Pembrolizumab in MMR-Proficient Metastatic Colorectal Cancer Pharmacologically Primed to Trigger Dynamic Hypermutation Status
Phase: PHASE2
Conditions: Colorectal Neoplasms, Microsatellite Instability
Sponsor: IFOM ETS - The AIRC Institute of Molecular Oncology

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PEMBROLIZUMAB IN MMR-PROFICIENT METASTATIC COLORECTAL 
CANCER PHARMACOLOGICALLY PRIMED TO TRIGGER DYNAMIC 
HYPERMUTATION STATUS. 
THE ARETHUSA TRIAL 
 
IMP Identifiers: 
 
 
 
       PEMBROLIZUMAB 
 TEMOZOLOMIDE 
 
Protocol Number: 
 
 
                    IFOM-CPT002/2018/PO001   
 
 
 
 
 
EudraCT Number: 
 
 
 
       2018-001441-14 
 
 
Protocol Version (Date):  
 
 
       V3.1,  20th Nov 2019 
 
 
Sponsor                                                                       IFOM – Istituto FIRC di Oncologia Molecolare 
 
 
CONFIDENTIAL 
This document contains confidential information belonging to Sponsor. Except as may be otherwise agreed to in 
writing, by accepting or reviewing these materials, you agree to hold such information in confidence and not to disclose 
it to others (except where required by applicable law), nor to use it for unauthorized purposes. In the event of actual or 
suspected breach of this obligation Sponsor should be promptly notified. 
 

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SIGNATURE PAGE ....................................................................................................................................................4 
1.0 
STUDY PERSONNEL.....................................................................................................................................9 
2.0 
SUMMARY ............................................................................................................................................... 10 
3.0 
SYNOPSIS................................................................................................................................................. 11 
4.0 
TRIAL DESIGN........................................................................................................................................... 17 
4.1 
OVERALL TRIAL DESIGN .......................................................................................................................................... 17 
4.1.1 
SCREENING Phase ........................................................................................................................................ 17 
4.1.2 
PRIMING Phase ........................................................................................................................................... 17 
4.1.3 
TRIAL Phase ................................................................................................................................................. 18 
4.2 
TRIAL DIAGRAM .................................................................................................................................................... 19 
5.0 
OBJECTIVES OF THE TRIAL ......................................................................................................................... 20 
5.1 
PRIMARY OBJECTIVES ............................................................................................................................................. 20 
5.2 
SECONDARY OBJECTIVES ......................................................................................................................................... 20 
5.3 
EXPLORATORY OBJECTIVES ...................................................................................................................................... 20 
5.4 
PRIMARY ENDPOINT ............................................................................................................................................... 20 
5.5 
SECONDARY ENDPOINTS ......................................................................................................................................... 20 
5.6 
EXPLORATORY ENDPOINTS ....................................................................................................................................... 21 
6.0 
BACKGROUND & RATIONALE .................................................................................................................... 21 
6.1 
BACKGROUND ....................................................................................................................................................... 21 
6.1.1 
Translational background............................................................................................................................ 21 
6.1.2 
Pharmaceutical and Therapeutic Background ............................................................................................ 23 
6.1.3 
Preclinical and Clinical Trial Data ................................................................................................................ 23 
6.2 
RATIONALE........................................................................................................................................................... 24 
6.2.1 
Translational rationale for ARETHUSA enrichment strategy ....................................................................... 24 
6.2.2 
Justification for Dose ................................................................................................................................... 25 
6.2.3 
Rationale for Endpoints ............................................................................................................................... 26 
7.0 
METHODOLOGY ....................................................................................................................................... 26 
7.1 
ENTRY CRITERIA .................................................................................................................................................... 26 
7.1.1 
Entry criteria for SCREENING Phase............................................................................................................. 26 
7.1.2 
Entry Criteria for PRIMING Phase ................................................................................................................ 27 
7.1.3 
Entry Criteria for TRIAL Phase...................................................................................................................... 28 
7.1.4 
Adequate Organ Function Laboratory Values ............................................................................................. 28 
7.2 
EXCLUSION CRITERIA .............................................................................................................................................. 29 
7.2.1 
Lifestyle Restrictions .................................................................................................................................... 31 
7.2.2 
Pregnancy .................................................................................................................................................... 31 
7.2.3 
Use in Nursing Women ................................................................................................................................ 31 
7.3 
TRIAL TREATMENTS ................................................................................................................................................ 31 
7.3.1 
Temozolomide - Dose Selection/Modification ............................................................................................. 32 
7.3.1 
Pembrolizumab - Dose Selection/Modification ........................................................................................... 34 
7.3.2 
Timing of Dose Administration .................................................................................................................... 34 
7.3.2 
Dose Modification and toxicity management for immune-related AEs associated with pembrolizumab ... 35 
7.3.3 
Dose modification and toxicity management of infusion-reactions related to pembrolizumab ................. 39 
7.3.4 
Other allowed dose interruption for pembrolizumab .................................................................................. 42 
7.3.5 
Second Course ............................................................................................................................................. 42 
7.4 
RANDOMIZATION OR TREATMENT ALLOCATION............................................................................................................ 43 
7.5 
STRATIFICATION .................................................................................................................................................... 43 
7.6 
CONCOMITANT MEDICATIONS/VACCINATIONS (ALLOWED & PROHIBITED) ......................................................................... 43 
7.6.1 
Acceptable Concomitant Medications ......................................................................................................... 43 
7.6.2 
Prohibited Concomitant Medications .......................................................................................................... 44 
7.6.3 
Rescue Medications & Supportive Care ....................................................................................................... 45 

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7.7 
PARTICIPANT WITHDRAWAL/DISCONTINUATION CRITERIA ............................................................................................. 45 
7.8 
PARTICIPANT REPLACEMENT STRATEGY ...................................................................................................................... 46 
7.8.1 
PRIMING Phase ........................................................................................................................................... 46 
7.8.2 
TRIAL Phase ................................................................................................................................................. 46 
7.9 
CLINICAL CRITERIA FOR EARLY TRIAL TERMINATION ...................................................................................................... 46 
8.0 
TRIAL FLOW CHART .................................................................................................................................. 47 
9.0 
TRIAL PROCEDURES .................................................................................................................................. 49 
9.1 
TRIAL PROCEDURES ................................................................................................................................................ 49 
9.1.1 
Administrative Procedures .......................................................................................................................... 49 
9.1.2 
Clinical Procedures/Assessments ................................................................................................................. 51 
9.1.3 
Tumor Imaging and Assessment of Disease ................................................................................................ 52 
9.1.4 
Tumor Tissue Collection and Correlative Studies Blood Sampling ............................................................... 55 
9.1.5 
Laboratory Procedures/Assessments .......................................................................................................... 58 
9.1.6 
Other Procedures ......................................................................................................................................... 60 
9.1.7 
Visit Requirements....................................................................................................................................... 60 
9.2 
SAFETY ASSESSMENT .............................................................................................................................................. 62 
9.2.1 
Definitions for Adverse Event and Serious Adverse Event ........................................................................... 62 
9.2.2 
Reporting of Adverse Events ........................................................................................................................ 63 
9.2.3 
Reporting of Serious Adverse Events ........................................................................................................... 64 
9.2.4 
Definition and Reporting of an Overdose for This Protocol ......................................................................... 65 
9.2.5 
Events of Clinical Interest (pembrolizumab) ................................................................................................ 66 
9.2.6 
Recording AEs/SAEs in the Case Report Form ............................................................................................. 67 
9.2.7 
Sponsor Responsibility for Reporting Adverse Events .................................................................................. 72 
10.0 
STATISTICAL CONSIDERATIONS ................................................................................................................. 72 
11.0 
LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL SUPPLIES ...................................................... 75 
11.1 
INVESTIGATIONAL PRODUCT..................................................................................................................................... 75 
11.2 
PACKAGING AND LABELING INFORMATION .................................................................................................................. 76 
11.3 
CLINICAL SUPPLIES DISCLOSURE ................................................................................................................................ 76 
11.4 
STORAGE AND HANDLING REQUIREMENTS .................................................................................................................. 76 
11.5 
RETURNS AND RECONCILIATION ................................................................................................................................ 76 
12.0 
ADMINISTRATIVE AND REGULATORY DETAILS ........................................................................................... 76 
12.1 
CONFIDENTIALITY .................................................................................................................................................. 76 
12.2 
DATA MANAGEMENT ............................................................................................................................................. 77 
12.2.1 
Record Retention .................................................................................................................................... 77 
13.0 
REFERENCES ............................................................................................................................................ 77 
APPENDICES .......................................................................................................................................................... 82 
APPENDIX 1: ECOG PERFORMANCE STATUS .............................................................................................................................. 82 
APPENDIX 2: COMMON TERMINOLOGY CRITERIA FOR ADVERSE EVENTS V4.03 (CTCAE) ................................................................... 82 
APPENDIX 3: CONTRACEPTION REQUIREMENTS........................................................................................................................... 82 
APPENDIX 4: RESPONSE ASSESSEMENT CRITERIA .......................................................................................................................... 84 
Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 ........................................................................................ 84 
Immune Response Evaluation Criteria in Solid Tumors (iRECIST)  Criteria for Evaluating Response in Solid Tumors . 84 
APPENDIX 5 RESULTS OF MGMT ANALYSIS BY IHC AND METHYL-BEAMING ..................................................................................... 88 
 
 
 
 
 
 

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Principal Investigator Agreement 
I have read the Protocol entitled “PEMBROLIZUMAB IN MMR-PROFICIENT 
METASTATIC COLORECTAL CANCER PHARMACOLOGICALLY PRIMED TO 
TRIGGER DYNAMIC HYPERMUTATION STATUS” and I agree to conduct the study as 
detailed herein and in compliance with ICH Guidelines for Good Clinical Practice and applicable 
regulatory requirements. I will provide all study personnel under my supervision with all 
information provided by the Sponsor and I will inform them about their responsibilities and 
obligations. 
 
 
 
Principal Investigator  
(printed name, Institution, Department  and location) 
 
 
 
 
Signature 
 
Date 
 
 
 
 
 
 
 
 
 
 

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LIST OF ABBREVIATIONS 
 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase (Serum Glutamic-pyruvic Transaminase) 
ANC 
Absolute Neutrophil Count 
aPTT    
Activated Partial Thromboplastin Time 
AST 
Aspartate Aminotransferase (Serum Glutamic-oxaloacetic Transaminase) 
B-ML     
Baseline Mutational Load 
CI         
Confidence Interval 
CL        
Clearance 
CR 
Complete Response 
CRC    
Colorectal Cancer 
CRF 
Case Report Form 
CRO     
Clinical Research Organization 
CT 
Computerized Tomography 
CTCAE  
Common Terminology Criteria for Adverse Events 
ctDNA   
Circulating tumor DNA 
DCL      
Data Clarification List 
D-ML 
Dynamic Mutational Load 
eCRF  
Electronic Case Report Form 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
EE        
Efficacy Evaluable 
iEE     
Immunotherapy Efficacy Evaluable 
EGFR 
Epidermal Growth Factor Receptor 
FACS 
Fluorescence activated cell sorting 
FFPE    
Formalin Fixed Paraffin Embedded 
FPI       
First Patient In 
FPFV 
First Patient First Visit 
GCP 
Good Clinical Practice 
G-CSF   
Granulocyte Colony Stimulating Factor 
IC         
Informed Consent 
IEC 
Independent Ethics Committee 
ICH       
International Conference on Harmonization 
IRB 
Institutional Review Board 
IV 
Intravenous 
LB-B-ML 
Liquid Biopsy-Baseline-Mutational Load 

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LB-D-ML 
Liquid Biopsy-Dynamic-Mutational Load 
LB-TMZ-ML 
Liquid Biopsy-Temozolomide-Mutational Load 
LDH 
Lactate Dehydrogenase 
LPI       
Last Patient In 
LPLV    
Last Patient Last Visit 
LVEF   
Left Ventricular Ejection Fraction  
mAb 
Monoclonal Antibody 
mCRC 
Metastatic Colorectal Carcinoma 
MGMT 
O6-methylguanine–DNA methyltransferase gene 
MMR 
Mismatch repair  
MMR-D 
Mismatch repair deficient 
MMR-P 
Mismatch repair proficient 
MRI 
Magnetic Resonance Imaging 
MSI 
microsatellite instable 
MSS 
microsatellite stable  
NCI       
National Cancer Institute  
NGS     
Next Generation Sequencing 
ORR 
Objective Response Rate 
OS 
Overall Survival  
PET      
Positron Emission Tomography  
PD 
Progressive Disease  
PD-ML 
Mutational Load assey performed at progression after pembrolizumab 
PFS 
Progression-Free Survival  
PR 
Partial Response 
QW, Q2W, Q3W, Q8W, Q9W  
Every Week, Every 2 Weeks, Every 3 Weeks, Every 8 Weeks, Every 9 Weeks 
RAS mCRC 
Extended RAS mutated CRC (Extended RAS= KRAS and NRAS) 
RBC    
Red Blood Cell 
RECIST  
Response Evaluation Criteria In Solid Tumors 
iRECIST 
Immune Response Evaluation Criteria In Solid Tumors 
ROC    
Receiver Operator Characteristic 
SAE 
Serious Adverse Event 
SAS      
Statistical Analysis System 
SD 
Stable Disease 
SE        
Safety Evaluable 
SUSAR  
Suspect Unexpected Serious Adverse Reaction 
TMF     
Trial Master File 
TMZ 
Temozolomide 

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ULN 
Upper Limit of Normal 
WBC    
White Blood Count / White Blood Cells  
WKS     
Weeks 
WT 
Wild Type 
 

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1.0 STUDY PERSONNEL 
 
STUDY CHAIRS 
 
CLINICAL 
Prof. Salvatore Siena 
Niguarda Cancer Center, Grande Ospedale Metropolitano – MILANO e Dipartimento di Oncologia ed Onco-
Ematologia Università degli Studi di Milano 
Telefono: 02 6444 2291; Email: salvatore.siena@ospedaleniguarda.it 
 
TRANSLATIONAL 
Prof. Alberto Bardelli 
Fondazione Piemontese per la Ricerca sul Cancro (FPRC) – CANDIOLO (TO) e Dipartimento di Oncologia 
Università degli Studi di Torino 
Telefono: 011 993 3548; Email: alberto.bardelli@ircc.it 
 
OPERATIONAL 
Dr.ssa Silvia Marsoni 
IFOM – Istituto FIRC di Oncologia Molecolare (MILANO) 
Telefono: 02 57430 3862; Email: silvia.marsoni@ifom.eu 
 
 
 
 
 
PROJECT MANAGER 
Dr. Paolo Luraghi 
IFOM – Istituto FIRC di Oncologia Molecolare (MILANO) 
Telefono: 02 57430 3799; Email: paolo.luraghi@ifom.eu 
 
 
 
BIOSTATICIAN   
Dr. Valter Torri 
Istituto Mario Negri – MILANO 
Telefono: 02 390 141; Email: valter.torri@marionegri.it 
 

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2.0 SUMMARY 
Abbreviated Title 
THE ARETHUSA TRIAL 
Trial Phase 
2 
Clinical Indication 
Metastatic colorectal cancer  
Trial Type 
Interventional 
Type of control 
NA 
Route of administration 
Intravenous Pembrolizumab and oral Temozolomide 
Trial Blinding 
NA 
Treatment Groups  
TMZ priming followed by Pembrolizumab 
Number 
of 
trial 
participants 
54 in TRIAL Phase | 67 PRIMING Phase | 670 SCREENING 
Phase 
Estimated 
enrollment 
period 
24 months 
Estimated duration of trial 36 months (40 months to final report) 
Duration of Participation 
36 months + Follow Up 
Estimated average length 
of treatment per patient 
6 months (Pembrolizumab); 3 months (Temozolomide) 
 

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3.0 SYNOPSIS 
Protocol 
Number 
IFOM-CPT002/2018/PO001   
Title 
PEMBROLIZUMAB IN MMR-PROFICIENT METASTATIC 
COLORECTAL CANCER PHARMACOLOGICALLY PRIMED TO 
TRIGGER DYNAMIC HYPERMUTATION STATUS 
Brief title THE ARETHUSA TRIAL 
Sponsor 
IFOM - Istituto FIRC di Oncologia Molecolare 
Clinical 
Phase 
Phase II  
Study 
Rationale 
ARETHUSA has been designed on the basis of four translational findings, namely 
that:  i) pembrolizumab is active only in MMR deficient mCRC patients;  ii) the 
DNA alkylator temozolomide is active only in mCRC patients whose tumor 
harbours a silenced O-6-methylguanine-DNA methyltransferase (MGMT), a DNA 
repair enzyme that removes alkylated adducts from the DNA. MGMT is silenced 
by gene promoter hypermethylation, a favourable context of susceptibility to DNA 
damage (Amatu et al., 2016; Esteller and Herman, 2004); iii) in the tumor 
specimens of mCRC patients with MMR proficient tumors collected at treatment 
progression we observed that resistance to TMZ correlated with inactivation of 
MMR and increased mutational loads (Germano et al., 2017) iv) TMZ can cause 
inactivation of DNA repair, increased mutational burden and effective immune 
surveillance in preclinical models, de facto turning a MMR-P tumors into a ‘MMR-
D like’ (Germano et al., 2017). 
Moreover, mutational load has been correlated with response to immune checkpoint 
blockade. In particular clinical evidence, mostly derived from melanoma patients 
treated with ipilimumab, suggests that the mutational burden associated with 
clinical benefit is between 10 and 20 mutations per MB (Lauss et al., 2017; Snyder 
et al., 2014; Van Allen et al., 2015). Indeed a recent study (Panda et al., 2017) in 
224 colorectal cancer shows that 100% of 193 cases with < 20 mutations were 
MMR proficient and that  100% of 31 cases with > 20 mutations were either MMR-
deficient (n 28) or POLE mutated (n 3).  Thus, an absolute mutational load cutoff 
of 20 mutations per MB was able, in CRC patients, to detect with digital sensitivity 
and specificity the subset of patients most likely to achieve a clinical response from 
immunotherapy.  
Collectively these results suggest that an increase in mutational load (and the 
ensuing neo-antigen burden) induced by TMZ treatment, might sensitize to immune 
checkpoint blockade, and drive the rationale of the ARETHUSA trial.   A 20 

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mutations/MB has been defined as the absolute post-temozolomide cut-off value 
for patient eligibility on the basis of the studies reported above.  
Primary 
Objective 
To assess the objective response rate of pembrolizumab in metastatic MMR 
proficient, extended RAS mutated CRC patients who have failed standard therapies, 
with MGMT-negative tumors treated with TMZ, given with the intent of increasing 
the mutational load of their tumors (cohort P). 
Secondar
y 
Objective
(s) 
• To assess the  objective response rate in MMR deficient patients (cohort D)  
identified during the MMR screening. 
• To assess the Progression Free Survival (PFS) and Overall Survival (OS)  both 
P and D cohorts  patients.  
• To assess the safety and tolerability of pembrolizumab after TMZ-priming 
therapy (cohort P). 
• To assess the safety and tolerability of pembrolizumab (cohort D). 
Translatio
nal 
Objective
(s) 
• To validate the assessment of mutational load in plasma using the tissue 
mutational load as ‘gold’ standard.  
• To assess the relationship between selected immune biomarkers in archival 
and/or fresh tumor tissue and blood with response to pembrolizumab.  
• To compare these immunomarkers profiles prior and after TMZ in cohort P 
patients, and prior and after pembrolizumab treatment in all patients (cohort D 
and P).  
• To assess the relationship of RAS status in MMR deficient patients (cohort D) 
with response to Pembrolizumab. 
Primary 
Endpoint 
Objective response rate (ORR) to pembrolizumab in cohort P according to RECIST 
(as specified in table 9.1.3). 
Secondar
y 
Endpoint(
s) 
 
• ORR to pembrolizumab in cohort D according to RECIST (as specified in table 
9.1.3).  
• PFS and OS in both cohorts P and D.  
• Safety and tolerability according to CTCAE version 4.03. 
Translatio
nal 
endpoint(
s) 
 
• Validation of tumor mutational load assessment in plasma.  
• Expression of  immune markers and their correlation with response to 
temozolomide and pembrolizumab.  
• ORR to pembrolizumab in RAS and non-RAS mutant stratified cohort D 
according to RECIST. 
Trial 
Design 
2-cohort, open label, transformative, precision medicine, phase II trial in extended 
RAS-mutated MMR-Proficient metastatic CRC (RAS mCRC) and unselected 
MMR-Deficient mCRC patients refractory to standard treatments. Extended RAS 

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mutations include KRAS and NRAS mutations. The trial consists of 3 different 
phases: 
Screening Phase: MMR-Deficient (MMR-D) patients proceed directly to TRIAL 
Phase (cohort D) to be treated with pembrolizumab. RAS mutant MMR-Proficient 
(MMR-P) patients, instead, are further tested for O6-methylguanine–DNA 
methyltransferase gene expression (MGMT) status in tissue (MGMT protein IHC 
and MGMT promoter methylation). MGMT IHC negative and promoter methylated 
patients will proceed to PRIMING phase.  
Priming Phase: MMR-P patients showing negative MGMT protein and high levels 
of MGMT promoter methylation in tissues will receive TMZ therapy until 
progression. Two tumor biopsies will be taken prior to starting therapy and at 
progression to determine the mutational load. Patients with a mutational load < 20 
mutations/megabase will go off-study. Patients with a mutational load >20 
mutations/megabase, will proceed to trial phase no longer than week 5 post TMZ-
ML.  
Trial Phase: Eligible patients, i.e. MMRD patients (cohort D) and patients with a 
TMZ-ML > 20 mutations per megabase at TMZ-ML (cohort P), will be treated with 
pembrolizumab 200 mg administered as an intravenous infusion over 30 minutes 
every 3 weeks until disease progression, unacceptable toxicity, or up to 24 months 
in patients without disease progression.  
Inclusion 
Criteria 
Patient selection criteria are progressively restrictive according to the 3-step 
selection strategy of the trial and are reported separately for each phase. 
 
SCREENING Phase  
 
1. Histologically confirmed diagnosis of metastatic colorectal cancer. 
2. Documented RAS extended mutations in the archival sample (cohort P 
only). 
3. ECOG performance status 0-1. 
4. SCREENING phase informed consent signed. 
5. Understanding and accepting the need for undergoing two tumor biopsies if 
eligible for PRIMING Phase. 
6. Age ≥ 18 years. 
7. Availability of all diagnostic FFPE blocks (primary tumor and or 
metastases), or at least 20 slides (primary tumor and/or metastases). 
Formalin-fixed, paraffin embedded (FFPE) tissue blocks are preferred to 
slides.   

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8. Normal organ functions. 
PRIMING Phase 
1. Fulfilment of all the SCREENING inclusion criteria; 
2. PRIMING informed consent signed;  
3. Confirming the willingness to undergo two tumor biopsies,  
4. Acceptance that, if the mutational load determination is unfeasible for 
technical reasons (not enough tissue, substandard test performance, etc.), 
access to TRIAL phase will not be possible.   
5. Imaging documented failure of previous standard CRC therapies including 
fluoropyrimidine, oxaliplatin, irinotecan plus or minus antiangiogenics 
agents (Bevacizumab, Aflibercept, Regorafenib, others). 
6. At least one measurable tumor lesion as per RECIST v1.1. Lesions in 
previously irradiated areas or those that have received other loco-regional 
therapies (i.e. percutaneous ablation) should not be considered measurable 
unless there is clear documented evidence of progression of the lesion since 
therapy. Imaging must be performed maximum within 28 days prior to 
enrolment. 
7. ECOG performance status 0 or 1; 
8. Following results in the SCREENING Phase tests: 
• Proficient MMR status assessed by IHC  or  MSI-Low  status defined by 
PCR (Bethesda panel); 
• Negative score for the MGMT protein expression IHC test; 
• Positive score for the MGMT promoter methylation performed on Tissue. 
9. Women with childbearing potential should complete a pregnancy test and 
be willing to use highly effective contraceptive methods. 
10. Normal organ functions.   
TRIAL Phase 
1. Fulfilment of all the SCREENING inclusion criteria and Deficient MMR 
status (IHC) or MSI-High status (PCR) (cohort D only).  
2. Fulfilment of all the SCREENING and PRIMING inclusion criteria (cohort 
P only).  
3. TRIAL Phase informed consent signed (both cohorts).  
4. Imaging documented PD to TMZ (cohort P only).  
5. A mutational load value > 20 mutations/MB at TMZ-ML assay (cohort P 
only). 

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6. Imaging documented failure of previous standard CRC therapies including 
fluoropyrimidine, oxaliplatin, irinotecan plus or minus targeted agents 
(Bevacizumab, Aflibercept, Regorafenib, Cetuximab, Panitumumab, 
others) (cohort D only). 
7. At least one measurable tumor lesion as per RECIST v1.1. Lesions in 
previously irradiated areas or those that have received other loco-regional 
therapies (i.e. percutaneous ablation) should not be considered measurable 
unless there is clear documented evidence of progression of the lesion since 
therapy. Imaging must be performed maximum within 28 days prior to 
enrolment (both cohorts). 
8. Woman with childbearing potential should complete a pregnancy test and 
be willing to use highly effective contraceptive methods (both cohorts). 
9. Normal organ functions.  Blood specimens must be collected within 10 days 
prior to the start of study treatment (both cohorts). 
See exclusion criteria in the protocol 
Treatmen
ts 
Drug 
Dose/Pote
ncy 
Dose 
Frequen
cy 
Route 
of 
Administra
tion 
Regimen/Treat
ment Period 
Use 
Temozolom
ide 
150 or 200 
or 100 mg/ 
(7.3.1.2) 
Q4W 
P.O. 
Days 
1-5  
every 28 days 
(PRIMING 
Phase) 
Experime
ntal 
 
Pembrolizu
mab 
200 mg 
Q3W 
IV infusion Day 1 of each 3 
week 
cycle 
(TRIAL Phase) 
Experime
ntal 
 
Sample 
Size 
A two-stage Green-Dahlberg design has been used to calculate the sample size for 
the P-cohort under the following assumptions:  
• The null hypothesis that the true (H0) response rate is 10% (not considered 
clinically compelling) will be tested against a one-sided alternative (H1).  
• In the first stage, 10 patients will be accrued. If no responses are observed, 
the study will be stopped. Otherwise, 10 additional patients will be accrued 
for a total of 20.  
• The null hypothesis will be rejected if 5 or more responses are observed in 
20 patients.  
• This design yields a type I error rate of 0.05, and power of 90% when the 
true response rate is 40%.  
• In order to recruit 20 patients in the P-cohort the number of MMR 
unselected patients to be pre-screened for MMR status MGMT promoter 

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methylation and the number of MGMT-PM+ to be primed with TMZ are 
reported in the Table below  
Number of Patients to be: 
N 
Expected 
Percent 
RAS mutant MMR proficient cases to be screened 
670 
A  
expected MGMT IHC negative  
335 
B = 50% of A 
expected promoter methylated in tissue  
67 
C = 20% of B  
and 10% of A 
expected with increase of mutational load ≥ 20 
mutations/Mb (PRIMING Phase) 
20 
D= 30% of C 
 
For cohort D a precise sample size has NOT been calculated since the ORR in 
deficient patients is a secondary objective of the trial. We will enroll up to 34 MMR-
D cases according to an estimated prevalence of 4-5%. These patients will 
constitute the D-cohort. We expect a response rate between 40% (95% C.I .18-.71) 
and 33%  (95% C.I .21-.46) as reported by Le et al (2015 & 2019) with 11-14 
responses accordingly. 
 
 
 
 
 
 
 
 
 
 
 
 

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4.0 TRIAL DESIGN 
4.1 
Overall Trial Design 
This is a 2-cohort, open label, transformative, precision medicine, phase II trial in extended RAS- 
mutated metastatic CRC (RAS mCRC) patients refractory to standard treatments. Extended RAS 
mutations include KRAS and NRAS mutations.  
The design of Arethusa is based on two main observations (see 6.1.1). First that objective responses 
with pembrolizumab can only be achieved in MMR-deficient tumors with a high mutational load 
(and ensuing high neo-antigen burden)(Le et al., 2015). Second, that the low mutational load, 
typical of MMR-proficient tumors, can be increased in vitro and in in-vivo models (with an ensuing 
increase in the tumor’s neo-antigen burden) by a pre-treatment with DNA-alkylating agents, thus 
sensitizing the tumor to the immune checkpoint blockade (Germano et al., 2017). We have 
designed Arethusa accordingly with the purpose of i) identifying upfront MMR-deficient patients 
(cohort D); ii) using TMZ to exploit the favorable context of susceptibility to DNA damage of 
MGMT methylated tumors as an enrichment strategy for MMR-proficient patients (cohort P), and 
iii) using cohort D patients as a ‘natural positive comparator’ for cohort P results.  
On the basis of this translational rationale and due to the its proof of concept nature, Arethusa 
consists of three different phases - SCREENING Phase, PRIMING phase, and TRIAL Phase, as 
described below and reported in Trial Diagram (see 4.2).   
4.1.1 SCREENING Phase 
MMR-Deficient (MMR-D) patients proceed directly to TRIAL Phase (cohort D) to be treated with 
pembrolizumab. RAS mutant MMR-Proficient (MMR-P) patients, instead, are further tested for 
O6-methylguanine–DNA methyltransferase gene expression (MGMT) status in tissue (MGMT 
protein by IHC and MGMT promoter methylation). MGMT IHC negative and promoter 
methylated patients will proceed to PRIMING phase. MGMT IHC positive and/or MGM promoter 
non-methylated patients will go off-study. 
4.1.2 PRIMING Phase 
MMR-P patients who have failed standard therapies including fluoropyrimidines, oxaliplatin, 
irinotecan plus or minus antiangiogenic agents (Bevacizumab, Regorafenib, others), showing 
negative MGMT protein in tissues and high levels of MGMT promoter methylation in tissue, will 
undergo a tumor biopsy (non-mandatory for the enrollment) to retrospectively determine the 
mutational load of their tumor (Baseline Mutational Load = B-ML), and will then receive TMZ 
therapy (OS dx5,q28 days) until progression.  
At PD patients will be re-biopsied (mandatory for the enrolment) to prospectively determine the 
tumor mutational load (Post Temozolomide Mutational Load= TMZ-ML). Patients with a 
mutational load < 20 mutations/megabase will go off-study. Patients with a mutational load >20 
mutations/megabase, will proceed to trial phase no longer than week 5 post TMZ-ML. This time 
gap is related to the technical time requirements to perform and interpret the mutational load assay. 

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During the 5-week gap, standard palliative treatment with the exception of regorafenib, can be 
delivered, at physician discretion, for symptomatic disease control. 
Two liquid biopsies for the experimental NGS determination of the mutational load in plasma will 
also be performed concomitantly with the tissue biopsies: before treatment with TMZ  (LB-B-ML) 
and at PD following TMZ treatment (LB-TMZ-ML). 
4.1.3 TRIAL Phase 
Eligible patients, i.e. MMRD patients (cohort D) who have failed standard therapies including 
fluoropyrimidines, oxaliplatin, irinotecan plus or minus targeted agents (Bevacizumab, 
Aflibercept, Regorafenib, Cetuximab, Panitumumab, others), and patients with a TMZ-ML > 20 
mutations per megabase at TMZ-ML (cohort P), will be treated with pembrolizumab 200 mg 
administered as an intravenous infusion over 30 minutes every 3 weeks until disease progression, 
unacceptable toxicity, or up to 24 months in patients without disease progression.  
Two further tissue biopsies might be performed at each patient before (cohort D only, non-
mandatory) and after Pembrolizumab treatment (both cohorts, non-mandatory) to perform 
mutational load test (Dynamic and PD Mutational Load = D-ML and PD-ML).   
In addition, for the experimental NGS determination of mutational load in plasma, two liquid 
biopsies will also be performed concomitantly with expected D-ML and PD-ML tissue biopsies 
(LB-D-ML and LB-PD-ML). These will be done even in the case tissue biopsies will not be 
executed. 
Results from D-ML and LB-D-ML (if the liquid biopsy assay will result reliable, see point 5.3) 
will be used post-hoc to stratify patients according to the ratio of predicted private/shared antigens. 
Private antigens are defined as predicted neo-antigens present at one specific time point, while 
shared antigens are present in all the 3 time points collected (LB-B-ML; D-ML; LB-PD-ML).  PD-
ML and LB-PD-ML data will be used for correlative studies on primary and secondary resistance 
to pembrolizumab. 
 

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4.2 
Trial Diagram 
 
 
mCRC MMR-Deficient Patients
SCREENING PHASE
PRIMING PHASE
TRIAL PHASE
TMZ 
Priming 
to PD
PRIMARY CHEMOTHERAPY PD1
2
1
MGMT expression assessment on tissue
Tissue samples for NGS Analysis           
(Temozolamide Mutational Load TMZ-ML)4
ARETHUSA TRIAL
Is the patient MMR-Proficient and 
potentially  MGMT-methylated 
(IHC negative on tissue  = 50% of patients)?
YES
NO
OFF 
study
MGMT promoter methylation assessment on
tissue
Is the patient MGMT promoter methylation 
positive on tissue 
(20% of MGMT- IHC negative)?
NO
OFF 
study
YES
PD @ RECIST 1.13
670*
1: 1st line FOLFOXIRI or 1st line FOLFOXà 2nd line FOLFIRI or viceversa ;  ± bevacizumab, aflbercept or regorafenib.
2: B-ML is non-mandatory. 
3: Patients will enter the Pembrolizumab Phase once in PD (or if TMZ toxicity is unacceptable). 
4: TMZ-ML is mandatory for prospective patients enrichment.
335*
67*
P-Cohort
20*
* N° of pts
Tissue sampling for NGS Analysis           
(Baseline Mutational Load - B-ML )2 
WEEK 1 PRE TMZ
335*
268*
67*
D-Cohort
34*
@ PD
67*
3
Go to 
Trial Phase
NO
OFF 
study
Has the patient ≥ 20 mutations 
(30% of MGMT promoter methylation positive)?
NGS Analysis Results
YES
Pembro 
to 
PD
47*
PRIMARY 
CHEMOTHERAPY 
PD1
mCRC RAS-mut MMR-Proficient PATIENTS 
34*

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5.0 OBJECTIVES OF THE TRIAL 
5.1 
Primary Objectives  
The primary objective of ARETHUSA is to assess the objective response rate of 
pembrolizumab in metastatic MMR proficient, extended RAS mutated CRC patients with 
MGMT positive tumor treated with TMZ, given with the intent of increasing the mutational 
load of their tumors (cohort P), who have failed standard therapies. 
The primary hypothesis tested by ARETHUSA is whether immunoediting in MMR proficient 
cancers can be favourably disrupted by pharmacologically increasing the tumor mutational 
load with DNA alkylators, thus enabling the tumor to dynamically express proteins (neo-
antigens) that can positively affect T cell functions under checkpoint inhibition.   
5.2 
Secondary Objectives  
The secondary objectives of ARETHUSA are; 
1. To assess the  objective response rate in MMR deficient patients (cohort D)  identified 
during the MMR screening. 
2. To assess the Progression Free Survival (PFS) and Overall Survival (OS)  both P and 
D cohorts  patients.  
3. To assess the safety and tolerability of pembrolizumab after TMZ-priming therapy 
(cohort P). 
4. To assess the safety and tolerability of pembrolizumab (cohort D). 
5.3 
Exploratory Objectives 
The exploratory objective of ARETHUSA are: 
1. To validate the assessment of mutational load in plasma using the tissue mutational 
load as ‘gold’ standard.  
2. To assess the relationship between selected immune biomarkers in archival and/or 
fresh tumor tissue and blood with response to pembrolizumab.  
3. To compare these immunomarkers profiles prior and after TMZ in cohort P patients, 
and prior and after pembrolizumab treatment in all patients (cohort D and P).  
4. To assess the relationship of RAS status in MMR deficient patients (cohort D) with 
response to Pembrolizumab. 
5.4 
Primary Endpoint 
Objective response rate (ORR) to pembrolizumab in cohort P according to RECIST (as specified 
in table 9.1.3). 
5.5 
Secondary Endpoints 
1. ORR to pembrolizumab in cohort D according to RECIST (as specified in table 9.1.3). 
2. PFS and OS in both cohorts P and D.  

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3. Safety and tolerability according to CTCAE version 4.03. 
 
5.6 
Exploratory Endpoints 
1. Validation of tumor mutational load assessment in plasma.  
2. Expression of  immune markers and their correlation with response to temozolomide and 
pembrolizumab.  
3. ORR to pembrolizumab in RAS and non-RAS mutant stratified cohort D according to 
RECIST. 
 
6.0 BACKGROUND & RATIONALE 
6.1 
Background  
6.1.1 Translational background 
In Western countries, colorectal cancer (CRC) is the third common tumor type and the second 
leading cause of cancer-related death (Le et al., 2015). Even with a survival exceeding two years 
in genetically selected patients (Siegel et al., 2013), metastatic CRC remains mostly not curable. 
While the molecular landscape of CRC is well described (Douillard et al., 2013; Network, 2012), 
the unmet need for therapies targeting new oncogenic drivers and overcoming resistance to EGFR 
antagonists still remains. Insights from genetic studies have provided the impetus for efforts to 
target key signaling pathways, but have also led researchers to revisit established targets, such as 
HER2 (Bertotti et al., 2015), or vulnerability molecular contexts such as mismatch-repair (MMR) 
deficiency (Sartore-Bianchi et al., 2016a). 
CRCs with genetic defects in MMR pathways harbor hundreds to thousands of somatic mutations 
mostly in regions of repetitive DNA known as microsatellites (Boland and Shike, 2010; Lynch and 
de la Chapelle, 2003). The accumulation of mutations in these regions is termed microsatellite 
instability (MSI) and occurs in approximately 15% of CRC but only in less than 5% of metastatic 
tumors (Goldstein et al., 2014). It has been recently demonstrated that a high burden of mutations 
results in a greatly increased number of mutation-associated neoantigens which can be recognized 
by the patient autologous immune system (Segal et al., 2008). In this setting, treatment with the 
anti–programmed death ligand 1 (PD-1) immune checkpoint inhibitor pembrolizumab is effective 
in inducing durable objective responses in 40% of cases (Sartore-Bianchi et al., 2016a). These 
results should be regarded as quite remarkable, especially since clinical efficacy was independent 
from RAS mutations presence, a mutation greatly constraining the use of targeted treatment and 
negatively affecting prognosis in CRC (Sartore-Bianchi et al., 2016b).  
Given the low incidence of MMR-deficient mCRC limited to less than 5% of cases, it is crucial to 
understand if selected subsets of MMR-proficient patients could still benefit from checkpoint 
inhibition (CPi). In other tumor types MMR-proficient cancers with high mutational burden are 
susceptible to CPi (Rizvi et al., 2015; Yadav et al., 2016). Whether a MMR-proficient phenotype 
can be turned into ‘MMR-deficient like’, is thus a key question in CRC.  
We previously demonstrated that specific environmentally-derived carcinogens can cause genetic 
instability in CRC cell lines. Specifically, treatment with the alkylating agent N-methyl-N9-nitro-
Nnitrosoguanidine (MNNG) can select for tumor cells with MMR-deficient status (Bardelli et al., 

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2001). TMZ is known to induce loss of the mismatch repair protein MSH6 in human glioblastomas 
(Cahill et al., 2007) and an increase in G>A mutation incidence (Alexandrov et al., 2013). In 
parallel, other studies have demonstrated that TMZ side effects can influence the immune cell 
compartment by selectively depleting the immuno-suppressive T regulator lymphocytes (Tregs) 
(Coffield et al., 2004), and activating the immuno-active T cytotoxic lymphocytes (Tc) and natural 
killers (NK) (Hervieu et al., 2013).  
While alkylating agents represent the backbone of systemic treatment for many solid tumors, this 
is not the case for CRC where this class of compounds is of limited use. Indeed, only few data are 
available regarding treatment of CRC with alkylating agents. Specifically their use has been 
mainly based on the presence of a deficiency of O(6)-methylguanine-DNA-methyltransferase 
(MGMT), that occurs through gene promoter hypermethylation, as a favorable context of 
susceptibility to DNA damage (Amatu et al., 2016; Esteller and Herman, 2004). MGMT is the 
DNA repair enzyme in charge of removing DNA alkylated adducts caused by carcinogens and 
certain cytotoxics. We have previously reported that MGMT is hypermethylated at high levels in 
20% CRC samples (Barault et al., 2015). We have also described that alkylating agents, such as 
the iv dacarbazine or its oral form temozolomide (TMZ), have clinical efficacy in a subgroup of 
microsatellite stable (MSS) metastatic CRCs displaying MGMT promoter hypermethylation even 
though clinical benefit is limited in time (Amatu et al., 2013, 2016). Importantly, in these phase II 
studies, clinical specimens from five patients with MMR proficient tumors were collected and it 
was observed that resistance to TMZ correlated with inactivation of MMR and increased 
mutational loads in 2 of 5 cases. These clinical finding prompted a ‘reverse translational’ set of in 
vitro and in vivo experiments showing that TMZ,  in syngeneic mouse MMR proficient colon 
cancer models, can indeed causes inactivation of DNA repair, increased mutational burden and 
effective immune surveillance followed by dynamic mutational profiles, resulting in persistent 
renewal of neo-antigens.  TMZ untreated proficient models instead exhibit stable mutational loads 
and neo-antigen profiles over time. Similarly, MMR inactivation following TMZ resistance in 
human colorectal cancer cells also resulted in continuous renewal of neo-antigens (Germano et al., 
2017).  
Pembrolizumab is a potent humanized immunoglobulin G4 (IgG4) monoclonal antibody (mAb) 
with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting its 
interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-
L2).  Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor 
blocking activity for PD-1.  Pembrolizumab has an acceptable preclinical safety profile and is in 
clinical development as an intravenous (IV) immunotherapy for advanced malignancies.  
Keytruda® (pembrolizumab) is indicated for the treatment of patients across a number of 
indications because of its mechanism of action to bind the PD-1 receptor on the T cell.  For more 
details on specific indications refer to the Investigator brochure. 
On May 23, 2017, pembrolizumab (KEYTRUDA, Merck & Co.) was granted a first world-wide 
tissue/site-agnostic accelerated approval by the U.S. Food and Drug Administration for adult and 
pediatric patients with unresectable or metastatic, microsatellite instability-high (MSI-H) or 
mismatch repair deficient (dMMR) solid tumors that have progressed following prior treatment 
and who have no satisfactory alternative treatment options including MSI-H or dMMR colorectal 

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cancer that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan 
(see https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm560040.htm.) 
6.1.2 Pharmaceutical and Therapeutic Background 
The importance of intact immune surveillance function in controlling outgrowth of neoplastic 
transformations has been known for decades (Disis, 2010). Accumulating evidence shows a 
correlation between tumor-infiltrating lymphocytes in cancer tissue and favorable prognosis in 
various malignancies.  In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector 
T-cells/FoxP3+ regulatory T-cells (T-regs) correlates with improved prognosis and long-term 
survival in solid malignancies, such as ovarian, colorectal, and pancreatic cancer; hepatocellular 
carcinoma; malignant melanoma; and renal cell carcinoma.  Tumor-infiltrating lymphocytes can 
be expanded ex vivo and reinfused, inducing durable objective tumor responses in cancers such as 
melanoma (Dudley et al., 2005; Hunder et al., 2008). 
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune 
control.  The normal function of PD-1, expressed on the cell surface of activated T-cells under 
healthy conditions, is to down-modulate unwanted or excessive immune responses, including 
autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an immunoglobulin (Ig) superfamily 
member related to cluster of differentiation 28 (CD28) and cytotoxic T-lymphocyte-associated 
protein 4 (CTLA-4) that has been shown to negatively regulate antigen receptor signaling upon 
engagement of its ligands (PD-L1 and/or PD-L2)(Greenwald et al., 2005; Okazaki et al., 2001). 
The structure of murine PD-1 has been resolved (Zhang et al., 2004). PD-1 and its family members 
are type I transmembrane glycoproteins containing an Ig-variable–type (IgV-type) domain 
responsible for ligand binding and a cytoplasmic tail responsible for the binding of signaling 
molecules.  The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an 
immunoreceptor tyrosine-based inhibition motif, and an immunoreceptor tyrosine-based switch 
motif.  Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases, SHP-1 and SHP-2, 
to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, leading to the 
dephosphorylation of effector molecules such as CD3 zeta (CD3ζ), protein kinase C-theta (PKCθ), 
and zeta-chain-associated protein kinase (ZAP70), which are involved in the CD3 T-cell signaling 
cascade(Chemnitz et al., 2004; Okazaki et al., 2001; Riley, 2009; Sheppard et al., 
2004).  The mechanism by which PD-1 down-modulates T-cell responses is similar to, but distinct 
from, that of CTLA-4, because both molecules regulate an overlapping set of signaling proteins 
(Francisco et al., 2010; Parry et al., 2005). As a consequence, the PD-1/PD-L1 pathway is an 
attractive target for therapeutic intervention in extended RAS mutated mCRC.  
6.1.3 Preclinical and Clinical Trial Data 
Refer to the Investigator’s Brochure for Preclinical and Clinical data. 

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6.2 
Rationale 
6.2.1 Translational rationale for ARETHUSA enrichment strategy  
ARETHUSA has been designed on the basis of four translational findings, as described in the 
background. Namely that:  i) pembrolizumab is active only in MMR deficient mCRC patients;  ii) 
the DNA alkylator temozolomide is active only in mCRC patients whose tumor harbor a silenced 
O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that removes 
alkylated adducts from the DNA. MGMT is silenced by gene promoter hypermethylation, a 
favorable context of susceptibility to DNA damage (Amatu et al., 2016; Esteller and Herman, 
2004); iii) in the tumor specimens of mCRC patients with MMR proficient tumors collected at 
treatment progression we observed that resistance to TMZ correlated with inactivation of MMR 
and increased mutational loads (Germano et al., 2017) iv) TMZ can cause inactivation of DNA 
repair, increased mutational burden and effective immune surveillance in preclinical models, de 
facto turning a MMR-P tumors into a ‘MMR-D like’ (Germano et al., 2017). 
Moreover, mutational load has been correlated with response to immune checkpoint blockade. In 
particular clinical evidence, mostly derived from melanoma patients treated with ipilimumab, 
suggests that the mutational burden associated with clinical benefit is between 10 and 20 mutations 
per MB (Lauss et al., 2017; Snyder et al., 2014; Van Allen et al., 2015). Indeed a recent study 
(Panda et al., 2017)in 224 colorectal cancer shows that 100% of 193 cases with < 20 mutations 
were MMR proficient and that  100% of 31 cases with > 20 mutations were either MMR-deficient 
(n 28) or POLE mutated (n 3).  Thus, an absolute mutational load cutoff of 20 mutations per MB 
was able, in CRC patients, to detect with digital sensitivity and specificity the subset of patients 
most likely to achieve a clinical response from immunotherapy.  
Collectively these results suggest that an increase in mutational load (and the ensuing neo-
antigen burden) induced by TMZ treatment, might sensitize to immune checkpoint 
blockade, and drive the rational of the ARETHUSA trial.   A 20 mutations/MB has been 
defined as the absolute post-temozolomide cut-off value for patient eligibility on the basis of 
the studies reported above.   
Accordingly, we have designed a three-phased enrichment strategy for the trial (see trials design 
4.1) based on the following assumptions that have been adjusted according to the actual screening 
results (see Appendix 5 and Trial diagram, 4.2 for details); 
• Within a population of extended RAS mutated MMR-P patients, 50% will score MGMT 
negative in tissue. 
• Of these, 20% (10% of the initial population) will score MGMT promoter methylation 
positive (MGMT- PM+). Only these patients will be eligible for the TMZ priming phase. 
• At progression during treatment with TMZ, it is expected that 30% of patients will have a 
tissue mutational load of > 20 mutations/mb.  
• Only patients showing a > 20 mutations/mb mutational load might benefit from a therapy 
with pembrolizumab. 

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6.2.2 Justification for Dose  
The dose-dense regimen for TMZ has been chosen to maximize the targeting on DNA repair 
proteins. The tolerability of the dose dense schedule has been proven by our own previous phase 
II trial in chemorefractory mCRC patients (Amatu et al., 2016; Barault et al., 2015). 
Pembrolizumab will be used at FDA approved dose for mCRC. 
The planned dose of pembrolizumab for this study is 200 mg every 3 weeks (Q3W).  Based on the 
totality of data generated in the Keytruda development program, 200 mg Q3W is the appropriate 
dose of pembrolizumab for adults across all indications and regardless of tumor type.  As outlined 
below, this dose is justified by: 
• Clinical data from 8 randomized studies demonstrating flat dose- and exposure-efficacy 
relationships from 2 mg/kg Q3W to 10 mg/kg every 2 weeks (Q2W),  
• Clinical data showing meaningful improvement in benefit-risk including overall survival 
at 200 mg Q3W across multiple indications, and 
• Pharmacology data showing full target saturation in both systemic circulation (inferred 
from pharmacokinetic [PK] data) and tumor (inferred from physiologically-based PK 
[PBPK] analysis) at 200 mg Q3W 
Among the 8 randomized dose-comparison studies, a total of 2262 participants were enrolled with 
melanoma and non-small cell lung cancer (NSCLC), covering different disease settings (treatment 
naïve, previously treated, PD-L1 enriched, and all-comers) and different treatment settings 
(monotherapy and in combination with chemotherapy).  Five studies compared 2 mg/kg Q3W 
versus 10 mg/kg Q2W (KN001 Cohort B2, KN001 Cohort D, KN002, KN010, and KN021), and 
3 studies compared 10 mg/kg Q3W versus 10 mg/kg Q2W (KN001 Cohort B3, KN001 Cohort F2 
and KN006).  All of these studies demonstrated flat dose- and exposure-response relationships 
across the doses studied representing an approximate 5- to 7.5-fold difference in exposure.  The 2 
mg/kg (or 200 mg fixed-dose) Q3W provided similar responses to the highest doses studied.  
Subsequently, flat dose-exposure-response relationships were also observed in other tumor types 
including head and neck cancer, bladder cancer, gastric cancer and classical Hodgkin Lymphoma, 
confirming 200 mg Q3W as the appropriate dose independent of the tumor type.  These findings 
are consistent with the mechanism of action of pembrolizumab, which acts by interaction with 
immune cells, and not via direct binding to cancer cells. 
Additionally, pharmacology data clearly show target saturation at 200 mg Q3W.  First, PK data in 
KN001 evaluating target-mediated drug disposition (TMDD) conclusively demonstrated 
saturation of PD-1 in systemic circulation at doses much lower than 200 mg Q3W.  Second, a 
PBPK analysis was conducted to predict tumor PD-1 saturation over a wide range of tumor 
penetration and PD-1 expression.  This evaluation concluded that pembrolizumab at 200 mg Q3W 
achieves full PD-1 saturation in both blood and tumor. 
Finally, population PK analysis of pembrolizumab, which characterized the influence of body 
weight and other participant covariates on exposure, has shown that the fixed-dosing provides 
similar control of PK variability as weight-based dosing, with considerable overlap in the 
distribution of exposures from the 200 mg Q3W fixed dose and 2 mg/kg Q3W dose.  Supported 

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by these PK characteristics, and given that fixed-dose has advantages of reduced dosing 
complexity and reduced potential of dosing errors, the 200 mg Q3W fixed-dose was selected for 
evaluation across all pembrolizumab protocols. 
6.2.3 Rationale for Endpoints 
6.2.3.1 Efficacy Endpoints 
Objective response rate has been chosen as primary end-point for historical comparison with the 
seminal results of the first pembrolizumab monotherapy trial in MMR deficient mCRC patients 
(Le et al., 2015). On the same token we have chosen progression, death, and toxicity as secondary 
endpoints.    
6.2.3.2 Biomarker Research 
In addition to the MMR and MGMT markers, and the longitudinal mutational burden 
determination, which are an inherent component for the enrichment strategy of the trial, the 
following potential markers of pembrolizumab pharmacodynamics will be considered:   
Analysis of PD-L1 and PD1 expression in TRIAL Phase eligible patients: 
• PD-L1 will be measured on archival or newly obtained core or excisional biopsies by 
centralized IHC using QualTeck assay and 22C3 antibody clone (Merck). Positivity is 
defined as membranous PD-L1 expression in > 1% of cells in tumor and stroma.   
• PD1 and other lymphocyte activation markers will be measured in peripheral blood 
lymphocyte (PBL) post-treatment, and correlated with mutational load. PBL will be 
collected at baseline and with each treatment. PBL are isolated and stored frozen until use. 
Investigation of the immunomodulatory effect of TMZ:  
We plan to explore also the association of PD-1 positivity, and tumor infiltrating lymphocyte 
characteristics with clinical responses to subsequent Pembrolizumab treatment. In such cases 
archived tumor tissues and tissue obtained at biopsy will be compared. Archival and post-TMZ 
tumor biopsies will be analyzed for PD-1/PD-L1 expression as well as for a variety of potential 
immune-markers as described in 9.1.4.2.  
 
7.0 METHODOLOGY 
7.1 
Entry Criteria 
Patient selection criteria are progressively restrictive according to the 3-step selection strategy of 
the trial and are reported separately for each phase. 
7.1.1 Entry criteria for SCREENING Phase  
1. Histologically confirmed diagnosis of metastatic colorectal cancer. 

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2. Documented RAS extended mutations in the archival sample (cohort P only). 
3. ECOG performance status 0-1. 
4. SCREENING phase informed consent signed. 
5. Understanding and accepting the need for undergoing two tumor biopsies if eligible for 
PRIMING Phase. 
6. Age ≥ 18 years. 
7. Availability of all diagnostic FFPE blocks (primary tumor and or metastases), or at least 
20 slides (primary tumor and/or metastases). Formalin-fixed, paraffin embedded (FFPE) 
tissue blocks are preferred to slides.   
8. Normal organ functions (see Table 7.1.4 below). 
7.1.2 Entry Criteria for PRIMING Phase 
Participants are eligible to be included in the PRIMING Phase, only if all of the following criteria 
apply: 
1. Fulfilment of all the SCREENING inclusion criteria; 
2. PRIMING informed consent signed;  
3. Confirming the willingness to undergo two tumor biopsies,  
4. Acceptance that if the mutational load determination is unfeasible for technical reasons 
(not enough tissue, substandard test performance, etc.), access to TRIAL phase will not be 
possible.   
5. Imaging documented failure of previous standard CRC therapies including 
fluoropyrimidine, oxaliplatin, irinotecan plus or minus antiangiogenics agents 
(Bevacizumab, Aflibercept, Regorafenib, others). 
6. At least one measurable tumor lesion as per RECIST v1.1. Lesions in previously irradiated 
areas or those that have received other loco-regional therapies (i.e. percutaneous ablation) 
should not be considered measurable unless there is clear documented evidence of 
progression of the lesion since therapy. Imaging must be performed maximum within 28 
days prior to enrolment. 
7. ECOG performance status 0 or 1; 
8. Following results in the SCREENING Phase tests: 
• Proficient MMR status assessed by IHC  or  MSI-low  status defined by PCR (Bethesda 
panel); 
• Negative score for the MGMT protein expression IHC test; 
• Positive score for the MGMT promoter methylation performed on tissue. 
9. Women with childbearing potential should complete a pregnancy test and be willing to use 
highly effective contraceptive methods. 
10. Normal organ functions as defined in the Table 7.1.4 below.   

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7.1.3 Entry Criteria for TRIAL Phase 
1. Fulfilment of all the SCREENING inclusion criteria and deficient MMR status  (IHC) or 
MSI-High status (PCR) (cohort D only).  
2. Fulfilment of all the SCREENING and PRIMING inclusion criteria (cohort P only).  
3. TRIAL Phase informed consent signed (both cohorts).  
4. Imaging documented PD to TMZ (cohort P only).  
5. A mutational load value > 20 mutations/MB at TMZ-ML assay (cohort P only). 
6. Imaging documented failure of previous standard CRC therapies including 
fluoropyrimidine, oxaliplatin, irinotecan plus or minus targeted agents (Bevacizumab, 
Aflibercept, Regorafenib, Cetuximab, Panitumumab, others) (cohort D only). 
7. At least one measurable tumor lesion as per RECIST v1.1. Lesions in previously irradiated 
areas or those that have received other loco-regional therapies (i.e. percutaneous ablation) 
should not be considered measurable unless there is clear documented evidence of 
progression of the lesion since therapy. Imaging must be performed maximum within 28 
days prior to enrolment (both cohorts). 
8. Women with childbearing potential should complete a pregnancy test and be willing to use 
highly effective contraceptive methods (both cohorts). 
9. Normal organ functions as defined in the Table 7.1.4 below.  Blood specimens must be 
collected within 10 days prior to the start of study treatment. 
7.1.4 Adequate Organ Function Laboratory Values 
System 
Laboratory Value 
Hematological 
 
Absolute neutrophil count (ANC) 
≥1500/µL 
Platelets 
≥100.000/µL 
Hemoglobin 
≥9.0 g/dL or ≥5.6 mmol/La 
Renal 
 
Creatinine 
OR 
Measured or calculatedb creatinine clearance 
(GFR can also be used in place of creatinine or 
CrCl) 
≤1.5 × ULN OR 
≥30 mL/min for participant with creatinine 
levels >1.5 × institutional ULN 
Hepatic 
 
Total bilirubin 
≤1.5 ×ULN OR direct bilirubin ≤ULN for 
participants with total bilirubin levels 
>1.5 × ULN 
AST (SGOT) and ALT (SGPT) 
≤2.5 × ULN (≤5 × ULN for participants with 
liver metastases) 
Coagulation 
 
International normalized ratio (INR) OR 
prothrombin time (PT) 
Activated partial thromboplastin time (aPTT) 
≤1.5 × ULN unless participant is receiving 
anticoagulant therapy as long as PT or aPTT is 
within therapeutic range of intended use of 
anticoagulants 

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7.2 
Exclusion Criteria 
Participants are excluded from the study if any of the following criteria apply: 
1. A woman of child bearing potential who has a positive serum pregnancy test within 
72 hours prior to allocation (see Appendix 3).  
2. Has received prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or 
with an agent directed to another stimulatory or co-inhibitory T-cell receptor 
(eg, CTLA-4, OX-40, CD137). 
3. Has received prior systemic anti-cancer therapy including investigational agents 
within 4 weeks [could consider shorter interval for kinase inhibitors or other short 
half-life drugs] prior to enrollment. 
a. Note: Participants must have recovered from all AEs due to previous therapies 
to ≤Grade 1 or baseline. Participants with ≤Grade 2 neuropathy may be 
eligible. 
b. Note: If participant received major surgery, they must have recovered 
adequately from the toxicity and/or complications from the intervention prior 
to starting study treatment. 
4. Has received prior radiotherapy within 2 weeks of start of study treatment (with 
Pembrolizumab).  Participants must have recovered from all radiation-related 
toxicities, not require corticosteroids, and not have had radiation pneumonitis.  A 1-
week washout is permitted for palliative radiation (≤2 weeks of radiotherapy) to non-
CNS disease. 
5. Has received a live vaccine within 30 days prior to the first dose of study drug.  
Examples of live vaccines include, but are not limited to, the following:  measles, 
mumps, rubella, varicella/zoster (chicken pox), yellow fever, rabies, and typhoid 
vaccine.  Seasonal influenza vaccines for injection are generally killed virus vaccines 
and are allowed; however, intranasal influenza vaccines (eg, FluMist®) are live 
attenuated vaccines and are not allowed. 
6. Is currently participating in or has participated in a study of an investigational agent 
or has used an investigational device within 4 weeks prior to the first dose of study 
treatment. 
ALT (SGPT)=alanine aminotransferase (serum glutamic pyruvic transaminase); AST (SGOT)=aspartate 
aminotransferase (serum glutamic oxaloacetic transaminase); GFR=glomerular filtration rate; ULN=upper 
limit of normal. 
a Criteria must be met without erythropoietin dependency and without packed red blood cell (pRBC) 
transfusion within last 2 weeks.  
b Creatinine clearance (CrCl) should be calculated per institutional standard. 
Note: This table includes eligibility-defining laboratory value requirements for treatment; laboratory value 
requirements should be adapted according to local regulations and guidelines for the administration of specific 
therapies. 

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a. Note:  Participants who have entered the follow-up phase of an investigational 
study may participate as long as it has been 4 weeks after the last dose of the 
previous investigational agent. 
7. Has a diagnosis of immunodeficiency or is receiving chronic systemic steroid therapy 
(in dosing exceeding 10 mg daily of prednisone equivalent) or any other form of 
immunosuppressive therapy within 7 days prior to the first dose of study drug. 
 
8. Has a known additional malignancy that is progressing or has required active 
treatment within the past 3 years.  Note:  Participants with basal cell carcinoma of the 
skin, squamous cell carcinoma of the skin, or carcinoma in situ (e.g. breast carcinoma, 
cervical cancer in situ) that have undergone potentially curative therapy are not 
excluded. 
9. Has known active CNS metastases and/or carcinomatous meningitis.  Participants 
with previously treated brain metastases may participate provided they are 
radiologically stable, i.e. without evidence of progression for at least 4 weeks by 
repeat imaging (note that the repeat imaging should be performed during study 
screening), clinically stable and without requirement of steroid treatment for at least 
14 days prior to first dose of study treatment. 
10. Has severe hypersensitivity (≥Grade 3) to pembrolizumab and/or any of its excipients. 
11. Has severe hypersensitivity (≥Grade 3) to temozolomide and/or any of its excipients. 
12. Has active autoimmune disease that has required systemic treatment in the past 2 years 
(i.e. with use of disease modifying agents, corticosteroids or immunosuppressive 
drugs). Replacement therapy (eg., thyroxine, insulin, or physiologic corticosteroid 
replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a 
form of systemic treatment.  
13. Has a history of (non-infectious) pneumonitis that required steroids or has current 
pneumonitis. 
14. Has an active infection requiring systemic therapy. 
15. Has a known history of Human Immunodeficiency Virus (HIV).  Note: No HIV 
testing is required. 
16. Has a known history of Hepatitis B (defined as Hepatitis B surface antigen [HBsAg] 
reactive) or known active Hepatitis C virus infection. Note: no testing for Hepatitis B 
and Hepatitis C is required unless mandated by local health authority.  
17. Has a known history of active TB   (Bacillus Tuberculosis).  
18. Has a history or current evidence of any condition, therapy, or laboratory abnormality 
that might confound the results of the study, interfere with the subject’s participation 
for the full duration of the study, or is not in the best interest of the subject to 
participate, in the opinion of the treating investigator.  
19. Has known psychiatric or substance abuse disorders that would interfere with 
cooperation with the requirements of the trial 
20. Is pregnant or breastfeeding, or expecting to conceive or father children within the 
projected duration of the study, starting with the screening visit through 120 days after 
the last dose of trial treatment. 

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7.2.1 Lifestyle Restrictions 
7.2.1.1 Meals and Dietary Restrictions 
Participants should maintain a normal diet unless modifications are required to manage an AE such 
as diarrhea, nausea or vomiting. 
7.2.1.2 Contraception 
ICH M3 guidance requires precautions to be taken to minimize risk to fetus or embryo when 
including women of childbearing potential in clinical studies. 
Temozolomide and Pembrolizumab may have adverse effects on a fetus in utero.  Refer to 
Appendix 3 for approved methods of contraception. 
7.2.2 Pregnancy 
If a participant inadvertently becomes pregnant while on treatment with Temozolomide or 
pembrolizumab, the participant will be immediately discontinued from study treatment.  The site 
will contact the participant at least monthly and document the participant’s status until the 
pregnancy has been completed or terminated. The outcome of the pregnancy will be reported to 
delegated pharmacovigilance and to Merck Global Safety according to timelines as defined in  
section 9.2.3 if the outcome is a serious adverse experience (e.g., death, abortion, congenital 
anomaly, or other disabling or life-threatening complication to the mother or newborn).  The study 
Investigator will make every effort to obtain permission to follow the outcome of the pregnancy 
and report the condition of the fetus or newborn to Merck.  If a male participant impregnates his 
female partner, the study personnel at the site must be informed immediately and the pregnancy 
must be reported to delegated pharmacovigilance and to Merck Global Safety followed as 
described in section 9.2.3. 
7.2.3 Use in Nursing Women 
It is unknown whether pembrolizumab is excreted in human milk.  Since many drugs are excreted 
in human milk, and because of the potential for serious adverse reactions in the nursing infant, 
participants who are breast-feeding are not eligible for enrollment. 
7.3 
Trial Treatments 
Only qualified personnel familiar with procedures that minimize undue exposure to themselves 
and to the environment should undertake the preparation, handling, and safe disposal of 
chemotherapeutic agents.  
The treatment to be used in this trial is outlined below in Table 7.3. 

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Table 7.3  Trial Treatment 
Drug 
Dose/Potency 
Dose 
Frequency 
Route 
of 
Administration 
Regimen/Treatment 
Period 
Use 
Temozolomide 
150 or 200 or 
100 
mg/m2 
(7.3.1.2) 
Q4W 
P.O. 
Days 
1-5   
every 
28 
days 
(PRIMING Phase) 
Experimental 
 
Pembrolizumab 
200 mg 
Q3W 
IV infusion 
Day 1 of each 3 week 
cycle (TRIAL Phase) 
Experimental 
Trial treatment should begin as close as possible to the date on which treatment is 
allocated/assigned. 
7.3.1 Temozolomide - Dose Selection/Modification 
7.3.1.1 Dose Selection 
 The rationale for selection of doses to be used in this trial is provided in section 6.2.2 Justification 
for Dose.     
7.3.1.2 Temozolomide-Dose modification (Escalation/Titration/Other) 
The starting dose of TMZ is 150 mg/sq/day. The dose can be modified at the start of cycle 2. 
During treatment, a complete blood count should be obtained on Day 22 (21 days after the first 
dose of TMZ). According to day 22 CBC results and non-haematological toxicity observed during 
cycle 1 the dose of TMZ remain 150 mg/sq/day, or is modified as reported below. 
Table 7.3.1.2 Temozolomide - Dose modification 
 
Temozolomide 2nd cycle dose 
Toxicity 
Increase dose to 200 
mg/sq/day 
Reduce dose to 100 
mg/sq/day 
Discontinue TMZ 
 
Absolute Neutrophil Count 
day 22 cycle 1 
≥ 1.5 x 109/L, 
< 1.0 x 109/L 
Na 
Thrombocyte Count 
≥ 100 x 109/L. 
< 50 x 109/L 
Na 
CTC                                     
Non-Hematologic toxicity        
(except for alopecia, nausea, 
vomiting) 
Grade ≤ 2 (except for 
alopecia, 
nausea 
and 
vomiting), 
CTC Grade 3 
CTC Grade 4 
In the event of multiple toxicities, dose modifications should be applied based upon the most severe 
toxicity observed. 

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7.3.1.3 Patients with hepatic or renal impairment 
The pharmacokinetics of TMZ are comparable in patients with normal hepatic function and in 
those with mild or moderate hepatic impairment. No data are available on the administration of 
TMZ in patients with severe hepatic impairment (Child’s Class C) or with renal impairment. Based 
on the pharmacokinetic properties of TMZ, it is unlikely that dose reductions are required in 
patients with severe hepatic impairment or any degree of renal impairment. However, caution 
should be exercised when TMZ is administered in these patients. 
7.3.1.4 Elderly patients 
Based on a population pharmacokinetic analysis in patients 19-78 years of age, clearance of TMZ 
is not affected by age. However, elderly patients (> 70 years of age) appear to be at increased risk 
of neutropenia and thrombocytopenia. 
7.3.1.5 Contraindications to Temozolomide treatment 
• Hypersensitivity to the active substance or to any of the excipients. 
• Hypersensitivity to dacarbazine (DTIC). 
• Severe myelosuppression. 
Note: For warnings see the Temozolomide SmPC. 
 
7.3.1.6 TMZ-Timing of dose administration 
Temozolomide will be administered orally once daily at the dose of 150-200 mg/m2/day for 5 
consecutive days of each treatment cycle (see 7.3.1.2). A treatment cycle will comprise 5 days of 
Temozolomide administration (Day 1 to 5) followed by 23 days of rest for a total of 28 days (4 
weeks) period (dose-schedule: 150 mg/m2 day 1-5 q28). After an overnight fasting, with free 
access to water, patients will take the study drug with a large glass of plain water without ice. A 
light breakfast can be served 1.5-2 hours after study drug intake. 
Dose modification should be implemented according to recommendations detailed in section 
7.3.1.2. 
Each patient will remain on treatment until PD unless patient refusal, consent withdrawal, or the 
occurrence of unacceptable toxicity.  
The site personnel handling the study medication to patients must ensure that they fully understand 
that the treatment period (cycle) comprises 28 days, consisting of 5 days of daily administration, 
followed by a 23-day resting period, from Day 6 to Day 28. During each treatment cycle, extension 
of treatment period to Days >5 is not allowed, even in case of missed doses. 
Additionally, the patients should be informed: 

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• to take the study drug in fasting state. 
• to swallow the capsules whole with a glass of plain water without ice; capsules must not 
be opened or chewed; water is allowed during fasting period. 
• to take the study drug at the same time each day (1.5-2 hours before breakfast). 
• if the patient forgets to take the study drug at the usual time, to take it between meals at 
any time during the same day and record the event. 
• if the patient misses one day, to take the normal amount the next day, if scheduled. Not to 
double the daily prescribed dose of the study drug and to track the number of capsules 
missed and the reason for it. 
• if vomiting occurs during or after having taken the daily dose, not to take extra capsules 
that day and to take the normal amount the next day, if scheduled. Not to double the daily 
prescribed dose of the study drug and record the event to adhere to the principles of safe 
handling and storage of the study medication. 
• Breaking/crushing or opening the capsules must be avoided. In such cases, record of the 
broken capsules should be kept. In case of opening of the capsules, to avoid contact or 
inhalation. In case of skin contact, to wash the affected area with plenty of water or soap 
and water. In case of eye-contact, to rinse thoroughly with plenty of water and see medical 
advice as soon as possible. 
• To return all packaging as well as not-taken capsules (if applicable) to the site personnel. 
 
As much of this information as possible should first be given at the pre-treatment visit and 
reinforced on subsequent visits. A diary of TMZ assumption must be given to the patient every 
treatment cycle and the correct compilation must be revised by the investigator. 
7.3.1 Pembrolizumab - Dose Selection/Modification 
7.3.1.1 Dose Selection 
The rationale for selection of doses to be used in this trial is provided in section 4.0– Background 
and Rationale.   
7.3.2 Timing of Dose Administration 
Trial treatment should be administered on Day 1 of each cycle after all procedures/assessments 
have been completed as detailed on the Trial Flow Chart (section 6.0).   
• Pembrolizumab 200 mg will be administered on an outpatient basis as a 30 minute IV 
infusion every 3 weeks.  Sites should make every effort to target infusion timing to be as 
close to 30 minutes as possible.  However, given the variability of infusion pumps from 
site to site, a window of -5 minutes and +10 minutes is permitted (i.e., infusion time is 30 
minutes: -5 min/+10 min). 
• Pembrolizumab may be administered up to 3 days before or after the scheduled Day 1 of 
each cycle due to administrative reasons. 

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7.3.2 Dose Modification and toxicity management for immune-related AEs associated with 
pembrolizumab 
AEs associated with pembrolizumab exposure may represent an immunologic etiology. These 
immune-related AEs (irAEs) may occur shortly after the first dose or several months after the last 
dose of pembrolizumab treatment and may affect more than on body system simultaneously. 
Therefore, early recognition and initiation of treatment is critical to reduce complications.   
• Based on existing clinical study data, most irAEs are reversible and could be managed with 
interruptions of pembrolizumab, administration of corticosteroids and/or other supportive 
care. 
• For suspected irAEs, ensure adequate evaluation to confirm etiology or exclude other 
causes. Additional procedures or tests such as bronchoscopy, endoscopy, skin biopsy may 
be included as part of the evaluation. Based on the severity of irAEs, withhold or 
permanently discontinue pembrolizumab and administer corticosteroids.  
• Dose modification and toxicity management guidelines for irAEs associated with 
pembrolizumab are provided in Table 7.3.2.

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Table 7.3.2 Dose modification and toxicity management guidelines for immune-related AEs associated with pembrolizumab  
General instructions: 
1. Corticosteroid taper should be initiated upon AE improving to Grade 1 or less and continue to taper over at least 4 weeks. 
2. For situations where pembrolizumab has been withheld, pembrolizumab can be resumed after AE has been reduced to Grade 1 or 0 and 
corticosteroid has been tapered. Pembrolizumab should be permanently discontinued if AE does not resolve within 12 weeks of last dose or 
corticosteroids cannot be reduced to ≤10 mg prednisone or equivalent per day within 12 weeks.  
3. For severe and life-threatening irAEs, IV corticosteroid should be initiated first followed by oral steroid. Other immunosuppressive treatment 
should be initiated if irAEs cannot be controlled by corticosteroids. 
 
Immune-related 
AEs 
Toxicity grade or 
conditions 
(CTCAEv4.03) 
Action taken to 
pembrolizumab 
irAE 
management 
with 
corticosteroid 
and/or 
other 
therapies  
Monitor and follow-up  
Pneumonitis  
Grade 2 
Withhold  
• Administer corticosteroids (initial 
dose of 1-2 mg/kg prednisone or 
equivalent) followed by taper 
 
• 
Monitor participants for signs and symptoms 
of pneumonitis 
• 
Evaluate 
participants 
with 
suspected 
pneumonitis with radiographic imaging and 
initiate corticosteroid treatment 
• 
Add 
prophylactic 
antibiotics 
for 
opportunistic infections 
Grade 3 or 4, or 
recurrent Grade 2 
Permanently 
discontinue 
Diarrhea / Colitis  Grade 2 or 3 
Withhold 
• Administer corticosteroids (initial 
dose of 1-2 mg/kg prednisone or 
equivalent) followed by taper 
 
• 
Monitor participants for signs and symptoms 
of enterocolitis (ie, diarrhea, abdominal pain, 
blood or mucus in stool with or without fever) 
and of bowel perforation (ie, peritoneal signs 
and ileus). 

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Grade 
4 
or 
recurrent Grade 3 
(for =Colitis) 
Permanently 
discontinue 
• 
Participants with ≥ Grade 2 diarrhea 
suspecting 
colitis 
should consider 
GI 
consultation and performing endoscopy to 
rule out colitis.  
• 
Participants with diarrhea/colitis should be 
advised to drink liberal quantities of clear 
fluids.  If sufficient oral fluid intake is not 
feasible, fluid and electrolytes should be 
substituted via IV infusion. 
AST 
/ 
ALT 
elevation 
or 
Increased 
bilirubin 
Grade 2 
Withhold 
• Administer corticosteroids (initial 
dose of 0.5- 1 mg/kg prednisone or 
equivalent) followed by taper 
• 
Monitor with liver function tests (consider 
weekly or more frequently until liver enzyme 
value returned to baseline or is stable 
Grade 3 or 4  
Permanently 
discontinue 
• Administer corticosteroids (initial 
dose of 1-2 mg/kg prednisone or 
equivalent) followed by taper 
Type 1 diabetes 
mellitus (T1DM) 
or 
Hyperglycemia 
 
Newly 
onset 
T1DM or  
Grade 
3 
or 
4 
hyperglycemia 
associated 
with 
evidence of b-cell 
failure 
Withhold 
• Initiate insulin replacement therapy 
for participants with T1DM  
• Administer anti-hyperglycemic in 
participants with hyperglycemia  
 
• 
Monitor participants for hyperglycemia or 
other signs and symptoms of diabetes. 
Hypophysitis 
Grade 2 
Withhold 
• Administer 
corticosteroids 
and 
initiate hormonal replacements as 
clinically indicated.   
 
• 
Monitor for signs and symptoms of 
hypophysitis (including hypopituitarism and 
adrenal insufficiency)  
Grade 3 or 4  
Withhold 
or 
permanently 
discontinue1  
Hyperthyroidism  
Grade 2 
Continue  
• Treat with non-selective beta-
blockers 
(eg, 
propranolol) 
or 
thionamides as appropriate  
• 
Monitor for signs and symptoms of thyroid 
disorders.  
 
Grade 3 or 4  
Withhold or  

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permanently 
discontinue1 
Hypothyroidism 
Grade 2-4 
Continue  
• Initiate 
thyroid 
replacement 
hormones (eg, levothyroxine or 
liothyroinine) per standard of care 
• 
Monitor for signs and symptoms of thyroid 
disorders.  
 
Nephritis 
and 
Renal 
dysfunction 
Grade 2 
Withhold  
• Administer 
corticosteroids 
(prednisone 
1-2 
mg/kg 
or 
equivalent) followed by taper. 
• 
Monitor changes of renal function 
 
Grade 3 or 4  
Permanently 
discontinue 
Myocarditis 
Grade 1 or 2 
Withhold 
• Based on severity of AE administer 
corticosteroids 
• 
Ensure adequate evaluation to confirm 
etiology and/or exclude other causes 
 
Grade 3 or 4 
Permanently 
discontinue 
All 
other 
immune-related 
AEs 
Intolerable/ 
persistent Grade 2 
Withhold 
• Based on type and severity of AE 
administer corticosteroids  
• 
Ensure adequate evaluation to confirm 
etiology and/or exclude other causes 
 
Grade 3 
Withhold 
or 
discontinue based 
on the type of 
event.  Events that 
require 
discontinuation 
include 
and 
not 
limited to:  Gullain-
Barre 
Syndrome, 
encephalitis 
Grade 
4 
or 
recurrent Grade 3  
Permanently 
discontinue 
1. Withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating physician.   
NOTE:  For participants with Grade 3 or 4 immune-related endocrinopathy where withhold of pembrolizumab is required, pembrolizumab may be resumed 
when AE resolves to ≤ Grade 2 and is controlled with hormonal replacement therapy or achieved metabolic control (in case of T1DM).   

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7.3.3 Dose modification and toxicity management of infusion-reactions related to pembrolizumab 
Pembrolizumab may cause severe or life-threatening infusion-reactions including severe hypersensitivity or anaphylaxis. Signs and 
symptoms usually develop during or shortly after drug infusion and generally resolve completely within 24 hours of completion of 
infusion. Dose modification and toxicity management guidelines on pembrolizumab associated infusion reaction are provided in Table 
7.3.3.  
 
 
 
 
 
 
 
 
 
 
 
 
Table 7.3.3  Pembrolizumab Infusion Reaction Dose modification and Treatment Guidelines 

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NCI CTCAE Grade 
Treatment 
Premedication at Subsequent Dosing 
Grade 1  
Mild 
reaction; 
infusion 
interruption 
not 
indicated; 
intervention not indicated 
Increase monitoring of vital signs as medically indicated until the 
participant is deemed medically stable in the opinion of the investigator. 
None 
Grade 2 
Requires therapy or infusion 
interruption 
but 
responds 
promptly 
to 
symptomatic 
treatment (e.g., antihistamines, 
NSAIDs, narcotics, IV fluids); 
prophylactic 
medications 
indicated for ≤24 hrs 
Stop Infusion. 
Additional appropriate medical therapy may include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDs 
Acetaminophen 
Narcotics 
 
Increase monitoring of vital signs as medically indicated until the 
participant is deemed medically stable in the opinion of the investigator. 
If symptoms resolve within 1 hour of stopping drug infusion, the infusion 
may be restarted at 50% of the original infusion rate (e.g. from 100 mL/hr 
to 50 mL/hr).  Otherwise dosing will be held until symptoms resolve and 
the participant should be premedicated for the next scheduled dose. 
Participants who develop Grade 2 toxicity despite adequate 
premedication should be permanently discontinued from further study 
drug treatment 
Participant may be premedicated 1.5h (± 
30 minutes) prior to infusion of _____ 
with: 
Diphenhydramine 
50 
mg 
po 
(or 
equivalent dose of antihistamine). 
Acetaminophen 500-1000 mg po (or 
equivalent dose of analgesic). 

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Grades 3 or 4 
Grade 3: 
Prolonged (i.e., not rapidly 
responsive 
to 
symptomatic 
medication 
and/or 
brief 
interruption 
of 
infusion); 
recurrence 
of 
symptoms 
following initial improvement; 
hospitalization indicated for 
other clinical sequelae (e.g., 
renal impairment, pulmonary 
infiltrates) 
Grade 4: 
Life-threatening; pressor or 
ventilatory support indicated 
Stop Infusion. 
Additional appropriate medical therapy may include but is not limited to: 
Epinephrine** 
IV fluids 
Antihistamines 
NSAIDs 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Increase monitoring of vital signs as medically indicated until the 
participant is deemed medically stable in the opinion of the investigator. 
Hospitalization may be indicated. 
**In cases of anaphylaxis, epinephrine should be used immediately. 
Participant is permanently discontinued from further study drug 
treatment. 
No subsequent dosing 
Appropriate resuscitation equipment should be available at the bedside and a physician readily available during the period of drug administration. 
For further information, please refer to the Common Terminology Criteria for Adverse Events v4.03 (CTCAE) at http://ctep.cancer.gov 

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7.3.4 Other allowed dose interruption for pembrolizumab  
Pembrolizumab may be interrupted for situations other than treatment-related AEs such as 
medical / surgical events or logistical reasons not related to study therapy. Participants should 
be placed back on study therapy within 3 weeks of the scheduled interruption, unless otherwise 
discussed with the Sponsor. The reason for interruption should be documented in the patient's 
study record. 
7.3.5 Second Course  
All participants who stop study treatment with SD or better, may be eligible for up to an 
additional 17 cycles (approximately 1 year) of pembrolizumab treatment if they progress after 
stopping study treatment from the initial treatment phase.  This retreatment is termed the 
Second Course Phase of this study and is only available if the study remains open and the 
participant meets the following conditions: 
Either 
• Stopped 
initial 
treatment 
with 
study 
treatment 
after 
attaining 
an 
investigator-determined confirmed CR based on RECIST 1.1, and 
o Was treated with at least 8 cycles of study treatment before discontinuing treatment, 
and 
o Received at least 2 treatments with pembrolizumab beyond the date when the initial 
CR was declared 
OR 
• Had SD, PR, or CR and stopped study treatment after completion of 35 administrations 
(approximately 2 years) of study treatment for reasons other than disease progression 
or intolerability 
AND 
• Experienced an investigator-determined radiographic disease progression by 
RECIST 1.1 after stopping initial treatment, and 
o No new anticancer treatment was administered after the last dose of study 
treatment, and 
o The participant meets all of the safety parameters listed in the inclusion criteria and 
none of the safety parameters listed in the exclusion criteria, and 
o The study is ongoing 

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An objective response or disease progression that occurs during the Second Course Phase for 
a participant will not be counted as an event for the primary analysis of either endpoint in this 
study. 
7.4 
Randomization or Treatment Allocation 
MMR Deficient or MSI-H patients are allocated to Cohort D. Originally MMR Proficient or 
MSI-low patients at the end of the PRIMING phase either are allocated to Cohort P if TMZ-
ML > 20 mut/Megabase, or go off protocol  if TMZ-ML < 20 mut/Megabase. 
7.5 
Stratification 
For exploratory analysis purpose only, patients will be stratified post-hoc according to selected 
immune biomarkers (see 9.1.4.2) or other putative predictive markers. 
7.6 
Concomitant Medications/Vaccinations (allowed & prohibited) 
Medications or vaccinations specifically prohibited in the exclusion criteria (see 7.2) are not 
allowed during the ongoing trial.  If there is a clinical indication for one of these or other 
medications or vaccinations specifically prohibited during the trial, discontinuation from trial 
therapy or vaccination may be required.  The final decision on any supportive therapy or 
vaccination rests with the investigator and/or the participants's primary physician.  
7.6.1 Acceptable Concomitant Medications 
Throughout the study, investigators may prescribe any concomitant medications or treatments 
deemed necessary to provide adequate supportive care. 
Anticancer therapy (chemotherapy, biologic or radiation therapy, palliative radiotherapy 
covering > 25% of the bone marrow reserve, and surgery) must not be given to patients during 
the study treatment, except to TMZ during TMZ Priming Phase. 
Radiotherapy for palliation in non-target lesions with antalgic aim is allowed. Recombinant 
human granulocyte colony stimulating factor (G-CSF) or erythropoiesis stimulating agent use 
according to the current approved label or institutional guidelines is permitted. 
All treatments that the investigator considers necessary for a participant’s welfare may be 
administered at the discretion of the investigator in keeping with the community standards of 
medical care including palliative treatment (with the exception of regorafenib), delivered for 
symptomatic disease during the 5-week gap between progression to TMZ and start of 
pembrolizumab treatment (see 4.1.2)..  All concomitant medication will be recorded on the 
case report form (CRF) including all prescription, over-the-counter (OTC), herbal 
supplements, and IV medications and fluids.  If changes occur during the trial period, 
documentation of drug dosage, frequency, route, and date may also be included on the CRF. 

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All concomitant medications received within 28 days before the first dose of trial treatment 
and 30 days after the last dose of trial treatment should be recorded.  Concomitant medications 
administered after 30 days after the last dose of trial treatment should be recorded for SAEs 
and ECIs as defined in section 9.2. 
7.6.2 Prohibited Concomitant Medications 
Parcticipants are prohibited from receiving the following therapies during the Screening and 
Treatment Phase (including retreatment for post-complete response relapse) of this trial: 
• Antineoplastic systemic chemotherapy or biological therapy (with the exception 
described in 5.1.2 and 8.2.1) 
• Immunotherapy not specified in this protocol 
• Chemotherapy not specified in this protocol 
• Investigational agents other than TMZ (PRIMING Phase) and pembrolizumab (TRIAL 
Phase) 
• Radiation therapy other than required for palliation in non-target lesions or to a 
symptomatic brain metastasis.   
• Live vaccines within 30 days prior to the first dose of study treatment and while 
participating in the study.  Examples of live vaccines include, but are not limited to, the 
following: measles, mumps, rubella, varicella/zoster, yellow fever, rabies, BCG, and 
typhoid vaccine. Seasonal influenza vaccines for injection are generally killed virus 
vaccines and are allowed; however, intranasal influenza vaccines (eg, FluMist®) 
are live attenuated vaccines and are not allowed.  
• Systemic glucocorticoids for any purpose other than to modulate symptoms from an 
event of clinical interest of suspected immunologic etiology.  The use of physiologic 
doses of corticosteroids may be approved after consultation with the Sponsor. 
Participants who, in the assessment by the investigator, require the use of any of the 
aforementioned treatments for clinical management should be removed from the study.  All 
treatments that the Investigator considers necessary for a participant’s welfare may be 
administered at the discretion of the Investigator in keeping with the community standards of 
medical care.   
Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed 
during the ongoing study.  If there is a clinical indication for any medication or vaccination 
specifically prohibited during the study, discontinuation from study therapy or vaccination may 
be required.  The final decision on any supportive therapy or vaccination rests with the 
investigator and/or the participant's primary physician.  However, the decision to continue the 
participant on study treatment requires the mutual agreement of the investigator, the Sponsor 
and the participant. 
There are no prohibited therapies during the Post-Treatment Follow-up Phase (TRIAL phase).  

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7.6.3 Rescue Medications & Supportive Care 
Participants should receive appropriate supportive care measures as deemed necessary by the 
treating investigator.  Suggested supportive care measures for the management of AEs with 
potential immunologic etiology are outlined along with the dose modification guidelines in 
Table 7.3.3.  Where appropriate, these guidelines include the use of oral or IV treatment with 
corticosteroids, as well as additional anti-inflammatory agents if symptoms do not improve 
with administration of corticosteroids.  Note that several courses of steroid tapering may be 
necessary as symptoms may worsen when the steroid dose is decreased.  For each disorder, 
attempts should be made to rule out other causes such as metastatic disease or bacterial or viral 
infection, which might require additional supportive care.  The treatment guidelines are 
intended to be applied when the Investigator determines the events to be related to 
pembrolizumab.  
Note: If after the evaluation of the event, it is determined not to be related to pembrolizumab, 
the Investigator does not need to follow the treatment guidance.  Refer to Table 7.3.3 for 
guidelines regarding dose modification and supportive care.  
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or 
skin photography as part of evaluation of the event.  
7.7 
Participant Withdrawal/Discontinuation Criteria 
Participants may discontinue study treatment at any time for any reason or be dropped from 
the study treatment at the discretion of the investigator should any untoward effect occur.  In 
addition, a participant may be discontinued from study treatment by the investigator or the 
Sponsor if study treatment is inappropriate, the trial plan is violated, or for administrative 
and/or other safety reasons.  Specific details regarding procedures to be performed at study 
treatment discontinuation are provided in section 9.1.6 – Other Procedures. 
A participant must be discontinued from study treatment but continue to be monitored in the 
study for any of the following reasons: 
• The participant or participant’s legally acceptable representative requests to 
discontinue study treatment 
• Confirmed radiographic disease progression outlined in section 9.1.3.2. 
• Any progression or recurrence of any malignancy, or any occurrence of another 
malignancy that requires active treatment 
• Unacceptable adverse experiences as described in section 7.3.3. 
• The participant has a medical condition or personal circumstance which, in the opinion 
of the investigator and/or sponsor, placed the participant at unnecessary risk from 
continued administration of study treatment. 
• The participant has a confirmed positive serum pregnancy test 
• Noncompliance with study treatment or procedure requirements 
• Recurrent Grade 2 pneumonitis 

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• Discontinuation of treatment may be considered for participants who have attained a 
confirmed complete response (CR) and have been treated for at least 8 cycles (at least 
24 weeks), receiving at least 2 doses of pembrolizumab; beyond the date when the 
initial CR was declared. These participants may be eligible for second course treatment 
described in section 7.3.5. 
• The participant is lost to follow-up 
• Completion of 35 treatments (approximately 2 years) with pembrolizumab 
Note: The number of treatments is calculated starting with the first dose.  Participants who 
stop the combination or pembrolizumab after receiving 35 doses may be eligible for 
retreatment if they progress after stopping study treatment provided they meet the 
requirements detailed in section 7.3.5. Participants may be retreated in the Second Course 
Phase (Retreatment) for up to an additional 17 cycles (approximately 1 year). 
• Administrative reasons 
7.8 
Participant Replacement Strategy 
The following patients will be substituted:  
7.8.1 PRIMING Phase 
Patients not receiving at least two courses of TMZ for undue toxicity or consent withdrawal or 
patients refusing or failing to have a mutational load determined at post-TMZ biopsy 
(PRIMING Phase only) 
7.8.2 TRIAL Phase 
Patients that cannot complete the first administration of pembrolizumab due to severe infusion 
reactions requiring treatment suspension. 
7.9 
Clinical Criteria for Early Trial Termination 
Early trial termination will be the result of the criteria specified below: 
1. Quality or quantity of data recording is inaccurate or incomplete 
2. Poor adherence to protocol and regulatory requirements 
3. Incidence or severity of adverse drug reaction in this or other studies indicates a potential 
health hazard to participants 
4. Plans to modify or discontinue the development of the study drug 
In the event of Merck decision to no longer supply study drug, ample notification will be 
provided so that appropriate adjustments to participant treatment can be made. 

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8.0 TRIAL FLOW CHART 
 
 

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9.0  TRIAL PROCEDURES 
9.1 
Trial Procedures 
The Trial Flow Chart - section 8.0 summarizes the trial procedures to be performed at each 
visit.  Individual trial procedures are described in detail below.  It may be necessary to perform 
these procedures at unscheduled time points if deemed clinically necessary by the investigator. 
Furthermore, additional evaluations/testing may be deemed necessary by the Sponsor and/or 
Merck for reasons related to participant safety. In some cases, such evaluation/testing may be 
potentially sensitive in nature (e.g., HIV, Hepatitis C, etc.), and thus local regulations may 
require that additional informed consent be obtained from the participant.  In these cases, such 
evaluations/testing will be performed in accordance with those regulations. 
9.1.1 Administrative Procedures 
9.1.1.1 Informed Consent 
The Investigator must obtain documented consent from each potential participant prior to 
participating in a clinical trial. Due to the complex trial design of Arethusa, three Informed 
Consent (IC) will be obtained by each patient, and documented prior the patient’s entry to each 
subsequent phase of the trial (SCREENING, PRIMING and TRIAL), if appropriate. The 
specific set of conditions that have to be included in each IC form are defined by the eligibility 
criteria of the three phases (see 7.1 and 7.2) 
Consent must be documented by the participant’s dated signature or by the participant’s legally 
acceptable representative’s dated signature on a consent form along with the dated signature 
of the person conducting the consent discussion.  
Copies of the signed and dated consent forms should be given to the participant before 
participation in each Phase of the trial. 
The initial informed consent form, any subsequent revised written informed consent form and 
any written information provided to the participant must receive the IRB/ERC’s 
approval/favorable opinion in advance of use.  The participant or his/her legally acceptable 
representative should be informed in a timely manner if new information becomes available 
that may be relevant to the participant’s willingness to continue participation in the trial.  The 
communication of this information will be provided and documented via a revised consent 
form or addendum to the original consent form that captures the participant’s dated signature 
or by the participant’s legally acceptable representative’s dated signature. 
Specifics about a trial and the trial population will be added to the consent form template at 
the protocol level.   
The informed consent will adhere to IRB/ERC requirements, applicable laws and regulations 
and Sponsor requirements. 

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9.1.1.2 Inclusion/Exclusion Criteria 
All inclusion and exclusion criteria will be reviewed by the investigator or qualified designee 
to ensure that the participant qualifies for the trial.  
9.1.1.3 Medical History 
A medical history will be obtained by the investigator or qualified designee.  Medical history 
will include all active conditions, and any condition diagnosed within the prior 10 years that 
are considered to be clinically significant by the Investigator.  Details regarding the disease for 
which the participant has enrolled in this study will be recorded separately and not listed as 
medical history.   
9.1.1.4 Prior and Concomitant Medications Review 
9.1.1.4.1 Prior Medications 
The investigator or qualified designee will review prior medication use, including any 
protocol-specified washout requirement, and record prior medication taken by the participant 
within 28 days before starting the trial.  Treatment for the disease for which the participant has 
enrolled in this study will be recorded separately and not listed as a prior medication.  
9.1.1.4.2 Concomitant Medications 
The investigator or qualified designee will record medication, if any, taken by the participant 
during the trial.  All medications related to reportable SAEs and ECIs should be recorded as 
defined in section 9.2. 
All concomitant medications received within 30 days after the last dose of trial treatment 
should be recorded.  
9.1.1.5 Disease Details and Treatments 
9.1.1.5.1 Disease Details 
The investigator or qualified designee will obtain prior and current details regarding disease 
status.  
9.1.1.5.2 Prior Treatment Details 
The investigator or qualified designee will review all prior cancer treatments including 
systemic treatments, radiation and surgeries. 
9.1.1.5.3 Subsequent Anti-Cancer Therapy Status 
The investigator or qualified designee will review all new anti-neoplastic therapy initiated after 
the last dose of trial treatment.  If a participant initiates a new anti-cancer therapy within 30 
days after the last dose of trial treatment, the 30 day Safety Follow-up visit must occur before 

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the first dose of the new therapy.  Once new anti-cancer therapy has been initiated the 
participant will move into survival follow-up.  
9.1.1.6 Assignment of Screening Number 
Each patient enrolled in the SCREENING phase of the study will receive an univocal Subject 
code that will be assigned automatically by the eCRF. In the case that the patient will be 
enrolled in the PRIMING and TRIAL phases a Patient ID will be assigned by the Sponsor upon 
enrollment request of the participating centers.  
9.1.2 Clinical Procedures/Assessments 
9.1.2.1 Adverse Event (AE) Monitoring 
The investigator or qualified designee will assess each participant to evaluate for potential new 
or worsening AEs as specified in the Trial Flow Chart and more frequently if clinically 
indicated.  Adverse experiences will be graded and recorded throughout the study and during 
the follow-up period according to NCI CTCAE Version 4.03 (see Appendix 2).  Toxicities will 
be characterized in terms regarding seriousness, causality, toxicity grading, and action taken 
with regard to trial treatment.  
Please refer to section 9.2 for detailed information regarding the assessment and recording of 
AEs.   
9.1.2.2 Full Physical Exam 
The investigator or qualified designee will perform a complete physical exam during the 
screening period.  Clinically significant abnormal findings should be recorded as medical 
history.  A full physical exam should be performed during screening and during all on treatment 
and FU visit as reported in the Trial Flow Chart. For cycles that do not require a full physical 
exam per the Trial Flow Chart, the investigator or qualified designee will perform a directed 
physical exam as clinically indicated prior to trial treatment administration.   
9.1.2.3 Vital Signs 
The investigator or qualified designee will take vital signs at screening, prior to the 
administration of each dose of trial treatment and at treatment discontinuation as specified in 
the Trial Flow Chart (section 8.0).  Vital signs should include temperature, pulse, respiratory 
rate, weight and blood pressure.  Height will be measured at screening only. 
9.1.2.4 ECG 
The investigator or qualified designee will perform a 12-lead ECG. 

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9.1.2.5 Eastern Cooperative Oncology Group (ECOG) Performance Scale 
The investigator or qualified designee will assess ECOG status (see Appendix 1) at screening, 
prior to the administration of each dose of trial treatment and discontinuation of trial treatment 
as specified in the Trial Flow Chart (section 8.0).   
9.1.3 Tumor Imaging and Assessment of Disease 
9.1.3.1 Tumor Imaging 
Tumour imaging will be performed at each center by local radiologists according to RECIST 
and to iRECIST (see appendix 4). However CT-Scans will be cloud stored for post-trial 
centralized response revision.  
Tumor imaging is strongly preferred to be acquired by computed tomography (CT).  For the 
abdomen and pelvis, contrast-enhanced magnetic resonance imaging (MRI) may be used when 
CT with iodinated contrast is contraindicated, or when local practice mandates it.  MRI is the 
strongly preferred modality for imaging the brain.  The same imaging technique regarding 
modality, ideally the same scanner, and the use of contrast should be used in a participant 
throughout the study to optimize the reproducibility of the assessment of existing and new 
tumor burden and improve the accuracy of the assessment of response or progression based on 
imaging. 
Imaging assessment will be as follow: 
• SCREENING Phase: CT scans performed up to 28 days prior to screening eligibility 
check are considered valid.   
 
• PRIMING Phase: RECIST 1.1 will be used to assess response to TMZ. Patients in 
CR/PR/SD are reassessed every 8 weeks. However, in order to fulfil RECIST, the 
FIRST occurrence of CR/PR MUST be reconfirmed not earlier than 4 and not later than 
5 weeks later.  
 
• TRIAL Phase: Tumor imaging must be performed within 0 to 28 days prior to starting 
treatment with pembrolizumab.  Local reading (Investigator assessment with site 
radiology reading) will be used to determine eligibility. 
- Baseline for D-cohort patient; Imaging data from Screening phase can be used, 
provided timeframe is less than 28 days.  
- Baseline for P-cohort patients: the baseline correspond to the TMZ PD imaging 
assessment. 
- During trial for both cohorts: tumor assessment must be performed every 9 
weeks. However in order to fulfil RECIST, the FIRST occurrence of CR/PR 
MUST be reconfirmed not earlier than 4 and not later than 5 weeks later.  
- At RECIST PD clinically stable patients should not undergo treatment 
discontinuation until progression is confirmed locally 4 to 8 weeks later 
according to iRECIST (see Table 9.1.3 and Figure 9.1.3).  

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Participants who have unconfirmed disease progression may continue on 
treatment at the discretion of the Investigator until progression is confirmed by 
the site provided they are clinically stable.  Participants who receive 
confirmatory imaging do not need to undergo the next scheduled tumor imaging 
if it is less than 4 weeks later; tumor imaging may resume at the subsequent 
scheduled imaging time point, if clinically stable.  Participants who have 
confirmed disease progression by iRECIST, as assessed by the site, will 
discontinue study treatment.   
- In participants who discontinue study treatment, tumor imaging should be 
performed at the time of treatment discontinuation (±4-week window).  If 
previous imaging was obtained within 4 weeks prior to the date of 
discontinuation, then imaging at treatment discontinuation is not mandatory.  In 
participants who discontinue study treatment due to documented disease 
progression, this is the final required tumor imaging. 
- In case of discontinuation of treatment for any reason other than disease 
progression, patients will still be re-evaluated every 8 weeks until disease 
progression or start of a new treatment or death or patients refusal, whichever 
comes first.  
- After 1 year, the imaging time point will occur approximately every 12 weeks.  
- At trial end, all in-Trial tumor assessments radioimaging data will be reviewed 
centrally by two radiologists who will read the CT/MRI scans blinded using the 
Quibim Precision® software to collect, store, and guide the revision of the 
imaging results. The imaging review protocol and tumor assessment re-
conciliation report will be included in the final study report and or in the 
publication of the study. 
9.1.3.2 Disease assessment  
RECIST 1.1 (referencing a maximum of 5 target lesions in total and 2 per organ) will be used 
as a primary measure for assessment of tumor response and as date of disese progression.  
The Sponsor might also require, for research purpose, to enable a broader sampling of tumor 
burden to be conducted centrally.  
RECIST 1.1 Progression (PD) definition will be used for discontinuation of study treatment 
during the temozolomide PRIMING  phase, and in TRIAL phase whenever the Investigator at 
site decide NOT to use iRECIST criteria, as described below. iRECIST is based on RECIST 
1.1, but adapted to account for the unique tumor response seen with immunotherapeutic drugs.   
Disease assessment in the TRIAL phase for both Cohorts takes into account the observation 
that some patients treated with immunotherapy can have a transient tumor flare, which might 
be misconstrued for PD by RECISTs, in the first few months after the start of pembrolizumab, 
and then experience subsequent disease response.  Thus in the TRIAL phase, when tumor 
imaging shows PD by RECIST 1.1  the local investigator will re-assess the patient per 
iRECIST criteria according to  the flow-chart in figure 9.1.3, table 9.1.3.  

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Per iRECIST, disease progression should be confirmed by the site 4 to 8 weeks after first 
radiologic evidence of RECIST 1.1 PD in clinically stable participants.  Participants who have 
unconfirmed disease progression may continue on treatment at the discretion of the 
Investigator until progression is confirmed provided they have met the conditions detailed in 
Table 9.1.3 below. 
Patients who receive confirmatory imaging do not need to undergo the next scheduled tumor 
imaging if it is less than 4 weeks later; tumor imaging may resume at the subsequent scheduled 
imaging time point, if clinically stable.  Participants who have confirmed disease progression 
by iRECIST, as assessed by the site, will discontinue study treatment.  Exceptions are detailed 
in table 9.1.3. 
A description of the adaptations and iRECIST process is provided in Appendix 4, with 
additional detail in the iRECIST publication (Seymour et al., 2017). 
Table 9.1.3:  Imaging and Treatment after First Radiologic Evidence of Progressive Disease 
 
Clinically Stable 
Clinically Unstable 
Imaging 
Treatment 
Imaging 
Treatment 
Imaging and treatment options at first RECIST PD determination 
First radiologic 
evidence of PD by 
RECIST 1.1  
Repeat imaging at 
4 to 8 weeks to 
confirm PD. 
May continue study 
treatment at the 
Investigator’s 
discretion while 
awaiting confirmatory 
tumor imaging by site 
by iRECIST. 
Repeat imaging at 
4 to 8 weeks to 
confirm PD per 
Investigator’s 
discretion only. 
Discontinue treatment 
Imaging and treatment options upon iRECIST determination 
Repeat tumor 
imaging confirms 
PD (iCPD) by 
iRECIST per 
Investigator 
assessment 
No additional 
imaging required. 
Discontinue treatment 
(exception is possible 
upon consultation with 
Sponsor). 
No additional 
imaging required. 
Not applicable 
Repeat tumor 
imaging shows 
iUPD by iRECIST 
per Investigator 
assessment 
Repeat imaging at 
4 to 8 weeks to 
confirm PD.  May 
occur at next 
regularly 
scheduled imaging 
visit. 
Continue study 
treatment at the 
Investigator’s 
discretion. 
Repeat imaging at 
4 to 8 weeks to 
confirm PD per 
Investigator’s 
discretion only. 
Discontinue treatment 
Repeat 
tumor 
imaging shows iSD, 
iPR, or iCR by 
iRECIST 
per 
Investigator 
assessment. 
Continue regularly 
scheduled imaging 
assessments. 
Continue 
study 
treatment 
at 
the 
Investigator’s 
discretion. 
Continue regularly 
scheduled imaging 
assessments. 
May 
restart 
study 
treatment if condition has 
improved 
and/or 
clinically 
stable 
per 
Investigator’s discretion.  
Next tumor image should 
occur according to the 
regular imaging schedule. 
iCPD = iRECIST confirmed progressive disease; iCR = iRECIST complete response; iRECIST = modified Response 
Evaluation Criteria in Solid Tumors 1.1 for immune-based therapeutics; iSD = iRECIST stable disease; iUPD = iRECIST 
unconfirmed progressive disease; PD = progressive disease; RECIST 1.1 = Response Evaluation Criteria in Solid Tumors 
1.1.. 

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Figure 9.1.3 : Imaging and Treatment for Clinically Stable Participants after First Radiologic 
Evidence of PD Assessed by the Investigator 
 
 
9.1.4 Tumor Tissue Collection and Correlative Studies Blood Sampling 
Specific Standard Operating Procedures for sample collection will be used according to our 
Internal Sample Collections Guidelines following the schedule reported in section 8.0. 
Residual sample material available after completion of the designated analyses may be used in 
the future for identification of additional pharmacodynamic or predictive markers or to 
enhance understanding of disease biology. If biomarker samples are drawn but study drug(s) 
is not administered, samples will be retained.  
 
9.1.4.1  Mandatory assays 
• Assessment of MMR/MSI status and MGMT expression: during the screening 
phase all patients will be tested for the expression of MMR proteins by IHC on the 
diagnostic FFPE block or slides, or for microsatellite instability by PCR analysis of 
unstable microsatellite loci (Bethesda panel). All MMR proficient or MSI-low patients, 
will be assessed for expression of MGMT by IHC on diagnostic FFPE blocks or slides. 
MGMT-negative cases by IHC will be confirmed by MGMT promoter methylation 
analysis on tissue. Percent of methylation will be normalized on tumor content 

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evaluated by hematoxylin and eosin staining to correct for stromal infiltration present 
in the original slide used for DNA extraction.  
• Mutational Load determination: the mutational load (ML) will be determined in 
tissue (TMZ-ML mandatory) and plasma (translational) by NGS on Illumina platform 
with a minimum depth of 10X and using a proprietary bioinformatic pipeline (IDEA) 
to estimate the mutational burden normalized per mega base (MB). Only alterations 
supported by 5% significance level are considered. 
ML will be assessed in MGMT-methylated, MMR-Proficient patients enrolled in the 
TMZ PRIMING Phase in samples collected at different time points, as follows: 
– Baseline Mutational Load (B-ML):  Pre TMZ treatment, in tissue (non-
mandatory) and plasma (translational) samples collected before TMZ 
administration. 
– Priming Mutational Load (TMZ-ML): at PD post TMZ treatment to declare 
patients eligible or not for the pembrolizumab TRIAL Phase, in tissue 
(mandatory) and plasma (translational) samples . NOTE: Patients with a TMZ-
ML > 20/mB will be eligible for P- cohort of PEMBRO Trial Phase  
– Dynamic Mutational Load (D-ML): pre pembrolizumab first dose in either 
tissue (only D-cohort, whenever possible) or plasma (both cohorts, 
translational). 
– Progression Mutational Load (PD-ML): at PD during/after pembrolizumab 
treatment, in tissue (whenever possible) and plasma (translational) samples. 
Timepoints for tissue biopsy and blood sampling are reported in trial diagram (Figure 4.2) and  
detailed in section 8.0 – Trial Flow Chart.  
9.1.4.2 Companion translational assays. 
The following samples, in addition to the mandatory tissue sample (see 9.1.4.1), are collected, 
stored and used to perform complementary translational assays: 
• Diagnostic FFPE blocks which are collected for all patients and stored until the end 
of the trial. 
• Peripheral blood samples will be also collected along trial treatments following the 
schedule reported in section 8.0: in particular every 2 cycles for TMZ and before 
every treatment for pembrolizumab, from subjects enrolled in both D- and P- 
cohorts. 
• Vital tumor tissue from tumor biopsy which are collected, at baseline (B-ML) and 
at tumor progression after TMZ treatment (TMZ-ML), before (only Cohort-D) and 
at tumor progression (both Cohorts) after pembrolizumab treatment when possible 
(D-ML and PD-ML). 
Complementary translational assays may include analysis on:   

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Serum. To identify potential new serum markers with prognostic and/or predictive value for 
clinical outcomes (response, progression-free survival and overall survival, toxicity), soluble 
factors, such as cytokines, chemokines, soluble receptors, and antibodies to tumor antigens 
will be characterized and quantified by immunoassays in serum. Analyses may include, but 
not necessarily be limited to: soluble PD-1, Interleukines (1β, 2 , 4, 6, 10, 12α), Tumor 
Necrosis Factor (TNF), TGFbeta,  Interferon gamma (IFNγ), monocyte chemotactic 
protein (MCP-1), and others; 
Peripheral Blood Mononuclear Cells (PBMC).  To characterize circulating immune cells 
specific lymphocyte subsets will be isolated from PBMC and expression levels of T cell co-
stimulatory markers on PBMC will be quantified by flow cytometry. Analyses may also 
include, but not necessarily be limited to, the quantification of T (CD3; CD4; CD8 and the 
proportion of memory and effector T cell subset), B, NK (CD3-; CD56+) and TREG 
(CD3+/CD4+; CD25+; FOXP3+), MDSC (CD33+;CD11b, CD14-; HLA-DR low) cell-
subsets, and the evaluation of expression levels of PD-1, PD-L1, and others immune-
regulatory molecules on the same subsets of cells. To identify potential neo-antigens, T cell 
receptor (TCR) re-arrangements will be analyzed  by NGS analysis of PBMC or subsets of 
Lymphocytes isolated by FACS. Amplification and sequencing of CDR3 regions in rearranged 
TCR β-chains will be analyzed using the immunoSEQ Assay (Adaptive Biotechnologies). The 
assay combines multiplex PCR with high-throughput sequencing and a sophisticated 
bioinformatics pipeline for TCRβ CDR3 region analysis.  
To identify potential predictive signature(s) of response to pembrolizumab gene expression 
profiling will be determined from PBMC or circulating immune cell-subsets isolated by  FACS  
by RNASeq and/or Affymetrix gene array technology and/or quantitative real-time polymerase 
chain reaction (qPCR).  
Tumor Samples. Biopsy samples may be used for the following analysis:  
Characterization of tumor infiltrating lymphocytes (TILs) and tumor antigens. 
Immunohistochemistry (IHC) will be used to assess the number and composition of immune 
infiltrates in order to define the immune cell subsets present within formalin-fixed, paraffin 
embedded (FFPE) tumor tissue. These IHC analyses will include, but not necessarily be limited 
to, the following markers: CD4, CD8, CD45RO, FOXp3, PD-1, PD-L1, PD-L2, myeloid cell 
markers (CD33, CD68, CD11b) and others.  
Characterization of T cell repertoire. TCR sequencing (immunoseq) will be performed on 
tumor biopsies in order to characterize T cell clonality, either on  FFPE or RNA later-preserved 
samples or on subsets of immune cells isolated from fresh tumor tissue. 
Gene expression profiling. Tumor biopsies that are collected in RNAlater or equivalent fixative 
or FACS-sorted cell population isolated from fresh tumor biopsies will also be examined for 
mRNA gene expression by RNASeq and/or Affymetrix gene array technology and/or 
quantitative real-time polymerase chain reaction (qPCR) to detect expression of genes with 
possible prognostic or predictive value.  

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Immune cells isolated form blood (fresh isolated or vitally DMSO-cryopreserved PBMC or 
immune cell subsets) and biopsies (tumor infiltrating immune cells)  might undergo functional 
assays to assess specificneaontigen recognition and to investigate molecular markers of 
response.   
Biopsies at confirmed progression are strongly encouraged for the purposes of understanding 
mechanisms of resistance to therapy. 
Other translational studies.  
Microbiome analysis. The composition of gut microbiome had been correlated with 
pathogenesis and progression of colorectal cancer through several mechanisms, including 
induction of chronic inflammation. In addition, the composition of microbiome had been 
suggested to influence the innate and adaptive immune system composition and function 
possibly influencing the activity of pembrolizumab in colorectal cancer.  
Thus DNA and RNA may be extracted from fecal samples or buccal swabs taken prior to 
therapy and on-treatment, and microbiota might be isolated from the same samples for 
functional analysis, with the purpose of characterizing the gut microbiome and to correlate 
longitudinal changes in its composition with response to therapy. Patients will be provided 
with the kit and the instructions for the collection of stools, and they will be asked to fill a diary 
reporting diet information on the 3 days before collection.  
The relative priority of each individual assays might change over time depending on the 
availability of material, and according to the results of the currently prioritized marker 
analyses. 
9.1.5 Laboratory Procedures/Assessments 
Details regarding specific laboratory procedures/assessments to be performed in this trial are 
provided below.  
Laboratory tests for hematology, chemistry, urinalysis, and others are specified in Table 9.1.5   

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Table 9.1.5 Laboratory Tests  
Hematology 
Chemistry 
Urinalysis 
Other 
Hematocrit 
Albumin 
Blood 
Serum β-human chorionic gonadotropin† 
Hemoglobin 
Alkaline phosphatase 
Glucose 
  (β-hCG)† 
Platelet count 
Alanine aminotransferase (ALT) 
Protein 
PT (INR) 
WBC (total and differential) 
Aspartate aminotransferase (AST) 
Specific gravity  
aPTT 
Red Blood Cell Count 
Lactate dehydrogenase (LDH) 
Microscopic exam (If abnormal)  
Total thriiodothyronine (T3) 
Absolute Neutrophil Count 
Carbon Dioxide ‡ 
results are noted 
Free tyroxine (T4) 
Absolute Lymphocyte Count 
  (CO2 or biocarbonate) 
Serum pregnancy test † 
Thyroid stimulating hormone (TSH) 
 
Uric Acid 
 
 
 
Calcium 
 
 
 
Chloride 
 
Blood for correlative studies 
 
Glucose 
 
Stools 
 
Phosphorus 
 
Buccal Swab 
 
Potassium   
 
  
 
Sodium 
 
  
 
Magnesium 
 
  
 
Total Bilirubin 
 
  
 
Direct Bilirubin (If total bilirubin is 
elevated above the upper limit of 
normal) 
 
 
 
Total protein 
 
 
 
Blood Urea Nitrogen 
 
 
 
Creatinine/Creatinine Clearance 
 
 
† Perform on women of childbearing potential only.   
‡ If considered standard of care in your region. 
 
 
 

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Laboratory tests for screening or entry into the Second Course Phase should be performed within 
10 days prior to the first dose of treatment.  After Cycle 1, pre-dose laboratory procedures can 
be conducted up to 72 hours prior to dosing.  Results must be reviewed by the investigator or 
qualified designee and found to be acceptable prior to each dose of trial treatment.  
9.1.6 Other Procedures 
9.1.6.1 Withdrawal/Discontinuation 
When a participant discontinues/withdraws prior to trial completion, all applicable activities 
scheduled for the final trial visit should be performed at the time of discontinuation.  Any adverse 
events which are present at the time of discontinuation/withdrawal should be followed in 
accordance with the safety requirements outlined in section 9.2 - Assessing and Recording 
Adverse Events.  Participants who a) attain a CR or b) complete 24 months of treatment with 
pembrolizumab may discontinue treatment with the option of restarting treatment if they meet 
the criteria specified in section 7.3.5.  After discontinuing treatment following assessment of 
CR, these participants should return to the site for a Safety Follow-up Visit (described in section 
9.1.7.4.1) and then proceed to the Follow-Up Period of the study (described in section 9.1.7.4.2). 
9.1.6.2 Blinding/Unblinding 
This is an open-label trial; therefore, the Sponsor, investigator and subject will know the 
treatment administered. 
9.1.7 Visit Requirements 
Visit requirements are outlined in section 8.0 - Trial Flow Chart.   Specific procedure-related 
details are provided above in section 9.1 - Trial Procedures. 
9.1.7.1 Screening Phase period 
CT scans performed up to 28 days prior to screening eligibility check are considered valid.  From 
-28 to -1 days before the TMZ priming baseline visit as reported in section 8.0 - Trial Flow 
Chart. 
All patients at the baseline of the Screening Phase will be tested for MMR status on the 
diagnostic FFPE block or slides according to the IHC MSI test (Bethesda panel). 
All MMR proficient patients, will be assessed for immunohistochemical expression of MGMT 
as previously described (9.1.4.1). IHC negative cases will be assessed for MGMT promoter 
methylation status on tissue as described previously (9.1.4.1). Percent of methylation will be 
normalized on tumor content evaluated by hematoxylin and eosin staining to correct for stromal 
infiltration present in the original slide used for DNA extraction. Tumors with values below 63% 
will be considered MB-low, while those with values above or equal to 63% will considered MB-
high. 

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9.1.7.2 PRIMING  Phase Period 
Each patient will be treated until PD unless patient refusal, consent withdrawal, or the 
occurrence of unacceptable toxicity.  
The treatment period (cycle) comprises 28 days, consisting of 5 days of daily administration, 
followed by a 23-day resting period, from Day 6 to Day 28. During each treatment cycle, 
extension of treatment period to Days > 5 is not allowed, even in case of missed doses. A diary 
of TMZ assumption must be given to the patient every treatment cycle and the correct 
compilation must be revised by the investigator. 
9.1.7.3 TRIAL Phase Period 
Patients eligible for the TRIAL phase will be treated with Pembrolizumab. The drug will be 
administered every 3 weeks unless earlier progression, patient refusal, consent withdrawal, or 
the occurrence of unacceptable toxicity (see section 7.3). 
The treatment period (cycle) comprises 21 days, consisting of 1 day of daily administration, 
followed by a 20-day resting period, from Day 2 to Day 21.  
9.1.7.4 Post-Treatment TRIAL Phase Visits 
Patients who discontinue Pembrolizumab treatment should be scheduled for a safety follow-up 
visit within 30 days after the last dose of study or after the decision to discontinue study 
treatment. 
9.1.7.4.1 Safety Follow-Up TRIAL Phase Visit 
The mandatory Safety Follow-Up Visit should be conducted approximately 30 days after the 
last dose of study treatment or before the initiation of a new anti-cancer treatment, whichever 
comes first.  All AEs that occur prior to the Safety Follow-Up Visit should be recorded.  
Participants with an AE of Grade > 1 will be followed until the resolution of the AE to Grade 0-
1 or until the beginning of a new anti-cancer therapy, whichever occurs first.  SAEs that occur 
within 90 days of the end of treatment or before initiation of a new anti-cancer treatment should 
also be followed and recorded.  Participants who are eligible for retreatment with 
pembrolizumab (as described in 7.3.5) may have up to two safety follow-up visits, one after the 
Initial Treatment Period and one after the Second Course Treatment. 
9.1.7.4.2 Follow-up Visits 
Participants who discontinue study treatment for a reason other than disease progression will 
move into the Follow-Up Phase and should be assessed every 8 weeks (56 ± 7 days) by 
radiologic imaging to monitor disease status.  After 1 year, the imaging time point will occur 
every 12 weeks (± 7 days). Every effort should be made to collect information regarding disease 
status until the start of new anti-cancer therapy, disease progression, death, end of the study or 
if the participant begins retreatment with pembrolizumab as detailed in section 7.3.5.  

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Information regarding post-study anti-cancer treatment will be collected if new treatment is 
initiated. 
Participants who are eligible to receive retreatment with pembrolizumab according to the criteria 
in section 7.3.5 will move from the follow-up phase to the Second Course Phase when they 
experience disease progression.   
9.1.7.4.3 Survival Follow-up 
Participants who experience confirmed disease progression or start a new anticancer therapy, 
will move into the Survival Follow-Up Phase and should be contacted by telephone every 12 
weeks to assess for survival status until death, withdrawal of consent, or the end of the trial, 
whichever occurs first.  
9.2 
Safety Assessment 
 
All patients will be evaluable for safety and toxicity from the time of their first treatment with 
temozolomide and pembrolizumab. Toxicity will be assessed using NCI Clinical Trials Criteria 
for Adverse Events (CTCAE) Version 4.03 (see appendix 2). All adverse events (AEs) occurring 
after the last administration of study treatment will be recorded on the case report forms (CRF) 
as described below (section 9.2.6). The investigator will decide if those events are drug related 
(not related, not likely, possibly, probably, certainly) and this decision will be recorded on the 
forms for all AEs. Serious adverse events (SAEs), as defined by the GCP Guideline, must be 
immediately reported according to the procedure detailed below (section 9.2.1). 
 
 
9.2.1 Definitions for Adverse Event and Serious Adverse Event 
An Adverse Event (AE) is defined as any untoward medical occurrence in a patient or clinical 
investigation participant administered a medicinal product and which does not necessarily have 
to have a causal relationship with this treatment. An AE can therefore be any unfavourable and 
unintended sign (including an abnormal laboratory finding, for example), symptom, or disease 
temporally associated with the use of a medicinal product or protocol-specified procedure, 
whether or not considered related to the medicinal product or protocol-specified procedure.  Any 
worsening (i.e., any clinically significant adverse change in frequency and/or intensity) of a pre-
existing condition that is temporally associated with the use of the investigational products, is 
also an AE. 
AEs may occur during the course of the use of investigational product in clinical trials, or as 
prescribed in clinical practice, from overdose (whether accidental or intentional), from abuse 
and from withdrawal. 
 
A Serious Adverse Event (SAE) is any adverse event that: 
• Results in death; 
• Is life threatening; 
• Results in persistent or significant disability/incapacity; 

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• Results in or prolongs an existing inpatient hospitalization; 
• Is a congenital anomaly/birth defect; 
• Is another important medical event  
• Note: In addition to the above criteria, adverse events meeting either of the below 
criteria, although not serious per ICH definition, are reportable to the delegated 
pharmacovigilance service and to  Merck Global Safety (if concerning pembrolizumab, 
Attn: Worldwide Product Safety; FAX 215-661-6229) in the same timeframe as SAEs 
to meet certain local requirements. Therefore, these events are considered serious by 
Merck for collection purposes. 
• Is a new cancer (that is not a condition of the study); 
• Is associated with an overdose. 
 
Refer to Table 9.2 for additional details regarding each of the above criteria. 
 
 
9.2.2 Reporting of Adverse Events 
AEs will be collected from the time the first dose of study medication (either pembrolizumab or 
temozolomide) is administered until 30 days following discontinuation of study treatment. SAEs 
will be collected over the same time period as stated above for AEs of temozolomide, or up to 
90 days for pembrolizumab. In addition, any SAE assessed as related to study participation, 
study medication or concomitant medication must be recorded from the time a subject consents 
to participate in the study up to and including any follow-up contact. After discontinuation of 
study medication, the investigator will monitor all AEs/SAEs that are ongoing until resolution 
or stabilization of the event or until the subject is lost to follow-up. 
 
Reporting criteria for AEs/SAEs: 
 
- All AEs, SAEs and other reportable safety events that occur after the consent form is 
signed but before planned treatment initiation must be reported by the investigator if the 
event cause the participant to be excluded from the study.  
 
- All AEs must be reported by the investigator from the time of treatment allocation through 
30 days following cessation of study treatment.  
 
- SAEs regarding pembrolizumab must be reported by the investigator from the time of 
treatment allocation through 90 days (pembrolizumab) or 30 days (temozolomide) 
following cessation of study treatment, or 30 days following cessation of study treatment 
if the participant initiates new anticancer therapy, whichever is earlier.  
 
- All pregnancies must be reported by the investigator from the time of treatment allocation 
through 120 days (pembrolizumab) or 90 days (temozolomide) following cessation of 
study treatment, or 30 days following cessation of study treatment if the participant 
initiates new anticancer therapy, whichever is earlier.  
 

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- Any SAE brought to the attention of an investigator at any time outside of the time period 
specified above must be reported immediately by the investigator if the event is considered 
to be study drug-related. 
 
- A death on study requires reporting regardless of causality and attribution to treatment or 
other cause must be provided.  Death due to disease progression is not to be reported as 
an AE. Deaths that occur beyond 30 days after the end of study drug 
administration/initiation of an alternate therapy, do not qualify as SAEs. 
 
Note: Adverse events will not be collected for subjects during the SCREENING Phase period 
(for determination of archival tissue status) as long as that subject has not undergone any 
protocol-specified procedure or intervention.  Since we will use FFPE blocks and routine blood 
draws to determine both MMR and MGMT status the subject will first required to provide 
consent to the main study but AEs will NOT be captured. 
 
 
9.2.3 Reporting of Serious Adverse Events  
Each adverse event is to be classified by the investigator as SERIOUS or NON-SERIOUS. This 
classification of the seriousness of the event determines the reporting procedures to be followed.  
 
In order to comply with SAEs reporting responsibilities, the Sponsor of the study will appoint a 
delegated CRO as "Third Party Pharmacovigilance Provider". If a SAE occurs, the 
Pharmacovigilance has to be notified by fax or by e-mail, using the designated form, within 24 
hours of awareness of the event by the Investigator.  
 
Moreover, in case of any SAE, or follow up to a SAE, including death due to any cause whether 
or not related to the Merck product (pembrolizumab), it must be reported also to Merck Global 
Safety within 2 working days. Additionally, any SAE considered by the investigator to be related 
to pembrolizumab, which is brought to the attention of the investigator either at any time 
between informed consent to the end of the specified safety follow-up period, or at any time 
outside of the follow-up time period, must also be reported immediately to Merck Global Safety 
(Attn: Worldwide Product Safety; FAX 215-661-6229). 
Investigators are not obligated to actively seek AE or SAE or other reportable safety events in 
former study participants. However, if the investigator learns of any SAE, including a death, at 
any time after a participant has been discharged from the study, and he/she considers the event 
to be reasonably related to the study treatment or study participation, the investigator must 
promptly notify Merck Global Safety (Attn: Worldwide Product Safety; FAX 215-661-6229). 
 
The initial report should be followed by submission of more detailed adverse event information 
within 5 calendar days after the Investigator first became aware of the serious adverse event. 
Reporting requirements for adverse events are summarized in the table below.  
 
Gravity 
Reporting Time 
Type of Report 

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SERIOUS 
Within 24 hours from awareness by 
the investigator 
Initial report on SAE report form + 
case report form  
Within 5 calendar days from initial 
report 
Follow-up/Final report on SAE 
report form 
NON 
SERIOUS 
Per case report form submission 
procedure 
Case report form 
 
If for any reason the SAE form transmission is not possible, Pharmacovigilance should be 
informed by phone of the occurrence of the event. In this exceptional case, Pharmacovigilance 
will complete a SAE form with information received, which will be sent to the investigator for 
confirmation, and in the meanwhile Pharmacovigilance procedures will be initiated. 
 
SAEs should also be reported on the AE case report form (CRF). The form to be used for SAE 
expedited reporting is not the same as the AE CRF, but where the same data are collected, the 
forms must be completed in a consistent manner. For example, the same AE term should be used 
on both forms. 
 
Pharmacovigilance assesses each SAE reported by the study Investigators to identify any 
suspected unexpected serious adverse reactions (SUSAR), i.e. serious adverse events considered 
at least possible associated to the study treatment by the Investigator and not listed in the IMP 
reference document(s) and provide this information to the sponsor for the final evaluation. If a 
SAE is assessed as a possible SUSAR, the sponsor, through delegated Pharmacovigilance, may 
urgently require further information to the Investigator. Pharmacovigilance will issue a SUSAR 
notification whenever appropriate, and submit it to all concerned recipients according to current 
law and following sponsor’s indication. 
 
Follow-up information is to be reported on a new SAE form and transmitted to the same fax 
number as the initial report. A follow-up report is to be filled in, not only to complete the 
information provided on the initial report but also to modify any incorrect data. The SAE fax 
delivery confirmation sheets must be retained at the study sites. 
At the end of the study all original SAE report forms are to be collected in the Investigator File, 
while the corresponding copies must be retained by the CRO personnel and archived in the TMF. 
 
For the expectedness assessment of each case with pembrolizumab, the Sponsor will use the 
Investigator Brochure as the Reference Safety Information. For the expectedness assessment of 
each case with temozolomide, the Sponsor will use the SmPC (Undesirable Effects, section 4.8) 
and the safety data of the studies published in colorectal cancer (Amatu et al., 2013, 2016). 
 
 
9.2.4 Definition and Reporting of an Overdose for This Protocol 
Temozolomide 
Doses of 500, 750, 1,000, and 1,250 mg/m2 (total dose per cycle over 5 days) have been 
evaluated clinically in patients. Dose-limiting toxicity was haematological and was reported 
with any dose but is expected to be more severe at higher doses. An overdose of 10,000 mg 
(total dose in a single cycle, over 5 days) was taken by one patient and the adverse reactions 

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reported were pancytopenia, pyrexia, multiorgan failure and death. There are reports of patients 
who have taken the recommended dose for more than 5 days of treatment (up to 64 days) with 
adverse events reported including bone marrow suppression, with or without infection, in some 
cases severe and prolonged and resulting in death. In the event of an overdose, haematological 
evaluation is needed. Supportive measures should be provided as necessary.  
 
Pembrolizumab 
For purposes of this study, an overdose of pembrolizumab will be defined as any dose of 1,000 
mg or greater (≥5 times the indicated dose).  No specific information is available on the treatment 
of overdose of pembrolizumab. In the event of overdose, the participant should be observed 
closely for signs of toxicity.  Appropriate supportive treatment should be provided if clinically 
indicated. 
If an adverse event(s) is associated with (“results from”) the overdose of a Merck product, the 
adverse event(s) is reported as a serious adverse event, even if no other seriousness criteria are 
met. 
If a dose of Merck’s product meeting the protocol definition of overdose is taken without any 
associated clinical symptoms or abnormal laboratory results, the overdose is reported as a non-
serious Event of Clinical Interest (ECI), using the terminology “accidental or intentional 
overdose without adverse effect.” 
All reports of overdose with and without an adverse event must be reported within 24 hours to 
Pharmacovigilance and within 2 working days hours to Merck Global Safety (Attn: Worldwide 
Product Safety; FAX 215-661-6229). 
 
 
9.2.5 Events of Clinical Interest (pembrolizumab) 
Selected non-serious and serious adverse events are also known as Events of Clinical Interest 
(ECI) and must be reported within 2 working days to Merck Global Safety. (Attn: Worldwide 
Product Safety; FAX 215-661-6229). 
For the time period beginning when the consent form is signed until treatment 
allocation/randomization, any ECI, or follow up to an ECI, that occurs to any participant must 
be reported within 2 working days to Merck Global Safety if it causes the participant to be 
excluded from the trial, or is the result of a protocol-specified intervention, including but not 
limited to washout or discontinuation of usual therapy, diet, placebo treatment or a procedure. 
For the time period beginning at treatment allocation through 90 days following cessation of 
treatment, or 30 days following cessation of treatment if the participant initiates new anticancer 
therapy, whichever is earlier, any ECI, or follow up to an ECI, whether or not related to Merck 
product, must be reported within 2 working days to Merck Global Safety. 
Events of clinical interest for this trial include: 
1.  an overdose of Merck product, as defined in section 9.2.4 - Definition of an Overdose for 
This Protocol and Reporting of Overdose to the Sponsor, that is not associated with clinical 
symptoms or abnormal laboratory results.  
2.  an elevated AST or ALT lab value that is greater than or equal to 3X the upper limit of normal 
and an elevated total bilirubin lab value that is greater than or equal to 2X the upper limit of 
normal and, at the same time, an alkaline phosphatase lab value that is less than 2X the upper 
limit of normal, as determined by way of protocol-specified laboratory testing or unscheduled 
laboratory testing.* 

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*Note:  These criteria are based upon available regulatory guidance documents. The purpose 
of the criteria is to specify a threshold of abnormal hepatic tests that may require an 
additional evaluation for an underlying etiology.  
 
 
9.2.6 Recording AEs/SAEs in the Case Report Form 
AEs can be assessed directly by the Investigator during a clinical visit or based on 
laboratory/Instrumental examinations or can be referred by the patient. 
 
Pre-existing Conditions 
A pre-existing condition (i.e., a disorder starting before the adverse event reporting period) 
should not be reported as an adverse event unless the condition worsens during the adverse event 
reporting period. 
 
Procedures 
Diagnostic and therapeutic procedures, such as surgery, should not be reported as adverse 
events, while the medical condition for which the procedure was performed should be reported 
if it meets the definition of an adverse event. For example, an appendectomy performed for an 
acute appendicitis occurring during the adverse event reporting period should not be reported 
as adverse event; while “acute appendicitis” is to be reported as adverse event. If a patient 
undergoes a surgical procedure that was planned prior to entry into the trial, and surgery is not 
performed due to a worsening of a baseline condition, this baseline condition should not be 
reported as an adverse event. 
 
Symptoms of Targeted Disease 
Tumor-related signs and symptoms will be followed at each visit. Although a measure of 
efficacy, these will always be reported as pre-existing conditions at baseline and during 
treatment only if they meet the definition of adverse event. 
For all adverse events the Investigator will be asked to assess its relationship with the study 
treatment. 
 
Causality Assessment and Grading of Adverse Event Severity 
The assessment of relationship to study drug will be done according to the following causality 
scale based on the WHO definitions: 
• Certain: A clinical event, including laboratory test abnormality, occurring in a plausible 
time relationship to drug administration, and which cannot be explained by concurrent 
disease or other drugs or chemicals. The response to withdrawal of the drug (de-
challenge) should be clinically plausible. The event must be definitive 
pharmacologically or phenomenologically, using a satisfactory re-challenge procedure 
if necessary 
• Probable: A clinical event, including laboratory test abnormality, with a reasonable time 
sequence to administration of the drug, unlikely to be attributed to concurrent disease or 
other drugs or chemicals, and which follows a clinically reasonable response on 
withdrawal (rechallenge). Rechallenge information is not required to fulfil this 
definition. 

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• Possible: A clinical event, laboratory test abnormality, with a reasonable time sequence 
to administration of the drug, but which could also be explained by concurrent disease 
or other drugs or chemicals. Information on drug withdrawal may be lacking or unclear 
• Unlikely: A clinical event, laboratory test abnormality, with a temporal relationship to 
drug administration which makes a causal relationship improbable, and in which other 
drugs, chemicals or underlying disease provide plausible explanations Severity grading 
of adverse events and pre-existing conditions will be done according to the National 
Cancer Institute (NCI) Common Toxicity Criteria (CTCAE) V. 4.03. 
AEs that are not defined in the NCI CTCAE should be evaluated for severity according to the 
following scale: 
• Grade 1 = Mild – transient or mild discomfort; no limitation in activity; no medical 
intervention/therapy required; 
• Grade 2 = Moderate – mild to moderate limitation in activity, some assistance may be 
needed; no or minimal medical intervention/therapy required; 
• Grade 3 = Severe – marked limitation in activity, some assistance usually required; 
medical intervention/therapy required, hospitalization is possible; 
• Grade 4 = Life threatening – extreme limitation in activity, significant assistance 
required; significant medical intervention/therapy required, hospitalization or hospice 
care probable; 
• Grade 5 = Death - the event results in death. 
Note the distinction between the gravity and the severity of an adverse event. Severe is a measure 
of intensity; thus, a severe reaction is not necessarily a serious reaction. For example, a headache 
may be severe in intensity but would not be classified as serious unless it meets one of the criteria 
for serious events listed above. 
 
Pregnancy Reporting 
Although pregnancy and infant exposure during breast feeding are not considered adverse 
events, it is the responsibility of investigators or their designees to report any pregnancy or 
lactation in a participant (spontaneously reported to them) that occurs during the study using the 
“exposure in utero” form. 
Pregnancies and infant exposures during breastfeeding that occur after the consent form is signed 
but before treatment allocation/randomization must be reported by the investigator if they cause 
the participant to be excluded from the trial, or are the result of a protocol-specified intervention, 
including but not limited to washout or discontinuation of usual therapy, diet, placebo treatment 
or a procedure.   
Pregnancies and infant exposures during breastfeeding that occur from the time of treatment 
allocation/randomization through 120 days (pembrolizumab) or 90 days (temozolomide) 
following cessation of study product, or 30 days following cessation of treatment if the 
participant initiates new anticancer therapy, whichever is earlier, must be reported by the 
investigator to delegated Pharmacovigilance within 2 weeks of learning of its occurrence..  All 
reported pregnancies must be followed to the completion/termination of the pregnancy. 
Pregnancy outcomes of spontaneous abortion, missed abortion, benign hydatidiform mole, 
blighted ovum, fetal death, intrauterine death, miscarriage and stillbirth must be reported as 
serious events (Important Medical Events). If the pregnancy continues to term, the outcome 
(health of infant) must also be reported. 

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Such events must be reported within 24 hours to delegated Pharmacovigilance and within 2 
working days hours to Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215-661-
6229). 
 
Overdose 
Reporting if any overdose (accidental or intentional) which results in serious adverse reactions 
is to be handled following the SAE procedures. This includes reports related to drug intake 
with suicidal intentions and consequent drug overdose. 
Overdose reporting even not associated with adverse reactions shall be anyhow reported 
immediately to delegated Pharmacovigilance using the most rapid type of communication 
(phone, e-mail). 
 
Follow-up of Unresolved Adverse Events 
All adverse events should be followed at least until 30 days following the last dose of IMP. 
Drug-related and serious adverse events ongoing at the end of this observation period must be 
recorded until they are resolved or the investigator assesses them as chronic or the subject is lost 
to follow-up or starts a new anti-cancer treatment, whichever occurs earlier. 

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Table 9.2 Evaluating Adverse Events 
An investigator who is a qualified physician, will evaluate all adverse events as to: 
V4.03 
CTCAE 
Grading 
Grade 1 
Mild; asymptomatic or mid symptoms; clinical or diagnostic observations only; intervention not indicated. 
 
Grade 2 
Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate instrumental ADL. 
 
Grade 3 
Severe or medically significant but not immediately life-threatening; hospitalization or prolongation or hospitalization indicated; disabling; limiting 
self-care ADL. 
 
Grade 4 
Life threatening consequences; urgent intervention indicated. 
 
Grade 5 
Death related to AE 
Seriousness 
A serious adverse event is any adverse event occurring at any dose or during any use of Merck product that: 
 
†Results in death; or 
 
†Is life threatening; or places the participant, in the view of the investigator, at immediate risk of death from the event as it occurred (Note: This does not include an adverse 
event that, had it occurred in a more severe form, might have caused death.); or 
 
†Results in a persistent or significant disability/incapacity (substantial disruption of one’s ability to conduct normal life functions); or 
 
†Results in or prolongs an existing inpatient hospitalization (hospitalization is defined as an inpatient admission, regardless of length of stay, even if the hospitalization is a 
precautionary measure for continued observation. (Note: Hospitalization for an elective procedure to treat a pre-existing condition that has not worsened is not a serious adverse 
event.  A pre-existing condition is a clinical condition that is diagnosed prior to the use of a Merck product and is documented in the patient’s medical history.); or 
 
†Is a congenital anomaly/birth defect (in offspring of participant taking the product regardless of time to diagnosis);or 
 
Is a new cancer (that is not a condition of the study) (although not serious per ICH definition, is reportable to the Sponsor within 24 hours and to Merck within 2 working days 
to meet certain local requirements); or 
 
Is an overdose (whether accidental or intentional).  Any adverse event associated with an overdose is considered a serious adverse event for collection purposes. An overdose 
that is not associated with an adverse event is considered a non-serious event of clinical interest and must be reported within 24 hours to the Sponsor and to Merck within 2 
working days.. 
 
Other important medical events that may not result in death, not be life threatening, or not require hospitalization may be considered a serious adverse event when, based upon 
appropriate medical judgment, the event may jeopardize the participant and may require medical or surgical intervention to prevent one of the outcomes listed previously 
(designated above by a †). 
Duration 
Record the start and stop dates of the adverse event. If less than 1 day, indicate the appropriate length of time and units 
Action taken  
Did the adverse event cause Merck product to be discontinued? 
Relationship to 
Merck Product  
Did Merck product cause the adverse event? The determination of the likelihood that Merck  product caused the adverse event will be provided by an investigator who is a 
qualified physician. The investigator’s signed/dated initials on the source document or worksheet that supports the causality noted on the AE form, ensures that a medically 
qualified assessment of causality was done. This initialed document must be retained for the required regulatory time frame. The criteria below are intended as reference 
guidelines to assist the investigator in assessing the likelihood of a relationship between the test drug and the adverse event based upon the available information. 
The following components are to be used to assess the relationship between Merck product and the AE; the greater the correlation with the components and their respective 
elements (in number and/or intensity), the more likely Merck product caused the adverse event (AE): 
 
Exposure 
Is there evidence that the participant was actually exposed to Merck product such as: reliable history, acceptable compliance assessment (pill count, diary, 
etc.), expected pharmacologic effect, or measurement of drug/metabolite in bodily specimen? 
 
Time Course 
Did 
the 
AE 
follow 
in 
a 
reasonable 
temporal 
sequence 
from 
administration 
of 
Merck 
product?  
Is the time of onset of the AE compatible with a drug-induced effect (applies to trials with investigational medicinal product)? 
 
Likely Cause 
Is the AE not reasonably explained by another etiology such as underlying disease, other drug(s)/vaccine(s), or other host or environmental factors 

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Relationship 
The following components are to be used to assess the relationship between the test drug and the AE: (continued) 
to 
Merck 
Product 
(continued)  
Dechallenge 
Was Merck product discontinued or dose/exposure/frequency reduced? 
If yes, did the AE resolve or improve? 
If yes, this is a positive dechallenge. If no, this is a negative dechallenge. 
(Note: This criterion is not applicable if: (1) the AE resulted in death or permanent disability; (2) the AE resolved/improved despite continuation of 
the Sponsor’s product; or (3) the trial is a single-dose drug trial); or (4) Sponsor’s product(s) is/are only used one time.) 
 
Rechallenge 
Was the participant re-exposed to Merck product in this study? 
If yes, did the AE recur or worsen? 
If yes, this is a positive rechallenge. If no, this is a negative rechallenge. 
(Note: This criterion is not applicable if: (1) the initial AE resulted in death or permanent disability, or (2) the trial is a single-dose drug trial); or (3) 
Sponsor’s product(s) is/are used only one time). 
NOTE: IF A RECHALLENGE IS PLANNED FOR AN ADVERSE EVENT WHICH WAS SERIOUS AND WHICH MAY HAVE BEEN 
CAUSED BY MERCK PRODUCT, OR IF REEXPOSURE TO MERCK PRODUCT POSES ADDITIONAL POTENTIAL SIGNIFICANT RISK 
TO THE PARTICIPANT, THEN THE RECHALLENGE MUST BE APPROVED IN ADVANCE BY THE SPONSOR AS PER DOSE 
MODIFICATION GUIDELINES IN THE PROTOCOL. 
 
Consistency 
with 
Trial 
Treatment 
Profile 
Is the clinical/pathological presentation of the AE consistent with previous knowledge regarding Merck product or drug class pharmacology or 
toxicology? 
The assessment of relationship will be reported on the case report forms /worksheets by an investigator who is a qualified physician according to his/her best clinical judgment, including 
consideration of the above elements. 
Record one of the following 
Use the following scale of criteria as guidance (not all criteria must be present to be indicative of Merck product relationship). 
Yes, 
there 
is 
a 
reasonable 
possibility of Merck product 
relationship. 
There is evidence of exposure to Merck product.  The temporal sequence of the AE onset relative to the administration of Merck product is reasonable.  
The AE is more likely explained by Merck product than by another cause. 
No, there is not a reasonable 
possibility of Merck product 
relationship 
Participant did not receive the Merck  product OR temporal sequence of the AE onset relative to administration of Merck product is not reasonable 
OR the AE is more likely explained by another cause than the Merck product.  (Also entered for a participant with overdose without an associated 
AE.) 

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9.2.7 Sponsor Responsibility for Reporting Adverse Events 
All Adverse Events will be reported to regulatory authorities, IRB/IECs and investigators in 
accordance with all applicable global laws and regulations.  
 
10.0 STATISTICAL CONSIDERATIONS 
10.1 General considerations 
The hypothesis tested by ARETHUSA is whether immunoediting in MMR proficient cancers 
can be disrupted by increasing the tumor mutational load with TMZ, enabling the tumor to 
dynamically express neo-antigens that positively affect T cell functions under check-point 
inhibition.  
The main analysis thus will concern the response to pembrolizumab in MMR proficient 
patients (cohort P) with measurable disease at baseline who have received at least 1 cycle of 
pembrolizumab (iEE population, see 10.3.1).   
10.2 Sample size calculation 
A two-stage Green-Dahlberg design has been used [Green SJ and Dahlberg S Statistics in 
Medicine 11:853-862,1992], to calculate the sample size for the P-cohort under the following 
assumptions:  
• The null hypothesis that the true (H0) response rate is 10% (not considered clinically 
compelling) will be tested against a one-sided alternative (H1).  
• In the first stage, 10 patients will be accrued. If no responses are observed, the study 
will be stopped. Otherwise, 10 additional patients will be accrued for a total of 20.  
• The null hypothesis will be rejected if 5 or more responses are observed in 20 patients.  
• This design yields a type I error rate of .05, and power of 90% when the true response 
rate is 40%.  
• In order to recruit 20 patients in the P-cohort the number of MMR unselected patients 
to be pre-screened for MMR status MGMT promoter methylation and the number of 
MGMT-PM+ to be primed with TMZ are reported in Table 10.1. below as well as in  
Study Diagram,  section 4.2. 
 
 
 
Table 10.1 Number of patients required in each trial Phase. 

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Number of Patients to be: 
N 
Expected Percent 
RAS mut MMR proficient cases to be screened 
670 
A 
expected MGMT IHC negative  
335 
B = 50% of A 
expected promoter methylated in tissue  
67 
C=20% of B               
and 10% of A 
expected with increase of mutational load ≥ 20 mutations/Mb 
(PRIMING Phase) 
20 
D= 30% of C 
 
For cohort D, a  precise sample size has NOT been calculated  since the ORR in deficient 
patients is a secondary objective of the trial We will enroll up to 34 MMR-D cases according 
to an estimated prevalence of 4-5 %. These patients will constitute the D-cohort. We expect a 
response rate between 40% (95% C.I .18-.71) and 33%  (95% C.I .21-.46) as reported by Le et 
al., (2015 & 2019) with 11-14 responses accordingly.  
 
10.3 Statistical Analysis Plan 
10.3.1 Populations considered for the analysis 
All patients signing the informed consent for the PRIMING and TRIAL Phases will be 
accounted for. The number of patients who will not result eligible, who died or withdrew before 
these phases, will be specified.  
A patient will be considered to be PRIMING and TRIAL Phases eligible if he/she did not have 
any deviation from the patient entry criteria listed in section 7.1.2 (PRIMING) and 7.1.3 
(TRIAL). Potential eligibility problems will be assessed by the Clinical Study Coordinator at 
time of medical review.  
The final efficacy analysis will occur after 20 patients entered in the P-Cohort are evaluable 
for response according to RECIST 1.1. The distribution of follow-up time in both cohorts (P 
and D) will be described and the number of patients lost to follow-up will be reported. 
Conclusions will be based on all eligible patients in each cohort. Further analysis may be 
performed excluding those patients for whom major protocol deviations have been identified 
(e.g., early death due to other reasons, early discontinuation of treatment, major protocol 
violations, etc.). However, these sub-analyses may not serve as the basis for drawing 
conclusions concerning treatment efficacy, and the reasons for excluding patients from the 
analysis should be clearly reported. 
Three populations will be considered for the analysis, as follows: 

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• 
The Safety Evaluable (SE) population defined as all treated patients (i.e. eligible as 
decided at the time of registration that receives at least 1 dose of any study treatment i.e. TMZ 
and Pembrolizumab). An incorrect treatment schedule or drug administration or an early 
termination of treatment does not result in exclusion of patients from this population. This 
population will be the object of the Safety and Tolerability analysis. Patients with major 
deviations from the eligibility criteria affecting safety or from the treatment schedule at cycle 
1 for reasons other than toxicity may be presented in separate tables/listings. 
• 
The Efficacy Evaluable (EE) population defined as all patient treated with 
pembrolizumab in P-cohort, with no major deviations from the eligibility criteria affecting 
efficacy evaluation, for which the tumor response could be evaluated at least once while on 
treatment. These patients should have received at least 2 cycles after treatment starts, unless 
disease progression occurs within this period. 
• 
The immunotherapy Efficacy Evaluable (iEE) population. Anti-tumor immune 
responses such as those associated with pembrolizumab may result in objective responses that 
are delayed and that can be preceded by initial apparent radiological progression. This initial 
apparent progression may occur as a result of either delayed anti-tumor activity and/or robust 
tumor immune cell infiltration with a concomitant increase in tumor size. In addition, lesions 
that might otherwise be undetectable with conventional imaging may increase in size as a result 
of these processes and be recorded as new lesions. Conventional RECIST 1.1 response criteria 
may not be thus adequate to capture and describe the anti-tumor activity  of pembrolizumab. 
Therefore, modified response criteria have been developed that account for the possible 
appearance of new lesions and allow radiological progression to be confirmed at subsequent 
assessment. For these reason a second efficacy analysis will be conducted (iEE analysis) on 
the same population used for the EE analysis using the Modified RECIST criteria. 
10.3.2 Primary analysis  
The main analysis will focus on the objective response rate (ORR) of the P Cohort.   
An objective response is defined as either a CR or PR assessed, as outlined in sections 9.1.3 
and 10.3.1 (iEE population), first by  RECIST 1.1 and then by iRECIST criteria at the 
discretion of the local Investigator.  
Best objective response of either type will be centrally confirmed using the Quibim Precision®   
software (the cloud-based repository for radioimaging). In case of disagreement between local 
and central evaluation, the central response evaluation will be used for the analysis. The 
duration of response will be calculated accordingly.  
An estimate of ORR and its 95% CIs will be calculated using the Clopper-Pearson method. 
Discrepant results, impinging in the acceptance of rejection of the null hypothesis, will be 
discussed and agreed upon between the Protocol Chair and the Sponsor.   
10.3.3 Secondary analyses  
A secondary analysis will also estimate the ORR, as defined above, in the D-Cohort and 
compare it to the ORR in the P-cohort.  CIs for the difference in ORRs between the two cohorts 

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will be determined using the normal approximation to the binomial distribution. The ORRs 
will be compared between the two cohorts with use of the stratified Cochran-Mantel-Haenszel 
test. 
PFS and OS, for both cohorts,  will be estimated using the Kaplan-Meier product-limit method. 
Estimates at weekly intervals will be tabulated with their standard error; medians will be 
provided with 95% confidence interval.  
DCR is defined as the rate of patients with complete or partial response as best response or 
stable disease maintained for ≥ 18 weeks per RECIST v1.1. The analysis methods for DCR 
will be the same as those for the analysis of ORR. 
Duration of response, defined as the time between the date of the first occurrence of a complete 
or partial response and the date of first documented progressive disease or death is documented, 
will also be estimated using Kaplan-Meier methodology above. Patients who have not 
progressed or who have not died by the data cutoff date for analysis will be censored at the 
time of last tumor assessment date. If no tumor assessments were performed after date of the 
first occurrence of a complete or partial response, DOR will be censored at the date of the first 
occurrence of a complete or partial response plus 1 day. 
Adverse events will reported as proportions.  
10.3.4 Exploratory  analysis 
Exploratory biomarker analyses will be performed in order to understand the association of 
these markers with study drug response, including efficacy and/or adverse events. 
Exploratory biomarker analyses will include assessments of biomarkers in both tumor tissue 
and blood. Changes in biomarkers will be listed by cohort and response status. The 
pharmacodynamic biomarker analyses will include patients with at least one predose and one 
postdose biomarker assessment. 
 
11.0 LABELING, PACKAGING, STORAGE AND RETURN OF CLINICAL 
SUPPLIES 
11.1 Investigational Product  
The investigator shall take responsibility for and shall take all steps to maintain appropriate 
records and ensure appropriate supply, storage, handling, distribution and usage of 
investigational product in accordance with the protocol and any applicable laws and 
regulations. 
Pembrolizumab will be provided by Merck and Temozolomide by the Sponsor through 
Niguarda Pharmacy, as summarized in Table 11. 
Table 11. Product Descriptions 

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Product Name & Potency 
Dosage Form 
Pembrolizumab 100 mg/ 4mL 
Solution for Injection 
Temozolomide 5, 20, 100, 250 mg 
Hard Capsules 
 
11.2 Packaging and Labeling Information 
Supplies will be labeled in accordance with regulatory requirements. Pembrolizumab will be 
labelled as MK-3475. 
11.3 Clinical Supplies Disclosure 
This trial is open-label; therefore, the participant, the trial site personnel, the Sponsor and/or 
designee are not blinded to treatment. Drug identity (name, strength) is included in the label 
text; random code/disclosure envelopes or lists are not provided. 
11.4 Storage and Handling Requirements 
Clinical supplies must be stored in a secure, limited-access location under the storage 
conditions specified on the label.   
Receipt and dispensing of trial medication must be recorded by an authorized person at the 
trial site. 
Clinical supplies may not be used for any purpose other than that stated in the protocol. 
11.5 Returns and Reconciliation 
The investigator is responsible for keeping accurate records of the clinical supplies received 
from Merck or designee, the amount dispensed to and returned by the participants and the 
amount remaining at the conclusion of the trial.  
Upon completion or termination of the study, all unused and/or partially used investigational 
product will be destroyed at the site per institutional policy. It is the Investigator’s 
responsibility to arrange for disposal of all empty containers, provided that procedures for 
proper disposal have been established according to applicable federal, state, local and 
institutional guidelines and procedures, and provided that appropriate records of disposal are 
kept. 
12.0 ADMINISTRATIVE AND REGULATORY DETAILS 
12.1 Confidentiality 
Sponsor assures that the key design element of this protocol will be posted in a publicly 
accessible database such as EUDRACT and clinicaltrial.gov; in addition, upon study 

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completion, the results of this study will be submitted for publication and posted in publicly 
accessible database for clinical trial studies. 
All information regarding study drug supplied by Sponsor to the investigator is privileged and 
confidential information. The investigator agrees to use this information to accomplish the 
study and will not use it for other purposes without consent from Sponsor. 
It is understood that there is an obligation to provide Sponsor with complete data obtained 
during the study. The investigator agrees to keep in confidence all the results obtained from 
the study. Such information shall not be disclosed to third parties without prior written 
permission from Sponsor, except to regulatory authority(ies), when requested. 
Individual investigators may present results of the study at scientific meetings. However prior 
to the submission, the Sponsor will have the opportunity to review and comment the abstracts 
for a period of up to 15 calendar days prior to the submission. 
12.2 Data Management 
Data Management will be carried out by a delegated CRO. Medical terms are coded according 
to the MedDRA dictionary. Data will be analyzed using SAS® System currently used at CRO. 
Data cleaning will include both visual and computer-driven procedures in order to minimize 
logical inconsistencies and errors within the collected data. The data are checked for 
completeness, accuracy and consistency. The errors detected will be rectified by means of Data 
Clarification List (DCL) that will be used by the monitor for resolution of queries.  
12.2.1 Record Retention 
To enable evaluation and/or audits and/or regulatory authorities inspections, the Investigator 
agrees to keep records, including the identity of all participating subjects (“Subject 
identification Log”), all original signed informed consent forms, copies of all case report 
forms, source documents, detailed records of treatment disposition as well as the 
documentation included in the Investigator File according to local regulations or as specified 
in the Clinical Trial Agreement. 
If the Investigator relocates, retires, or for any reason withdraws from the study, Sponsor 
should be prospectively notified. The study records must be transferred to an acceptable 
designee, such as another investigator, another institution, or to delegated CRO. The 
investigator must obtain Sponsor’s written permission before disposing of any records. 
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APPENDICES 
  Appendix 1: ECOG Performance Status 
Grade 
Description 
0 
Normal activity. Fully active, able to carry on all pre-disease 
performance without restriction. 
1 
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). 
2 
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. 
3 
In bed >50% of the time. Capable of only limited self-care, confined 
to bed or chair more than 50% of waking hours. 
4 
100% bedridden. Completely disabled. Cannot carry on any self-care. 
Totally confined to bed or chair. 
5 
Dead. 
* As published in Am. J. Clin. Oncol.:  Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., 
McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology 
Group. Am J Clin Oncol 5:649-655, 1982. The Eastern Cooperative Oncology Group, Robert Comis 
M.D., Group Chair. 
Appendix 2: Common Terminology Criteria for Adverse Events V4.03 (CTCAE) 
The descriptions and grading scales found in the revised NCI Common Terminology Criteria 
for Adverse Events (CTCAE) version 4.03 will be utilized for adverse event reporting. 
(https://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03/CTCAE_4.03_2010-06-
14_QuickReference_5x7.pdf) 
 
Appendix 3: Contraception Requirements 
Female Participants: 
Female participants of childbearing potential are eligible to participate if they agree to use one 
of the contraception methods described in Table A3 consistently and correctly during the 
protocol-defined time frame in section 8.0 and 9.0. 
Table  A3: 
Contraceptive Methods 
Acceptable Contraceptive Methods   
Failure rate of >1% per year when used consistently and correctly.  
● Male or female condom with or without spermicide 
● Cervical cap, diaphragm or sponge with spermicide 

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Highly Effective Contraceptive Methods That Are User Dependent a  
Failure rate of <1% per year when used consistently and correctly. 
● Combined (estrogen- and progestogen- containing ) hormonal contraception b 
○ Oral  
○ Intravaginal  
○ Transdermal  
○ Injectable 
● Progestogen-only hormonal contraception b 
○ Oral  
○ Injectable  
Highly 
Effective 
Methods 
That 
Have 
Low 
User 
Dependency 
Failure rate of <1% per year when used consistently and correctly. 
● Progestogen- only contraceptive implant b, c  
● Intrauterine hormone-releasing system (IUS) b  
● Intrauterine device (IUD) 
● Bilateral tubal occlusion 
 
● Vasectomized partner  
A vasectomized partner is a highly effective contraception method provided that the partner is the sole 
male sexual partner of the WOCBP and the absence of sperm has been confirmed. If not, an additional 
highly effective method of contraception should be used.  
● Sexual abstinence  
Sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual 
intercourse during the entire period of risk associated with the study treatment. The reliability of sexual 
abstinence needs to be evaluated in relation to the duration of the study and the preferred and usual 
lifestyle of the participant.)  
Notes:  
Use should be consistent with local regulations regarding the use of contraceptive methods for 
participants of clinical studies. 
 
a) Typical use failure rates are lower than perfect-use failure rates (i.e. when used consistently and 
correctly).  
b) If hormonal contraception efficacy is potentially decreased due to interaction with study treatment, 
condoms must be used in addition to the hormonal contraception during the treatment period and for at 
least 60 days after the last dose of study treatment . 
c) If locally required, in accordance with Clinical Trial Facilitation Group (CTFG) guidelines, acceptable 
hormonal contraceptives are limited to those which inhibit ovulation.  
 
 
Pregnancy Testing: WOCBP should only be included after a negative highly sensitive urine 
or serum pregnancy test. 

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Pregnancy testing will be performed whenever an expected menstrual cycle is missed or when 
pregnancy is otherwise suspected. 
 
Appendix 4: Response assessement criteria 
Response Evaluation Criteria in Solid Tumors (RECIST) 1.1  
RECIST version 1.1* will be used in this study for assessment of tumor response. While either 
CT or MRI may be utilized, as per RECIST 1.1, CT is the preferred imaging technique in this 
study. 
* As published in the European Journal of Cancer: 
 
E.A. Eisenhauer, P. Therasse, J. Bogaerts, L.H. Schwartz, D. Sargent, R. Ford, J. 
Dancey, S. Arbuck, S. Gwyther, M. Mooney, L. Rubinstein, L. Shankar, L. Dodd, R. Kaplan, 
D. Lacombe, J. Verweij. New response evaluation criteria in solid tumors: Revised RECIST 
guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228-47. 
In addition, volumetric analysis will be explored by central review for response assessment. 
 
Immune Response Evaluation Criteria in Solid Tumors (iRECIST)  Criteria for 
Evaluating Response in Solid Tumors 
iRECIST will be used in this study for assessment of tumor response. While either CT or MRI 
may be utilized, as per iRECIST, CT is the preferred imaging technique in this study. 
In addition, volumetric analysis will be explored by central review for response assessment. 
 
Assessment at Screening and Prior to RECIST 1.1 Progression 
Until radiographic progression based on RECIST 1.1, there is no distinct iRECIST assessment. 
Assessment and Decision at RECIST 1.1 Progression 
In participants who show evidence of radiological PD by RECIST 1.1 the Investigator will 
decide whether to continue a participant on study treatment until repeat imaging is obtained.  
This decision by the Investigator should be based on the participant’s overall clinical condition. 
Clinical stability is defined as the following:  

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• Absence of symptoms and signs indicating clinically significant progression of disease 
• No decline in ECOG performance status  
• No requirements for intensified management, including increased analgesia, radiation, 
or other palliative care 
Any participant deemed clinically unstable should be discontinued from study treatment at 
site-assessed first radiologic evidence of PD, and is not required to have repeat tumor imaging 
for confirmation of PD by iRECIST. 
If the Investigator decides to continue treatment, the participant may continue to receive study 
treatment and the tumor assessment should be repeated 4 to 8 weeks later to confirm PD by 
iRECIST, per Investigator assessment.  I 
Tumor flare may manifest as any factor causing radiographic progression per RECIST 1.1, 
including: 
• Increase in the sum of diameters of target lesion(s) identified at baseline to ≥ 20% and 
≥ 5 mm from nadir 
o Please note: the iRECIST publication uses the terminology “sum of 
measurements”, but “sum of diameters” will be used in this protocol, consistent 
with the original RECIST 1.1 terminology. 
• Unequivocal progression of non-target lesion(s) identified at baseline 
• Development of new lesion(s) 
iRECIST defines new response categories, including iUPD (unconfirmed progressive disease) 
and iCPD (confirmed progressive disease).  For purposes of iRECIST assessment, the first visit 
showing progression according to RECIST 1.1 will be assigned a visit (overall) response of 
iUPD, regardless of which factors caused the progression. 
At this visit, target and non-target lesions identified at baseline by RECIST 1.1 will be assessed 
as usual. 
New lesions will be classified as measurable or non-measurable, using the same size thresholds 
and rules as for baseline lesion assessment in RECIST 1.1.  From measurable new lesions, up 
to 5 lesions total (up to 2 per organ), may be selected as New Lesions – Target.  The sum of 
diameters of these lesions will be calculated, and kept distinct from the sum of diameters for 
target lesions at baseline.  All other new lesions will be followed qualitatively as New Lesions 
– Non-target. 
Assessment at the Confirmatory Imaging 
On the confirmatory imaging, the participant will be classified as progression confirmed (with 
an overall response of iCPD), or as showing persistent unconfirmed progression (with an 
overall response of iUPD), or as showing disease stability or response (iSD/iPR/iCR). 
Confirmation of Progression 

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Progression is considered confirmed, and the overall response will be iCPD, if ANY of the 
following occurs: 
• Any of the factors that were the basis for the initial iUPD show worsening 
o For target lesions, worsening is a further increase in the sum of diameters of 
≥ 5 mm, compared to any prior iUPD time point 
o For non-target lesions, worsening is any significant growth in lesions overall, 
compared to a prior iUPD time point; this does not have to meet the “unequivocal” 
standard of RECIST 1.1 
o For new lesions, worsening is any of these: 
§ An increase in the new lesion sum of diameters by ≥ 5 mm from a prior iUPD 
time point  
§ Visible growth of new non-target lesions  
§ The appearance of additional new lesions 
• Any new factor appears that would have triggered PD by RECIST 1.1 
Persistent iUPD 
Progression is considered not confirmed, and the overall response remains iUPD, if: 
• None of the progression-confirming factors identified above occurs AND 
• The target lesion sum of diameters (initial target lesions) remains above the initial PD 
threshold (by RECIST 1.1) 
Additional imaging for confirmation should be scheduled 4 to 8 weeks from the scan on which 
iUPD is seen.  This may correspond to the next visit in the original visit schedule.  The 
assessment of the subsequent confirmation scan proceeds in an identical manner, with possible 
outcomes of iCPD, iUPD, and iSD/iPR/iCR. 
Resolution of iUPD 
Progression is considered not confirmed, and the overall response becomes iSD/iPR/iCR, if: 
• None of the progression-confirming factors identified above occurs, AND 
• The target lesion sum of diameters (initial target lesions) is not above the initial PD 
threshold.  
The response is classified as iSD or iPR (depending on the sum of diameters of the target 
lesions), or iCR if all lesions resolve. 
In this case, the initial iUPD is considered to be pseudo-progression, and the level of suspicion 
for progression is “reset”.  This means that the next visit that shows radiographic progression, 
whenever it occurs, is again classified as iUPD by iRECIST, and the confirmation process is 
repeated before a response of iCPD can be assigned. 
Management Following the Confirmatory Imaging 

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If repeat imaging does not confirm PD per iRECIST, as assessed by the Investigator, and the 
participant continues to be clinically stable, study treatment may continue and follow the 
regular imaging schedule.  If PD is confirmed, participants will be discontinued from study 
treatment. 
NOTE: If a participant has confirmed radiographic progression (iCPD) as defined above, but 
the participant is achieving a clinically meaningful benefit, an exception to continue study 
treatment may be considered.  In this case, if study treatment is continued, tumor imaging 
should continue to be performed following the intervals as outlined in section 8.0. 
Detection of Progression at Visits After Pseudo-progression Resolves 
After resolution of pseudo-progression (ie, achievement of iSD/iPR/iCR), iUPD is indicated 
by any of the following events: 
• Target lesions 
o Sum of diameters reaches the PD threshold (≥ 20% and ≥ 5 mm increase from 
nadir) either for the first time, or after resolution of previous pseudo-progression.  
The nadir is always the smallest sum of diameters seen during the entire trial, either 
before or after an instance of pseudo-progression. 
• Non-target lesions 
o If non-target lesions have never shown unequivocal progression, their doing so for 
the first time results in iUPD. 
o If non-target lesions had shown previous unequivocal progression, and this 
progression has not resolved, iUPD results from any significant further growth of 
non-target lesions, taken as a whole. 
• New lesions 
o New lesions appear for the first time 
o Additional new lesions appear 
o Previously identified new target lesions show an increase of ≥ 5 mm in the new 
lesion sum of diameters, from the nadir value of that sum 
o Previously identified non-target lesions show any significant growth 
If any of the events above occur, the overall response for that visit is iUPD, and the iUPD 
evaluation process (see Assessment at the Confirmatory Imaging above) is repeated.  
Progression must be confirmed before iCPD can occur. 
The decision process is identical to the iUPD confirmation process for the initial PD, except in 
one respect. If new lesions occurred at a prior instance of iUPD, and at the confirmatory scan 
the burden of new lesions has increased from its smallest value (for new target lesions, their 
sum of diameters is ≥5 mm increased from its nadir), then iUPD cannot resolve to iSD or iPR. 
It will remain iUPD until either a decrease in the new lesion burden allows resolution to iSD 
or iPR, or until a confirmatory factor causes iCPD. 
Additional details about iRECIST are provided in the iRECIST publication (Seymour et al., 
2017). 

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Appendix 5 Results of MGMT analysis by IHC and methyl-Beaming 
Results obtained from ARETHUSA data accrual at the 20th of November 2019 on MGMT 
screening: MGMT IHC results show a ~50% of negativity (77 negative over 160 tested); 
MGMT promoter methylation results (by methyl-Beaming) show a positivity of ~20% (15 
positive over 73 tested). 
 
 
 
 
 
 
 
 
83; 52%
77; 48%
MGMT-IHC
Pos
Neg
15; 21%
58; 79%
MGMT-Methylation
Pos
Neg
a
b