing or disease tendencies, or misattributed paternity. Patients will be clearly informed that the data related to DNA sequencing and genetic analysis is coded, investigational and will not be shared with patients, family members or health care providers. 12.4.2 Known Potential Benefits The study drug may help to control the disease. The results may help the investigators learn more about the disease and develop new treatments for patients with this disease 12.4.3 Assessment of Potential Risks and Benefits BTC treatment needs improved therapy options. Preclinical studies suggest that study therapy may have tremendous anti-tumor efficacy. A number of clinically appropriate strategies to minimize risks to patients have been built into the protocol through the means of inclusion/exclusion criteria, monitoring strategies, and management guidelines. Overall, the potential benefit of the study treatment in subjects with BTC outweigh the risks associated with study drugs. The potential benefit to a patient that participates in this study is better control of their tumor growth and disease recurrence which may or may not have a favorable impact on symptoms and/or survival. Potential adverse reactions attributable to the administration of the study drug utilized in this trial are discussed in Sections **1.2.5.2 and 1.2.6.2**. All care will be taken to minimize side effects, but they can be unpredictable in nature and severity.
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12.5 consent process and documentation

The informed consent document will be provided as a physical or electronic document to the participant or consent designee(s) as applicable for review prior to consenting. A designated study investigator will carefully explain the procedures and tests involved in this study, and the  
associated risks, discomforts and benefits. In order to minimize potential coercion, as much time as is needed to review the document will be given, including an opportunity to discuss it with friends, family members and/or other advisors, and to ask questions of any designated study investigator. A signed informed consent document will be obtained prior to entry onto the study. The initial consent process as well as re-consent, when required, may take place in person or remotely (e.g., via telephone or other NIH approved remote platforms used in compliance with policy, including HRPP Policy 303) per discretion of the designated study investigator and with the agreement of the participant/consent designee(s). Whether in person or remote, the privacy of the subject will be maintained. Consenting investigators (and participant/consent designee, when in person) will be located in a private area (e.g., clinic consult room). When consent is conducted remotely, the participant/consent designee will be informed of the private nature of the discussion and will be encouraged to relocate to a more private setting if needed.  
Consent will be documented with required signatures on the physical document (which includes the printout of an electronic document sent to participant) or as described below, with a manual (non-electronic) signature on the electronic document. When required, witness signature will be obtained similarly as described for the investigator and participant.
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manual (non-electronic) signature on electronic

When a manual signature on an electronic document is used for the documentation of consent at the NIH Clinical Center, this study will use the following to obtain the required signatures:  
Adobe platform (which is not 21 CFR Part 11 compliant); or, iMedConsent platform (which is 21 CFR Part 11 compliant)
During the consent process, participants and investigators will view individual copies of the approved consent document on screens at their respective locations (if remote consent); the same screen may be used when in the same location but is not required. Both the investigator and the subject will sign the document using a finger, stylus or mouse. Note: Refer to the CCR SOP PM-2, Obtaining and Documenting the Informed Consent Process for additional information (e.g., verification of participant identity when obtaining consent remotely) found here. 12.5.1 Consent Process for Adults Who Lack Capacity to Consent to Research Participation For participants addressed in section **12.3**, an LAR will be identified consistent with Policy 403 and informed consent obtained from the LAR, as described in Section **12.5**
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13 regulatory and operational considerations

This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable cause. Written notification, documenting the reason for study suspension or termination, will be provided by the suspending or terminating party to study participants. If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination or suspension. Study participants will be contacted, as applicable, and be informed of changes to study visit schedule.  
Circumstances that may warrant termination or suspension include, but are not limited to:  
Determination of unexpected, significant, or unacceptable risk to participants Demonstration of efficacy that would warrant stopping Insufficient compliance to protocol requirements Data that are not sufficiently complete and/or evaluable Determination that the primary endpoint has been met Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
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13.2 quality assurance and quality

The clinical site will perform internal quality management of study conduct, data and biological specimen collection, documentation and completion. An individualized quality management plan will be developed to describe a site's quality management. Quality control (QC) procedures will be implemented beginning with the data entry system and data QC checks that will be run on the database will be generated. Any missing data or data anomalies will be communicated to the site(s) for clarification/resolution. Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is conducted and data are generated and biological specimens are collected, documented (recorded), and reported in compliance with the protocol, International Conference on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). The investigational site will provide direct access to all trial related sites, source data/documents, and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory authorities.
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13.3 conflict of interest policy

The independence of this study from any actual or perceived influence, such as by the pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be required to have such conflicts managed in a way that is appropriate to their participation in the design and conduct of this trial. The study leadership in conjunction with the NCI has established policies and procedures for all study group members to disclose all conflicts of interest and will establish a mechanism for the management of all reported dualities of interest.
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13.4 confidentiality and privacy

Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, and the sponsor. This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participants. Therefore, the study protocol, documentation, data, and all other information generated will be held in strict confidence.  
No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor. All research activities will be conducted in as private a setting as possible. The study monitor, other authorized representatives of the sponsor, representatives of the Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the participants in this study. The clinical study site will permit access to such records. The study participant's contact information will be securely stored at each clinical site for internal use during the study. At the end of the study, all records will continue to be kept in a secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. Study participant research data will not include the participant's contact or identifying information. Rather, individual participants and their research data will be identified by a unique study identification number. The study data entry and study management systems used by clinical sites will be secured and password protected. At the end of the study, all study databases will be archived at the NIH Clinical Center. To further protect the privacy of study participants, a Certificate of Confidentiality has been issued by the National Institutes of Health (NIH). This certificate protects identifiable research information from forced disclosure. It allows the investigator and others who have access to research records to refuse to disclose identifying information on research participation in any civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or local level. By protecting researchers and institutions from being compelled to disclose information that would identify research participants, Certificates of Confidentiality help achieve the research objectives and promote participation in studies by helping assure confidentiality and privacy to participants.
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14 pharmaceutical information 14.1 trametinib

Commercial supplies of trametinib will be supplied by Novartis 14.1.2 Acquisition and Accountability Trametinib will be provided by Novartis and delivered directly to the NIH Pharmacy. Individual bottles with tablets will be prepared for each study participant according to assigned dose by NIH
Pharmacy personnel. Patients will pick up bottles at NIH Pharmacy and will return bottles and notused tablets after completion of every cycle together with Medication Diary to Study Coordinator.  
After review of leftover tablets and Medication Diary, unused tablets will be returned to pharmacy and disposed by pharmacy personnel. 14.1.3 Formulation, Appearance, Packaging, and Labeling  
Please refer to the package insert for additional information.
14.1.4 Product Storage and Stability Please refer to the package insert for additional information. 14.1.5 Preparation N/A.
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14.2 hydroxychloroquine (generic)

14.2.1 Source Hydroxychloroquine will be provided by the NIH Clinical Center Pharmacy according to standard pharmacy procedures 14.2.2 Acquisition and Accountability Hydroxychloroquine will be delivered directly to the NIH Pharmacy. Individual bottles with tablets will be prepared for each study participant according to assigned dose by NIH Pharmacy personnel. Patients will pick up bottles at NIH Pharmacy and will return bottles and not-used tablets after completion of every cycle together with Medication Diary to Study Coordinator. After review of leftover tablets and Medication Diary, unused tablets will be returned to pharmacy and disposed by pharmacy personnel. 14.2.3 Formulation, Appearance, Packaging, and Labeling Please refer to the package insert for additional information. 14.2.4 Product Storage and Stability Please refer to the package insert for additional information. 14.2.5 Preparation N/A.
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14.3 trusight™️ oncology 500 panel

TruSight™ Oncology 500 panel assay is not FDA approved; it is being used as a treatment determining in-vitro diagnostic device in this study. According to 21 CFR 812.3(m), a significant risk device presents a potential for serious risk to the health, safety and welfare of a subject and meets the significant risk criteria listed in the table below along with the sponsor's conclusions with regard to the applicability of these criteria to the current study. The device has been assessed by the sponsor as non-significant risk (NSR) per the below.  
| Significant Risk Criteria                          | Applicable  to current  study                                                                   | Justification                                                    |
|----------------------------------------------------|-------------------------------------------------------------------------------------------------|------------------------------------------------------------------|
| Is an implant                                      | No                                                                                              | TruSight™ Oncology 500 panel is not  introduced into the subject |
| Is used in supporting or                           | No                                                                                              | The device is diagnostic                                         |
| sustaining human life Is of substantial importance | No                                                                                              | While the device is diagnostic, we do not                        |
| in diagnosing mitigating or                        | believe in presents a potential for serious risk  to the health and welfare of the subject. The |                                                                  |  
| Significant Risk Criteria      | Applicable  to current  study                                                                                                                                                                                                                                           | Justification                                                                                                                                                                           |
|--------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| treating disease or preventing | assessment of KRAS mutation status is only                                                                                                                                                                                                                              |                                                                                                                                                                                         |
| impairment of human health     | used to help to increase the possibility that all  persons enrolling on the study might derive  benefit from therapy. Persons that are deemed  ineligible to enroll on the basis of this test are  eligible for studies within TGMB that are not  reliant on this test. |                                                                                                                                                                                         |
| Otherwise poses a risk         | No                                                                                                                                                                                                                                                                      | Testing will be performed on fresh sample  that is collected at screening for confirmation  of diagnosis. No additional collection of  sample will occur for purposes of KRAS  testing. |
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15 references

1. Bridgewater, J., et al., *Guidelines for the diagnosis and management of intrahepatic* cholangiocarcinoma. J Hepatol, 2014. 60(6): p. 1268-89.  
2. Siegel, R.L., K.D. Miller, and A. Jemal, *Cancer statistics, 2018.* CA Cancer J Clin, 2018.  
68(1): p. 7-30.  
3. Valle, J.W., et al., *New Horizons for Precision Medicine in Biliary Tract Cancers.* Cancer Discov, 2017. 7(9): p. 943-962.  
4. Wang, S.J., et al., *Nomogram for predicting the benefit of adjuvant chemoradiotherapy* for resected gallbladder cancer. J Clin Oncol, 2011. 29(35): p. 4627-32.  
5. Waterhouse, P., et al., *Lymphoproliferative disorders with early lethality in mice deficient* in Ctla-4. Science, 1995. 270(5238): p. 985-8.  
6. Lee, G.W., et al., Combination chemotherapy with gemcitabine and cisplatin as first-line treatment for immunohistochemically proven cholangiocarcinoma. Am J Clin Oncol, 2006. 29(2): p. 127-31.  
7. Lee, J., et al., *Comparison of the Efficacy between Gemcitabine-Cisplatin and* Capecitabine-Cisplatin Combination Chemotherapy for Advanced Biliary Tract Cancer. Cancer Res Treat, 2015. 47(2): p. 259-65.  
8. Ulahannan, S.V., et al., *Identification of active chemotherapy regimens in advanced* biliary tract carcinoma: a review of chemotherapy trials in the past two decades. Hepat Oncol, 2015. 2(1): p. 39-50.  
9. Waters, A.M. and C.J. Der, *KRAS: The Critical Driver and Therapeutic Target for* Pancreatic Cancer. Cold Spring Harb Perspect Med, 2018. 8(9).  
10. Cox, A.D., et al., *Drugging the undruggable RAS: Mission possible?* Nat Rev Drug Discov, 2014. 13(11): p. 828-51.  
11. Prior, I.A., P.D. Lewis, and C. Mattos, *A comprehensive survey of Ras mutations in* cancer. Cancer Res, 2012. 72(10): p. 2457-67.  
12. Churi, C.R., et al., *Mutation profiling in cholangiocarcinoma: prognostic and therapeutic* implications. PLoS One, 2014. 9(12): p. e115383.  
13. Zhu, A.X., et al., Genomic profiling of intrahepatic cholangiocarcinoma: refining prognosis and identifying therapeutic targets. Ann Surg Oncol, 2014. 21(12): p. 3827-34.  
14. Robertson, S., et al., The frequency of KRAS and BRAF mutations in intrahepatic cholangiocarcinomas and their correlation with clinical outcome. Hum Pathol, 2013.  
44(12): p. 2768-73.  
15. Chen, T.C., Y.Y. Jan, and T.S. Yeh, *K-ras mutation is strongly associated with* perineural invasion and represents an independent prognostic factor of intrahepatic cholangiocarcinoma after hepatectomy. Ann Surg Oncol, 2012. **19 Suppl 3**: p. S675-81.  
16. Javle, M., et al., *Phase II Study of BGJ398 in Patients With FGFR-Altered Advanced* Cholangiocarcinoma. J Clin Oncol, 2018. 36(3): p. 276-282.  
17. Lowery, M.A., et al., *Safety and activity of ivosidenib in patients with IDH1-mutant* advanced cholangiocarcinoma: a phase 1 study. Lancet Gastroenterol Hepatol, 2019. 4(9): p. 711-720.  
18. El-Khoueiry, A.B., et al., *Nivolumab in patients with advanced hepatocellular carcinoma*
(CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet, 2017. 389(10088): p. 2492-2502.  
19. Zhu, A.X., et al., *Pembrolizumab in patients with advanced hepatocellular carcinoma* previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial. Lancet Oncol, 2018. 19(7): p. 940-952.  
20. Gou, M., et al., *Efficacy and safety of nivolumab for metastatic biliary tract cancer.* Onco Targets Ther, 2019. 12: p. 861-867.  
21. Ueno, M., et al., Pembrolizumab for advanced biliary adenocarcinoma: Results from the multicohort, phase 2 KEYNOTE-158 study. Annals of Oncology, 2018. 29(suppl_8): p. 1.  
22. Xie, C., et al., *Tremelimumab in Combination With Microwave Ablation in Patients With* Refractory Biliary Tract Cancer. Hepatology, 2019. 69(5): p. 2048-2060.  
23. Papke, B. and C.J. Der, *Drugging RAS: Know the enemy.* Science, 2017. 355(6330): p.  
1158-1163.  
24. Bekaii-Saab, T., et al., *Multi-institutional phase II study of selumetinib in patients with* metastatic biliary cancers. J Clin Oncol, 2011. 29(17): p. 2357-63.  
25. Bridgewater, J., et al., A phase 1b study of Selumetinib in combination with Cisplatin and Gemcitabine in advanced or metastatic biliary tract cancer: the ABC-04 study. BMC Cancer, 2016. 16: p. 153.  
26. Sahu, N., et al., Cotargeting of MEK and PDGFR/STAT3 Pathways to Treat Pancreatic Ductal Adenocarcinoma. Mol Cancer Ther, 2017. 16(9): p. 1729-1738.  
27. Hanahan, D. and R.A. Weinberg, *Hallmarks of cancer: the next generation.* Cell, 2011.  
144(5): p. 646-74.  
28. Yang, Z. and D.J. Klionsky, *Eaten alive: a history of macroautophagy.* Nat Cell Biol, 2010. 12(9): p. 814-22.  
29. Tsukada, M. and Y. Ohsumi, *Isolation and characterization of autophagy-defective* mutants of Saccharomyces cerevisiae. FEBS Lett, 1993. 333(1-2): p. 169-74.  
30. Behrends, C., et al., *Network organization of the human autophagy system.* Nature, 2010.  
466(7302): p. 68-76.  
31. Mizushima, N. and B. Levine, *Autophagy in mammalian development and differentiation.*
Nat Cell Biol, 2010. 12(9): p. 823-30.  
32. Levine, B. and D.J. Klionsky, Autophagy wins the 2016 Nobel Prize in Physiology or Medicine: Breakthroughs in baker's yeast fuel advances in biomedical research. Proc Natl Acad Sci U S A, 2017. 114(2): p. 201-205.  
33. Cadwell, K., Crosstalk between autophagy and inflammatory signalling pathways:
balancing defence and homeostasis. Nat Rev Immunol, 2016. 16(11): p. 661-675.  
34. Eisenberg-Lerner, A. and A. Kimchi, The paradox of autophagy and its implication in cancer etiology and therapy. Apoptosis, 2009. 14(4): p. 376-91.  
35. White, E., *Deconvoluting the context-dependent role for autophagy in cancer.* Nat Rev Cancer, 2012. 12(6): p. 401-10.  
36. Yang, Z.J., et al., *The role of autophagy in cancer: therapeutic implications.* Mol Cancer Ther, 2011. 10(9): p. 1533-41.  
37. Sasaki, M., et al., Autophagy may occur at an early stage of cholangiocarcinogenesis via biliary intraepithelial neoplasia. Hum Pathol, 2015. 46(2): p. 202-9.  
38. O'Dell, M.R., et al., *Kras(G12D) and p53 mutation cause primary intrahepatic* cholangiocarcinoma. Cancer Res, 2012. 72(6): p. 1557-67.  
39. Huang, J.L. and A.F. Hezel, *Autophagy in intra-hepatic cholangiocarcinoma.*
Autophagy, 2012. 8(7): p. 1148-9.  
40. Bryant, K.L., et al., *KRAS: feeding pancreatic cancer proliferation.* Trends Biochem Sci, 2014. 39(2): p. 91-100.  
41. Pylayeva-Gupta, Y., E. Grabocka, and D. Bar-Sagi, *RAS oncogenes: weaving a* tumorigenic web. Nat Rev Cancer, 2011. 11(11): p. 761-74.  
42. White, E., *Exploiting the bad eating habits of Ras-driven cancers.* Genes Dev, 2013.  
27(19): p. 2065-71.  
43. Hippert, M.M., P.S. O'Toole, and A. Thorburn, *Autophagy in cancer: good, bad, or both?*
Cancer Res, 2006. 66(19): p. 9349-51.  
44. Fujii, S., et al., *Autophagy is activated in pancreatic cancer cells and correlates with* poor patient outcome. Cancer Sci, 2008. 99(9): p. 1813-9.  
45. Guo, J.Y., et al., Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev, 2011. 25(5): p. 460-70.  
46. Yang, S., et al., *Pancreatic cancers require autophagy for tumor growth.* Genes Dev, 2011. 25(7): p. 717-29.  
47. Kinsey, C.G., et al., *Protective autophagy elicited by RAF-->MEK-->ERK inhibition* suggests a treatment strategy for RAS-driven cancers. Nat Med, 2019.  
48. Wolpin, B.M., et al., Phase II and pharmacodynamic study of autophagy inhibition using hydroxychloroquine in patients with metastatic pancreatic adenocarcinoma. Oncologist, 2014. 19(6): p. 637-8.  
49. Shimizu, S., et al., Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma. Int J Cancer, 2012. 131(3): p. 548-57.  
50. Nakamura, O., et al., The combination of rapamycin and MAPK inhibitors enhances the growth inhibitory effect on Nara-H cells. Int J Mol Med, 2014. 33(6): p. 1491-7.  
51. Infante, J.R., et al., A randomised, double-blind, placebo-controlled trial of trametinib, an oral MEK inhibitor, in combination with gemcitabine for patients with untreated metastatic adenocarcinoma of the pancreas. Eur J Cancer, 2014. 50(12): p. 2072-81.  
52. Simon, R., *Optimal two-stage designs for phase II clinical trials.* Control Clin Trials, 1989. 10(1): p. 1-10.
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16 appendices 16.1 appendix a

| Grade                               | Descriptions                                                                                                                                                                                                                                             |
|-------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Normal activity. Fully active, able |                                                                                                                                                                                                                                                          |
| 0                                   | to carry on all pre-disease  performance without restriction. Symptoms, but ambulatory.  Restricted in physically strenuous  activity, but ambulatory and able  to carry out work of a light or  sedentary nature (e.g., light  housework, office work). |
| 1                                   | In bed <50% of the time.  Ambulatory and capable of all  self-care, but unable to carry out  any work activities. Up and about  more than 50% of waking hours.                                                                                           |
| 2                                   | In bed >50% of the time. Capable  of only limited self-care, confined  to bed or chair more than 50% of  waking hours.                                                                                                                                   |
| 3                                   | 100% bedridden. Completely  disabled. Cannot carry on any  self-care. Totally confined to bed  or chair.                                                                                                                                                 |
| 5                                   | Dead.                                                                                                                                                                                                                                                    |
| 4                                   |                                                                                                                                                                                                                                                          |  
ECOG Performance Status Scale  
| Points assigned   |                 |                 |           |
|-------------------|-----------------|-----------------|-----------|
| Parameter         | 3               |                 |           |
| 1                 | 2               | Moderate        |           |
| Ascites           | Absent          | Slight          |           |
| Bilirubin         | <2 mg/dL        | 2-3 mg/dL       | > mg/dL   |
| (34.2             | (34.2 to 51.3   | (>51.3          |           |
| micromol/liter)   | micromol/liter) | micromol/liter) |           |
| Albumin           | >.3.5 g/dL (35  | 2.8-3.5 g/dL    | <2.8 g/dL |
| g/liter)          | (28 to 35       | ( <28 g/liter)  |           |
| g/liter)          |                 |                 |           |
| Prothrombin time  |                 |                 |           |
| Seconds over      | <4              | 4-6             | >6>       |
| control           |                 |                 |           |
| INR               | <1.7            | 1.7-2.3         | >2.3      |
| Ence phalopathy   | None            | Grade 1-2       | Grade 3-4 |  
16.2 Appendix B Child-Pugh Classification System Modified Child-Pugh classification of the severity of liver disease according to the degree of ascites, the plasma concentrations of bilirubin and albumin, the prothrombin time, and the degree of encephalopathy. A total score of 56 is considered grade A (well-compensated disease); 7-9 is grade B (significant functional compromise); and 10-15 is grade C (decompensated disease). These grades correlate with one- and two-year patient survival: grade A - 100 and 85 percent; grade B - 80 and 60 percent; and grade C - 45 and 35 percent.
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instructions to the patient

1. Complete one form for each cycle of treatment 2. You will take trametinib once a day and HCQ twice a day every day during 28-day cycle 3. HCQ should be taken every 12 hours at approximately the same times each day. Doses should be  
taken within 2 hours of the scheduled time.  
4. In case of a missed dose (more than 2 hours late) or vomiting after taking HCQ, do not make up  
the missed dose of HCQ.  
5. Take trametinib on empty stomach: at least 1 hour before or 2 hours after a meal with a glass of  
water. Take HCQ with a meal or a glass of milk.  
6. Record the date, the number of tablets that you took, and when you took them. 7. If you have any comments or notice any side effects, please record them in the comment's  
column.  
8. Please bring this form and your bottles (even it is empty) when you come for your clinic visits.
of oral hcq tablets
instructions to the patient

taken  
Day Date (every 12 hours)  
| 8.                            | Please bring this form and your bottles (even it is empty) when you come for your clinic visits. # of Oral HCQ Tablets  taken   |                  |                       |                           |
|-------------------------------|---------------------------------------------------------------------------------------------------------------------------------|------------------|-----------------------|---------------------------|
| Day                           | Date                                                                                                                            | (every 12 hours) | # of Oral  trametinib | Comments (side effects or |
| Tablets taken                 | missed doses)                                                                                                                   |                  |                       |                           |
| AM                            | PM                                                                                                                              |                  |                       |                           |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 |                                                                                                                                 |                  |                       |                           |  
| Version Date: 04/06/2022 # of Oral HCQ Tablets  taken Day Date (every 12 hours)                                       | # of Oral  trametinib   | Comments (side effects or   |
|-----------------------------------------------------------------------------------------------------------------------|-------------------------|-----------------------------|
| Tablets taken                                                                                                         | missed doses)           |                             |
| AM                                                                                                                    | PM                      |                             |
| 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Patient's signature: ___________________________________________________ |                         |                             |
of oral hcq tablets
16.4 appendix d modified immune-related

This new classification is based on the recent learning from clinical studies with cancer immunotherapies that even if some new lesions appear at the beginning of a treatment or if the total tumor burden does not increase substantially, tumor regressions or stabilizations might still occur later. The irRC were created using bi-dimensional measurements (as previously widely used in the World Health Organization criteria). For this trial, the concepts of the irRC are combined with RECIST 1.1 to come up with the modified irRC. For modified irRC, only target and measurable lesions are taken into account. In contrast to the RECIST 1.1 criteria, the modified irRC criteria (a) require confirmation of both progression and response by imaging at 6 weeks after initial imaging and (b) do not necessarily score the appearance of new lesions as progressive disease if the sum of lesion diameters of target lesions (minimum of 10 mm per lesion, maximum of 5 target lesions, maximum of 2 per organ) and measurable new lesions does not increase by ≥ 20%.  
The same method of assessment and the same technique should be used to characterize each identified and reported target lesion(s) at baseline, during the trial, and at the end of trial visit. All measurements should be recorded in metric notation. The modified irRC based on RECIST 1.1 are displayed below.