Treatment With Nivolumab and All-trans Retinoic Acid for Patients With Refractory Pancreatic Cancer
Phase: EARLY_PHASE1
Conditions: Effect of Drug
Sponsor: China Medical University Hospital

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Treatment with Nivolumab and All-Trans 
Retinoic Acid for Patients with Refractory 
Pancreatic Cancer 
 
 
Protocol 
 
 
 
 
 
 
 
Date: October 7, 2022 
 

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Table of content 
 
1. Abstract 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 
2. Background 
 
 
 
 
 
 
 
 
 
 
 
 
4 
3. Objectives  
 
 
 
 
 
 
 
 
 
 
 
 
11 
4. Drug information 
 
 
 
 
 
 
 
 
 
 
 
12 
5. Selection of patients 
 
 
 
 
 
 
 
 
 
 
14 
  5.1. Inclusion criteria 
 
 
 
 
 
 
 
 
 
 
14 
  5.2. Exclusion criteria 
 
 
 
 
 
 
 
 
 
 
14 
6. Study treatment 
 
 
 
 
 
 
 
 
 
 
 
16 
  6.1. Overall study design 
 
 
 
 
 
 
 
 
 
16 
  6.2. Dosage modification and treatment delay  
 
 
 
 
16 
  6.3. Concomitant therapy 
 
 
 
 
 
 
 
 
 
17 
  6.4. Off study criteria 
 
 
 
 
 
 
 
 
 
 
18 
7. Study calendar  
 
 
 
 
 
 
 
 
 
 
 
19 
8. Efficacy assessment 
 
 
 
 
 
 
 
 
 
 
22 
9. Serious adverse effect and reporting  
 
 
 
 
 
 
23 
10. Statistical consideration and patient accrual 
 
 
 
 
 
26 
11. Ethical, legal, and administrative aspects  
 
 
 
 
 
27 
  11.1 Data collection  
 
 
 
 
 
 
 
 
 
 
27 
  11.2 Study records retention  
 
 
 
 
 
 
 
 
27 
12. References 
 
 
 
 
 
 
 
 
 
 
 
 
28 
 
 

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1. Abstract 
Pancreatic cancer is an aggressive and lethal disease. Even two 
combination regimens, FOLFIRINOX and albumin-bound paclitaxel in 
combination with gemcitabine, are superior to gemcitabine alone as the 
first-line treatments, the prognosis of patients with locally advanced or 
metastatic pancreatic cancer remains poor, with a median overall survival of 8 
to 11 months and an estimated 2-year survival of only 2%. Even more, there is 
currently no available treatment proven beneficial for patients who fail the 
second-line therapy with liposomal irinotecan in combination with 5-FU.  
 
Immune checkpoint inhibitors targeting PD-1 or PD-L1 represent a 
popular treatment option for patients with some kinds of cancers, but previous 
studies in pancreatic cancer have failed. One critical challenge for 
immunotherapy in pancreatic cancer is to find efficient therapeutic approaches 
to reverse this “cold” tumor into a “hot” tumor. Our previous basic studies 
revealed that all-trans retinoic acid repressed pancreatic adenocarcinoma cell 
growth and colony formation. Set-to-set genetic analysis revealed that genes 
regulated by all-trans retinoic acid were predominantly involved in immune 
response including interleukin-6, tumor necrotic factor-α, IFNs signaling, and 
PD-L1. Furthermore, mice bearing pancreatic cancer treated with combination 
of anti-PD-1 and all-trans retinoic acid had a significantly longer survival 
compared to mice treated with anti-PD-1 or all-trans retinoic acid alone. 
 
Based on the previous findings, we propose this pilot, compassionate use 
of combination therapy with all-trans retinoic acid and nivolumab for patients 
with locally advanced or metastatic pancreatic adenocarcinoma who are 
refractory to current available treatment. This study will also examine ADAR1 
as a novel prognostic and predictive biomarker for pancreatic adenocarcinoma 
patients. This study will help to delineate the role of ADAR1 as a predictive 
biomarker for immunotherapy, all-trans retinoic acid as a suppressor of 
ADAR1, and most importantly, the combination of all-trans retinoic acid and 
nivolumab as an effective anticancer therapy for patients with pancreatic 
adenocarcinoma.

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2. Background 
2.1. Pancreatic adenocarcinoma 
The incidence of pancreatic cancer is rising, and it is predicted to become 
the second leading cause of cancer-related death by 2030 in the world 
(Kenner et al. 2016). The prognosis of patients with advanced or metastatic 
pancreatic cancer is poor, especially for those who have refractory pancreatic 
adenocarcinoma. Pancreatic adenocarcinoma is characterized by early 
metastasis, extensive desmoplasia (Armstrong et al. 2004) and 
non-immunogenic tumor microenvironment (Riquelme et al. 2018), which are 
the reasons leading to poor response to treatments. 
Currently, potential therapeutic options for pancreatic adenocarcinoma 
are surgery, radiation, chemotherapy, targeted therapy, and immunotherapy. A 
chemotherapy drug, gemcitabine, is the standard therapy for pancreatic 
adenocarcinoma, although the resistance to gemcitabine has been a major 
challenge (Amrutkar et al. 2017). Gemcitabine resistance comes from several 
causes including abnormalities in drugs metabolism and desmoplastic reaction 
caused impairment of drug delivery (Ghaneh et al. 2008). Furthermore, the fact 
that immunosuppressive tumor microenvironment consistently impedes the 
functional activation of immune cells (Principe et al. 2016; Beatty et al. 2015), 
contributes to the low response of pancreatic adenocarcinoma cells for 
treatments and represents an additional problem in the design of novel 
therapies for this disease. Thus, one critical challenge of pancreatic 
adenocarcinoma treatment is to find efficient therapeutic approaches to 
reverse a “cold” into a “hot” tumor, priming immune response during pancreatic 
adenocarcinoma progression. The current proposal aims to identify a 
promising therapeutic approach, with a focus on boosting tumor-specific 
immunity for pancreatic adenocarcinoma patients. 
 
2.2. Immunotherapy in pancreatic adenocarcinoma 
Interferons (IFNs) are cytokines that play a critical role in limiting infectious 
and suppressing tumor progression due to interferon-stimulated genes 
expression such as TNF-α, IL-6 and IFNs per se (Schoggins et al. 2019). 
However a subset of interferon-stimulated genes act as negative pathway 
regulators and immunosuppressive factors to extinguish signaling and limit 
therapeutic efficiency of cancers, for example, PD-L1 (Benci et al. 2016). To 

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date, immune checkpoint inhibitors targeting PD-1 or PD-L1 represent a 
popular treatment option for patients with certain cancers (Wu et al. 2019). 
However, a part of cancer patients including pancreatic adenocarcinoma 
endure resistance to anti-PD1/PD-L1 immunotherapies because of loss of 
IFNs signaling (de Erauso et al. 2020).  
 
2.3. Adenosine deaminase acting on RNA 1 (ADAR1) 
Recently, emerging data suggest that a member of interferon-stimulated 
genes, double-stranded RNA-specific adenosine deaminase (ADAR1) which is 
an RNA editing enzyme and a negative regulator of IFNs signaling (Figure 2), 
promotes immune silencing and tumor viability (Herbert et al. 2019). Several 
reports indicated reducing ADAR1 expression in tumors increases sensitivity 
to checkpoint inhibitors (Liu, et al. 2019; Gannon et al. 2018), suggesting that 
suppression of ADAR1 may be a promising strategy for tumor immunotherapy 
for cancer patients. However, there is limited understanding of how to inhibit 
ADAR1 for application of cancer therapy. 
 
2.4. Retinoic acid and pancreatic adenocarcinoma 
Retinoic acid plays important roles in cell development, differentiation 
(Gudas et al. 2011), immune regulation (Larange et al. 2016) as well as tumor 
progression (Tang et al. 2011). Except for its cytotoxic effect on tumor cells 
(Shilkaitis et al. 2015), it has been reported that retinoic acid enhances 
protective antitumor immunity through mechanisms such as induction of cell 
differentiation and inhibition of immunosuppressive cells (Malkovský et al. 
1983; Walkley et al. 2002). In 2004, all-trans retinoic acid (Vesanoid) was 
approved by FDA to induce remission in patients with acute promyelocytic 
leukemia (Küley-Bagheri et al. 2018). Regarding the present clinical trials of 
all-trans retinoic acid in solid tumors, for example, breast cancers, all-trans 
retinoic acid does not have significant activity in patients with 
hormone-refractory, metastatic breast cancer (Sutton et al. 1997). These 
unexpected results of clinical trials emerging may be due to the ignored 
immune checkpoints up-regulated by all-trans retinoic acid. This proposal is 
raised for development of a potential therapeutic approach for pancreatic 
adenocarcinoma patients by regulating tumor immune response. Recently, our 
laboratory has found several important evidence to support the use of all-trans 
retinoic acid in pancreatic cancer. 

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2.5. Key evidence available to support the use of all-trans 
retinoic acid in pancreatic adenocarcinoma 
 
2.5.1. All-trans retinoic acid represses pancreatic adenocarcinoma 
cells growth and triggers IFNs signaling 
 
In our previous studies, all-trans retinoic acid represses pancreatic 
adenocarcinoma cell growth and colony formation (Figure 1A and 1B). 
Interestingly, set-to-set analysis of the significant gene sets regulated by 
all-trans retinoic acid revealed a strong overrepresentation of gene sets 
involved in immune response including interleukin-6, tumor necrotic factor 
(TNF)-α and IFNs signaling (Figure 1C). 
 
Figure 1. Retinoic acid represses pancreatic adenocarcinoma cells growth and triggers 
interferons signaling. (A) The effects of retinoic acid on pancreatic adenocarcinoma viability. (B) 
Colony formation assay. (C) GSEA leading-edge analysis of identified significant gene sets 
regulated by retinoic acid in MDA-MB231 cells. RA, retinoic acid. 
 
2.4.2. All-trans retinoic acid and PD-L1 in pancreatic 
adenocarcinoma: all-trans retinoic acid enhances PD-L1 but 
represses ADAR1 expression 
 
Due to IFNs signaling stimulated by all-trans retinoic acid, we further 
investigate the effect of all-trans retinoic acid on the downstream of 
IFNs-stimulated genes. Intriguingly, all-trans retinoic acid specifically induces 
the expression of PD-L1 instead of other immune checkpoints regulated by 
IFNs (Figure 2A and 2B). More importantly, ADAR1, a member of 
interferon-stimulated genes and a negative regulator of IFNs signaling, is 
decreased by all-trans retinoic acid (Figure 2B). The set-to-set analysis of the 

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significant gene sets indicates that knockdown of ADAR1 enriches immune 
gene profiles which are similar to those regulated by all-trans retinoic acid 
(Figure 2C), implying that all-trans retinoic acid triggers an intense IFNs 
signaling due to blockage of a negative regulator, ADAR1. 
 
Figure 2. Retinoic acid enhances PD-L1 but represses ADAR1 expression. (A) Representative 
immune checkpoint array comparing vehicle control (DMSO) versus retinoic acid in HPAC 
cells. Quantitative data for the relative PD-L1 expression are determined with ImageJ software 
and represented at the right. (B) Western blot analysis of ADAR1, PD-L1 and β-actin. Cells 
were treated with retinoic acid with the dose as indicated for 24 h. (C) GSEA leading-edge 
analysis of identified significant gene sets in ADAR1-knockout H196 cells (GSE122168). RA, 
retinoic acid. 
 
 
2.4.3. Combination treatment of anti-PD1 and all-trans retinoic acid 
improves survival rate in murine pancreatic cancer model 
 
Since all-trans retinoic acid induced PD-L1 and inhibited ADAR1 
expression, we further tested for any synergistic effect between anti-PD-1 
blockade and all-trans retinoic acid. To this end, we analyzed the in vivo 
efficacy of all-trans retinoic acid and anti-PD-1 combination treatment in the 
murine syngeneic Pan02 pancreatic cancer model (Figure 3A). Mice treated 
with anti-PD-1 antibody and all-trans retinoic acid had significantly smaller 
tumors compared to mice receiving either monotherapy (Figure 3B). 
Furthermore, combination treatment was led to a significant improvement in 
survival compared to both anti-PD-1 and all-trans retinoic acid monotherapy 
groups (Figure 3C). The average mouse body weight did not differ significantly 
among all four groups (Figure 3D), suggesting that these regimens did not 
cause significant toxicity effects. The phenomenon that all-trans retinoic acid 
inhibit ADAR1 expression can be confirmed in syngeneic Pan02 pancreatic 
cancer model (Figure 3E). 

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Figure 3. Anti-PD1 antibody and retinoic acid combination treatment improves antitumor effect 
in murine PDAC model. (A) The outline of schemed treatment strategy and tumor challenge. 
(B) Left, representative images showing tumors harvested from mice bearing Pan02 tumor 
cells received indicated treatments. Scale bar, 1.2 cm. Right, tumor weight. (C) Survival rate of 
mice after systemic therapy. (D) Body weight from all groups. (E) Western blot analysis of 
adar1 and vinculin protein expression levels in Pan02 cells from subcutaneous murine PDAC 
model treated with indicated regimens. 
 
2.4.4. ADAR1 positively correlates with poor survival in cancer 
patients 
 
The data from The Cancer Genome Atlas (TCGA) database shows that 
ADAR1 mRNA is upregulated in various cancers including pancreatic 
adenocarcinoma (Figure 4A). ADAR1 proteins positively correlates with poor 
survival among either breast cancer (p=0.0162 and p=0.0373 in two cohorts, 
respectively; Figure 4B; Liu et al. 2014) or pancreatic adenocarcinoma patients 
(p=0.0045; Figure 4C and 4D). Addition of gemcitabine, a cornerstone of 
pancreatic adenocarcinoma treatment, induces the expression of both ADAR1 
and PD-L1 in vitro; however, all-trans retinoic acid can inhibit ADAR1 in 
gemcitabine treated pancreatic adenocarcinoma cells (Figure 4E). Next, we 
conducted GSEA to investigate which gene sets are associated with 
gemcitabine-resistant pancreatic adenocarcinoma. The results indicated 
gemcitabine-resistant pancreatic adenocarcinoma have negatively enriched 
hallmark gene sets including interferon-α response (NES = -1.195, p < 0.0001; 
Figure 4F). Since all-trans retinoic acid triggers interferon-α signaling and 
suppresses ADAR1 as well as induces PD-L1, combination treatment of 
all-trans retinoic acid and anti-PD1 might be a promising therapeutic strategy 

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for pancreatic adenocarcinoma patients who have failed gemcitabine 
chemotherapy. 
 
Figure 4. ADAR1 positively correlates with poor survival in cancer patients. (A) ADAR1 gene 
expression profiled by GEPIA. Tumor (T), red dots; normal tissues (N), green dots. BRCA, 
breast invasive carcinoma; ESCA, esophageal carcinoma; HNSC, head and Neck squamous 
cell carcinoma; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; PAAD, 
pancreatic adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach 
adenocarcinoma; TGCT, testicular germ cell tumors. (B) The correlation between ADAR1 
protein expression and breast cancer patients' survival in two cohorts (left, Liu et al., 2014; 
right, TCGA-RPPA). (C) The correlation between ADAR1 protein expression and PDAC 
patients' survival in MDA cohort. (D) Representative images of the immunohistochemical 
staining of ADAR1 in PDAC patients. (E) Western blot analysis showing the effects of 
combination treatment with gemcitabine and RA. BxPC3 
 
In summary, all-trans retinoic acid represses ADAR1, a member of 
interferon-stimulated genes and a negative regulator of IFNs signaling. On the 
other hand, although all-trans retinoic acid simultaneously increases PD-L1 
expression for cancer cells to evade immune surveillance, it can be overcome 
by blockade of PD1/PD-L1 immune checkpoints (Figure 5). Since combination 
of anti-PD1 antibody and all-trans retinoic acid may thoroughly block 
self-regulating negative feedback loops of IFNs response, this therapeutic 
strategy may improve outcomes of pancreatic adenocarcinoma patients. 

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Figure 5. The schema of hypothetic model. 
 
2.5. Proposal of all-trans retinoic acid in patients with 
refractory pancreatic adenocarcinoma 
 
Based on the previous findings, we propose this pilot, compassionate use 
of combination therapy with all-trans retinoic acid and nivolumab for patients 
with locally advanced or metastatic pancreatic adenocarcinoma who are 
refractory to current available treatment. This study will also examine ADAR1 
as a novel prognostic and predictive biomarker for pancreatic adenocarcinoma 
patients. The major goals of the study is to examine:  
1. The anticancer activity of the combination therapy with all-trans retinoic acid 
and nivolumab in patients with advanced or metastatic pancreatic 
adenocarcinoma 
2. The expression and change of ADAR1 as a prognostic and predictive 
biomarkers of immunotherapy for pancreatic adenocarcinoma. 
This study will help to delineate the role of ADAR1 as a predictive 
biomarker for immunotherapy, all-trans retinoic acid as a suppressor of 
ADAR1, and most importantly, the combination of all-trans retinoic acid and 
nivolumab as an effective anticancer therapy for patients with pancreatic 
adenocarcinoma. In addition to be a proof of concept study, we hope this 
combination can be beneficial for patients with refractory pancreatic 
adenocarcinoma.  

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3. Objective 
3.1. To evaluate the therapeutic efficacy of all-trans retinoic acid in 
combination with nivolumab in patients with advanced or metastatic pancreatic 
cancer. 
3.2. To evaluate the role of ADAR1 as the predictive biomarker of 
immunotherapy for pancreatic cancer. 
 
 

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4. Drug information 
4.1. Nivolumab 
ONO-4538 is a human monoclonal antibody that targets the human PD-1 
receptor and is produced in CHO cells by recombinant deoxyribonucleic acid 
(DNA) technology. The drug substance is an aqueous solution of ONO-4538 
that is clear or opalescent and colorless or pale yellow; fine particles may be 
slightly observed in the solution. The drug product is stored away from light at 
2°C–8°C. ONO-4538 specifically binds to the PD-1 receptor and has shown 
high affinity for human PD-1 and simian PD-1 with dissociation constant (KD) 
values of 3.06 nmol/L and 3.92 nmol/L, respectively. On the basis of the above, 
ONO-4538 is expected to exert its antitumor effect by inhibiting the binding 
between PD-1 and PD-1 ligand and thereby augmenting the activity of 
antigen-specific T cells, which results in an enhanced tumor-specific immune 
response. The safety profile of ONO-4538 monotherapy has shown no 
substantial difference across the tumor types without dose dependency in the 
incidence, severity, or causal relationship of adverse events and adverse drug 
reactions. Ongoing clinical studies are evaluating the safety of the combination 
use of ONO-4538 with other agents, including ipilimumab, cytotoxic 
chemotherapy agents, angiogenesis inhibitors, and molecular-targeted 
agents.  
Currently, nivolumab has been proved by US FDA for the treatment of: 
melanoma, non-small cell lung cancer, small cell lung cancer, renal cell 
carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the 
head and neck, urothelial carcinoma, microsatellite instability-high (MSI-H) or 
mismatch repair deficient (dMMR) metastatic colorectal cancer, and 
hepatocellular carcinoma. 
 
4.2. All-trans retinoic acid (Vesanoid) 
Chemically, vesanoid is all-trans retinoic acid (tretinoin) and is related to 
retinol (Vitamin A). Vesanoid induces maturation of acute promyelocytic 
leukemia cells. It is available in a 10 mg soft gelatin capsule for oral 
administration. Each capsule also contains beeswax, butylated hydroxyanisole, 
edetate disodium, hydrogenated soybean oil flakes, hydrogenated vegetable 
oils and soybean oil. The gelatin capsule shell contains glycerin, yellow iron 

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oxide, red iron oxide, titanium dioxide, methylparaben and propylparaben. It is 
a yellow to light orange crystalline powder with a molecular weight of 300.44. 
Vesanoid is not a cytolytic agent but instead induces cytodifferentiation 
and decreased proliferation of acute promyelocytic leukemia cells in culture 
and in vivo. In acute promyelocytic leukemia patients, vesanoid treatment 
produces an initial maturation of the primitive promyelocytes derived from the 
leukemic clone, followed by a repopulation of the bone marrow and peripheral 
blood by normal, polyclonal hematopoietic cells in patients achieving complete 
remission. The exact mechanism of action of vesanoid in acute promyelocytic 
leukemia is unknown. 
The reported adverse effects of vesanoids include (US-FDA package 
inserts): 
(1) Incidence10%：headache, fever, malaise, dry skin, bone soreness, 
hemorrhage, infection, edema, chest tightness, nausea, vomiting, skin rash, 
mucositis, itching, sweating, visual acuity abnormality, hair loss, abdominal 
pain, diarrhea, fullness, arrhythmia, flushing, hypotension, hypertension, 
dizziness, insomnia, depression  
(2) Incidence 1-10%：visual field abnormality, flank soreness, cellulitis, skin 
palor, hypothermia, ascites, acidosis, dyspnea, pleural effusion, asthma, 
pulmonary edema, laryngeal edema, hepatosplenomegaly, hepatitis, oral ulcer, 
heart failure, anxiety, hallucination, unsteady gait, dysuria, renal dysfunction, 
enlarged prostate,  
(3) Incidence0.1-1%：shearing impairment, hypercalcemia, pancreatitis, 
myositis, organomegaly, hyperbasophilemia 

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5. Selection of patients 
5.1. Inclusion criteria: 
Patients will be included in the study if they meet all of the following criteria: 
(1) Patients with age ≥ 20 years old 
(2) Histologically confirmed pancreatic adenocarcinoma 
(3) Unresectable locally advanced, recurrent or metastatic diseases ineligible 
or unsuitable for further surgical or radiation interventions 
(4) Documented disease progression within 6 months after standard 
chemotherapies or no available standard chemotherapy. The standard 
chemotherapies include gemcitabine, nab-paclitaxel, S-1, and FOLFIRINOX. 
Patient who has prior anti-PD1/anti-PD-L1 treatment will not be eligible. 
(5) ECOG Performance Status 0-2 
(6) Documented measurable disease as defined by RECIST v1.1 
(7) Adequate hematologic parameters, and hepatic and renal functions defined 
as 
a.absolute neutrophil count ≥ 1,000/μL 
b.platelets 
≥ 75,000/μL 
c.total bilirubin ≤ 2.5X ULN (≤ 5X ULN if attributable to liver metastases) 
d.AST/ALT ≤ 2.5X ULN (≤ 5X ULN if attributable to liver metastases) 
e.serum creatinine ≤ 2 mg/dL or creatinine clearance ≥ 30 mL/min (by 
calculated or 24-hour urine collection) 
(8) Normal ECG or ECG without any clinical significant findings 
(9) Able to understand and sign an informed consent (or have a legal 
representative who is able to do so) 
 
5.2. Exclusion criteria: 
Patients will be excluded from the study if they meet any of the following 
criteria: 
(1) History of allergic reaction to all-trans retinoic acid or nivolumab 
(2) Patient with liver cirrhosis with Child-Pugh score ≥ 8 (Late Child-Pugh B 
and Child-Pugh C) 
(3) Active CNS metastasis defined by clinical symptoms, cerebral edema, 
steroid or anti-convulsant requirement, or progressive growth.  Patients with a 
history of CNS metastasis or cord compression are allowed in the study if they 
have been treated and are clinically stable 

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(4) With clinically significant gastrointestinal disorder including bleeding, 
inflammation, occlusion or diarrhea > grade 1 
(5) With uncontrolled intercurrent illness that could limit study compliance or 
judged to be ineligible for the study by the investigators including, but not 
limited to, any of the following: 
a. ongoing or active infection requiring antibiotic treatment 
b. symptomatic congestive heart failure, unstable angina pectoris, or cardiac 
arrhythmia 
c. psychiatric illness or social situation that would preclude study compliance 
(6) Pregnant or breast feeding women (a urine pregnancy test must be 
performed on all patients who are of childbearing potential before entering the 
study, and the result must be negative) 
(7) Patients taking the following medications: immunosuppressants, 
corticosteroids with the exception of administration topically (e.g., external, 
intra-articular, intranasal, ophthalmic, or inhalational use) or temporarily (e.g., 
for treatment or prophylaxis of contrast medium allergy or adverse events), 
antitumor therapies (e.g., chemotherapies, molecular-targeted therapies, 
immunotherapies),  radiopharmaceuticals with the exception of diagnostic 
purposes, transplant therapies, vitamin A, antifibrinolytic agents (tranexamic 
acid, aminocaproic acid, aprotinin), inducers (rifampicin, glucocorticoids, 
phenobarbital and pentobarbital) or inhibitors (ketoconazole, cimetidine, 
erythromycin, verapamil, diltiazem and cyclosporine) of the hepatic P450 
system, and other unapproved drugs (e.g., investigational use of drugs, 
unapproved combined formulations, unapproved dosage forms). 

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6. Study treatment 
6.1. Overall study design 
6.1.1. Number of subject: 10 
6.1.2. Study period: 2021.2.1-2022.12.31  
6.1.3. Treatment plan 
6.1.3.1. Patient will receive oral all-trans retinoic acid (Vesanoid) 45 mg/m2 on 
days 1-14, and nivolumab 3mg/kg intravenously on day 1. The treatment cycle 
will repeat every 2 weeks.  
 
 
 
 
6.1.3.2. The treatment will continue till disease progression, unacceptable 
toxicity, patients refusal, withdraw of consent, death, or not beneficial for 
patient by treating physician‟s opinion. 
 
6.2. Dosage modification and treatment delay 
6.2.1. The dose of nivolumab will be kept without modification. The 
management of immune-related adverse effects and further use of nivolumab 
is according to the treatment guideline by The National Comprehensive 
Cancer Network (NCCN). 
6.2.2. The treatment of all-trans retinoic acid will be hold if patient encounter 
grade 3 or higher hematological or non-hematological toxicities except 
alopecia or diarrhea which can be controlled by further medication. After 
resume, the dose of all-trans retinoic acid can be reduced to a lower level if 
patient has grade 3 non-hematological toxicity or recurrent grade 2 
hematological toxicity.  
6.2.3. All-trans retinoic acid dose modifications are permanent; no dose 
escalations are allowed after dose reduction. Any patient who has 2 dose 
reductions and experiences a toxicity that would cause a third dose reduction 
must be discontinued from the study drug which is causing the toxicity. 

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6.2.4 Dose of any drug omitted for toxicity is not replaced or restored; instead, 
the patient should resume the planned treatment cycles. 
6.2.5. Dose modification for non-serious and no-life-threatening toxicities such 
as alopecia, altered taste, or nail changes may not be required. The final 
decision is left to the discretion of the treating investigator. 
6.2.6. In situations where concomitant toxicities of varying severity exist, dose 
modification will be tailored by the toxicity with highest National Cancer 
Institute (NCI) Common Terminology Criteria for Adverse Effect (CTCAE) 
grade. 
6.2.7. If there is delay or modification in administration of study drug due to 
toxicity, treatment with the other study agent should be discontinued. 
6.2.8. If a toxicity related to any component of chemotherapy does not resolve 
in the same treatment cycle, the administration of that component can be 
delayed up to 28 days from the next planned dose of the component. If the 
toxicity does not resolve within 28 days, patient will be withdraw from the 
study.  
 
6.3. Concomitant therapy 
6.3.1. Prohibited therapies through study period 
The following treatments are prohibited throughout the study period (i.e., from 
informed consent through completion of the protocol-specified final 
examination), unless absolutely necessary for medical reasons. 
(1) Immunosuppressants  
(2) Corticosteroids with the exception of administration topically (e.g., external, 
intra-articular, intranasal, ophthalmic, or inhalational use) or temporarily (e.g., 
for treatment or prophylaxis of contrast medium allergy or adverse events). 
(3) Antitumor therapies (e.g., chemotherapies, molecular-targeted therapies, 
immunotherapies), including antibody products, with the exception of 
protocol-specified standard therapies  
(4) Surgical therapies for malignant tumors 
(5) Radiotherapies to the targeted lesions 
(6) Radiopharmaceuticals with the exception of diagnostic purposes 
(7) Transplant therapies  
(8) Inducers (rifampicin, glucocorticoids, phenobarbital and pentobarbital) or 
inhibitors (ketoconazole, cimetidine, erythromycin, verapamil, diltiazem and 
cyclosporine) of the hepatic P450 system are suggested to be avoid. Although 
vesanoid is metabolized by the hepatic P450 system, to date there are no data 

18 
 
to suggest that co-use with these medications increases or decreases either 
efficacy or toxicity of Vesanoid.  
(9) Vitamin A 
(10) Antifibrinolytic agents (tranexamic acid, aminocaproic acid, aprotinin) 
(11) Other unapproved drugs (e.g., investigational use of drugs, unapproved 
combined formulations, unapproved dosage forms)  
 
6.3.2. Permitted therapies through study period 
(1) For subjects who have experienced infusion-related reactions to the 
investigational product and may experience recurrences of infusion related 
reactions, prophylactic medication with acetaminophen or diphenhydramine is 
recommended before administration of the investigational product.  
(2) Corticosteroids may be used in the usual dosage and mode of 
administration in the study site for subjects in whom chemotherapy-induced 
nausea/vomiting is concerned. 
 
6.3.3. No data on the effect of food on the absorption of VESANOID are 
available. The absorption of retinoids as a class has been shown to be 
enhanced when taken together with food. 
 
6.4. Off study criteria 
6.4.1. Radiological evidence of disease progression, 
6.4.2. Delayed recovery of treatment-related toxicities, which prohibits the 
administration of drug in next cycle of treatment on day 29, 
6.4.3. Presence of NCI grade 4 non-hematological toxicity, 
6.4.4. Presence of NCI grade 3 or higher non-hematologic toxicity after 2 dose 
reductions, 
6.4.5. Presence of severe allergic reaction or anaphylaxis, 
6.4.6.Treatment-related event that is deemed life-threatening if the event is 
considered possibly related to any components of study therapy, 
6.4.7.Death of the patient, 
6.4.8.The patient requests to be withdrawn from study treatment, 
6.4.9. Available potential better alternative treatment at the discretion of 
in-charged physicians. 
 

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7. Study calendar 
7.1. The following tests should be performed within one week before the 
initiation of therapy (as the baseline data) and then be repeated as clinical 
needed following the initiation of therapy till the end of therapy visit. Baseline 
image studies (scan and x-film) must be done within 4 weeks prior to the start 
of therapy. 
7.2. Physical examination, comprehensive history, performance status 
(ECOG score), complete blood cell count, differential blood cell count 
(CBC/DC), albumin, liver function test (total bilirubin, AST, ALT, alkaline 
phosphatase, r-GT), serum creatinine, glomerular filtration rate (GFR), fasting 
blood sugar, electrolyte (Na, K), lipid profile (cholesterol, triglyceride), and 
coagulation profile (prothrombin time, activated partial thromboplastin time). 
Other laboratory tests are on the physician‟s decision clinically. 
7.3. GFR is calculated according to Cockcroft-Gault equation [(140-age) x 
body weight (kg) x (1 if male or 0.85 if female) / (72 x serum creatinine level, 
mg/dl)] every treatment cycle. 
7.4. Serum level of CEA and CA 19-9 will be checked every 2 cycles of 
treatment. 
7.5. Image study (chest X-film + abdominal CT/MRI, or chest + abdominal 
CT/MRI) will be examined every 8 weeks. For patients with radiographic 
objective response, scan should be repeated to document tumor response 4 
weeks later. 
7.6. Chest X-film can be omitted if chest computed tomography is done. 
7.7. Bone scan will be done in patients with clinically suspected or evident 
bony metastases at screening, and be repeated every 4 cycles of treatment 
afterward. 
7.8. Exploratory biomarker 
7.8.1. To evaluate the change of cytokine (IFNs, IL-6, IL-2, etc.), 10 ml of blood 
will be collected during the screening period and every 2 cycles of treatment till 

20 
 
disease progression, end of treatment due to other reasons, or 12 months with 
whatever comes first. 
7.8.2. Tumor tissue will be assessed for the expression of PD-L1 and 
immune-related genes (PD-L1, ADAR1, etc.) immunohistochemically. Two to 5 
slides of tissue from archived tumor are needed. 
7.8.3. The tumor tissue and blood for cytokine and immune-related genes 
determination will be managed at Center for Molecular Medicine at China 
Medical University Hospital, and be stored for 15 years.  
7.9. Flow chart of evaluation 
 
 
Baseline 
Day 8 
q 1 cycle q 2 cycles q 4 cycles q 8 week 
EOTf 
Follow-up 
History / physical 
examination 
X 
X 
X 
 
 
 
X 
 
Performance 
Status, ECOG 
X 
X  
X 
 
 
 
X 
 
Body weight 
X 
 
X 
 
 
 
X 
 
CBC and 
differential count 
X 
X  
X 
 
 
 
X 
 
Albumin 
X 
 
 
X 
 
 
X 
 
Liver function testa 
X 
X 
X 
 
 
 
X 
 
Serum Creatinine / 
Fasting blood 
sugar / Electrolytes 
/ Lipid profile / 
Coagulation 
X 
 
X 
 
 
 
X 
 

21 
 
profileb 
GFR (glomerular 
filtration rate) 
X 
 
X 
 
 
 
X 
 
CEA, CA19-9 
X 
 
 
X 
 
 
X 
 
Chest X-filmc,e 
X 
 
 
 
 
X 
 
 
Abdominal (± 
chest) CT / MRIe 
X 
 
 
 
 
X 
Xg 
Xh 
Bone scand,e 
X 
 
 
 
 
X 
 
 
Tissue sampling 
X 
 
 
 
 
 
 
 
Serum sampling 
X 
 
 
X 
 
 
X 
 
 
aliver function test include total bilirubin, AST, ALT, alkaline phosphatase, -GT 
belectrolytes include Na and K, lipid profile include cholesterol and triglyceride, coagulation profile include 
prothrombin time and activated partial thromboplastin time 
cchest X-film can be omitted if chest tomography is done 
dfor patients with clinically suspected or evident bony metastases 
echest X-film, abdominal (± chest) CT / MRI and bone scan (if needed) are permitted to have the examination 
on schedule  5 days 
fend of treatment 
gfor patients without disease progression by last CT/MRI which is examined more than 2 weeks before 
off-study 
hevery 2 months till disease progression or death 
 

22 
 
8. Efficacy assessment 
8.1. The objective tumor response and duration of response are evaluated 
according to RECIST v1.1. Response rate and disease control rate will be 
calculated. Intervals of duration of response, progression-free survival, time to 
tumor progression and overall survival will also be collected. Tumor marker 
response will be evaluated.  
8.2. The overall survival will be calculated from the date of registration to the 
date of patient„s death. 
8.3. Time to tumor progression will be calculated from the date of registration 
to the first date of disease progression. 
8.4. Progression-free survival will be calculated from the date of registration 
to the date of disease progression or patient„s death of whatever cause. 
 

23 
 
9. Serious adverse effect and reporting 
9.1. Definition of Adverse Events 
9.1.1. An adverse event is defined as any unfavorable or unintended sign 
(including an abnormal laboratory value), symptom, or disease temporally 
associated with the use of the investigational product, regardless of whether 
the event is related to the investigational product.  
9.1.2. Worsening of the underlying disease, an accompanying symptom of the 
underlying disease, or a concurrent disease is handled as an adverse event if 
the worsening is beyond its natural course as medically confirmed.  
9.1.3. Tumor enlargement or appearance of a new lesion (except for malignant 
tumor histologically different from the primary lesion) after the start of the study 
is thought as an adverse event if the enlargement/appearance is beyond its 
natural course as medically confirmed. 
 
9.2. Definition of Serious Adverse Events 
A serious adverse events is defined as an event that meets the definition 
of adverse events and: 
(1) results in death 
(2) is life-threatening 
(3) requires inpatient hospitalization for treatment or prolongation of existing 
hospitalization 
(4) results in significant or permanent disability/incapacity 
(5) is a congenital anomaly 
(6) is otherwise a medically important event 
 
9.3. Criteria for Grading Adverse Events 
Adverse events will be graded using the definition by CTCAE 5.0  
 
9.4. Reporting of Serious Adverse Events 
Following the subject‟s written consent to participate in the study, all 
serious adverse events (SAEs) that occur between the start of the study 
treatment and 100 days after the end of the study treatment, whether related or 
not to the investigational product, must be collected, including those thought to 

24 
 
be associated with protocol-specified procedures. If applicable, SAEs must be 
collected that relate to any later protocol-specified procedure (e.g., a follow-up 
skin biopsy). 
The investigator should also report any SAE occurring after these time 
periods that is believed to be related to any investigational product or 
protocol-specified procedures. 
An SAE report should be completed for any event where doubt exists 
regarding its status of seriousness. 
If the investigator believes that an SAE is not related to any investigational 
product, but is potentially related to the conditions of the study (such as 
withdrawal of previous therapy or a complication of a study procedure), the 
relationship should be specified in the narrative section of the SAE Report 
Form. 
SAEs including overdose, whether related or not to the investigational 
product, and pregnancies must be reported to the sponsor (or designee) within 
24 hours. SAEs must be recorded on the SAE Report Form; pregnancies on a 
Pregnancy Surveillance Form. 
The report forms are to be transmitted via email or confirmed fax 
transmission to the following, and the original report forms are to remain on 
site. 
 
9.5. Reporting of Pregnancies 
Following initiation of the study treatment, if it is subsequently discovered 
that a study subject has become pregnant within 28 days after the end of the 
study treatment, or may have been pregnant during investigational product 
exposure, the investigational products should be permanently discontinued in 
an appropriate manner (e.g., dose tapering if necessary for subject safety). 
The investigator must immediately notify the sponsor (or designee) of this 
event, and complete and forward a Pregnancy Surveillance Form to the 
sponsor (or designee) within 24 hours. 
Follow-up information regarding the course of the pregnancy, including 
perinatal and neonatal outcomes and, if possible, newborn information must 
be reported on the Pregnancy Surveillance Form. 
Any pregnancy that occurs in a female partner of a male study participant 
should be reported to the sponsor. Information on this pregnancy will be 
collected on the Pregnancy Surveillance Form. 

25 
 
 
9.6. Reporting of Overdose 
An overdose is defined as the accidental or intentional administration of 
any dose of a drug product that is considered both excessive and medically 
important. All occurrences of overdose must be reported as an SAE (see 
Section 9.8 for reporting details). 
 
 
 

26 
 
10. Statistical consideration and patient accrual 
10.1. This is a compassionate use of combination therapy with all-trans 
retinoic acid and nivolumab for patients with locally advanced or metastatic 
pancreatic adenocarcinoma who are refractory to current standard of therapies. 
As a pilot and prove-of-concept study, the target number of patients is ten.  
 
10.2. Considering the current evidence, the study is worthy to expand to next 
step of trial if there is 2 or more patients who have response to the combination 
therapy. 
 

27 
 
11. Ethical, legal, and administrative aspects 
11.1. Data collection 
11.1.1. All clinical patient data collected according to the protocol will be stored 
by PI at Division of Hematology and Oncology, Department of Internal 
Medicine, China Medical University Hospital.  
11.1.2. Patient clinical source documents would include documents such as 
(although not be limited to) the following: patient hospital/clinic records, 
physician‟s and nurse‟s notes, original laboratory reports, ECG, 
electroencephalogram, x-ray,pathology and special assessment reports, 
consultant letters, and screening and enrollment log, etc. 
 
11.2. Study records retention 
11.2.1. Following closure of the study, the investigator must maintain all study 
records in a safe and secure location. The document will be stored in a locked 
cabinet and the electric data will be stored in a computer with secured 
password.  
11.2.2. The records must be maintained to allow easy and timely retrieval, 
when needed (eg, audit or inspection), and, whenever feasible, to allow any 
subsequent review of data in conjunction with assessment of the facility, 
supporting systems, and personnel.  
11.2.3. Where permitted by local laws/regulations or institutional policy, some 
or all of these records can be maintained in a format other than hard copy (eg, 
microfiche, scanned, electronic); however, caution needs to be exercised 
before such action is taken. The investigator must assure that all reproductions 
are legible, are a true and accurate copy of the original, and meet accessibility 
and retrieval standards, including regenerating a hard copy, if required. 
Furthermore, the investigator must ensure there is an acceptable backup of 
these reproductions and that an acceptable quality control process exists for 
making these reproductions. 
11.2.4. The minimum retention time will meet the strictest standard applicable 
to that study center for the study, as dictated by any institutional requirements 
or local laws or regulations, or the sponsor‟s standards/procedures; otherwise, 
the retention period will default to 15 years. 
 

28 
 
12. References 
Amrutkar M, et al. Pancreatic cancer chemoresistance to gemcitabine. 
Cancers. 2017 Nov 16;9(11):157. 
Armstrong T, et al. Type I collagen promotes the malignant phenotype of 
pancreatic ductal adenocarcinoma. Clin Cancer Res. 2004 Nov 
1;10(21):7427–37. 
Beatty GL, et al. Exclusion of T Cells from pancreatic carcinomas in mice is 
regulated by Ly6C(low) F4/80(+) extratumoral macrophages. 
Gastroenterology. 2015 Jul;149(1):201–10. 
Benci JL, et al. Tumor interferon signaling regulates a multigenic resistance 
program to immune checkpoint blockade. Cell. 2016 Dec 
1;167(6):1540–54. 
de Erauso LC, et al. Resistance to PD-L1/PD-1 Blockade Immunotherapy. A 
Tumor-Intrinsic or Tumor-Extrinsic Phenomenon? Front Pharmacol. 2020 
Apr 7;11:441. 
Ferry C, et al. Cullin 3 mediates SRC-3 ubiquitination and degradation to 
control the retinoic acid response. Proc Natl Acad Sci U S A. 2011 Dec 
20;108(51):20603–8. 
Gannon HS, et al. Identification of ADAR1 adenosine deaminase dependency 
in a subset of cancer cells. Nat Commun. 2018 Dec 21;9(1):5450. 
Germain P, et al. International Union of Pharmacology. LX. Retinoic acid 
receptors. Pharmacol Rev. 2006 Dec;58(4):712–25. 
Ghaneh P, et al. Biology and management of pancreatic cancer. Postgrad Med 
J. 2008 Sep;84(995):478–97. 
Gudas LJ, et al. Retinoids regulate stem cell differentiation. J Cell Physiol. 
2011 Feb;226(2):322–30. 
Herbert A, et al. ADAR and immune silencing in cancer. Trends Cancer. 2019 
May;5(5):272–82. 
Kenner BJ, et al. Early detection of pancreatic cancer–a defined future using 
lessons from other cancers. Pancreas. 2016 Sep;45(8):1073–9. 
Küley-Bagheri Y, et al. Effects of all-trans retinoic acid (ATRA) in addition to 
chemotherapy for adults with acute myeloid leukaemia (AML) (non-acute 
promyelocytic leukaemia (non-APL)). Cochrane Database Syst Rev. 2018 
Aug 6;8(8):CD011960. 
Larange A, et al. Retinoic acid and retinoic acid receptors as pleiotropic 
modulators of the immune system. Annu Rev Immunol. 2016 May 
20;34:369–94. 

29 
 
Liu H, et al. Tumor-derived IFN triggers chronic pathway agonism and 
sensitivity to ADAR loss. Nat Med. 2019 Jan;25(1):95–102. 
Liu NQ, et al. Comparative proteome analysis revealing an 11-protein 
signature for aggressive triple-negative breast cancer. J Natl Cancer Inst. 
2014 Feb;106(2):djt376. 
Malkovský M, et al. Enhancement of specific antitumor immunity in mice fed a 
diet enriched in vitamin A acetate. Proc Natl Acad Sci U S A. 1983 
Oct;80(20):6322–6. 
Principe DR, et al. TGFβ signaling in the pancreatic tumor microenvironment 
promotes fibrosis and immune evasion to facilitate tumorigenesis. Cancer 
Res. 2016 May 1;76(9):2525–39. 
Riquelme E, et al. Immunotherapy for pancreatic cancer: more than just a gut 
feeling. Cancer Discov. 2018 Apr;8(4):386–8. 
Royal RE, et al. Phase 2 trial of single agent Ipilimumab (anti-CTLA-4) for 
locally advanced or metastatic pancreatic adenocarcinoma. J Immunother. 
2010 Oct;33(8):828-33. 
Schoggins JW, et al. Interferon-stimulated genes: what do they all do? Annu 
Rev Virol. 2019 Sep 29;6(1):567–84. 
Shilkaitis A, et al. Retinoids induce cellular senescence in breast cancer cells 
by RAR-β dependent and independent pathways: Potential clinical 
implications (Review). Int J Oncol. 2015 Jul;47(1):35–42. 
Sutton LM, et al. Pharmacokinetics and clinical impact of all-trans retinoic acid 
in metastatic breast cancer: a phase II trial. Cancer Chemother Pharmacol. 
1997;40(4):335–41. 
Tang XH, et al. Retinoids, retinoic acid receptors, and cancer. Annu Rev 
Pathol. 2011;6:345–64. 
Upadhrasta S, et al. Strategies in developing immunotherapy for pancreatic 
cancer: recognizing and correcting multiple immune “defects” in the tumor 
microenvironment. J Clin Med. 2019 Sep 16;8(9):1472. 
Walkley CR, et al. Retinoic acid receptor antagonism in vivo expands the 
numbers of precursor cells during granulopoiesis. Leukemia. 2002 
Sep;16(9):1763–72. 
Wang XJ, et al. Identification of retinoic acid as an inhibitor of transcription 
factor Nrf2 through activation of retinoic acid receptor alpha. Proc Natl 
Acad Sci U S A. 2007 Dec 4;104(49):19589–94. 
Watanabe I, et al. Advanced pancreatic ductal cancer: fibrotic focus and 
beta-catenin expression correlate with outcome. Pancreas. 2003 
May;26(4):326–33.  

30 
 
Wu Y, et al. PD-L1 distribution and perspective for cancer 
immunotherapy-blockade, knockdown, or inhibition. Front Immunol. 2019 
Aug 27;10:2022.