Phase II Study to Evaluate the Efficacy and Safety of Sirolimus in Subjects With Metastatic, Mismatch Repair Deficient Solid Tumors After Immunotherapy
Phase: PHASE2
Conditions: Metastatic dMMR Solid Cancer, Solid Tumor, Cancer, Metastatic Solid Tumor
Sponsor: Albert Einstein College of Medicine

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Phase II study to evaluate the Efficacy and Safety of Sirolimus in subjects with 
metastatic, mismatch repair deficient solid tumors after
immunotherapy.
Principal Investigator:
Chaoyuan Kuang, MD
Department of Medical Oncology 
Albert Einstein College of Medicine/Montefiore Medical Center
1695 Eastchester Road
Bronx, NY 10461
Email: chaoyuan.kuang@einsteinmed.edu
Co- Investigators:
Winfried Edelmann, PhD
Professor, Department of Cell Biology and Genetics 
Albert Einstein College of Medicine
Michael F. Price Center
1301 Morris Park Avenue, R277 Bronx, NY 10461
E mail: winfried.edelmann@einsteinmed.edu
Study Statistician:
Mimi Kim, ScD
Professor, Department of Epidemiology and Population Health 
Albert Einstein College of Medicine
Jack and Pearl Resnick Campus 
1300 Morris Park Avenue
Bronx, NY 10461
Email: mimi.kim@einstein.yu.edu 
Document version (date)
Version 5.0 (01/2023)
Commercially available agent
Sirolimus
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-To assess safety of sirolimus trough evaluation of incidence and severity of adverse events by CTCAE.
-To determine other efficacy mesures (PFS, OS) of sirolimus in dMMR solid tumors
Secondary Objectives:
-To evaluate the efficacy of sirolimus by estimating the ORR as assessed by RECIST 1.1 in dMMR solid tumors
Primary Objective:
- Sirolimus improves ORR by 20% in patients with dMMR solid tumors
Hypothesis:
Expected drop-out rate: 15% Calculated N: 19 patients
Type 1 error: 0.05
Power: 80%
Statistical Plan:
Effect size: 20% improvement in ORR (i.e., from 5% to 25%)
TRIAL SCHEMA
“Open-label, Single arm Phase II study of Sirolimus in metastatic, mismatch-repair deficient solid tumors after immunotherapy”
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Contents
1.
ABSTRACT ......................................................................................................5
2.
INTRODUCTION..............................................................................................6
2.1 Background....................................................................................................6
2.2 mTOR signaling pathway in cancer ...............................................................7
2.3 Pre-clinical Data.............................................................................................7
2.3.1 In-vitro data..............................................................................................7
2.3.2 Animal model data...................................................................................8
2.4 Clinical experience with mTOR inhibitors ....................................................11
2.5. Relevance and Significance........................................................................12
3.
OBJECTIVES .................................................................................................12
3.1 Primary objective:.........................................................................................12
3.2 Secondary objectives:..................................................................................13
4.
MATERIAL AND METHODS..........................................................................13
4.1 Study design ................................................................................................13
4.2 Patient selection...........................................................................................13
4.2.1. Inclusion criteria....................................................................................13
4.3 Treatment plan.............................................................................................14
4.4 Dosing delays/modifications.........................................................................15
4.5 Response Evaluation Plan...........................................................................15
4.6 Biomarker studies ........................................................................................15
4.6.1 Prior to treatment initiation:....................................................................15
4.6.2 During treatment:...................................................................................16
5.
STATISTICAL CONSIDERATIONS ...............................................................16
5.1 Endpoints.....................................................................................................16
5.1.1 Primary Endpoint...................................................................................16
5.1.2 Secondary Endpoints ............................................................................16
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5.2.Sample size .................................................................................................17
5.3.Analysis Plan ...............................................................................................17
6.
ETHICAL CONSIDERATIONS.......................................................................18
6.1 Patient Recruitment .....................................................................................18
6.2 Patient confidentiality...................................................................................18
6.3 Local Regulations ........................................................................................19
6.4 Informed Consent ........................................................................................19
6.5 Data storage ................................................................................................20
6.6 Data and Safety Monitoring Plan .................................................................20
7.
BENEFIT/RISK ASSESSMENT .....................................................................20
7.1 Benefits: .......................................................................................................20
7.2 Risks: ...........................................................................................................21
8.
REFERENCES...............................................................................................22
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1. ABSTRACT
Despite 
recent 
therapeutic 
strategies, 
including 
immunotherapy, 
treatment 
alternatives for patients with metastatic mismatch-repair deficient (dMMR) solid 
tumors remain scarce. Pre-clinical data suggests that dMMR tumors are susceptible 
to rapamycin (sirolimus), a mTOR inhibitor. In these tumors, characterized by higher 
levels of oxidative stress, sirolimus can exert a cytotoxic effect, led by the failure to 
repair DNA damage by inhibition of antioxidant enzymes such as FOXO3a triggered 
by Akt hyperactivation.
This proposal presents a phase 2 clinical trial designed to evaluate the efficacy of 
sirolimus in patients with dMMR solid tumors after immunotherapy. We hypothesize 
that sirolimus will increase the overall response rate (ORR) by 20%. To detect such a 
difference with 80% power and a type-1 error rate of 0.05, 16 participants are 
required. Accounting for a 15% drop-out rate, we aim to enroll 19 subjects.
After informed consent, adult patients with histologically-proven metastatic solid 
cancer, dMMR by immunohistochemistry (IHC) or next-generation sequencing, a 
performance status of ≤ 2 by ECOG, at least one measurable lesion based on 
Response Evaluation Criteria in Solid Tumors (RECIST v1.1), hemoglobin ≥9 g/dL, 
absolute neutrophil count ≥1,500 mm3, platelet count ≥75,000 mm3, adequate liver 
and renal function (≤ G1 by CTCAE) will be eligible.
Sirolimus to reach a serum trough level target ≥ 8 ng/ml will be administered orally in 
28-day cycles and will be continued until progression of disease evidenced by 
imaging, unacceptable toxicity or patient withdrawal. Imaging within 21 days of 
treatment initiation will be considered as baseline. Response to treatment will be 
assessed by imaging after 8 weeks of achieving therapeutic sirolimus levels, and 
every 8 weeks thereafter. ORR will be defined as the proportion of patients who 
achieve complete response or partial response, as assessed by our independent 
radiology team based on RECIST v1.1. Corresponding exact 95% confidence 
intervals will also be computed for the true ORR. Side effects will be evaluated and 
graded using CTCAE at every clinic visit and will be reported as frequencies.
Historical tumor samples will be requested for evaluation of potential biomarkers. In 
cases where archival samples are not available, an optional biopsy prior to treatment 
will be offered. All cases with available baseline biopsy, will be offered a repeated 
biopsy 8 weeks after achieving rapamycin serum target level. Expression levels of 
Akt, pAkt, Foxo3a, pFoxo3a and 8OxoG will be determined by IHC and reported as 
1+ (low), 2+ (intermediate) or 3+ (high). An increase in biomarker expression will be 
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defined as a change from 1+ at baseline to 3+ after treatment for each specific 
biomarker. Tumor mutational burden and gene mutations will be evaluated by next-
generation sequencing in baseline biopsies. Exploratory analysis will be performed to 
compare biomarkers between patients with and without objective response.
2. INTRODUCTION
2.1Background
Deficient mismatch repair (dMMR) is a molecular fingerprint that results from the 
accumulation of mutations at DNA elements named microsatellites which are 
characterized by short nucleotide-repetitive sequences in the coding or non-coding 
regions (1). dMMR has been reported in different solid cancer types with an estimated 
frequency of 2.2% among more than 50 different cancer types; most commonly 
represented by colorectal and endometrial cancer (2). It can present as hereditary or 
sporadic (2). In the former, it is driven by a germline mutation in the mismatch repair 
(MMR) genes which predispose to colorectal cancer (CRC), endometrial cancer and 
others such as pancreatic, gastric, urinary tract, brain and skin cancer in Lynch 
syndrome (3). In the latter, dMMR is generated by a biallelic somatic mutation in the 
MMR genes or by epigenetic changes, which most commonly cause MLH1 promoter 
hypermethylation. (4) (5).
Independent of cancer type, dMMR tumors showed an important overall response 
rate (ORR) with an extended duration of response to programmed-death ligand 1 
(PDL1) inhibitors such as pembrolizumab (6). Prior studies have reported ORRs 
ranging between 31-47.9% and durable responses of more than 6 months in two- 
thirds of responders. Consequently, pembrolizumab was approved in 2017 by the 
United States Food and Drug Administration (USFDA) for adult and pediatric patients 
with unresectable or metastatic dMMR solid tumors that had exhausted other 
therapeutic options (7). However, treatment options for patients with dMMR solid 
cancer after immunotherapy, due to progression of disease or inability to tolerate 
treatment for immune-related adverse events, are limited and represent the focus of 
current pre-clinical and clinical studies.
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2.2mTOR signaling pathway in cancer
mTOR is a serine/threonine kinase that belongs to the phosphoinositide 3-kinase 
related protein kinase (PIKK) family and has two different complexes named mTOR 
complex-1 (mTORC1) and mTOR complex-2 (mTORC2). mTOR signaling regulates 
cell growth, metabolism, proliferation and survival through activation of transcriptional 
factors and inhibition of autophagy. It has been reported that this pathway is 
overactivated in more than 70% of cancers (8). Consequently, mTOR inhibitors have 
been developed with currently 3 different FDA-approved drugs in the market: 
sirolimus, temsirolimus and everolimus. This phase 2 trial will study the prototype 
mTOR inhibitor, sirolimus, which is a direct mTORC1 inhibitor.
mTORC1 has important components such as the associated protein regulator named 
RAPTOR and the negative regulator called PRAS40. mTORC1 pathway can be 
activated via the PI3K/AKt pathway or the ras-MAPK pathway as a response to 
different stimuli including oncogenic growth factors, insulin, nutrients and hypoxia. In 
the former, Akt induces phosphorylation that leads to dissociation and inactivation of 
the TSC2/TSC1 complex that negatively regulates RHEB, an mTORC1 activator. 
Additionaly, Akt can directly phosphorylate and inactivate PRAS40, which releases 
RAPTOR that activates mTORC1. In the latter, the ras-MAPK pathway causes 
activation of MEK 1/2, ERK 1/2 and RSK which can phosphorylate TSC2 causing its 
dissociation from TSC1 leading to mTORC1 activation (8).
The overactivation of mTOR signaling in cancer follows three different mechanisms:
1) mutations in the mTOR gene; 2) mutations in the components of mTORC1 and 
mTORC2 or 3) Mutations in suppressor genes or oncogenes. In this regard, PTEN is 
a tumor suppressor gene that blocks the PI3K pathway. Mutations of PTEN are 
commonly reported in different solid tumors and are frequently mutated in dMMR 
tumors (8).
2.3 Pre-clinical Data
2.3.1
In-vitro data
Vilar et al. analyzed 5 different gene expression signatures in CRC aiming to compare 
the gene expression profile between dMMR and pMMR CRC tumors. This information 
was used to create an intersection signature, which included genes that were 
differentially expressed in all the signatures, and a union signature, which included 
genes that were differentially expressed in any signature. A connectivity map was 
used to select candidate compounds from 164 tested compounds which allowed the
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recognition of 2 drugs (rapamycin and a phosphoinositide 3-kinase named LY- 
294002) that were selected for testing in-vitro using 8 CRC cell lines. Of these, 2 were 
pMMR (HT-29 and SW-480) and 6 were dMMR (HCT-116 and Lovo that had MLH1 
and MSH2 mutations; HCT-15 and HCT-8 that had MSH6 mutation; SW-48 and RKO 
that displayed hypermethylation in the promoter of MLH1 gene).
At rapamycin concentrations ranging from 0.6 to 17 uM, 5 of 6 dMMR CRC cell lines 
were sensitive to mTOR inhibition compared to MSS cell lines with a 6x 
difference in IC50 (p=0.002). Of note, cell lines with mutation in the MMR proteins 
MSH2 and MSH6 were the most sensitive to mTOR inhibition. These results were 
confirmed in proliferation data and highlighted the potential relevance of mTOR 
inhibition as a targeted therapy among dMMR CRC tumors (Figure 1).
2.3.2
Animal model data
Preclinical genetic dMMR mouse models and dMMR patient-derived xenografts were 
developed in the laboratory of Dr. Edelmann in Einstein. In order to generate mice 
with tissue-specific inactivation of MSH2 called VCMsh2loxP, Cre-LoxP recombination 
was used to target MSH2 loss to the intestinal mucosa of Msh2loxP mice using a Villin- 
cre transgene. Patient tumor cells were injected subcutaneously into the right flank 
side. All VCMsh2loxP mice spontaneously developed intestinal adenocarcinomas 6-8 
months after the inoculation and died 4-6 weeks after diagnosis in the absence of 
treatment. Rapamycin was injected subcutaneously 3-5 times per week into tumor-
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bearing VCMsh2loxP mice. Treatment response was assessed by micro PET-CT at 14 
days of treatment and tumor volume measured using the 6”/150 mm Vernier caliper 
W/fin adj. (Geneva Gage, INC)
In the absence of treatment, dMMR xenografts and pMMR mice evidenced increases 
in tumor volume and in mean standardized uptake values measured by micro PET- 
CT at day 14. When rapamycin was administered, all treated tumors (n>100) showed 
complete regression after 2-3 weeks of treatment and no tumor re-growth was 
observed in dMMR xenografts (Figure 2); while the tumor continued to increase in 
pMMR mice (Figure 3 and 4).
mTOR inhibitors, including rapamycin and analogs, have a well-established cytostatic 
effect. In addition, a cytotoxic effect can be exerted in dMMR xenografts as a 
consequence of higher levels of oxidative stress generated by Akt hyper- 
activation and failure to repair the resulting oxidative DNA damage in dMMR tumor 
cells. mTOR inhibition by rapamycin leads to Akt activation by phosphorylation to 
pAkt. FOXO transcription factors, including FOXO3a, promote resistance to oxidative 
stress by induction of antioxidant enzymes. pAkt induces phosphorylation of FOXO3a 
leading to its inhibition. (Figure 5, A and B). This leads to accumulation of ROS levels
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as evidenced by 8-oxoG a common DNA lesion resulting from ROS leading to 
autophagy and cell death as evidenced by Beclin-1 expression (Figure 5 C).
To show that cell death resulting from inability to repair oxidative DNA damage was 
restricted to dMMR cell lines; we performed experiments with two cell lines: HT29 
(pMMR) and LOVO (dMMRI). These showed that pAkt and pFOXO3a and ROS levels 
increase in dMMR and pMMR cell lines treated with rapamycin; but, 8-oxoG levels 
increase only in dMMR cell lines. To show the role of pAkt in cell death; experiments 
adding an Akt inhibitor (Akti) and N-acetylcysteine (an antioxidant) were perfomed, 
showing that 8oxoG levels do not increase when dMMR cell lines are treated with 
concomitant rapamycin and Akti or with rapamycin and NAC (Figure 6D). Cell viability 
is reduced in dMMR cell lines treated with rapamycin; however, the cytotoxic effect is 
attenuated when Akti or NAC are added (Figure 6E).
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2.3.3
Safety Data
Sirolimus was FDA-approved in 2010 for the prevention of organ rejection in patients 
receiving kidney transplants after proving superiority to azathioprine in this setting. 
Additionally, 
it 
was 
FDA-approved 
in 
2015 
for 
the 
management 
of 
lymphangioleiomyomatosis after showing improvement in lung function in a 
randomized clinical trial.
A pilot study that included 4 patients (gastric cancer:3, and cholangiocarcinoma:1) 
assessed the safety and efficacy of rapamycin among patients with refractory solid 
tumors with PIK3CA amplification/mutation. In this study, sirolimus was administered 
at 1 mg daily in 28-day cycles. Two patients received 2 cycles and one patient each 
received 1 and 4 cycles, respectively. Dose reductions/modifications were not 
required, grade 3 or greater were not observed and there were no treatment-related 
deaths. Three patients had stable disease (SD) after one cycle and one patient had 
SD after 4 cycles
2.4Clinical experience with mTOR inhibitors
In the United States, there are three FDA approved mTOR inhibitors. Of these, 2 of 
them (everolimus and temsirolimus) have been approved in the treatment of cancer 
and for organ transplantation rejection prophylaxis in kidney recipients; while the other 
(sirolimus) has been approved for the latter indication. In general, the most common 
adverse events are rash, asthenia, and mucositis. Common laboratory abnormalities 
include anemia, hyperglycemia, hyperlipidemia, and hypertriglyceridemia.
Temsirolimus was FDA-approved for the treatment of patients with advanced renal 
cell carcinoma (RCC) in 2007 after a randomized clinical trial showed superiority of 
the combination of temsirolimus and interferon versus interferon alone. Everolimus is 
FDA-approved for the treatment of patients with advanced renal cell carcinoma 
refractory, neuroendocrine tumors of gastrointestinal, pancreatic or lung origin, renal 
angiomyolipoma, tuberous sclerosis, subependymal giant cell astrocytoma and 
postmenopausal women with metastatic, hormone-positive breast cancer in the 
second-line setting. Sirolimus was FDA-approved in 2010 for the prevention of organ 
rejection in patients receiving kidney transplants after proving superiority to 
azathioprine in this setting. Additionally, it was FDA-approved in 2015 for the 
management of lymphangioleiomyomatosis after showing improvement in lung 
function in a randomized clinical trial.
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A pilot study that included 4 patients (gastric cancer:3 and cholangiocarcinoma:1) 
assessed the safety and efficacy of rapamycin among patients with refractory solid 
tumors with PIK3CA amplification/mutation. In this study, sirolimus was administered 
at 1 mg daily in 28-day cycles. Two patients received 2 cycles and one patient each 
received 1 and 4 cycles, respectively. Dose reductions/modifications were not 
required, grade 3 or greater were not observed and there were no treatment-related 
deaths. Three patients had stable disease (SD) after one cycle and one patient had 
SD after 4 cycles (9).
2.5Relevance and Significance
Despite exciting therapeutic improvement in dMMR metastatic solid tumors, effective 
treatment options after immunotherapy remain scarce. This study is relevant, as it will 
evaluate a potentially effective drug targeted to this patient population. This clinical 
trial has bench-to-bedside translational potential that could provide the foundation for 
an entirely new therapeutic option for patients with dMMR solid tumors that have run 
out of other options.
Currently, multiple clinical trials are evaluating the efficacy of mTOR inhibitors alone 
or in combination for the treatment of different cancer types (10-15); however, none of 
them have selected dMMR tumors. Therefore, this proposal is highly innovative, as it 
will focus the intervention to a subgroup of patients that is more likely to benefit, as 
suggested by our pre-clinical data. Furthermore, the evaluation of tissue biomarkers 
will confirm whether sirolimus can exert a cytotoxic effect (which has never been 
reported before) limited to dMMR tumors.
3. OBJECTIVES
3.1Primary objective:

To evaluate the efficacy of sirolimus by estimating the overall response rate 
(ORR) as assessed by Response Evaluation Criteria In Solid Tumors versión 
1.1 (RECIST v1.1) in patients with metastatic dMMR solid cancer after 
immunotherapy (either due to disease progression or to inability to tolerate 
treatment).
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3.2Secondary objectives:

To assess potential tissue biomarkers (Akt, pAkt, FOXO3a, pFOXO3a, 8OxoG 
expression by immunohistochemistry, tumor mutational burden/gene mutations 
by next-generation sequencing), the effect of sirolimus treatment on these 
biomarkers, and possible correlation between these biomarkers and clinical 
response.

To evaluate other clinical end-points such as progression-free survival, 
response duration and overall survival of sirolimus in patients with metastatic 
dMMR solid cancer after immunotherapy (either due to disease progression or to 
inability to tolerate treatment).

To evaluate tolerability and safety of sirolimus using Common Terminology 
Criteria for Adverse Events (CTCAE) grading scale.
4. MATERIAL AND METHODS
4.1Study design
Open-label, single arm, phase II clinical trial among patients with metastatic, dMMR 
solid tumors refractory/unable to tolerate immunotherapy.
4.2 Patient selection
4.2.1
Inclusion criteria

Metastatic solid cancer tumor after immunotherapy (either due to progression 
of disease or inability to tolerate treatment)

dMMR by immunohistochemistry (IHC) defined as the loss of expression in 
any of the four major MMR proteins (MLH1, MSH2, MSH6 and PMS2) or by next- 
generation sequencing (NGS)

Age older than 18 at the time of informed consent

Eastern Cooperative Oncology Group performance status of 0-2

≥1 measurable lesion based on RECIST, version 1.1 (16)

Absolute neutrophil count (ANC) ≥1,500 mm3

Platelet count ≥75,000 mm3

Hemoglobin ≥ 9 g/dl

Aspartate aminotransferase (AST) ≤3.0 times the upper normal limit (UNL)

Alanine aminotransferase (ALT) ≤3.0 times the upper normal limit (UNL)
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
Bilirubin ≤1.5 times the UNL

Serum creatinine ≤1.5 times the UNL
4.2.2
Exclusion criteria

Received immunotherapy in the prior 21 days.

Have not recovered from toxicities of prior treatments to at least grade 1.

Symptomatic central nervous system (CNS) metastases

Pregnancy or Breast-feeding.
4.3Treatment plan
Our reason for selecting sirolimus as the choice of mTOR inhibition for this clinical 
trial is primarily since all the pre-clinical experiments have been performed with it, and 
it is feasible to obtain within our estimated budget. Sirolimus has been commercially 
available since 1999, and fortunately, a generic version has been available since 
January 2019. Although never commercially approved as an anti-cancer agent, there 
is ample pre-clinical evidence to support such a role, and there are ongoing cancer 
clinical trials using this agent (17-19). Moreover, as if often the case, drugs do not get 
tested appropriately in time to meet the rigors of USFDA approval, nor do they have 
sufficient patent time remaining to justify the costs to be borne by a pharmaceutical 
corporation. The very robust pre-clinical data generated by us points to the efficacy of 
mTOR inhibition in dMMR tumors, and is “pathway driven”, rather than “tissue of 
origin” driven. We therefore propose to test this hypothesis in a tumor agnostic 
manner, and will enroll all solid tumors. This approach is similar to the tumor agnostic 
approach used to test pembrolizumab that led to its subsequent USFDA approval (6).
Based on pre-clinical models, IC50 concentrations in cell viability experiments using 
dMMR cell lines lie between 8-10 uM, as shown in Fig 3E (3) and is our intended 
target. As described in its package insert (20), at a daily dose  of  2 mg,  the steady 
state concentration is between 7.6 (± 3.1) and 15 (± 4.9) ng/ml. Factoring in the 
molecular weight of sirolimus (914); a dose of 2 mg is adequate to reach our target 
concentration. Hence, sirolimus will be started at 2 mg daily. Sirolimus dosing will be 
titrated to meet serum trough levels of >8 ng/ml, assayed at 7 days after starting a 
new dose, by chromatography/nmass spectrometry. Once adequate  serum levels 
are met (≥8 ng/ml), the same dosing will be continued until progression of disease as 
evidenced by imaging, or unacceptable toxicity.
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4.4Dosing delays/modifications
Each participant will be evaluated in the oncology cancer center by a physician every 
2 weeks for the first 2 cycles and on the first day of each subsequent cycle. The 
evaluation will include history and physical. Laboratory parameters including cell 
blood count (CBC), basic metabolic panel (BMP) and liver tests will be checked in 
every encounter to determine dosing delay/modification. Each cycle is of 28 days in 
duration starting once therapeutic trough sirolimus levels are achieved in blood.
Side effects will be evaluated using the National Cancer Institute Common 
Terminology Criteria for Adverse Events, version 4.0. Dose delay and modifications 
will be required for thrombocytopenia (<75K), neutropenia (ANC<1), or any persistent 
non-hematologic grade 2 or 3 toxicity. Discontinuation of sirolimus will be required for 
any grade 4 toxicity, or any toxicity requiring treatment interruption for ≥ 1 cycle.
4.5Response Evaluation Plan
Each participant will be required to have a recent imaging (CT or PET-CT) within 21 
days of treatment initiation which will be considered as baseline. In C1D1, the patient 
will be assessed in the oncology clinic to document baseline symptoms and laboratory 
exams including CBC, BMP, liver tests will be performed and repeated at every visit.
Imaging (CT or PET-CT) within 21 days of treatment initiation will be considered as 
baseline. Response to treatment will be assessed by imaging (CT-CAP or PET-CT) 
after after 8 weeks of achieving therapeutic sirolimus levels, and every 8 weeks 
thereafter. Images will be reviewed by the radiology team based on RECIST v1.1 
criteria (16).
4.6Biomarker studies
4.6.1
Prior to treatment initiation:

Historical tumor samples will be requested for all participants for evaluation of 
potential biomarkers. In cases where archival biopsies are not available, the 
patient will be offered a biopsy prior to treatment initiation. This will be optional and 
will not affect enrollment into the clinical trial. These samples will be used to 
evaluate the baseline expression of Akt, pAkt, FOXO3a, pFOXO3a, 8OxoG 
expression will be evaluated by immunohistochemistry in the laboratory of Dr. 
Edelmann.

We anticipate that most of the patients would have had next-generation 
sequencing requested before our evaluation, so, the results will be directly 
requested. In cases where it was not done; it will be ordered. This will be used to 
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explore potential mutations that could correlate with treatment response.
4.6.2
During treatment:

Repeated biopsies after 8 weeks of treatment will be offered to all participants 
that had an evaluable biopsy prior to treatment initiation. This will be optional 
and will not affect continuation into the clinical trial. Changes in the expression of 
Akt, pAkt, FOXO3a, pFOXO3a, 8OxoG expression will be evaluated by 
immunohistochemistry in the laboratory of Dr. Edelmann.
5. STATISTICAL CONSIDERATIONS
5.1Endpoints
5.1.1
Primary Endpoint

Objective response rate (ORR) defined as the proportion of patients who achieve a 
complete response (disappearance of all target tumors) or a partial response 
(≥30% decrease in the sum of the longest diameters of target tumors) based on 
RECIST V1.1 (16).
5.1.2
Secondary Endpoints

Progression free survival (PFS): PFS is defined as the time from treatment initiation 
to progression of known metastases or new metastatic site, or death from any 
cause after a timeframe of 24 weeks.

Response duration (RD): RD is defined as time from documentation of tumor 
response to disease progression.

Overall survival (OS): OS is defined as the time from treatment initiation to death 
from any cause.
5.2Sample size
This study is designed as a single arm, single-stage phase II trial to test the null 
hypothesis that Ho: ORR ≤ 0.05 vs HA: ORR > 0.05. The target sample size of 16 
subjects was determined assuming that the true ORR = 25% with sirolimus, i.e. an 
absolute improvement in ORR of 20%. If the number of responses at the end of the 
trial is 3 or more out of 16, we will conclude that sirolimus is beneficial for patients with 
dMMR tumors and further studies of this treatment will be conducted. If the drug is 
actually not effective (ORR ≤ 0.05), there is a 5% probability of erroneously concluding 
that it is effective (Type I error rate). If the drug is truly effective (ORR ≥ 0.25), there is 
at least 80% probability of correctly concluding that it is (power). To account for a 
15% drop-out rate, 19 patients will be enrolled. Because of the limited total sample 
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size, this trial is designed as a single stage study that does not allow for early stopping 
due to lack of efficacy.
We anticipate that it will be feasible to enroll 19 participants in a 10-month period 
based on the patient load treated at Montefiore/Einstein Cancer Center and the 
commitment of collaborators from other cancer centers in New York City (Lincoln 
Hospital, Jacobi Medical Center, St. Barnabas hospital, Cornell Cancer Center) to 
refer eligible patients for this trial. This study will be funded by the Montefiore/Einstein 
Cancer Center
5.3Analysis Plan
The ORR will be estimated as the proportion of patients with metastatic dMMR solid 
cancer refractory to immunotherapy who achieved the endpoint of objective response 
after treatment with sirolimus. Corresponding exact 95% confidence intervals will also 
be computed for the true ORR. The distribution of progression-free survival and 
overall survival will be estimated using the Kaplan-Meier method. Greenwood’s 
method will be applied to estimate confidence intervals.
As a secondary aim, we will evaluate other clinical efficacy end-points such as PFS 
(time from target serum sirolimus levels to progression of known metastases or new 
metastatic site, or death from any cause), response duration (time from 
documentation of tumor response to disease progression.) and overall survival (OS, 
time from target serum sirolimus levels to death from any cause). The distribution of 
PFS, DR and OS will be estimated using the Kaplan-Meier method. Greenwood’s 
method will be applied to estimate confidence intervals.
Provided the number of patients who achieve objective response is sufficient, 
exploratory analyses to assess the association between ORR and the expression of 
tissue biomarkers will be performed. An exact test for trend for ordered contingency 
tables will be used to compare the frequency of subjects with low, moderate and high 
biomarker expression between patients with and without objective response; Fisher’s 
exact test will be used to compare the frequency of subjects that had an increase in 
biomarker expression after treatment between patients with and without objective 
response. Gene mutations will be reported as frequencies and compared between 
patients with and without objective response by Fisher’s exact test. The two sample 
Wilcoxon rank sum or T-test will be conducted to compare the tumor mutational 
burden between patients with and without objective response.
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6. ETHICAL CONSIDERATIONS
6.1Patient Recruitment 
This is a single-center clinical trial. Patients will be recruited in the medical oncology 
clinic at Montefiore Einstein Cancer Center. Eligible patients will be informed about 
the clinical trial by the Principal Investigator and will be provided a copy of the ICF in 
the medical oncology clinic on the day of the initial visit. The Principal Investigator will 
be responsable for ensuring adequate patient’s understanding about the 
voluntariness of participation, visit and laboratory schedule, benefits and risk of the 
study and will answer all patient’s questions in the initial visit and throughout the 
study period. 
6.2Patient confidentiality
The Principal Investigator affirms and uphold the principle of the patient’s right to 
protection against invasion of privacy. To ensure this, each patient will be assigned a 
participant subject number at the time of consent which will be used throughout the 
study.  A patient’s source data must only be linked to the clinical study database or 
documentation via this unique identification number. 
6.3Local Regulations
The study fully adheres to the principles outlined in “Guideline for Good Clinical 
Practice” (GCP) ICH E6 Tripartite Guideline (January 1997) and remain consistent 
with the most recent version of the Declaration of Helsinki. The investigators will 
ensure that the conduct of the study complies with the basic principles of GCP 
as outlined in the current version of 21 CFR, subpart D, Part 312, 
“Responsibilities of Sponsors and Investigators” Part 50, “Protection of Human 
Subjects” and Part 56, “Institutional Review Boards.”
6.4Informed Consent
The participation in the study is voluntary. The ICF for this study is in compliance with 
ICH GCP, local regulatory requirements, and legal requirements .The ICF used in this 
study, and any changes made during the course of the study, must be prospectively 
approved by both the IRB before use.
The Principal Investigator must ensure that each study patient is fully informed about 
the nature and objectives of the study and possible risks associated with participation. 
The Principal Investigator must obtain written informed consent from each patient 
before any study-specific activity is performed. A copy of the informed consent form 
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19
(ICF) will be provided to the subject. If applicable, the ICF will be provided in a 
certified translation of the subject’s language. Signed ICFs will remain in each 
subject’s study file and will be available for verification at any time.
This study will not request for a waiver of informed consent.
The Principal Investigator will: 

Ensure each subject is given full and adequate oral and written information about 
the nature, purpose, possible risk and benefit of the study 

Ensure each subject is notified that they are free to discontinue from the study at 
any time 

Ensure that each subject is given the opportunity to ask questions and allowed 
time to consider the information provided 

Ensure each subject provides signed and dated informed consent before 
conducting any procedure specifically for the study 

Ensure the original, signed Informed Consent Form(s) is/are stored in the 
Investigator’s Study File 

Ensure a copy of the signed Informed Consent Form is given to the subject 

Ensure that any incentives for subjects who participate in the study as well as any 
provisions for subjects harmed as a consequence of study participation are 
described in the informed consent form that is approved by an IRB/IEC 
6.5Data storage
Data for this clinical trial will be stored in clinical research forms (CRFs) that will be 
designed by the Principal Investigator. The Principal Investigator has ultimate 
responsibility for the accuracy, authenticity, and timely collection and reporting of all 
clinical, safety, and laboratory data entered on the CRFs and any other data 
collection forms. 
Source documentation supporting the CRF data must indicate the patient’s 
participation in the study and must document the dates and details of study 
procedures, AEs, other observations, and patient status.
6.6Data and Safety Monitoring Plan
The study data including efficacy, major toxicities, and serious adverse events will be 
monitored by the Einstein DSMB (data and safety monitoring board). This committee 
is an integral part of the Albert Einstein Cancer Center, and meets and reviews data 
every month 
IRB NUMBER: 2019-10724
IRB APPROVAL DATE: 02/03/2023

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7. BENEFIT/RISK ASSESSMENT 
7.1Benefits: 
Patients will benefit from inclusion to this clinical trial as they will receive treatment 
with a drug (sirolimus) potentially efffective for their cancer type based on preclinical 
studies done at Albert Einstein Medical School. Only patients that have progressed or 
have been unable to tolerate the standard of care for MSI solid tumors 
(immunotherapy) will be eligible for this trial. 
Inclusion into this trial will not provide monetary compensation, remuneration, 
reimbursement or pro-rating scales for visits. Inclusion into the trial will not incur in 
additional charges to the patients. The study drug (sirolimus) will be provided without 
fee to the study patients. All other ancillary studies will be covered by either insurance 
or the cancer center: Imaging (CT/PET-CT scans) and routine laboratory studies 
(CBC, BMP, liver tests) at every visit are considered standard of care and will be 
covered by insurance. Repeated biopsies will be offered to patients (voluntary) and 
will be covered by Einstein Cancer Center. 
7.2Risks:
This study carries risk of side effects as patients will be receiving a medication. 
Sirolimus has been FDA-approved to prevent rejection in patients receiving renal 
transplants and in patients with lymphangioleiomyomatosis. Other drugs from the 
same class (everolimus and temsirolimus) are used in patients with breast cancer 
and renal cell carcinoma. To minimize this risk, patients will be monitored by the 
Investigators for any side effects which will be graded based on CTCAE v5.0. Based 
on side effects, the Investigators will decide whether it is safe to continue with the 
medication or if any changes are required. 
To prevent the risk of breach of confidentiality, each subject will be provided with a 
study ID under which all the clinical information will be stored. Only the research team 
for this clinical trial will have access to identifiers for the purpose of data collection 
and data verification. 
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IRB APPROVAL DATE: 02/03/2023

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IRB NUMBER: 2019-10724
IRB APPROVAL DATE: 02/03/2023