Targeting Triple Negative BREAst Cancer Metabolism With a Combination of Chemoimmunotherapy and a FASTing-like Approach in the Preoperative Setting: the BREAKFAST 2 Trial
Phase: PHASE2
Conditions: Breast Cancer, Triple Negative Breast Cancer, Dietary Exposure, Fasting
Sponsor: Fondazione IRCCS Istituto Nazionale dei Tumori, Milano

BREAKFAST 2 Protocol 
 
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CONFIDENTIAL 
 
 
  
Targeting triple negative BREAst cancer metabolism 
with a combination of chemoimmunotherapy and a 
FASTing-like approach in the preoperative setting: 
the BREAKFAST 2 trial 
 
 
 
 
EudraCT Number 
Not Applicable 
Acronym 
BREAKFAST 2 
Initial Protocol Version and Date 
Version 1.0 – October, 1st, 2022 
Protocol Version and Date Amendment (if 
applicable) 
 
Sponsor 
Fondazione 
IRCCS 
‘’Istituto 
Nazionale dei Tumori’’ – Milan   
 
 
 

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PROTOCOL SIGNATURE PAGE 
 
Targeting triple negative BREAst cancer metabolism with 
a combination of chemoimmunotherapy and a FASTing-
like approach in the preoperative setting: the 
BREAKFAST 2 trial 
 
A Study of Fondazione IRCCS Istituto Nazionale dei Tumori 
I confirm that the Sponsor will comply with all obligations as detailed in the study 
protocol and in all applicable regulations and guidelines. I ensure that the Investigators 
will be informed of all relevant information that become available during the conduct 
of this protocol. 
 
PRINCIPAL INVESTIGATOR SIGNATURE 
                                                              Signature 
 
                      Date 
Prof. Filippo G. de Braud             __________________                       ____________                         
CO-PRINCIPAL INVESTIGATOR SIGNATURE 
                                                              Signature 
 
                      Date 
Dott. Claudio Vernieri        
     __________________                            ____________    
 
 
 
 
 
 
 
 
 
 
 
 

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DECLARATION OF THE INVESTIGATOR 
 
Targeting triple negative BREAst cancer metabolism with 
a combination of chemoimmunotherapy and a FASTing-
like approach in the preoperative setting: the 
BREAKFAST 2 trial 
 
A Study of Fondazione IRCCS Istituto Nazionale dei Tumori 
I have read and understood the contents of the clinical protocol for the BREAKFAST 
2 Trial dated October 1st, 2022 and agree to perform the study according to the study 
protocol and all applicable regulations and guidelines. I will ensure that the 
Investigators are informed of all relevant information becoming available during the 
conduction of this study. 
 
Principal investigator name: 
Institution: 
Signature: 
Date: 
 
 
 
 
 
 
 
 
 
 
 

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Principal Investigators: 
PI: Filippo de Braud, MD. Tel: +39 02 23903066, Fondazione IRCCS Istituto 
Nazionale 
dei 
Tumori 
(Milan, 
Italy) 
e-mail 
address: 
filippo.debraud@istitutotumori.mi.it 
 
Co-PI: Claudio Vernieri, MD, PhD. Tel: +39 02 23903066, e-mail address: 
claudio.vernieri@istitutotumori.mi.it; claudio.vernieri@ifom.eu 
 
 
Other Institutions and local PIs participating in the trial: 
 
Division of Oncology 2, Istituto Oncologico Veneto, Via Gattamelata 64,  
35128 Padova, Italy 
Maria Vittoria Dieci, MD. Tel: +39 049 8215295, e-mail address: 
mariavittoria.dieci@iov.veneto.it 
 
Department of Medical Oncology, U.O. Clinica di Oncologia Medica, IRCCS 
Ospedale Policlinico San Martino, Largo Rosanna Benzi 10, 16132 Genova, Italy 
Matteo Lambertini, MD, PhD. Tel: +39 010 5554258, e-mail address: 
matteo.lambertini@unige.it 
 
Department of Medical Oncology, University of Naples Federico II, Via Sergio 
Pansini 5, 80131 Naples, Italy  
Sabino De Placido, MD. Tel: +39 081 7464272, e-mail address: sdponco@unina.it 
 
Medical Senology Division, IEO Istituto Europeo di Oncologia, Via Ripamonti 435, 
20132 Milano, Italy 
Monica Iorfida, MD. Tel: +39 02 57489970, e-mail address: monica.iorfida@ieo.it 
 
Department of Medical Oncology, Humanitas Research Hospital, Via Alessandro 
Manzoni 56, 20089 Rozzano, Italy 
Alberto 
Zambelli, 
MD. 
Tel: 
+39 
02 
82243163, 
e-mail 
address: 
alberto.zambelli@hunimed.eu 
 
Department of Medical Oncology, A.O. “Istituti Ospitalieri”, Viale Concordia 1, 
26100 Cremona, Italy 
Carla Strina, MD. Tel: +39 0372 408349, e-mail address: carla.strina@asst-
cremona.it 
 
Department of Clinical and Molecular Oncology, "Sapienza" University of Rome, 
00185, Rome, Italy 
Andrea Botticelli, MD, PhD. E-mail address: andrea.botticelli@uniroma1.it 
 
 
 

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Co-investigators: 
 
Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, 
via Venezian 1, 20133, Milan, Italy 
Francesca 
Ligorio, 
MD. 
Tel: 
+39-(0)2-23903066, 
e-mail 
address: 
francesca.ligorio@istitutotumori.mi.it 
Giovanni 
Fucà, 
MD. 
Tel: 
+39-(0)2-23903066, 
e-mail 
address: 
giovanni.fuca@istitutotumori.mi.it 
Lucrezia Zanenga (biologist, nutritionist), Tel: +39-(0)2-23903066, e-mail address: 
lucrezia.zanenga@istitutotumori.mi.it  
Leonardo 
Provenzano, 
MD. 
Tel: 
+39-(0)2-23903066, 
e-mail 
address: 
leonardo.provenzano@istitutotumori.mi.it  
Caterina 
Sposetti, 
MD. 
Tel: 
+39-(0)2-23903066, 
e-mail 
address: 
caterina.sposetti@istitutotumori.mi.it  
Riccardo 
Lobefaro, 
MD. 
Tel: 
+39-(0)2-23903066, 
e-mail 
address: 
riccardo.lobefaro@istitutotumori.mi.it 
Daniele 
Presti, 
MD. 
Tel: 
+39-(0)2-23903066, 
e-mail 
address: 
daniele.presti@istitutotumori.mi.it 
Giulia 
Valeria 
Bianchi, 
MD. 
Tel: 
+39-(0)2-23902286, 
e-mail 
address: 
giulia.bianchi@istitutotumori.mi.it 
Giuseppe 
Capri, 
MD, 
PhD. 
Tel: 
+39-(0)2-23902595, 
e-mail 
address: 
giuseppe.capri@istitutotumori.mi.it  
 
 
Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, via 
Venezian 1, 20133, Milan, Italy 
Secondo 
Folli, 
MD. 
Tel: 
+39-(0)2-23902996, 
e-mail 
address: 
secondo.folli@istitutotumori.mi.it 
Cristina 
Ferraris, 
MD. 
Tel: 
+39-(0)2-23903711, 
e-mail 
address: 
cristina.ferraris@istitutotumori.mi.it 
Gabriele 
Martelli, 
MD. 
Tel: 
+39-(0)2-23903436, 
email 
address: 
gabriele.martelli@istitutotumori.mi.it 
Massimiliano 
Gennaro, 
MD. 
Tel: 
+39-(0)2-23903246, 
e-mail 
address: 
massimiliano.gennaro@istitutotumori.mi.it 
 
 
 
Department of Diagnostic Pathology and Laboratory, Fondazione IRCCS Istituto 
Nazionale dei Tumori, via Venezian 1, 20133, Milan, Italy 

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Giancarlo 
Pruneri, 
MD. 
Tel: 
+39-02-23903480, 
e-mail 
address: 
giancarlo.pruneri@istitutotumori.mi.it 
Andrea 
Vingiani, 
MD. 
Tel: 
+39-02-23902615, 
email 
address: 
andrea.vingiani@istitutotumori.mi.it 
Antonino 
Belfiore, 
PhD. 
Tel: 
+39-0_2-23903016, 
email 
address: 
antonino.belfiore@istitutotumori.mi.it  
 
Daniele 
Morelli, 
MD. 
 
Tel: 
(+39) 
02 
2390 
2054, 
email 
address: 
daniele.morelli@istitutotumori.mi.it 
 
 
 
 
 
Clinical Epidemiology and Trials Organization Unit, Fondazione IRCCS Istituto 
Nazionale dei Tumori, via Venezian 1, 20133, Milan, Italy 
Luigi 
Mariani, 
MD. 
Tel: 
+39-02-23903199, 
e-mail 
address: 
luigi.mariani@istitutotumori.mi.it 
 
Breast Imaging Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, via 
Venezian 1, 2013, Milan, Italy 
Gianfranco 
Scaperrotta, 
MD. 
Tel: 
+39-02-23902789, 
e-mail 
address: 
gianfranco.scaperrotta@istitutotumori.mi.it  
Catherine 
Depretto, 
MD. 
Tel: 
+39-02-23903144, 
e-mail 
address: 
catherine.depretto@istitutotumori.mi.it  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
PROTOCOL SIGNATURE PAGE ............................................................................... 2 
DECLARATION OF THE INVESTIGATOR ............................................................. 3 
TABLE OF CONTENTS .............................................................................................. 7 
1 
ABBREVIATIONS AND DEFINITIONS OF TERMS ................................... 12 
2 
BACKGROUND .................................................................................................. 14 
3 
Standard preoperative therapy and unmet clinical needs in triple-negative 
breast cancer (TNBC) treatment ................................................................................ 14 
3.1 
Rationale for targeting TNBC metabolism in combination with neoadjuvant 
chemoimmunotherapy ...................................................................................................... 16 
3.2 
The Fasting-Like Approach (FLA) as a strategy to target cancer metabolism 
and to improve the tolerability of cytotoxic chemotherapy ........................................... 17 
Effects of nutrient starvation in TNBC ......................................................................................... 17 
Rationale for fasting during cancer treatment ............................................................................... 17 
In vivo evidence for combining fasting with cytotoxic chemotherapy ......................................... 18 
The Fasting-Like Approach (FLA) as a safe alternative to complete fasting ............................... 19 
Safety, feasibility and metabolic effects of the FLA in cancer patients ........................................ 19 
Rationale for combining calorie restriction with immunotherapy ................................................ 21 
3.3 
Study rationale and purpose ............................................................................... 22 
4 
STUDY DESIGN................................................................................................. 23 
4.1 
Description of study design ................................................................................. 23 
5 
OBJECTIVES AND ENDPOINTS .................................................................... 24 
5.1 
Objectives .............................................................................................................. 24 
Primary objective .......................................................................................................................... 24 
Secondary objectives .................................................................................................................... 24 
Exploratory objectives .................................................................................................................. 25 
6 
ENDPOINTS ....................................................................................................... 25 
6.1 
Primary endpoints................................................................................................ 25 

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6.2 
Secondary endpoints ............................................................................................ 25 
Efficacy endpoints ........................................................................................................................ 25 
Compliance endpoints ................................................................................................................... 26 
Safety endpoints ............................................................................................................................ 26 
Exploratory endpoints ................................................................................................................... 26 
7 
STUDY POPULATION ...................................................................................... 26 
7.1 
Patient population ................................................................................................ 26 
7.2 
Inclusion criteria .................................................................................................. 27 
7.3 
Exclusion criteria ................................................................................................. 28 
8 
EXPERIMENTAL INTERVENTION ............................................................... 30 
8.1 
Study intervention ................................................................................................ 30 
8.2 
Chemo-immunotherapy ....................................................................................... 30 
8.2.1 
Pembrolizumab prescribing ................................................................................ 31 
8.3 
FLA scheme (arm B) ............................................................................................ 31 
8.4 
Dietary recommendation for patients randomized to arm A and maintenance 
diet during the refeeding periods for patients randomized to arm B ........................... 33 
8.5 
Trial Blinding/Masking and treatment allocation ............................................ 34 
8.6 
Stratification ......................................................................................................... 34 
8.7 
Concomitant Medications (Allowed & Prohibited) .......................................... 34 
Acceptable Concomitant Medications .......................................................................................... 34 
Prohibited Concomitant Medications ............................................................................................ 34 
Rescue Medications & Supportive Care ....................................................................................... 35 
9 
VISIT SCHEDULE AND ASSESSMENTS ...................................................... 35 
9.1 
Screening and baseline (DAY -28 – DAY 0) ...................................................... 47 
9.2 
On Treatment Procedures  .................................................................................. 48 
9.3 
Before Surgery...................................................................................................... 49 
9.4 
Surgery .................................................................................................................. 49 
9.5 
Adjuvant treatment.............................................................................................. 49 

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9.6 
End of treatment .................................................................................................. 50 
9.7 
Additional therapy and follow-up....................................................................... 51 
9.8 
Tumor recurrence ................................................................................................ 51 
10 STUDY PROCEDURES ..................................................................................... 51 
10.1 
Screening procedures ........................................................................................... 51 
Informed consent .......................................................................................................................... 51 
Inclusion/Exclusion Criteria ......................................................................................................... 52 
Demographics and Medical History.............................................................................................. 52 
Prior and Concomitant Medications.............................................................................................. 52 
Randomization procedure/Assignment of Randomization Number .............................................. 52 
10.2 
Clinical procedures/assessments ......................................................................... 52 
Disease evaluation ........................................................................................................................ 52 
Multidisciplinary visit ................................................................................................................... 53 
Physical examination and Vital signs ........................................................................................... 53 
12-Lead Electrocardiogram and LVEF evaluation ....................................................................... 53 
ECOG Performance Status............................................................................................................ 53 
Pregnancy assessment ................................................................................................................... 54 
Height, weight and body mass index (BMI) ................................................................................. 54 
Nutritional evaluation and study of body composition ................................................................. 54 
Food diaries ................................................................................................................................... 54 
Phone/e-mail assessments ............................................................................................................. 55 
10.3 
Quality of Life assessments ................................................................................. 55 
10.4 
Laboratory Procedures/Assessments ................................................................. 55 
Metabolic evaluations ................................................................................................................... 56 
Hepatitis serology ......................................................................................................................... 57 
Urinalysis ...................................................................................................................................... 57 
Saliva and Stool analysis .............................................................................................................. 57 
Immunological analysis (including PBMCs) ................................................................................ 57 
Evaluation of pathological complete response (pCR) in surgical specimens ................................ 57 
Other analyses in tumor samples................................................................................................... 58 
10.5 
Withdrawal from study participation ................................................................ 58 
11 ADVERSE EVENTS (AEs) ................................................................................ 58 

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11.1 
Serious Adverse Events (SAEs) .......................................................................... 59 
11.2 
Adverse Event Intensity and Relationship to Study Intervention ................... 60 
11.3 
AEs collection and reporting ............................................................................... 60 
SAE collection and reporting ........................................................................................................ 60 
Non-serious Adverse Events collection and reporting .................................................................. 61 
11.4 
Laboratory test abnormalities ............................................................................ 61 
12 COMPLIANCE TO THE EXPERIMENTAL TREATMENT .......................... 62 
12.1 
Food diary during the FLA periods ................................................................... 62 
12.2 
Food diary outside the FLA periods ................................................................... 62 
13 DISCONTINUATION/MODIFICATION OF THE EXPERIMENTAL 
TREATMENT ............................................................................................................. 62 
13.1 
Discontinuation of chemo-immunotherapy ....................................................... 62 
13.2 
Modification of chemotherapy administration .................................................. 63 
Dose modifications for paclitaxel and carboplatin ........................................................................ 64 
Dose modifications for doxorubicin/epirubicin and cyclophosphamide ....................................... 67 
13.3 
Pembrolizumab discontinuation ......................................................................... 71 
13.4 
FLA discontinuation (arm B) .............................................................................. 73 
Criteria for FLA discontinuation based on weight loss ................................................................. 75 
14 BIOLOGICAL EVALUATIONS ........................................................................ 77 
14.1 
Blood analyses ...................................................................................................... 77 
Immunological parameters (20 ml of peripheral blood required) ................................................. 77 
Blood cell count (8 ml of peripheral blood required) .................................................................... 78 
Standard blood chemistry (12 ml of peripheral blood required) ................................................... 78 
Quantification of plasma amino acids (5 ml) ................................................................................ 78 
Lipid profile of whole blood, plasma and red blood cell membranes (3 ml) ................................ 78 
Metabolic evaluations and Growth factor analyses (3 ml) ............................................................ 78 
14.2 
Urine analyses ....................................................................................................... 79 
14.3 
Saliva and Stool analyses ..................................................................................... 79 
Oral and Fecal Microbiota analysis............................................................................................... 79 
Bacterial metabolites in feces ....................................................................................................... 79 

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14.4 
Biological evaluations on tumor tissue samples ................................................. 79 
Gene expression analysis .............................................................................................................. 80 
Genomic assays ............................................................................................................................. 81 
Peritumoral adipose tissue analysis ............................................................................................... 81 
15 STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN ................. 81 
15.1 
Definition of study populations ........................................................................... 81 
15.2 
Statistical plan and sample size calculation ....................................................... 82 
15.3 
Statistical analyses................................................................................................ 82 
Primary endpoint analysis ............................................................................................................. 82 
Secondary efficacy analyses ......................................................................................................... 82 
Safety analyses and unacceptable toxicity monitoring .................................................................. 83 
16 ETHICAL AND ADMINISTRATIVE CONSIDERATIONS ........................... 83 
16.1 
Independent Ethics Committee (IEC)  ............................................................... 83 
16.2 
Ethical conduct of the trial .................................................................................. 83 
16.3 
Informed Consent ................................................................................................ 83 
16.4 
Confidentiality of information ............................................................................ 84 
17 QUALITY CONTROL ........................................................................................ 85 
17.1 
Responsibilities of the investigators .................................................................... 85 
17.2 
Study Monitoring ................................................................................................. 85 
17.3 
Audits and inspections ......................................................................................... 85 
17.4 
Retention of Records ............................................................................................ 86 
18 STUDY CONDUCTION ..................................................................................... 86 
18.1 
Administrative Structure .................................................................................... 86 
18.2 
Training of study site personnel ......................................................................... 86 
18.3 
Study timetable and end of study ....................................................................... 86 
18.4 
Data Handling ...................................................................................................... 86 
18.5 
Publication of Data............................................................................................... 87 

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19 REFERENCES ................................................................................................... 87 
 
1 
ABBREVIATIONS AND DEFINITIONS OF TERMS 
AEs 
Adverse Event 
BIA 
Bioelectric Impedance Analysis 
BMI 
Body Mass Index  
CESM 
Contrast-enhanced mammography 
CRF 
Case Report Form  
CT 
Computed Tomography  
CTCAE 
Common Terminology For Adverse Events  
DMFS 
Distant Metastasis-Free Survival  
DFS 
Disease Free Survival 
EC 
Ethics Committee 
ECOG 
Eastern Cooperative Oncology Group  
EFS  
Event Free Survival 
EORTC 
European Organization For Research And Treatment Of 
Cancer  
EOT 
End Of Treatment  
ER 
Estrogen Receptor 
FFPE 
Formalin-Fixed Paraffin-Embedded  
FLA 
Fasting-Like Approach 
IEC 
Independent Ethics Committee  
IEO 
European Institute Of Oncology  
IGF-1 
Insulin-Like Growth Factor 1 
IHC 
Immunohistochemistry 
INT 
Fondazione IRCCS Istituto Nazionale Dei Tumori  
ISH 
In Situ Hybridization  
KM 
Kaplan-Meier  
LVEF 
Left Ventricular Ejection Fraction  
mTORC  
Mechanistic Target Of Rapamycin Complex 1  
CE-MRI 
MUGA 
Contrast-Enhanced Magnetic Resonance Imaging 
Multi-Gated Scintigraphic Scan  
NGS 
Next Generation Sequencing  
OS 
Overall Survival 
OTC 
Over-The-Counter  

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PBMCs 
Peripheral Blood Mononuclear Cells  
pCR 
Pathologic Complete Response 
PgR 
Progesteron Receptor 
PP2A 
Protein Phosphatase 2 A 
PS 
Performance Status 
SAE 
Serious Adverse Events 
SCFAs 
Short-Chain Fatty Acids  
SNLs 
Sentinel Lymphonodes  
TILs 
Tumor Infiltrating Lymphocytes  
WCRF 
World Cancer Research Fund  
WHO 
World Health Organization 
 
 

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2 
BACKGROUND 
3 
Standard preoperative therapy and unmet clinical needs in 
triple-negative breast cancer (TNBC) treatment 
Breast cancer (BC) is the most commonly diagnosed neoplasm, with an estimated 2,3 
million new cases diagnosed in 2020 worldwide. In Europe, BC accounts for 27.8.% of 
female cancers and 1 out of 12 women is estimated to develop breast cancer before the 
age of 75. Moreover, BC represents the leading cause of cancer-related deaths in 
women in the majority of European countries1. BC prevalence is rising, mainly as a 
result of increased incidence and population ageing but also of early detection and 
improvements in treatment efficacy. 
The majority (∼90%) of patients with BC are diagnosed with localized disease;2 
however, about 20-30% of resected BCs eventually recur with metastatic disease, 
which is almost invariably incurable.  
Approximately 15%-20% of all BCs are classified as Triple-Negative Breast Cancer 
(TNBC), as defined by absent or minimal (<1%) expression of oestrogen receptor (ER) 
and progesterone receptor (PgR) at immunohistochemistry (IHC), as well as by absence 
of HER2 overexpression at IHC and HER2 gene amplification. TNBC is characterized 
by an aggressive behavior, high metastatization potential and fast acquisition of 
resistance to chemotherapy. These features portend the worst prognosis among BC 
subgroups3,4. This is in part due to the lack of ER/PgR expression, which makes 
endocrine manipulations ineffective, but mostly to its intrinsic biological 
characteristics, resulting in fast acquisition of resistance to chemotherapy and tendency 
to spread to vital organs (such as liver, lungs and brain) in addition to lymph nodes and 
bones5.  
Due to high proliferation rate and common alterations in DNA repair enzymes, TNBC 
is exquisitely sensitive to cytotoxic chemotherapy, in particular to anthracyclines, 
cyclophosphamide and taxanes. The administration of systemic chemotherapy before 
surgery (neoadjuvant or preoperative chemotherapy) is the recommended approach for 
TNBC patients with clinically/radiologically node-positive (N+) disease and/or 
primary tumors larger than 1 cm in their maximum diameter6. 
In the preoperative setting, TNBC presents higher response rates when compared to 
hormone-sensitive neoplasms7.  
The use of preoperative chemotherapy is associated with several potential advantages. 
Firstly, it is associated with higher rates of breast-conserving surgery8. Secondly, it 
gives the opportunity to rapidly test tumor sensitivity to standard chemotherapy 
(plus/minus experimental treatments). Moreover, it provides prognostic indications; 
indeed, achieving pCR with preoperative chemotherapy is associated with significantly 
better long-term outcomes in patients with TNBC, including relapse free survival 
(RFS), distant-metastasis free survival (DMFS) and overall survival (OS)9. Finally, it 
allows to test the predictive role of biological (patient- or tumor-related) biomarkers of 
sensitivity to the proposed treatment in the pre-chemotherapy tissue, and to study how 
these biomarkers are modulated during the treatment in patients who fail to achieve 
pCR. 

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The standard preoperative treatment for TNBC has consisted for years in the sequential 
administration of anthracyclines- and taxanes-based chemotherapy regimen, which is 
associated with an expected pCR rate in the range of 31%-48%10-12.  
Several phase III trials recently tested the addition of carboplatin to standard 
anthracycline-taxane preoperative chemotherapy in patients with TNBC.  
By promoting DNA interstrand cross-linking, carboplatin induces DNA breaks that 
result in enhanced cytotoxicity in tumors with impaired DNA damage response, 
including the subgroup of patients with germline or somatic inactivating mutations of 
BRCA1/213. Clinical evidence comes from the work of Tutt et al. where carboplatin 
monotherapy demonstrated to double the response rate compared to docetaxel 
monotherapy in patients with advanced TNBC holding germline BRCA1/2 mutation14. 
In the early-stage disease treatment setting, several trials have been carried out to assess 
the efficacy of adding platinum compounds to standard anthracycline and taxane-based 
preoperative chemotherapy in TNBC. A meta-analysis10 of 9 different randomized 
controlled trials (RCT) showed a consistent increase of pCR rate from 37% to 52.1% 
with the addition of carboplatin. Of note, in this context, the presence of inactivating 
BRCA 1/2 mutations did not predict higher pCR rates with the addition of platinum 
compounds to standard preoperative chemotherapy10. A recent update of this meta-
analysis showed that platinum-based neoadjuvant chemotherapy, as compared with 
platinum-free regimens, is associated with significantly longer EFS (HR 0.70, 95% CI 
0.56-0.89) and with a non-significant 18% reduction in the risk of death (HR 0.82, 95% 
CI 0.64-1.04)15. Based on the reported improvement of TNBC patient EFS with the 
addition of carboplatin to anthracycline-taxane-based neoadjuvant chemotherapy, the 
chemotherapy backbone of neoadjuvant treatment in patients with stage II-III TNBC 
(node positive and/or primary tumors of maximum diameter higher than 2 cm) should 
contain an anthracycline, a taxane and carboplatin. In particular, 12 weekly 
administration of carboplatin-paclitaxel doublet, followed by for triweekly/biweekly 
injections of anthracyclines (doxorubicin/epirubicin)-cyclophosphamide have emerged 
as the most effective chemotherapy backbone in the neoadjuvant treatment setting. 
Since the efficacy of neoadjuvant carboplatin is not associated with the 
presence/absence of BRCA1/2 alterations, this chemotherapy regimen has become the 
standard-of-care backbone chemotherapy in several European and US Institutions in 
recent years6.   
Among BC subtypes, TNBC is the most immunogenic one, as it is characterized by a 
relatively high mutational burden and a tumor microenvironment characterized by 
immune cell infiltration; in addition, tumor infiltrating lymphocytes represent a strong 
prognostic factor in early-stage TNBC16. For these reasons, several clinical trials 
investigated the antitumor activity of adding immune checkpoints inhibitors (ICIs) in 
combination with neoadjuvant chemotherapy in patients with advanced TNBC. Among 
these studies, the Impassion 130 and the Keynote 355 trials demonstrated a clinically 
meaningful benefit from the addition of immunotherapy to standard first line treatment 
in advanced TNBC expressing high PDL1 levels17,18. 
Recently, several clinical trials showed that the addition of ICIs inhibitors to standard 
cytotoxic chemotherapy is effective also in the neoadjuvant setting, leading to a 
significant increase in pathological complete response (pCR) rates19-21. Among these 
trials, both the phase II GeparNuevo22, which investigated Atezolizumab in 

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combination with Nab-paclitaxel followed by Epirubicin plus Cyclophosphamide, and 
the phase III KEYNOTE 522 trial, in which Pembrolizumab was combined with 
anthracycline, taxane and carboplatin-based chemotherapy, showed an improvement in 
terms of pCR rates and long-term efficacy outcomes, such as DFS and EFS, 
respectively, in patients receiving ICIs. Importantly, the clinically meaningful EFS 
improvement demonstrated by the KEYNOTE-522 (HR 0.63; P = 0.0003), set the new 
standard for stage II-III early-stage TNBC. 
However, despite these improvements, a non-negligible proportion of TNBC patients 
treated in the early-stage setting will eventually recur, especially among those not 
achieving pCR at surgery. For instance, in the KEYNOTE-522 trial, the 3-year EFS 
rate in pembrolizumab-treated patients with residual disease at surgery is only 67.4%, 
highlighting the urgent need to develop new effective therapeutic strategies to improve 
treatment efficacy for TNBC patients.  
In addition, selecting patients more likely to benefit from the addition of 
immunotherapy to neoadjuvant chemotherapy remains a clinical challenge. Indeed, 
differently from the metastatic setting, PD-L1 positivity do not seem to have a 
predictive value in TNBC patients treated with neoadjuvant ICIs, as patients with PD-
L1+ TNBC obtain a higher pCR rate than those with PD-L1– cancers independently 
from the treatment received. 
 
3.1 Rationale for targeting TNBC metabolism in combination with 
neoadjuvant chemoimmunotherapy 
When compared to other breast cancer subtypes, TNBC is characterized by higher 
histologic grade, higher proliferation rate and clinical aggressiveness. These biological 
and clinical characteristics correspond, from a metabolic point of view, to highly 
glycolytic tumors; indeed, enhanced glycolysis represents the major source of anabolic 
precursors for the biosynthesis of amino acids and nucleotides, which are required by 
rapidly growing and proliferating cancer cells to synthesize or repair macromolecules 
(proteins, DNA). For instance, serine biosynthesis from the glycolysis intermediate 3-
phosphoglycerate is frequently upregulated in human TNBC, while inhibition of serine 
synthesis results in inhibition of cancer cell growth and proliferation23. 
However, TNBC are heterogeneous neoplasms from a metabolic point of view. For 
instance, Basal-Like 2 (BL2) tumors, as defined according to the classification of 
Lehmann24, are characterized by upregulation of genes involved in glycolysis. On the 
other hand, mesenchymal-like TNBC mostly rely on lipid and cholesterol metabolism.  
More recently, a multi-omics analysis of TNBC tumor samples identified three 
metabolic subtypes of TNBCs, characterized by distinct metabolic gene expression, 
metabolite abundance, genomic drivers, and sensitivity to various metabolic 
inhibitors25.  
Based on this heterogeneity, different metabolic interventions could produce 
differential anticancer activity depending on the specific metabolic tumor 
dependencies.  

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3.2 The Fasting-Like Approach (FLA) as a strategy to target cancer 
metabolism and to improve the tolerability of cytotoxic 
chemotherapy 
Effects of nutrient starvation in TNBC 
In in vitro experiments, reducing the concentration of glucose and serum - the major 
sources of growth factors and amino acids- in cell growth media strongly sensitizes 
models of TNBC to different chemotherapeutical agents, including doxorubicin, 
cyclophosphamide and platinum compounds26,27. This effect mainly results from the 
fact that rapidly growing and proliferating cancer cells are exquisitely sensitive to 
metabolic deprivation, especially when they are exposed to DNA-damaging agents. 
Indeed, under such conditions, they are unable to activate repair processes that require 
energy units (in the form of ATP) and anabolites. 
Rationale for fasting during cancer treatment 
Cyclic fasting is the simplest and most effective way to recapitulate in vivo the 
metabolic effects of in vitro starvation of glucose, amino acids and growth factors.  
In particular, water-only fasting reduces the blood concentration of metabolites (e.g., 
glucose) and growth factors (e.g., insulin and insulin-like growth factor or IGF-1) that 
foster tumor growth and proliferation, and it also increases the blood concentration of 
metabolites that inhibit energy production in cancer cells (e.g., ketone bodies) 28.  
Preclinical in vitro and in vivo evidence suggests that starvation/fasting is more toxic 
to highly replicating cancer cells than to normal tissues29. This phenomenon, known as 
differential stress response (DSR), is the consequence of the intrinsically different 
proliferative and metabolic characteristics of most cancer cells when compared to their 
normal counterpart. When deprived of glucose, growth factors (e.g., insulin, IGF-1) and 
other metabolites (e.g., glutamine), most normal cells typically enter a quiescent 
proliferative state that is characterized by reduced energy expenditure and inhibition of 
anabolic functions (such as DNA, protein and lipid synthesis), and by a parallel 
activation of catabolic processes, such as autophagy, which result in the replenishment 
of essential metabolites. This response to nutrient starvation allows normal cells to 
fulfill essential biological functions, such as DNA-repair and de novo synthesis of 
damaged proteins, lipids and macromolecular structures (e.g., ribosomes, 
mitochondria). These protective activities become especially important when cells need 
to repair chemotherapy-induced damage to macromolecules and other cellular 
structures. 
On the contrary, constitutive activation of oncogenic pathways in cancer cells makes 
them less capable of halting growth and proliferation during nutrient starvation; indeed, 
most cancer cells go on growing and proliferating irrespective of nutrient availability 
in the extracellular environment.  As a consequence of this uncoupling between cell 
proliferation and the concentration of extracellular metabolites, cancer cells are 
exposed to rapid energy/metabolite depletion that leads to energetic crisis and the 
impossibility to synthesize new DNA, protein and lipid molecules to sustain their 
growth.  

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The in vitro-observed DSR between normal and cancer cells predicts that fasting during 
ChT administration could reduce ChT-induced damage to normal tissues, while 
improving its in vivo anticancer activity. 
In vivo evidence for combining fasting with cytotoxic chemotherapy 
Consistent with in vitro experiments, cycles of short-term fasting (STF), which 
recapitulate in vitro starvation by reducing the concentration of blood glucose, insulin 
and IGF-1, significantly reduced the in vivo growth of several tumor models, including 
orthotopic models of TNBC (including syngeneic ones). Moreover, cyclic fasting also 
synergized with different cytotoxic agents, including anthracyclines and platinum 
compounds, against murine tumors, while protecting healthy tissues from ChT-induced 
damage23. These results are consistent with the DSR hypothesis and indicate that fasting 
has the potential to improve the therapeutic index of some chemotherapeutic agents by 
potentiating their anticancer activity and protecting normal tissues from chemotherapy-
induced AEs. The fact that the fasting-chemotherapy combination not only slowed 
down tumor growth, but also prolonged animal survival and rescued the lethality of 
high-dose chemotherapy, further supports the concept of a DSR, and indicates the 
potential safety and clinical usefulness of this combination. 
So far, only a few, small studies have been performed in cancer patients to test the 
tolerability of STF. The first published case series suggested that water-only fasting for 
1-5 consecutive days during ChT is quite well tolerated by patients with different tumor 
types, and could also reduce hematological and gastrointestinal toxicities associated 
with cytotoxic treatments, consistent with preclinical experiments30. Then, a small 
randomized study in women with breast cancer showed that 48 hour-water only fasting 
in combination with (neo)adjuvant ChT is associated with a reduction of ChT-induced 
hematological toxicities, including thrombocytopenia and anemia31. Another 
randomized trial showed that 72 hour fasting in combination with platinum-based ChT 
is more effective than 24- or 48-hour fasting in modifying systemic metabolism and in 
reducing DNA damage to peripheral blood neutrophils and lymphocytes; these data are 
indicative of a protective effect of fasting during ChT, and also support a positive 
correlation between fasting duration and normal tissue preservation32. Although the 
population of patients enrolled in this trial was heterogeneous in terms of tumor types, 
stage and treatment received, the fasting-chemotherapy combination was associated 
with a high rate of tumor responses, which was superior to what expected on the basis 
of published studies. While this could simply be the result of selecting patients with 
better prognostic features (e.g., patients who were not malnourished), it is important to 
note that fasting did not seem to be detrimental in the population of patients considered.  
Finally, one randomized trial performed in patients with breast or ovarian carcinoma 
recently showed that 48-hour fasting may improve the quality of life of patients 
receiving cytotoxic ChT.33 
 
Several concerns have been raised about the use of complete (water-only) fasting in 
cancer patients, because of the risk of inducing progressive weight loss or sarcopenia. 
This could be especially relevant for those tumors that frequently induce anorexia (such 
as tumors of the head and neck tract or the esophagus) or cachexia (such as pancreatic 
or gastric cancers), especially in the advanced stage. Conversely, cachexia is not 

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commonly observed in patients with limited-stage TNBC, and it is unlikely that cycles 
of fasting, alternated with refeeding periods, could directly induce cachexia. Indeed, 
intentional fasting induces systemic metabolic changes that significantly differ from 
metabolic changes occurring during cachexia. During intentional short-term fasting, the 
appetite is conserved, loss of lean mass and sarcopenia do not occur, and fat reserves 
are used to mobilize energy sources in the form of triglycerides and fatty acids. 
Conversely, cachexia is characterized by an intense systemic inflammatory response, 
which leads to loss of appetite and increased degradation of proteins in muscle cells, 
and finally results in loss of lean tissues and sarcopenia.  
The Fasting-Like Approach (FLA) as a safe alternative to complete fasting 
Since complete fasting is unlikely to be feasible for the vast majority of cancer patients, 
studies conducted in recent years have been focused on less drastic dietary interventions 
that are able to produce metabolic changes comparable to those induced by complete 
fasting. In particular, fasting-like approaches (FLAs), i.e., plant-based, severely calorie-
restricted, low-carbohydrate, low-protein diets, have recently emerged as an equally 
effective, and potentially better tolerated alternative to complete fasting.   
Studies performed in murine TNBC models have demonstrated that cycles of three-
days FLA enhance the anticancer activity of anthracycline- or platinum-based 
chemotherapy, while at the same time promoting intratumor infiltration by cytotoxic 
CD8+ lymphocytes26 and reducing tumor infiltration by immunosuppressive regulatory 
T cells (Tregs). Even more importantly, the depletion of CD8+ T cells in these 
experiments resulted in impaired efficacy of FLA, thus demonstrating that an active 
immune system is essential to mediate the antitumor effects of cyclic FLA in syngeneic 
mouse TNBC models. Moreover, we have recently shown that cycles of FLA deplete 
TNBC stem cells, and cancer stem depletion makes TNBCs exquisitely responsive to 
the inhibition of the PI3K/AKT/mTORC1 axis34. 
Based on these data, as well as on results of other preclinical works35,36, the FLA can 
be considered a valuable alternative to water-only fasting in preclinical tumor models, 
including models of TNBC. 
Safety, feasibility and metabolic effects of the FLA in cancer patients 
Cyclic FLA has been recently tested in relatively large clinical trials in cancer patients. 
The first of these studies, namely the DIRECT trial, was a multicentric, phase II-III, 
randomized clinical trial that investigated the antitumor activity of a severely calorie-
restricted (~1200 Kcal on day 1; ~200 Kcal on days 2-4), 4-day, plant-based, low 
amino-acid diet combined with neoadjuvant chemotherapy in 131 breast cancer 
patients. Unfortunately, the study was prematurely interrupted because of poor patient 
compliance with the experimental diet; nevertheless, promising antitumor activity was 
observed in the subset of patients who were compliant with the dietary intervention37. 
The results of this trial highlighted the importance of a proper management of patients 
undergoing severe calorie restriction to monitor their compliance with experimental 
dietary regimen38.  
The second trial was a phase Ib study, in which a less severely calorie-restricted dietary 
intervention regimen (~1100 Kcal on day 1; ~700 Kcal on days 2-5) was combined 
with standard antitumor therapies in a heterogeneous population of 90 cancer patients. 

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In this study, the experimental diet was well tolerated, metabolically active, and 
associated with promising antitumor activity when combined with standard anticancer 
therapies in a subset of BC patients35,39. 
The third, and most recently published trial, has been conducted by our group at 
Fondazione IRCCS Istituto Nazionale dei Tumori between January 2017 and January 
202040. The study, directed by Dr. Claudio Vernieri and Prof. Filippo de Braud, 
investigated the safety, feasibility and biological effects of a severely calorie-restricted 
FLA regimen in combination with their standard anticancer therapies in a 
heterogeneous cohort of 101 patients with different tumor types. Specifically, the FLA 
scheme used in this study consisted in a 5-day, vegetal-based, low-calorie (600 Kcal on 
day 1, 300 Kcal on days 2 to 5), low-carbohydrate, low-protein diet, with no restriction 
in the intake of non-caloric beverages.  
Among 101 patients enrolled in the study, 100 patients were able to complete at least 
one cycle of FLA. The trial met its primary endpoint, with an incidence of severe FLA-
related AEs of 12.9%, i.e., lower than the hypothesized 20%. The most common FLA-
related AE was fatigue, which occurred in 90.2% of patients and was G3/G4 in 4% of 
patients. Other severe (G3/G4) FLA-related AEs consisted of hypoglycemia (5%), 
nausea (1%), dizziness (1%) and increased AST levels (1%). Serious adverse events 
(SAEs) occurred in 4 patients (4%), and in 2 cases they were FLA-related (syncope, 
severe fatigue). Patient compliance to the prescribed FLA regimen was globally 
excellent, with a total number of 23 minor deviations (22.8% of all patients) and 6 
major deviations (5.9%) to the prescribed FLA regimen. Overall, 72 (71.3%) patients 
were fully compliant with the FLA (absence of major and minor deviations during all 
FLA cycles), while 95 (94.1%) patients were fully compliant, or they reported only 
minor deviations. Considering individual FLA cycles in all patients, the global rate of 
compliance was 91.8% (404 out of 440 cycles), with minor and major deviations being 
reported in 29 (6.6%) and 7 (1.6%) cycles, respectively. 
During the first FLA cycle, in 99 evaluable median plasma glucose levels were reduced 
by 18.6% (range: [-63.1%; + 67.8%]), serum insulin by 50.7% (range: [-91.3%; + 
697%]) and IGF-1 by 30.3% (range: [-72.3%; +139.8%]), while there was an increase 
of average urinary ketones (from 0.18 mg/dl to 59.9 mg/dl). Similar metabolic changes 
occurred in 9 healthy volunteers following the same 5-day FLA regimen, but not in a 
control population of 13 breast cancer patients receiving ChT alone. Of note, we 
observed qualitatively and quantitatively similar modifications of key metabolic 
parameters during eight subsequent FLA cycles, thus excluding the occurrence of 
metabolic adaptation to the FLA. Similar metabolic changes occurred in patients with 
different tumor types, and regardless of concomitant treatment (ChT vs other treatment 
types) or tumor stage (limited-stage vs advanced). 
In addition to promising biological and immunomodulatory effects40, cyclic FLA 
resulted in complete and long-lasting tumor remissions in some patients with highly 
aggressive, deadly and treatment-resistant neoplasms, such as pancreatic cancers, lung 
cancers and triple-negative breast cancer41. These “excellent tumor responses” were 
observed when cyclic FLA was combined with chemotherapy or immunotherapy.  
Based on these promising results, on the rationale for using the FLA as an 
investigational dietary intervention to boost the antitumor efficacy of chemotherapy in 
TNBC, and also based on results of preclinical studies showing that calorie restriction 

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synergizes with the antidiabetic compound metformin to cause metabolic crisis and to 
induce apoptosis in cancer cells42, in June 2020 we initiated a phase II, randomized 
clinical trial (BREAKFAST; NCT04248998) in which we aimed at investigating the 
antitumor activity of combining neoadjuvant anthracycline-taxane chemotherapy with 
cyclic FLA, plus/minus metformin, in patients with stage I-III TNBC. Between June 
2020 and February 2022, the study has enrolled 30 patients, and it was amended to be 
transformed into a multicentric study to accelerate patient enrollment. However, after 
changes in the standard-of-care therapy of stage II-III TNBC patients, and in particular 
after the addition of carboplatin and pembrolizumab to neoadjuvant anthracycline-
taxane regimens, we decided to prematurely interrupt the BREAKFAST study, and to 
design a new trial to combine standard chemoimmunotherapy with cyclic FLA (please 
see below).  
 
Rationale for combining calorie restriction with immunotherapy 
Preclinical studies showed that restricting the dietary intake of carbohydrates or 
proteins affects the frequency and activation status of myeloid and lymphoid cells in 
peripheral organs and in tumor microenvironment. For instance, in 4T1 and Py8119 
TNBC cell mouse transplants, glycolysis inhibition prevented G-CSF and GM-CSF 
production by cancer cells, thus reducing myeloid-derived suppressor cells (MDSCs) 
mobilization and their recruitment to the tumor site43. In addition, short-term fasting 
can downmodulate monocytes in peripheral blood and spleen of healthy mice by 
inhibiting systemic CCL2 production44. Coherently, in a syngeneic murine 4T1 TNBC 
model, the combination of FLA and chemotherapy (doxorubicin) determined the 
accumulation of cytotoxic CD3+/CD8+ T cells in the tumor bed, which were shown to 
be crucial to mediate the synergic effect between FLA and chemotherapy.26 As 
observed in a preclinical proof-of-concept study, short-term starvation could synergize 
with PD-1 blockade in murine models of lung cancer45 by promoting a decrease of Treg 
cells and an increase of effector T cells in the tumor microenvironment. Of note, the 
observed enhancement of tumor sensitivity to PD-1 blockade occurring by short-term 
starvation was mediated by an antitumor CD8 T cell response and was strictly 
dependent upon the inhibition of IGF-1–IGF-1R signaling in tumor cells. 
These desirable immunomodulatory effects have been recently confirmed and largely 
expanded in cancer patients enrolled in the recently published NCT03340935 trial40.  
Indeed, at the end of the first FLA cycle, flow cytometry analysis of peripheral blood 
mononuclear cells (PBMCs) revealed a significant decrease of total monocytes 
(CD14+) and of two highly immunosuppressive monocyte subsets, i.e., those lacking 
HLA-DR expression (CD14+HLA-DRneg), acknowledged as monocytic myeloid-
derived suppressor cells (M-MDSCs) and CD14+ cells expressing PD-L1 (CD14+PD-
L1+). The FLA also reduced low-density CD15+ granulocytes, which include 
polymorphonuclear MDSCs (PMN-MDSCs). Interestingly, the observed modifications 
in myeloid sub-populations were similar in patients undergoing the FLA in combination 
with ChT or with other standard antitumor therapies, but not in a control population of 
BC patients treated with ChT alone, which even resulted in a boost of CD14+PD-L1+ 
cells. In 8 healthy volunteers the FLA reduced CD14+, CD14+HLA-DRneg, 
CD14+PD-L1+ and CD15+ cells similarly to what observed in cancer patients.  FLA-

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induced reduction of myeloid cell subsets was paralleled by an increase of activated 
CD8+ T cells (co-expressing PD1 and CD69) and cytolytic CD3negCD16+CD56dim 
NK cells, while CD3+ T cells expressing the high affinity IL-2 receptor (CD3+CD25+ 
cells), which can be associated with Treg activity, were reduced. As in the case of 
myeloid cells, changes in lymphocytic populations occurred independently of 
concomitant antitumor therapies, and they were similarly observed in 8 healthy 
volunteers in a similar way as observed in patients. During the second FLA cycle, 
myeloid and lymphoid PBMC populations were modulated in a similar way as during 
the first FLA cycle, thus excluding the adaptation to FLA-induced systemic 
immunological effects. Inflammatory cytokines such as CCL2, G-CSF and IL-6, which 
are involved in myeloid cell mobilization from the bone marrow, were significantly 
reduced after the FLA. Finally, and even more importantly, an unplanned, interim 
analysis of the ongoing DigesT trial (NCT03454282) confirmed most of these systemic 
immunological modifications at the tumor level in 22 BC patients, where the FLA 
resulted in an increase of tumor-infiltrating T cells, activated T cells, lymphocytes with 
a memory phenotype, NKT and activated dendritic cells, paralleled by a decrease of 
M2 macrophages. Together, these data indicate that the FLA, alone or in combination 
with standard antitumor therapies, down-regulates immunosuppressive myeloid cell 
subsets, while at the same time increasing effector cells with an activated phenotype.  
3.3 Study rationale and purpose 
TNBC is the most aggressive subtype of breast cancer. TNBC patients who achieve 
pCR during neoadjuvant chemo-immunotherapy have significantly lower rates of 
disease recurrence or death. Preclinical studies indicate that combining nutrient 
starvation, in the form of cycles of FLA, with anthracycline- or platinum-based 
chemotherapy remarkably increases the therapeutic index of chemotherapy against 
murine and human models of breast cancer, including models of TNBC. In particular, 
the chemotherapy-fasting/FLA combination increases the anticancer activity of 
chemotherapy, while reducing treatment-related adverse events (AEs). Moreover, the 
FLA has demonstrated potent and desirable immunomodulatory effects both in in vivo 
studies and in patients with cancer, and the activation of antitumor immunity is a crucial 
mediator of the anticancer effects of the FLA, either alone or in combination with 
chemotherapy. Therefore, there is a strong biological rationale to combine cyclic FLA 
with ICIs in cancer therapy.   
Based on these data, we hypothesize that combining the FLA with standard-of-care, 
preoperative, anthracycline-taxane-carboplatin chemotherapy plus Pembrolizumab can 
increase the rate of pCR in a population of patients with stage II-III TNBC.  
 

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4 
STUDY DESIGN 
4.1 Description of study design 
This is an Italian, multicenter, open-label, two-arm, comparative, randomized phase II 
study. This study is designed to investigate if the addition if the experimental metabolic 
intervention consisting in cycles of FLA, as administered every three weeks up to a 
maximum of 8 consecutive cycles, is able to increase the anticancer activity of standard 
preoperative chemo-immunotherapy consisting of antracycline-taxane-carboplatin-
based chemotherapy plus pembrolizumab in patients with treatment naïve, localized 
(tumor stage T1c AND nodal stage N1-2, or tumor stage T2-4 AND nodal stage N0-2) 
invasive Triple Negative Breast Cancer (HER2 negative, ER <1%, PgR <1%). Bilateral 
and/or multifocal primary tumor is allowed, as well as inflammatory breast cancer, and 
the tumor with the most advanced T stage should be used to assess the eligibility. If 
multi-focal/multi-centric disease, TNBC needs to be confirmed for each focus. The 
primary study endpoint is pathologic complete response (pCR). 
Patients will be randomly allocated to one of the following treatment arms:  
 
- 
Arm A (control arm): 12 consecutive cycles of weekly paclitaxel plus 
carboplatin (PCb) combined with 4 triweekly cycles of Pembrolizumab, 
followed by 4 consecutive cycles of triweekly anthracycline (doxorubicin or 
epirubicin)-cyclophosphamide (AC or EC) chemotherapy combined with 4 
triweekly cycles of Pembrolizumab. This combination treatment will be further 
referred to as “standard treatment”.  
- 
Arm B (experimental arm): standard treatment in combination with up to a 
maximum of 8 consecutive triweekly cycles of 5-day FLA. 
Enrolled patients will be randomized in a 1:1 ratio and stratified according to a) disease 
stage: stage II (T1N1, T2N0, T2N1, T3N0) vs. stage III (T3N1; any T4; any N2); b) 
patient body mass index (BMI ≥25 kg/m2 vs <25 kg/m2). 
After completion of the experimental preoperative protocol, patients will undergo 
surgery between 14 and 28 days after the last chemotherapy administration. 
After surgery, patients will receive 9 additional triweekly pembrolizumab 
administration at the same dosage, and regardless of the pathologic tumor response 
(pCR yes vs. no). After surgery, patients may receive local radiotherapy, depending on 
the pathological stage and according to local and international guidelines.  
 
Figure 1: Study design. 

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5 
OBJECTIVES AND ENDPOINTS 
5.1 Objectives 
Primary objective  
To investigate if the experimental treatment, consisting of the administration of 
preoperative chemo-immunotherapy (PCb plus Pembrolizumab, followed by AC or EC 
plus Pembrolizumab) in combination with cyclic triweekly FLA, is able to increase the 
rate of pCR when compared to standard preoperative chemo-immunotherapy (PCb plus 
Pembrolizumab, followed by AC or EC plus Pembrolizumab).  
Secondary objectives 
- 
To assess the tolerability of the experimental treatment when compared to the 
control treatment, and in particular to evaluate the incidence of severe (grade 
3/4) adverse events (AEs) 
- 
To evaluate the safety of the experimental treatment, and in particular to 
evaluate treatment-related AEs 
- 
To study patient compliance, as defined as the ability to adhere to the prescribed 
FLA regimen in combination with chemoimmunotherapy pharmacological 
treatment based on the analysis of daily food diaries filled during each of 5 days 
while on FLA 
- 
To estimate clinical tumor responses, as assessed through clinical examination, 
breast US and, when available, breast MRI, according to RECIST 1.1 criteria 
after the first part of the neoadjuvant treatment (PCb plus Pembrolizumab 

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plus/minus FLA) and before surgery, in the experimental treatment arm when 
compared to the control arm 
- 
To estimate patient disease-free survival (DFS) in the experimental treatment 
arm when compared to the control arm 
- 
To estimate patient event-free survival (EFS) in the experimental treatment arm 
when compared to the control arm 
- 
To evaluate distant metastasis-free survival (DMFS) in the experimental 
treatment arm when compared to the control arm 
- 
To evaluate Overall Survival (OS) in the experimental treatment arm when 
compared to the control arm 
Exploratory objectives 
- 
To study the short-term (intra-cycle) and long-term (across subsequent cycles) 
effects of the experimental treatment on systemic metabolism by evaluating 
several blood metabolites, including plasma glucose, insulin, insulin-like 
growth factor 1 (IGF-1) levels, serum amino acids and whole blood and plasma 
lipid profile. 
- 
To study the short-term (intra-cycle) and long-term (across subsequent cycles) 
effects of the experimental treatment on blood immune populations. 
- 
To correlate experimental treatment-induced modifications of systemic 
metabolism and immunity with treatment activity, in terms of pCR, DFS, EFS, 
DMFS and OS. 
- 
To correlate tumor gene expression profiles with the efficacy of the 
experimental treatment, defined on the basis of rates of pCR.  
- 
To correlate the tumor mutational status with the efficacy of the experimental 
treatment, defined as the rate of pCR.  
- 
To evaluate the differential activity of FLA in subgroups with different 
pathologic residual cancer burden (RCB). 
- 
To explore the use of point-of-care ultrasound in the assessment of patients 
sarcopenia during anticancer treatments. 
- 
To evaluate patient-reported tolerability of the experimental treatment, and in 
particular treatment-related AEs (PROs), and compare it with physician-
reported tolerability. 
6 
ENDPOINTS 
6.1 Primary endpoints  
- 
Pathologic complete response (pCR), as defined as the absence of residual 
tumor cells in both breast tissue and axillary lymph nodes (ypT0/ypTis ypN0).  
6.2 Secondary endpoints 
Efficacy endpoints 

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- 
Disease free survival (DFS), as defined as the time from surgery to tumor 
recurrence, either local or distant, or death 
- 
Event-free survival (EFS), as defined as the time from the date of randomization 
to the first documentation of progressive disease or death  
- 
Distant metastasis free survival (DMFS), as defined as the time from surgery to 
the occurrence of distant metastases or death 
- 
Overall Survival (OS), as defined as the time from randomization to the date of 
death. Patients alive at the time of data cut-off and analysis will be censored at 
their last contact date 
Compliance endpoints 
- 
Dose-intensity, that is the dose of effective drug administrated per unit of time 
(e.g., mg/m2/week) 
- 
Percentage of patients with drug dose and/or time modifications 
- 
Percentage of patients with experimental dietary regimen modifications 
- 
Percentage of premature withdrawals 
Safety endpoints 
- 
Incidence, nature, severity and seriousness of AEs, according of NCI-CTCAE, 
version 5.0 
- 
Maximum toxicity grade experienced by each patient for each specific toxicity  
- 
Percentage of patients experiencing grade 3-4 toxicity for each specific toxicity 
- 
Patients with at least a SAE 
Exploratory endpoints 
- 
Metabolic biomarkers (e.g., plasma glucose, insulin, insulin-like growth factor 
1 (IGF-1) levels, serum amino acids and whole blood and plasma lipid profile, 
fecal microbiota) assessed at baseline and at each chemotherapy cycle  
- 
Role of key DNA repair, metabolic, autophagy and immunologic pathways in 
the efficacy of the experimental treatments, defined as the rate of pCR.  
7 
STUDY POPULATION 
7.1 Patient population 
This study will enroll patients with treatment-naïve, stage II-III (tumor stage T1c AND 
nodal stage N1-2, or tumor stage T2-4 AND nodal stage N0-2), invasive Triple 
Negative invasive Breast Cancer (HER2 negative, ER <1%, PgR <1%) who are 
candidate to receive neoadjuvant carboplatin-paclitaxel-anthracycline-pembrolizumab 
chemoimmunotherapy. Bilateral and/or multifocal primary tumor is allowed, as well as 
inflammatory breast cancer, and the tumor with the most advanced T stage should be 
used to assess the eligibility. If multi-focal/multi-centric disease, TNBC needs to be 
confirmed for each focus. 

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The investigators involved in the study must ensure that only patients who meet all the 
following inclusion and none of the exclusion criteria will be offered the experimental 
treatment. 
All young women with childbearing potential will be proposed to undergo fertility 
preservation methods before study treatment initiation and to accept the use of adequate 
contraceptive measures up to six months after the last treatment administration. In case 
of pregnancy desire, they will also be offered the use of GnRH analog in combination 
with the experimental study treatment to reduce the risk of premature ovarian failure 
and to reduce damage on fertility46. 
Patients enrolled in this study are not allowed to participate in additional parallel 
investigational drug or device studies. 
7.2 Inclusion criteria 
Patients eligible for inclusion in this study must meet all of the following criteria: 
1. Female sex 
2. Age ≥ 18 and ≤ 75 years. 
3. Evidence of a personally signed and dated informed consent document (ICD) 
indicating that the patient has been informed of all pertinent aspects of the study 
before enrollment  
4. Willingness and ability to comply with the prescribed FLA regimen, the 
scheduled visits, treatment plans, laboratory tests and other procedures. 
5. Histologically confirmed diagnosis of invasive TNBC candidate to neoadjuvant 
chemo-immunotherapy and subsequent curative surgery. On the basis of 
International Guidelines, TNBC is defined by absent or minimal (<1%) 
expression of oestrogen and progesterone receptors at IHC, and absence of 
HER2 protein over-expression and HER2 gene amplification, as defined as an 
IHC score of 0, 1+, or an IHC score of 2+ with in situ hybridization (ISH) 
analysis excluding HER2 gene amplification. The expression of hormone 
receptors (ER and PgR) and HER2 will be evaluated through 
immunohistochemistry (IHC), according to International Guidelines47,48 
6. Availability of a formalin-fixed, paraffin-embedded (FFPE) block containing 
tumor tissue, or at least 7 unstained tumor slides.  
7. Patients with tumor stage T1c AND nodal stage N1-2, or tumor stage T2-4 AND 
nodal stage N0-2 according to TNM.  
8. Presence of an Eastern Cooperative Oncology Group (ECOG) performance 
status 0 or 1. 
9. Presence of adequate bone marrow and organ function as defined by the 
following laboratory values:   
a. ANC ≥ 1.5 x 103/l 
b. platelets ≥ 100 x 103/l 
c. hemoglobin ≥ 9.0 g/dl 
d. calcium (corrected for serum albumin) within normal limits or ≤ grade 1 
according to NCI-CTCAE version 5.0 if not clinically significant 
e. potassium within the normal limits, or corrected with supplements 
f. creatinine < 1.5 ULN 

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g. blood uric acid < 10 mg/dl  
h. ALT and AST ≤ 2 x ULN  
i. total bilirubin < 1.5 ULN except for patients with Gilbert syndrome who may 
only be included if the total bilirubin is < 3.0 x ULN or direct bilirubin < 1.5 
x ULN 
j. Fasting glucose ≤ 250 mg/dl. 
10. Female patients of childbearing potential must agree to sexual abstinence or to 
use two highly effective methods of contraception throughout the study and for 
at least six months after the end of the FLA. Abstinence is only acceptable if it 
is in line with the preferred and usual lifestyle of the patient. Examples of 
contraceptive methods with a failure rate of < 1% per year include tubal ligation, 
male sterilization, hormonal implants, established, proper use of combined oral 
or injected hormonal contraceptives, and certain intrauterine devices. 
Alternatively, two methods (e.g., two barrier methods such as a condom and a 
cervical cap) may be combined to achieve a failure rate of < 1% per year. Barrier 
methods must always be supplemented with the use of a spermicide. A patient 
is of childbearing potential if, in the opinion of the Investigator, she is 
biologically capable of having children and is sexually active. 
11. Female patients are not of childbearing potential if they meet at least one of the 
following criteria:  
a. Have undergone a documented hysterectomy and/or bilateral 
oophorectomy 
b. Have medically confirmed ovarian failure  
c. Achieved post-menopausal status, defined as: ≥ 12 months of non-
therapy-induced amenorrhea or surgically sterile (absence of ovaries); 
in women <45 years of age FSH level in the postmenopausal range may 
be used to confirm a post-menopausal state in women not using 
hormonal contraception or hormonal replacement therapy.  
 
7.3 Exclusion criteria 
Patients eligible for this study must not meet any of the following criteria: 
1. Prior systemic treatment for breast cancer or other malignancies within 5 years 
of treatment enrollment, except for adequately treated basal cell or squamous 
skin cancer or in situ cervical cancer. Other malignancies diagnosed more than 
5 years before the diagnosis of breast cancer must have been radically treated 
without evidence of relapse at the moment of patient enrollment in the trial. 
2. Prior treatment with anthracyclines 
3. Prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent or with an 
agent directed to another co-inhibitory T-cell receptor (e.g., CTLA-4, OX-40, 
CD137)  
4. Body mass index (BMI) < 19 kg/m2. 
5. History of alcohol abuse. 
6. Non-intentional weight loss ≥ 5% in the previous 3 months, unless the patient 
has a BMI > 22 kg/m2 and weight loss has been lower than 10% at the time of 

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enrollment in the study; or non-intentional weight loss of ≥ 10% in the previous 
3 months, unless the patient has a BMI > 25 kg/m2 and weight loss has been 
lower than 15% at the time of the enrollment in the study. In both cases, weight 
must have been stable for at least one month before study enrollment. 
7. Active pregnancy or breast feeding. 
8. Known active B or C hepatitis or human immunodeficiency virus (HIV) 
infection, or occasional finding of active hepatitis B/C infection during 
screening tests before chemotherapy initiation, as defined as positive 
polymerase chain reaction (PCR) testing for HBV-DNA and HCV-RNA and 
qualitative PCR for HIV-RNA, or requiring active treatment at study 
enrollment. 
9. Serious infections in the previous 4 weeks before the FLA initiation, including, 
but not limited to, potential hospitalizations for complications of infections, 
bacteriemia or serious pneumonitis.  
10. Active autoimmune diseases requiring systemic treatments (e.g., systemic 
steroids or immune suppressants). Replacement therapy (e.g., thyroxine, 
insulin, or physiologic corticosteroid replacement therapy for adrenal or 
pituitary insufficiency) is not considered a form of systemic treatment.  
11. Active chronic therapy with systemic steroids at a dose ≥ 10 mg per day of 
prednisone or equivalent at study enrollment. 
12. Diagnosis of type 1 or 2 diabetes mellitus requiring pharmacologic therapy 
(including, but not limited to, insulin or insulin secretagogues), with the 
exception of metformin. A diagnosis of type 2 diabetes mellitus not requiring 
pharmacological treatments, or only requiring treatment with metformin, based 
on the judgment of a diabetologist, is compatible with patient enrollment in the 
trial. 
13. Anamnesis of clinically significant heart disease including: 
a. angina pectoris, coronary bypass, symptomatic pericarditis, myocardial 
infarction in the previous 12 months from the beginning of experimental 
therapy; 
b. congestive heart failure (NYHA III-IV). 
14. Anamnesis of clinically meaningful cardiac arrhythmias, such as ventricular 
tachycardia, chronic atrial fibrillation, complete bundle branch block, high 
grade atrio-ventricular block like bi-fascicular block, type II Mobitz and third 
grade atrio-ventricular block, nodal arrhythmias, supra-ventricular arrhythmia. 
15. Left ventricular ejection fraction lower than 50% at the cardiac scan with 
radionuclides or at echocardiography. 
16. Previous episodes of symptomatic hypotension leading to loss of consciousness. 
17. History of eating disorders (anorexia, bulimia). 
18. Baseline plasma fasting glucose ≤ 60 mg/dL. 
19. Medical or psychiatric comorbidities rendering the patient not candidate to the 
clinical trial, according to the investigator’s judgement. 
20. Other cardiac, liver, lung or renal comorbidities, not specified in the previous 
inclusion or exclusion criteria, but potentially exposing the patient to a high risk 
of lactic acidosis. 
21. Known history of active TB (Bacillus Tuberculosis). 

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8 
EXPERIMENTAL INTERVENTION 
8.1 Study intervention 
All patients enrolled in this study will receive preoperative chemo-immunotherapy, 
consisting in 12 consecutive cycles of weekly paclitaxel (at a dosage of 80 mg/m2) plus 
carboplatin (area under the curve -AUC- of 1.5 mg/mL per min), plus triweekly 
Pembrolizumab (200 mg), followed by 4 triweekly cycles of doxorubicin (at a dosage 
of 60 mg/m2) or epirubicin (at a dosage of 90 mg/m2) plus cyclophosphamide (at a 
dosage of 600 mg/m2 plus Pembrolizumab (200 mg). At enrollment, patients will be 
randomized in a 1:1 ratio to receive (arm B) or not (arm A) additional treatment 
consisting of triweekly 5-day FLA, up to a maximum of 8 consecutive cycles. At 
randomization, patients will be stratified according to tumor stage (II vs. III) and patient 
BMI (≥25 kg/m2 vs <25 kg/m2). 
8.2 Chemo-immunotherapy 
Patients 
will 
receive 
a 
sequential 
carboplatin-taxane-pembrolizumab 
and 
anthracycline-cyclophosphamide-pembrolizumab neoadjuvant chemo-immunotherapy 
program, consisting of two parts: 
1) Paclitaxel 80 mg/m2 plus Carboplatin AUC 1.5, weekly for 12 cycles (PCb) plus 
Pembrolizumab 200 mg flat dose every three weeks (part 1: approximately three 
months of therapy).  
2) Doxorubicin 60 mg/m2 or Epirubicin 90 mg/m2 plus Cyclophosphamide 600 
mg/m2 (AC or EC) plus Pembrolizumab 200 mg flat dose on day 1 every 21 days 
for 4 cycles (part 2: approximately another three months of therapy). 
All chemotherapy drugs will be administered i.v.. Patients may receive oral netupitant 
plus palonosetron (Akynzeo) plus 8 mg of i.v. dexamethasone the day of anthracycline-
containing chemotherapy administration to prevent the occurrence of nausea and 
vomiting. Prophylaxis for potential paclitaxel-induced allergic reactions may include 
an oral antihistamine on the night before chemotherapy (e.g., cetirizine 10 mg), as well 
as an oral antihistamine plus 8 mg of i.v. dexamethasone on the day of treatment 
administration. However, premedication schemes used at each participating center can 
be chosen at the discretion of the treating physician of that specific site, with the 
exception of dexamethasone, which should be administered at 8 mg dosage only on day 
1 of each treatment administration, and eventually escalated only in the case of adverse 
events (e.g., severe nausea, severe vomiting, allergic reactions). 
 
NOTE: To avoid interference with the metabolic effects induced by the FLA 
(including a reduction of blood glucose and insulin levels), carboplatin must be 
dissolved in saline (0.9% NaCl solution), and not in 5% glucose-containing 
solution. 

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8.2.1 Pembrolizumab prescribing 
Pembrolizumab has been approved by international regulatory agencies (i.e. FDA, 
EMA) as part of neoadjuvant treatment for early-stage TNBC (tumor stage T1c, nodal 
stage N1-2, or tumor stage T2-4, nodal stage N0-2). In Italy this treatment is actually 
prescribed through a compassionate use program. Centers will administer 
Pembrolizumab within the study through this compassionate program, after obtaining 
the specific additional informed consent provided by the pharmaceutical company. In 
case of formal registration and reimbursement from Italian regulatory agency (AIFA) 
during the course of the study, Pembrolizumab will be administered through National 
Health System. 
8.3 FLA scheme (arm B) 
Patients randomized to Arm B (experimental arm) will receive up to a maximum of 
eight cycles of FLA in combination with chemo-immunotherapy. 
Each FLA cycle will consist of 5 consecutive days of a specific FLA scheme, which 
will be repeated with a three-week interval. The FLA will consist of a plant-based, low-
calorie (about 600 Kcal on day 1; about 300 Kcal on day 2 to 5), low-protein, low-
carbohydrate diet. This is the same FLA scheme whose safety, feasibility, metabolic 
and immunological effects have been evaluated in the recently conducted 
NCT03340935 trial at our Institution35,40,41, and which has been combined with 
preoperative chemotherapy in the recently interrupted BREAKFAST trial 
(NCT04248998). 
All patients will be prescribed the same FLA regimen. No modifications nor 
personalization of the prescribed FLA regimen are allowed. 
The first FLA cycle will start two days prior to the day of first chemo-immunotherapy 
cycle administration and will continue for two more days after chemotherapy. In the 
absence of significant contraindications or severe adverse events, subsequent FLA 
cycles will recur with three-week intervals and will maintain the same timing with 
respect to chemo-immunotherapy administration. In case that the FLA is delayed for 
any reason, chemo-immunotherapy will be administered according to the schedules 
program; in case of FLA re-start, the FLA must be necessarily administered 
concomitantly with cycles 1st, 4th, 7th or 10th of weekly PCb, and concomitantly with 
triweekly AC/EC, and it must be started two days before chemotherapy administration. 
In particular, the FLA cannot be combined with cycles 2nd, 3rd, 5th, 6th, 8th, 9th, 11th or 
12th of PCb, and it cannot be initiated in days other than -2 of each triweekly AC/EC 
cycle. 
The full FLA scheme, with the approximated amount of calories contained in each 
food/beverage, is reported in Table 1. Mean caloric intake from individual FLA 
components were obtained from the Food Composition Database for Epidemiological 
Studies in Italy (“Food Composition Database for Epidemiological Studies in Italy” by 
Gnagnarella P, Salvini S, Parpinel M. Version 1.2015 Website http://www.bda-ieo.it/). 
Table 1 

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The FLA diet scheme 
Day 
Meal 
Food/beverage 
Day 1 
Breakfast 
One tea or non-caloric tisanes at the choice of the patient (without 
sugar or other sweeteners). In addition, possibility to drink one 
coffee (without sugar or other sweeteners). 
Lunch 
A dish of mixed vegetables (up to a total of 300 grams), including 
spinach, cabbage, savoy cabbage, cauliflower, broccoli, salad or 
zucchini, consumed as raw, boiled or steamed (72 KCal), together 
with a tablespoon (12 grams) of extra virgin olive oil (108 KCal). 
100 grams of whole wheat bread (224 KCal). 
Dinner 
100 grams of salad (lettuce, valerian salad, rocket salad, endive 
salad, chard, red and green radicchio, chicories, friarielli; 30 
KCal) with a teaspoon (6 grams) of extra virgin olive oil (54 
KCal); 250 grams of orange, apple or pear (100 KCal). 
Days 2-3-4 
 
Breakfast 
One tea or non-caloric tisanes at the choice of the patient, without 
sugar or other sweeteners. 
In addition, possibility to drink one coffee (without sugar or other 
sweeteners). 
Lunch 
Around 200 grams of salad (lettuce, valerian salad, rocket salad,  
endive salad, chard, red and green radicchio, chicories, friarielli; 
40 KCal) with a teaspoon of extra virgin olive oil (54 KCal).  
As an alternative, an equivalent amount of the following 
vegetables consumed as raw, boiled or steamed: spinach, cabbage, 
savoy 
cabbage, 
cauliflower, 
broccoli, 
zucchini, 
onions, 
artichokes.  
Dinner 
25 grams of shelled walnuts or 30 grams of almonds (180 KCal). 
Day 5 
Breakfast 
One tea or non-caloric tisanes at the choice of the patient (without 
sugar or other sweeteners). In addition, possibility to drink one 
coffee (without sugar or other sweeteners). 
Lunch 
A dish of mixed vegetables (up to a total of 300 grams), including 
spinach, cabbage, savoy cabbage, cauliflower, broccoli, salad or 
zucchini, consumed as raw, boiled or steamed (75 KCal), together 
with a teaspoon (6 grams) of extra virgin olive oil (54 KCal). 200 
grams of orange, apple or pear (100 KCal). 

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Dinner 
100 grams of salad (lettuce, valerian salad, rocket salad,  endive 
salad, chard, red and green radicchio, chicories, friarielli; 20 
KCal) with a teaspoon of extra virgin olive oil (54 KCal). 
 
Any food/beverage, including spices, not explicitly contained in this list must be 
avoided. 
Water intake will not be limited. Consuming around 2 liters of non-caloric beverages 
in the autumn-winter-spring seasons, and 3 liters during summer will be strongly 
recommended. A maximum of 2 tea cups, or 2 coffee cups, or unlimited herbal teas per 
day, as long as they are calorie-free, will be allowed. 
A maximum of half lemon is allowed during each of the five FLA days. 
The FLA will be repeated every 3 weeks, up to a maximum of 8 consecutive cycles, 
unless side effects requiring its temporary or permanent discontinuation do occur.  
8.4 
Dietary recommendation for patients randomized to arm A and 
maintenance diet during the refeeding periods for patients 
randomized to arm B 
All patients enrolled in this study will be recommended to follow a diet based on 
established dietary guidelines for cancer prevention and cancer survivors regardless of 
the randomization arm (World Cancer Research Fund (WCRF) 2018, American Cancer 
Society 2012, European Code Against Cancer 2014).  
Dietary recommendations will include: 
- 
eating plenty of whole grains, legumes, non-starchy vegetables and fruits; 
- 
choosing whole-grain breads, pasta and cereals (such as barley and oats) instead 
of the same products made from refined grains, and brown rice instead of white 
rice; 
- 
limiting high-calorie foods (high in sugar or fat) and avoiding sugary drinks; 
- 
avoiding processed meat and limiting red meat and foods high in salt; 
- 
choosing fish, poultry, or beans instead of red meat (beef, pork and lamb); 
- 
limiting alcohol intake. 
Arm B patients are invited to follow these recommendations as a maintenance diet 
between FLA cycles, while Arm A patients will be recommended to adhere to these 
guidelines for the whole treatment period. In the experimental arm, this maintenance 
diet is aimed at avoiding rebound increase in blood glucose and growth factor (e.g., 
insulin, IGF-1) concentration after the FLA completion, while at the same time 
stimulating regain of the weight lost during the FLA. Weight regain after the FLA is 
important to prevent progressive weight loss and loss of lean tissue. Preventing loss of 
lean mass is especially important to ensure the maintenance of patient health status, and 
to prevent the progressive degradation of muscle proteins, which may result in 
increased blood amino acid concentration and in tumor overfeeding. Therefore, no 
quantitative restrictions in food intake (i.e., total amounts and calorie content) will be 
imposed during the maintenance diet. Moreover, since 8 consecutive FLA could induce 
significant weight loss, patients will be monitored by an expert team of oncologists and 

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nutritionists (Dr. C. Vernieri, dr. G. Fucà, dr. F. Ligorio, dr. L. Zanenga) throughout 
the study protocol in order to modify dietary recommendations according to weight 
kinetics, body composition and treatment-related adverse events (i.e., diarrhea, nausea, 
vomiting). 
8.5 Trial Blinding/Masking and treatment allocation 
This is an open-label trial. Patients will be randomized to arm A or arm B in a 1:1 ratio. 
8.6 Stratification 
At randomization, patients will be stratified according to the two following factors: a) 
clinical disease stage according to TNM classification: stage II (T1N1, T2N0, T2N1, 
T3N0) vs. stage III (T3N1; any T4; any N2); b) patient BMI  (≥25 kg/m2 vs <25 kg/m2). 
8.7 Concomitant Medications (Allowed & Prohibited) 
Medications specifically prohibited in the exclusion criteria are not allowed during the 
trial. If there is a clinical indication for any of the prohibited medications, 
discontinuation from trial treatment may be required.  
Acceptable Concomitant Medications 
All treatments deemed necessary for a patient’s welfare may be administered at the 
discretion of the investigator in keeping with the community standards of medical care. 
Granulocyte colony stimulating factors may be administered if deemed necessary by 
treating oncologists and according to National/International Guidelines. All 
concomitant medications will be recorded on the case report form (CRF) including all 
prescription, over-the-counter (OTC), and i.v. medications and fluids. Documentation 
of drug dosage, frequency, route, and date may also be included on the CRF in case of 
changes occurring during the trial period. 
Prohibited Concomitant Medications 
Patients are prohibited from receiving the following therapies during the Screening and 
Treatment Phase of this trial: 
- 
Antineoplastic systemic treatments, except for the chemoimmunotherapy regimen 
explicitly specified in the study protocol. 
- 
Investigational treatments, including chemical, physical and dietary interventions, 
other than study treatment.  
- 
Chronic immunosuppressive drugs. 
- 
Hyperosmotic Laxatives (e.g., lactulose, sorbitol, polyethylene glycol), unless 
absolutely required for the treatment of acute constipation occurring during the 
study. 
- 
Herbal preparations/medications. These herbal medications include, but are not 
limited to: St. John’s wort, Kava, ephedra (ma huang), gingko biloba, Aloe Vera 
and its derivatives; artemisinin and its derivatives; dehydroepiandrosterone 

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(DHEA), yohimbe, ginseng and any type of dietary supplements whose chemical 
composition is unknown. Patients should stop using these herbal medications at 
least 10 days prior to treatment initiation. 
- 
Vitamin or food supplements, with the exception of Vitamin D. If the patient 
chronically takes dietary, salt- or food-containing supplements at the moment of 
study enrollment, she should discontinue them at least 10 days prior to treatment 
initiation. Continuation of chronic supplement intake during the FLA is only 
permitted if the supplements do not contain calories or high-dose vitamins and are 
limited to salts or low dosages of vitamins (below 1/10 of the Daily Recommended 
Dose). Any supplement with unknown chemical composition must be discontinued.  
- 
Therapeutic doses of warfarin sodium (Coumadin®). Therapeutic anticoagulation 
may be accomplished using low-molecular weight heparin. 
- 
Inhibitors of pancreatic alpha-amylase and lipase (e.g., Orlistat).  
- 
Anorectic drugs (e.g., dexamphetamine, phenmetrazine and benzphetamine). 
Rescue Medications & Supportive Care 
Patients should receive appropriate supportive care measures according to the 
investigator’s judgment as per clinical practice.  
9 
VISIT SCHEDULE AND ASSESSMENTS 
The Study Flow Chart (Table 2) lists the study procedures and indicates with an “X” 
the time points for visits and other evaluations to be performed. All screening 
assessments have to be completed no more than 28 days before initiation of the 
experimental treatment, with the exception of radiological disease evaluation (within 
28 days). Blood, urine, saliva and stool samples will be collected for metabolic, 
immunological, and microbiota analyses at scheduled time points. These procedures 
may also be performed at unscheduled time points if deemed clinically necessary by 
the investigator.

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Table 2: Study Flow Chart 
Tab 2a 
Assessment 
Screening 1 
Neoadjuvant 
Therapy 
(Tab 2b, 2c) 
Before Surgery 
(2- 3 weeks from the 
last dose of CT) 
SURGERY 
(within 4 weeks 
from the last 
dose of CT) 
Adjuvant 
Pembrolizumab 
(Tab 2d) 
EOT 
(within 4 weeks 
after the last 
Pembrolizumab 
administration) 
FOLLOW 
UP 20 
 
Signed Informed Consent2 
X 
 
 
 
 
 
 
Demographic data3 
X 
 
 
 
 
 
 
Breast Tumor Core Biopsy4 
X 
 
 
 
 
 
X22 
Complete Medical History 5 
X 
 
 
 
 
 
 
Physical Examination 6 
X 
 
X 
 
 
X 
X 
Weight, BMI and assessment 
of food intake7 
X 
 
X 
 
 
X 
 
Vital signs8 
X 
 
X 
 
 
X 
 
ECOG Performance status 
X 
 
X 
 
 
X 
 

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Hematology 9 
X 
 
X19 
 
 
X 
X21 
Blood chemistry 10 
X 
 
X19 
 
 
X 
X21 
T3, FT4 and TSH 
X 
 
X 
 
 
X 
X 
Serology 11 
X 
 
 
 
 
 
 
Urinalysis 12 
X 
 
X19 
 
 
X 
X21 
Saliva and Stool samples14 
 
 
X19 
 
 
X 
X21 
PBMC15 
and 
other 
immunological analysis 
 
 
X19 
 
 
X 
X21 
Metabolite and growth factor 
assessment15 
 
 
X19 
 
 
X 
X21 
Breast imaging 16 
X 
 
X 
 
 
 
X20 
(annually) 
Systemic 
radiological 
staging17 
X 
 
X 
 
 
 
 
Cardiac Examination (ECG 
and LVEF) 
X 
 
X 
 
 
 
 

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Pregnancy Test18 
X 
 
 
 
 
 
 
QoL Assessment 
X 
 
X 
 
 
X 
 
Food diaries22  
X22 
 
Throughout the study22 
 
 
Surgery23 
 
 
 
 
X 
 
 
Concomitant Medications 
 
Throughout the study 
 
 
Adverse events 
 
Throughout the study 
 
 
 
 
1. Screening/Baseline Assessments:  must be completed within 28 days prior to study treatment start unless otherwise specified as shown in the schedule of 
events above. Study treatment start is considered day 1 of the first FLA cycle 
2. Informed Consent: must be obtained prior to undergoing any study specific procedures. After enrollment, all patients will perform a visit (which can be a 
televisit) at the study Sponsor Institution (Fondazione IRCCS Istituto Nazionale dei Tumori), during which the patient will meet the multidisciplinary team 
responsible for the management of the FLA in all participating centers. 
3. Demographic: date of birth. 
4. For all the patients enrolled in the study, tumor specimen collected before the initiation of the experimental treatment must be submitted to the study Sponsor 
Institution to perform translational analyses. Preferably, a formalin-fixed, paraffin-embedded (FFPE) tumor tissue block should be provided. However, if it 
is not possible, the provision of at least seven (preferably 15) unstained slides with 4 μm-thick sections of the FFPE tumor tissue block is mandatory for 
study inclusion. An optional re-biopsy to collect fresh-frozen tumor material can be performed at the study Sponsor Institution, if technically feasible. 
Optionally, patients will be proposed to repeat a tumor biopsy at the study Sponsor Institution 14-21 days after the first FLA cycle completion, to collect 
FFPE and fresh-frozen tissue for early evaluation. A separate informed consent will be signed for optional biopsies.   
5. Medical History: Medical history should include history of disease process other than oncology (active or resolved) and concomitant illnesses.  

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6. Physical Exam: will include an assessment for emergent toxicities or changes from prior visits. These procedures may be conducted by the Investigator or 
his/her designee. 
7. Weight, BMI and food intake: weight will be measured in kilograms; BMI will be measured as weight (kg)/[height(m)]2 
8. Vital signs: will include blood pressure, body temperature and heart rate  
9. Hematology: to include platelet count, hemoglobin, and WBC count with 5- part differential. 
10. Blood chemistry: to include glucose, AST (SGOT), ALT (SGPT), serum creatinine, total bilirubin, alkaline phosphatase, LDH, chloride, uric acid, 
phosphorus, calcium, magnesium, potassium, sodium, BUN or urea. 
11. All patients will be tested for HBV and HCV. HBV serology will include HBsAg, antibodies against HBsAg, total HBcAg antibody (anti-HBcAb). HBV 
DNA should be obtained prior to the treatment phase in the case of HBsAg and/or anti-HBc positivity. HCV serology will include HCV antibody (anti-
HCV). HCV RNA should be obtained prior to the treatment phase if patient tests positive for anti-HCV.  
12. Urinalysis: physical-chemical analyses, including ketonuria. 
13. Saliva and Stool samples:  to be centralized at the Study Sponsor Institution. 
14. PBMC analysis: to include MDSCs, activated T cells, NK and DC cells. These analyses will be performed on samples centralized at the Study Sponsor 
Institution. 
15. Metabolite and growth factor assessment: total proteins, albumin, triglycerides; total and HDL cholesterol, prealbumin, transferrin, sideremia, ferritin, 
cholinesterase, cortisol, insulin, ®-hydroxybutyrate, IGF-1; IGFBP-1, IGFBP-2, IGFBP-3; VEGF-A, FGF; whole blood, plasma and red blood cell 
membrane lipid profiling; serum amino acid quantification. These analyses will be performed on samples centralized at the Study Sponsor Institution.  
16. Local staging: breast US is mandatory at each timepoint (screening, after PCb completion, before surgery); CE-MRI is mandatory at baseline and before 
surgery. Other radiological exams (mammography, contrast enhanced mammography (CESM), contrast enhanced MRI) can be performed at the discretion 
of the physician. 
17. Systemic radiological staging: at screening a CE total body CT scan (preferably) or, alternatively, a 18FDG Positron Emission Tomography (PET)/CT 
will be performed. Images will be centralized at the study Sponsor Institution. Before surgery, optionally, patient can undergo a CE total body CT scan to 
evaluate body composition.  
18. In women of childbearing potential: Serum is preferred at screening, but urine is allowed.  
19. These evaluations must be performed the day of surgery or no more than 24 hours before surgery 
20. Follow up: A visit will be performed every 6 months for the first 5 years, then a phone contact will be performed annually up to 10 years. Mammography 
will be performed annually as per local and international Guidelines.  
21. At disease recurrence At disease recurrence, if a diagnostic biopsy of a metastatic lesion is indicated as per clinical practice, optional core biopsy of the 
tumor will be performed for the collection of fresh-frozen tumor material. 

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22. All patients will be given food diaries to annotate the type and amount of food and beverage consumed at specific timepoints, i.e., i) during 7 consecutive 
days before treatment initiation ii) during one single day once a week (with the exception of the week in which patients receive chemotherapy plus 
Pembroluzumab plus/minus FLA) during the neoadjuvant treatment phase iii) during one single day every three weeks during the adjuvant treatment phase. 
In addition, patients randomized to arm B will receive a specific 5-day food diary to annotate the type and amount of food and beverage consumed during 
each FLA cycle; patients randomized to arm A will receive at the same timepoints 5-day food diaries to annotate the type and amount of food and beverage 
consumed. 
23. Surgical FFPE tumor specimens will be centralized to the study Sponsor Institution. Only for patients enrolled at the study Sponsor Institution, surgical 
fresh frozen tumor material will be collected as well. 
24. After surgery, patients will receive up to a maximum of nine triweekly Pembrolizumab cycles, according to local and international guidelines. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Tab 2b 
 
Neoadjuvant weekly Paclitaxel plus Carboplatin (PCb) x 12 cycles in combination with triweekly 
Pembrolizumab 
After 
PCb 
completion 
Cycles 
110, 4 
7; 10 
2; 3; 5; 6; 8; 9; 11; 12 
 
Day 
-2 
1 
3 
4 
-2 
1 
3 
1 
 
PCb (all arms) 
 
X 
 
 
 
X 
 
X 
 
Pembrolizumab (all arms) 
 
X 
 
 
 
X 
 
 
 
FLA (Arm B) 1 
X 
 
X 
 
 
Physical Examination 2 
X 
 
 
 
X 
 
 
 
 
Weight, BMI 
X 
 
 
 
X 
 
 
 
 
Hematology 3 
X 
 
 
X 
X 
 
 
X* 
 
Blood chemistry4 
X 
 
 
X 
X 
 
 
 
 
Urinalysis5 
X 
 
 
X 
 
 
 
 
 
 
Saliva and Stool samples6 
X 
 
 
X 
 
 
 
 
 
 

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PBMC7 and other immunological 
analysis 
X 
 
 
X 
 
 
 
 
 
 
Metabolite 
and 
growth 
factor 
assessment8 
X 
 
 
X 
 
 
 
 
 
 
LVEF 
 
 
 
 
 
 
 
 
 
X 
Breast imaging 9 
 
 
 
 
 
 
 
 
 
X 
QoL assessment 
 
 
 
 
 
 
 
 
 
X 
Food diaries11 
Throughout the study11 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Tab 2c 
 
Neoadjuvant AC or EC x 4 cycles (q3 weeks) 
Cycles 
1 
2-4 
Day 
-2 
1 
3 
4 
-2 
1 
3 
AC (all arms) 
 
X 
 
 
 
X 
 
Pembrolizumab (all arms) 
 
X 
 
 
 
X 
 
FLA (arm B) 1 
X 
 
X 
Physical Examination 2 
X 
 
 
 
X 
 
 
Weight, BMI  
X 
 
 
 
X 
 
 
Hematology 3 
X 
 
 
X 
X 
 
 
Blood chemistry4 
X 
 
 
X 
X 
 
 
T3, FT4 and TSH 
X 
 
 
 
 
 
 

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Urinalysis5 
X 
 
 
X 
 
 
 
Saliva and Stool samples6 
X 
 
 
X 
 
 
 
PBMC7 and other immunological analysis 
X 
 
 
X 
 
 
 
Metabolite and growth factor assessment8 
X 
 
 
X 
 
 
 
 
1. FLA Cycles will be performed every 3 weeks, and specifically during Cycles 1, 4, 7, 10 of weekly PCb plus pembrolizumab chemo-immunotherapy 
and every cycle of AC/EC plus pembrolizumab chemo-immunotherapy. Each FLA cycle begins at Day -2 and lasts until day 3 of every chemotherapy 
cycle (day -2; day -1; day 1, corresponding to the day of chemoimmunotherapy administration; day 2; day 3). Patients randomized to arm B will 
receive a specific 5-day food diary to annotate the type and amount of food and beverage consumed during each FLA cycle; patients 
randomized to arm A will receive at the same timepoints 5-day food diaries to annotate the type and amount of food and beverage consumed. 
2. Physical Examination will include an assessment for emergent toxicities or changes from prior visits, an evaluation of ECOG performance status, vital 
signs (blood pressure, body temperature and heart rate to be recorded in the sitting position after approximately 5 minutes of rest), weight, BMI, food 
intake.  Weight will be measured in kilograms; BMI will be measured as weight (kg)/[height(m)]2  
3. Hematology: to include platelet count, hemoglobin, and WBC count with 5- part differential (*allowed within 48 hours prior to the planned treatment 
administration)  
4. Blood chemistry: to include glucose, AST (SGOT), ALT (SGPT), serum creatinine, total and conjugate bilirubin, alkaline phosphatase, LDH, chloride, 
uric acid, phosphorus, calcium, magnesium, potassium, sodium, BUN or urea. 
5. Urinalysis: physical-chemical analyses, including ketonuria. 
6. Saliva and Stool samples:  to be centralized at the Study Sponsor Institution. 
7. PBMC analysis: to include MDSCs, activated T cells, NK and DC cells. These analyses will be performed on samples centralized at the Study Sponsor 
Institution. 

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8. Metabolite and growth factor assessment: total proteins, albumin, triglycerides; total and HDL cholesterol, prealbumin, transferrin, sideremia, ferritin, 
cholinesterase, cortisol, insulin, ®-hydroxybutyrate, IGF-1; IGFBP-1, IGFBP-2, IGFBP-3; VEGF-A, FGF; whole blood, plasma and red blood cell 
membrane lipid profiling; serum amino acid quantification. These analyses will be performed on samples centralized at the Study Sponsor Institution.  
9. Local staging: breast US is mandatory at each timepoint (screening, after PCb completion, before surgery). CE-MRI is mandatory at baseline and before 
surgery. Other radiological exams (mammography, contrast enhanced mammography (CESM), contrast enhanced MRI) may be performed at the 
discretion of the physician. 
10. An optional biopsy may be performed 14-21 days after the first PCb-pembrolizumab cycle  
11. All patients will be given food diaries to annotate the type and amount of food and beverage consumed at specific timepoints, i.e., i) during 7 consecutive 
days before treatment initiation ii) during one single day once a week (with the exception of the week in which patients receive chemotherapy plus 
Pembrolizumab plus/minus FLA) during the neoadjuvant treatment phase iii) during one single day every three weeks during the adjuvant treatment 
phase. In addition, patients randomized to arm B will receive a specific 5-day food diary to annotate the type and amount of food and beverage consumed 
during each FLA cycle; patients randomized to arm A will receive at the same timepoints 5-day food diaries to annotate the type and amount of food 
and beverage consumed. 
 
 
Tab 2d 
 
Adjuvant Pembrolizumab x 9 cycles (q3 weeks)* 
Cycles  
1 
2 
3 
4 
5 
6 
7 
8 
9 
Weight, BMI 
X 
 
 
X 
 
 
X 
 
 
Urinalysis1 
X 
 
 
X 
 
 
X 
 
 

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Saliva and Stool samples2 
X 
 
 
X 
 
 
X 
 
 
PBMC3 and other immunological analysis 
X 
 
 
X 
 
 
X 
 
 
Metabolite and growth factor assessment4 
X 
 
 
X 
 
 
X 
 
 
Food diaries5 
 
Throughout the study5 
*During the adjuvant phase, local blood analysis (hematology, biochemistry, thyroid function) will be performed at discretion of the physician, as per clinical 
practice. 
1. Urinalysis: physical-chemical analyses, including ketonuria. 
2. Saliva and Stool samples:  to be centralized at the Study Sponsor Institution. 
3. PBMC analysis: to include MDSCs, activated T cells, NK and DC cells. These analyses will be performed on samples centralized at the Study Sponsor 
Institution. 
4. Metabolite and growth factor assessment: total proteins, albumin, triglycerides; total and HDL cholesterol, prealbumin, transferrin, sideremia, ferritin, 
cholinesterase, cortisol, insulin, ®-hydroxybutyrate, IGF-1; IGFBP-1, IGFBP-2, IGFBP-3; VEGF-A, FGF; whole blood, plasma and red blood cell 
membrane lipid profiling; serum amino acid quantification. These analyses will be performed on samples centralized at the Study Sponsor Institution.  
5. All patients will be given food diaries to annotate the type and amount of food and beverage consumed during one single day every three weeks during 
the adjuvant treatment phase. 

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9.1 Screening and baseline (DAY -28 – DAY 0) 
The following procedures have to be performed at screening and/or baseline. Individual procedures 
are described in Section 10.  
- 
Written informed consent 
- 
Availability of Breast Tumor Core Biopsy (including the assessment of ER and PgR status by 
IHC, assessment of HER2 status by IHC and, in case, by ISH, assessment of tumor cell 
proliferation measurement through IHC of Ki-67, assessment tumor grade and TILs). For all 
the patients enrolled in the study, a tumor specimen collected before the initiation of the 
experimental treatment must be submitted to the study Sponsor Institution to perform 
translational analyses.  Preferably, a formalin-fixed, paraffin-embedded (FFPE) tumor tissue 
block should be provided. However, if it is not possible, the provision of at least seven 
(preferably 15) unstained slides with 4 μm-thick sections of the FFPE tumor tissue block is 
mandatory for study inclusion, An optional re-biopsy of the primary tumor can be performed 
at the study Sponsor Institution, if technically feasible, to collect fresh-frozen tumor material 
before study treatment(s) initiation. 
- 
Demographics and medical history 
- 
Disease details and diagnosis  
- 
Local staging: breast US is mandatory at each timepoint (screening, after PCb completion, 
before surgery). CE-MRI is mandatory at baseline and before surgery. Other radiological exams 
(mammography, contrast enhanced mammography (CESM), contrast enhanced MRI) may be 
performed at the discretion of the physician. 
- 
Systemic radiological staging: a CE total body CT scan (preferably) or, alternatively, a 
18FDG Positron Emission Tomography (PET)/CT will be performed. Images will be 
centralized at the study Sponsor Institution..  
- 
Eligibility criteria (Inclusion/Exclusion Criteria, see Paragraphs 7.2, 7.3) 
- 
Randomization procedure 
- 
Clinical and physical examination, assessment of blood pressure (BP), vital signs, 
nutritional/body composition 
- 
Cardiac examination: Electrocardiograph (ECG), Left ventricular ejection fraction (LVEF) 
by echocardiography or multi-gated scintigraphic scan (MUGA scan)  
- 
Laboratory tests including hematology, blood chemistry, TSH, T3 and T4, and hepatitis 
serology 
- 
Concomitant medications 
- 
Serum or urine pregnancy test in women of child-bearing potential (a negative serum 
pregnancy test outside the 14-days window must be confirmed with another negative 
pregnancy test)  
- 
Nutritional/body composition evaluation 
- 
Quality of life (QoL) assessment 
After enrollment and randomization all patients will perform a visit at the study Sponsor Institution 
(Fondazione IRCCS Istituto Nazionale dei Tumori), which can be also performed virtually (tele-
visit), during which the patient will meet the multidisciplinary team, composed of physicians and a 
biologist nutritionist, who will be responsible for the management of the FLA in all participating 

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centers. During this visit, the patient will receive detailed information about FLA composition, and 
how to prevent/manage adverse events related to the experimental treatment.  
In addition, each patient may undergo an optional core biopsy of the primary tumor at the study 
Sponsor Institution for the conduction of translational analyses in tumor samples in fresh or fresh-
frozen tumor specimens (see Section 14). 
9.2 On Treatment Procedures  
Patients randomized to arm B will undergo 5-day FLA cycles every 3 weeks, i.e., concomitantly with 
cycles n.1, 4, 7 and 10 of weekly PCb chemotherapy, i.e., at those cycles corresponding to the 
administration of pembrolizumab, and concomitantly with each of four triweekly AC/EC plus 
Pembrolizumab cycles.  
Before any systemic treatment administration, all patients will undergo: 
- 
assessment of blood tests including hematology tests 
- 
evaluation, recording and grading of treatment related-adverse events 
- 
evaluation of concomitant medications 
 
Before each FLA cycle, all patients will undergo: 
- 
Physical Examination, including an assessment for arising toxicities or changes from prior 
visits, an evaluation of ECOG PS, vital signs, weight, BMI, food intake.   
- 
Blood evaluations, including blood cell counts and chemistry laboratory tests in peripheral 
vein blood collected after at least 8-hour fasting 
- 
Assessment and grading of treatment-related adverse events 
- 
Evaluation of concomitant medications.  
Additionally, before and after the FLA cycles associated with the first and the fourth weekly 
administration of PCb (i.e., first and second Pembrolizumab cycles), as well as before and after the 
first AC/EC plus Pembrolizumab cycle (days -2 and +3 of these cycles), the following evaluations 
will be performed:  
- 
Quantification of blood metabolites and growth factors; immunological evaluations in 
peripheral blood after at least 8-hour fasting 
- Urinalysis with urinary ketone body dosage 
- Stool sampling  
- 
Saliva sampling 
After the completion of 12 PCb chemotherapy cycles (i.e., ~ after three months from treatment 
initiation), patients will undergo (Table 2): 
- 
Breast and regional nodes assessment with breast US. Other radiological exams 
(mammography, contrast enhanced mammography (CESM), contrast enhanced MRI) may be 
performed at the discretion of the physician. 
- 
Quality of life (QoL) assessment 
- 
TSH, T3 and T4 measurement (it can be performed with the blood collection performed before 
the first AC/EC cycle) 

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9.3 Before Surgery 
Before surgery, which will be scheduled between 2 to 4 weeks from the last dose of neoadjuvant 
chemotherapy, the following assessments should be performed: 
- 
Physical Examination including an assessment for arising toxicities or changes from prior 
visits, an evaluation of ECOG PS, vital signs, weight, BMI, food intake. 
- 
Breast US and CE-MRI are mandatory. Other radiological exams (mammography, contrast 
enhanced mammography (CESM), contrast enhanced MRI) may be performed at the 
discretion of the physician 
- 
Not mandatory: Contrast-enhanced total body CT scan. When the exam is performed, CT scan 
images will be centralized at the study Sponsor Institution. 
- 
Nutritional assessment 
- 
QoL assessment 
- 
LVEF evaluation 
 
Additionally, the day of surgery, or no more than 24 hours before surgery, the following assessments 
will be performed: 
- 
Blood tests including hematology and blood chemistry laboratory tests on peripheral blood 
after at least 8-hour fasting 
- 
Blood metabolites, and growth factor assessment; immunological evaluations on peripheral 
blood after at least 8-hour fasting 
- 
TSH, T3 and T4 measurement 
- Urinalysis 
- 
Stool sampling  
- 
Saliva sampling 
9.4 Surgery  
After completion of the preoperative treatment, patients will undergo breast conserving surgery or 
mastectomy in line with current standards of care and according to the recommendations of the 
multidisciplinary team, regardless of the study arm.  
Surgery must be performed within 28 days from the last administration of preoperative systemic 
treatment. 
 
9.5 Adjuvant treatment 
 
After surgery, patients will receive up to a maximum of nine triweekly Pembrolizumab cycles, 
according to local and international guidelines. 
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- 
Quantification of blood metabolites and growth factors; immunological evaluations in 
peripheral blood after at least 8-hour fasting 
- Urinalysis with urinary ketone body dosage 
- Stool sampling  
- 
Saliva sampling 
In addition, patients will be given food diaries to annotate the type and amount of food and beverage 
consumed during one single day every three weeks. 
 
Other clinical procedures (e.g., blood analyses) during this phase will be performed at the 
discretion of the physician as per clinical practice.  
 
9.6 End of treatment 
The end of the study for a single patient is defined as the date of definitive discontinuation of the 
experimental treatment. The end of the whole study is defined as the date of the last visit/phone 
contact, scheduled according to the study protocol. 
The End of Treatment (EOT) visit must be performed 21-28 days after the administration of the last 
dose of post-surgical Pembrolizumab. EOT visit requirements are outlined below:  
 
- 
Physical Examination, including an assessment for emergent toxicities or changes from prior 
visits, the evaluation of ECOG PS, vital signs, weight, BMI, food intake  
- 
Blood tests including hematology and blood chemistry laboratory tests on peripheral blood 
after at least 8-hour fasting 
- 
Blood metabolites, and growth factor assessment; immunological evaluations on peripheral 
blood after at least 8-hour fasting 
- 
TSH, T3 and T4 measurement 
- Urinalysis 
- 
Stool sampling  
- 
Saliva sampling 
- 
Treatment-related adverse events 
- 
Concomitant medications 
- 
QoL assessment 
 

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9.7 Additional therapy and follow-up 
Patients may receive local radiotherapy, depending on the pathological tumor response (pCR yes vs. 
no) and lymph node status, and according to local and international guidelines. All patients will 
undergo regular follow-up as per local and international guidelines.  
Only per protocol patients will enter the follow up period, which will initiate the day of the EOT visit. 
During the first 5 years of the follow-up period, patients will undergo a visit every 3-6 months, 
according to the physician judgment; then a phone contact will be performed annually up to 10 years. 
Mammography will be performed annually as per local and international Guidelines.  
 
9.8 Tumor recurrence 
In patients undergoing disease recurrence after surgery, if a diagnostic biopsy of a metastatic lesion 
is indicated as per clinical practice (e.g., TNBC biology confirmation), during the same procedure an 
additional, optional core biopsy of the tumor will be performed for the collection of fresh-frozen 
tumor material that will be used for subsequent biological evaluations. In addition, blood, urine, saliva 
and fecal samples will be collected at the detection of disease relapse for blood cell counts, standard 
biochemistry, immunologic (PBMC) and metabolic evaluations. 
10 STUDY PROCEDURES 
The Study Flow Chart (Table 2) summarizes the study procedures. Individual procedures are 
described in detail below. 
10.1 Screening procedures 
Screening procedures begin once a patient potentially candidate to be enrolled in the study has 
provided written informed consent. The following assessments have to be performed within 28 days 
prior to experimental treatment initiation.  
Informed consent 
The Investigator must obtain the documented consent from each patient (or his/her legal 
representative) considered for the trial and eligible for screening prior to performing any trial-related 
procedures. Informed consent should be asked once the report of the diagnostic core biopsy (including 
HER2 status, ER and PgR status and tumor proliferation measurement and/or tumor grade) is 
available at the local pathology site, and once the diagnosis of TNBC is confirmed. The Investigator 
will explain to all candidate participants (or their legal representative) the aims and characteristics of 
the study, as well as the potential benefits and risks associated with participation in the study.  The 
original consent form, signed and dated by the patient (or by the patient’s legal representative) and 
by the person who conducted the informed consent discussion, will be retained by the Investigator, 
and a copy will be given to the patient before enrollment in the trial. The informed consent form, any 
subsequent revised version and any other written information provided to the patients have to be 
approved by the IRB/EC’s and must adhere to applicable laws and regulations.  

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Inclusion/Exclusion Criteria 
All inclusion and exclusion criteria must be reviewed by the Investigator to ensure that the subject 
qualifies for the trial. 
Demographics and Medical History 
A detailed medical history has to be obtained by the Investigator. Medical history should include all 
active conditions, as well as any previously diagnosed condition that is considered to be clinically 
significant by the Investigator. Details regarding the subject’s tumor must be recorded separately and 
not listed as medical history.  
Prior and Concomitant Medications 
The Investigator will review the use of prior medications, including any protocol-specified washout 
requirement. Any medication taken by the participant within 28 days before the beginning of the 
experimental intervention should be recorded. 
Randomization procedure/Assignment of Randomization Number  
All eligible subjects will be allocated to one of the two experimental arms through a 1:1 random 
assignment, and they will be assigned a randomization number. At randomization, patients will be 
stratified according to a) disease stage based on TNM staging system (clinical stage II- versus clinical 
stage III) and b) patient BMI (i.e., ≥25 kg/m2 vs <25 kg/m2) to guarantee a balanced distribution of 
patients in the two experimental groups according to these two variables. Once a randomization 
number is assigned to a subject, it cannot be re-assigned to another subject.  
After enrollment, patients will perform a visit at the study Sponsor Institution, which can be 
performed virtually (tele-visit). During this visit, each patient will meet the multidisciplinary team 
responsible for the management of the FLA in all participating centers. 
10.2 Clinical procedures/assessments 
Disease evaluation 
The investigator, or a qualified designee, must obtain prior and current details regarding each 
subject’s tumor. Disease evaluation assessments have to be performed at screening/baseline and at 
the timepoints outlined in Table 2, and will include the following: 
- 
Clinical (by palpation) and radiological breast imaging. Breast US is mandatory at screening, 
after PCb completion, and before surgery. CE-MRI is mandatory at baseline and before 
surgery. Other radiological exams (mammography, contrast enhanced mammography 
(CESM), contrast enhanced MRI) can be performed at the discretion of the physician. 
- 
Total Body Computerized Tomography (CT) or, alternatively, 18FDG-PET/CT scan to rule 
out the presence of distant metastases.  
- 
HER2 status (based on local testing by IHC and, if necessary, FISH, CISH, or other 
evaluations) to exclude HER2 protein overexpression and HER2 gene amplification status 
based on International Guidelines50,51  

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- 
Hormone receptor status, as evaluated as the percentage of tumor cells staining positive for 
ER and PgR by IHC analysis 
- 
Tumor grade and/or Ki67 value or other tumor proliferation measures  
Optionally, a total body CT scan may be repeated before surgery. 
Multidisciplinary visit 
After enrollment, all patients will perform a visit at the study Sponsor Institution (Fondazione IRCCS 
Istituto Nazionale dei Tumori), which can be performed virtually (tele-visit), during which the patient 
will meet the multidisciplinary team responsible for the management of the FLA in all participating 
centers. During this visit, each patient will receive detailed information and advice for a proper 
management of the FLA in combination with standard chemoimmunotherapy, as well as for a timely, 
daily reporting of FLA tolerability and compliance to the multidisciplinary INT team during each day 
on the experimental dietary intervention.  
Physical examination and Vital signs 
The investigator, or a clinical designee, will perform a complete (full) physical examination during 
the screening/baseline visit, and as specified in the Study Flow Chart. Clinically significant abnormal 
findings emerging during the screening visit should be recorded as medical history, whereas new 
clinically significant abnormal findings detected after the first administration of study treatment 
should be recorded as AEs. 
Vital signs will be measured by the Investigator, or by a qualified designee, at screening visit, and as 
specified in the Study Flow Chart. Vital signs should include temperature, pulse and blood pressure. 
Height will be measured at screening only. 
12-Lead Electrocardiogram and LVEF evaluation 
A standard 12-lead ECG will be prescribed to all patients during the screening phase (if not performed 
within one month before enrollment) to exclude meaningful alterations of cardiac rhythm. Due to the 
potential cardiotoxic effect of the administered drugs (anthracycline), an evaluation of left ventricular 
ejection fraction (LVEF) by echocardiography or multi-gated scintigraphic scan (MUGA scan) will 
be also performed as specified in the Study Flow Chart (Table 2). 
Additional cardiological evaluations may be performed if clinically indicated (e.g., for AEs during 
the treatment administration).  
 
ECOG Performance Status 
Performance status will be assessed by the Investigator according to the Eastern Cooperative 
Oncology Group (ECOG, WHO) PS scale as specified in the Study Flow Chart (Table 3). 
 
 
Table 3  
ECOG (WHO) PS 

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Grade 0 
Fully active, able to carry on all pre-disease performance without restriction. 
Grade 1 
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 
Grade 2 
Ambulatory and capable of all self-care but unable to carry out any work 
activities. Up and about more than 50% of waking hours. 
Grade 3 
Capable of only limited self-care, confined to bed or chair more than 50% of 
waking hours. 
Grade 4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or 
chair. 
Grade 5 
Dead  
 
Pregnancy assessment 
Women of childbearing potential should be advised to use highly effective contraception methods 
while receiving the experimental treatment and for at least 6 months after the last administration of 
post-neoadjuvant Pembrolizumab. The same patients will undergo a serum or urine pregnancy test, 
before the first administration of the study treatment. 
Height, weight and body mass index (BMI) 
Height in centimeters (cm) and body weight (to the nearest 0.1 kilogram (kg) in indoor clothing, but 
without shoes), will be measured on the days outlined in Table 2. Patient body mass index (BMI) 
will be calculated at scheduled time points through the following formula: body weight (kg)/[height 
(m)]2. 
Nutritional evaluation and study of body composition 
Nutritional status will be assessed through the evaluation of anthropometric variables (weight, BMI) 
and, indirectly, through specific blood evaluations (dosages of transferrin, total cholesterol, 
prealbumin, absolute lymphocyte count and the neutrophil/lymphocyte ratio). A nutritional 
counseling will be provided by the study Sponsor Institution to all patients to maintain an appropriate 
diet, and to actively support patients undergoing unintentional, progressive low of body weight during 
the study treatment.  
Lean and fat mass at baseline will be estimated through CT scan evaluation, performed before the 
initiation of the experimental treatment and before surgery.  
 
 
Food diaries  
Caloric intake will be evaluated through food diaries, i.e., a self-reported account of all foods, 
beverages and dietary supplements consumed by a respondent over one or more days (National 

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Cancer Institute, 2016 https://dietassessmentprimer.cancer.gov/). Since the instrument is open-ended, 
there is no limit to the number of items that can be reported. Standard portion sizes will be used to 
help estimate serving sizes. Mean caloric intake will be determined using Food Composition Database 
for Epidemiological Studies in Italy (“Food Composition Database for Epidemiological Studies in 
Italy” by Gnagnarella P, Salvini S, Parpinel M. Version 1.2015 Website http://www.bda-ieo.it/). The 
absolute and relative intake of macronutrients (i.e., carbohydrates, proteins, lipids) and micronutrients 
will be extrapolated from the food diaries and related to the caloric need of every patient. 
All patients will be given food diaries to annotate the type and amount of food and beverage consumed 
at specific timepoints, i.e., i) during 7 consecutive days before treatment initiation ii) during one 
single day once a week (with the exception of the week in which patients receive chemotherapy plus 
Pembroluzumab plus/minus FLA) during the neoadjuvant treatment phase iii) during one single day 
every three weeks during the adjuvant treatment phase. 
In addition, patients randomized to arm B will receive a specific 5-day food diary to annotate the type 
and amount of food and beverage consumed during each FLA cycle; patients randomized to arm A 
will receive at the same timepoints 5-day food diaries to annotate the type and amount of food and 
beverage consumed. 
 
Phone/e-mail assessments 
During each of the five days of each FLA cycle (or during the corresponding timepoints in patients 
randomized to arm A), all patients will be contacted by the Investigator on a daily basis to 
communicate any arising toxicity, updated weight and blood pressure measurements, and specific 
information regarding the type and amount of food and beverage intake (according to the provided 
list).  
10.3 Quality of Life assessments 
All patients will be evaluated for quality of life at different time points as outlined in Table 2. Patient 
QoL will be evaluated through the European Organization for Research and Treatment of Cancer 
(EORTC) quality of life questionnaires (QLQ) C-30 version 3.0 and the EORTC QLQ - BR23. (see 
Appendix 1). The EORTC instruments will be scored according to the EORTC guidelines.  
 
10.4 Laboratory Procedures/Assessments  
All patients will undergo different laboratory tests as part of the screening/baseline assessment, and 
during the study course at pre-specified timepoints, as described in the Study Flow Chart (Table 4). 
These tests include standard blood cell counts, biochemical evaluations and additional metabolic and 
immunologic analyses, as outlined below. Exploratory analyses on gut microbiota will be conducted 
as well.  
 

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Table 4 – Hematology and blood chemistry.  
These evaluations will be performed locally at each study center. 
Hematology 
Blood chemistry 
Hemoglobin 
Glucose 
Alkaline phosphatase 
Hematocrit 
Creatinine 
LDH 
Platelet count 
BUN 
Uric acid 
WBC (total and differential) 
Alanine aminotransferase 
Potassium 
Red Blood Cell Count 
Aspartate aminotransferase 
Sodium 
Absolute Neutrophil Count 
Bilirubin 
Calcium 
Absolute Lymphocyte Count  
 
Absolute Monocyte Count 
 
 
 
Metabolic evaluations  
Metabolic assessment on patients’ blood will be conducted as listed in Table 5 on the days outlined 
in Table 2. To perform these analyses, samples will be centralized at the Study Sponsor Institution. 
Table 5 
Metabolic evaluations 
Insulin 
Insulin-like growth factor 1 
(IGF-1) 
Cortisol 
Sideremia 
Ferritin 
Transferrin 
Total protein 
Cholinesterase 
Prealbumin 
Total, HDL and LDL    
 cholesterol 
Triglyceride 
Albumin 
FGF 
Serum amino-acids 
Plasma fatty acids 

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VEGF-A 
PDGF 
®-hydroxybutyrate 
Serum IGFBP-1 
Serum IGFBP-2 
Serum IGFBP-3 
 
Hepatitis serology 
At screening all patients will be tested for HBV and HCV as by local clinical practice. HBV serology 
will include HBsAg and antibodies directed against HbS and HbC. HBV DNA should be obtained 
prior to the treatment phase in the case of HBsAg and/or anti-HBc positivity. HCV serology will 
include HCV antibody assessment (anti-HCV). HCV RNA should be obtained prior to treatment 
initiation if patient tests positive for anti-HCV. These analyses will be performed locally at each study 
center. 
 
TSH, T3 and T4  
Thyroid function will be evaluated as indicated in Table 2. These analyses will be performed locally 
at each study center. When needed, substitutive hormonal therapy should be initiated according to 
local and international guidelines. Unresolved abnormal labs that are drug-related AEs should be 
followed until resolution.  
 
Urinalysis 
Physical and chemical urinalysis, including the evaluation of urinary ketone concentration, will be 
performed on the days outlined in Table 2. These analyses will be performed locally at each study 
center. 
 
Saliva and Stool analysis 
Saliva and Fecal samples will be collected on the days outlined in Table 2. To perform these analyses, 
samples will be centralized at the Study Sponsor Institution Stool sample analysis will be performed 
following the procedure indicated in Section 14. 
 
Immunological analysis (including PBMCs) 
To perform these analyses, samples will be centralized at the Study Sponsor Institution. 
Assessments will be conducted on the days outlined in Table 2. For details see Section 14. 
 
Evaluation of pathological complete response (pCR) in surgical specimens 

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pCR is defined as the absence of residual invasive breast carcinoma, with or without ductal carcinoma 
in situ, AND the absence of any tumor deposit in sampled axillary nodes (ypT0/ypTis, ypN0, 
according to AJCC classification)52. 
Other analyses in tumor samples 
For all patients, diagnostic biopsy tissue and surgically removed primary tumor tissue (in the subset 
of patients not undergoing pCR) will be compared to assess changes in:  
- 
proliferation index (percentage of Ki67-positive cells on IHC); 
- 
tumor biology, including expression of estrogen and progesterone receptors by IHC, HER2 
protein by IHC (and, in case of equivocal value, by in situ hybridization analysis);  
Additional biological evaluations will be also performed, as outlined in Section 14. 
10.5 Withdrawal from study participation 
In accordance with the current revision of the Declaration of Helsinki and other applicable 
regulations, every patient has the right to withdraw from the study at any time and for any reason 
without prejudice to his or her future medical care by the physician or at the institution.  
Patients may discontinue the study protocol for any of the following reasons: 
- 
Adverse event(s) 
- 
Occurrence of an unacceptable toxicity 
- 
Loss to follow-up 
- 
General or specific changes in patient condition, that are considered unacceptable for further 
treatments in the judgment of the Investigator 
- 
Pregnancy 
- 
Protocol deviation 
- 
Technical problems 
- 
Patient decision 
- 
Lack of compliance to the experimental treatment 
 
At the time of study withdrawal, the Investigator should schedule the End of Treatment and Follow-
up visits in agreement with the patient.  
11 ADVERSE EVENTS (AEs) 
An Adverse Event (AE) is defined as any new untoward medical occurrence or worsening of a 
preexisting medical condition in a patient who is administered a study treatment; the event does not 
necessarily need to be causally related with that treatment. An AE can therefore be any unfavorable 
and unintended sign (such as an abnormal laboratory finding), symptom, or disease that occurs after 
a patient has signed an informed consent and that is temporally associated with the use of the study 
treatment (regardless of the treatment arm), whether or not considered related to the study therapy. 
AEs include the following: 

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- 
All suspected FLA-related adverse reactions 
- 
All chemo-immunotherapy-related adverse reactions 
- 
Apparently unrelated illnesses, including the worsening of a pre-existing illness 
- 
Injury or accidents. Note that if a medical condition is known to have caused an injury or 
accident (e.g., a fracture due to a fall secondary to dizziness), the medical condition (dizziness) 
and the injury (fracture) should be reported as two separate adverse events 
- 
Laboratory abnormalities and/or physical examination findings requiring clinical interventions 
(e.g., therapeutic measures, schedule changes) or further investigations (beyond ordering a 
repeat [confirmatory] test) or that are considered clinically significant by the investigator 
 
As far as possible, each adverse event should be evaluated to determine: 
1. The severity grade (CTCAE Grade 1-4) 
2. Its duration (Start and end dates) 
3. Its relationship to the study intervention (Reasonable possibility that AE is related: No, Yes)  
4. Action taken with respect to study treatment (none, dose adjusted, temporarily interrupted, 
permanently discontinued, unknown, not applicable) 
5. Whether medication or therapy was given (no concomitant medication/non-drug therapy, 
concomitant medication/non-drug therapy) 
6. Outcome 
(not 
recovered/not 
resolved, 
recovered/resolved, 
recovering/resolving, 
recovered/resolved with sequalae, fatal, unknown) 
7. Whether it is serious. Each AE has to be classified by the Investigator as serious or non-
serious. AEs reporting procedures are defined according to the classification of the gravity of 
the event.  
11.1 Serious Adverse Events (SAEs) 
The definition of seriousness is based on the patient/event outcome or action criteria associated with 
events that pose a threat to patient’s life or functioning. 
A Serious Adverse Event (SAE) is any untoward medical occurrence that at any dose meets one or 
more of the following definitions: 
- 
Results in death 
- 
Is life-threatening (defined as an event in which the participant was at risk of death at the time 
of the event; it does not refer to an event which hypothetically might have caused death if it 
were more severe) 
- 
Requires in-patient hospitalization or prolongation of existing hospitalization (see NOTE 
below) 
- 
Results in persistent or significant disability/incapacity 
- 
Is a congenital anomaly/birth defect  
- 
Suspected transmission of an infectious agent (e.g., pathogenic or nonpathogenic) via the study 
drug is a SAE.  
 
Important AEs not resulting in death, not being life-threatening, or not requiring hospitalization, can 
still be considered serious when, based on the Investigator’s medical judgment, they may jeopardize 
the patient or require medical or surgical intervention to avoid one of the unfavorable clinical 
outcomes listed above. Examples of such events include, but are not limited to, intensive treatment 

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in an emergency room or at home for allergic bronchospasm; blood dyscrasias or convulsions that do 
not result in hospitalization. 
 
NOTE: Hospitalizations are not considered SAEs if they fulfill the following criteria:  
- 
are elective and planned before entry into the study 
- 
are admissions as per protocol for a planned medical/surgical procedure 
- 
are emergency hospitalizations but do not result in overnight hospitalization, unless considered 
an important medical or life-threatening event 
11.2 Adverse Event Intensity and Relationship to Study Intervention 
All AEs, including SAEs, will be graded according to the NCI Clinical Toxicity Criteria [NCI-
CTCAE version 5.0]. 
The causal relationship to study drug is determined by a physician and should be used to assess all 
adverse events. The casual relationship can be one of the following: 
- 
Related: There is a reasonable causal relationship between the study intervention and the AE 
- 
Not related: There is not a reasonable causal relationship between study intervention and the AE 
The expression "reasonable causal relationship" is meant to convey in general that there are facts 
(e.g., evidence such as de-challenge/re-challenge) or other arguments to suggest a positive causal 
relationship.  
11.3 AEs collection and reporting 
AEs can be spontaneously reported or elicited during open-ended questioning, examination, or 
evaluation of a patient. In order to prevent reporting bias, patients should not be questioned regarding 
the specific occurrence of one or more AEs. 
All observed AEs must be reported in the CRF, regardless of treatment arm or suspected causal 
relationship to the study treatment. 
All AEs must be graded according to the NCI Common Terminology for Adverse Events (CTCAE), 
version 5.0. Any adverse event which changes CTCAE grade over the course of a given episode will 
have each change of grade recorded on the adverse event CRF/worksheets.  
For all AEs, the Investigator must obtain information that is adequate to determine the outcome of 
the AE, and to assess whether the AE meets the criteria for classification as a SAE. 
If known, the diagnosis of the underlying illness or disorder, rather than its individual symptoms, 
should be recorded. The following information should be captured for all AEs: onset, duration, 
intensity, seriousness, relationship to study intervention, action taken, and treatment required. If any 
treatment for the management of the AE was required, it should be recorded on the appropriate CRF 
page. Completion of supplemental CRFs may be requested for AEs and/or laboratory abnormalities 
that are reported/identified during the course of the study. 
SAE collection and reporting  
Following the Patient’s written consent to participate in the study, all SAEs must be collected, 
including those thought to be associated with protocol-specified procedures and those occurring 
within 30 days of study treatment discontinuation. The investigator should notify any SAE occurring 

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after this time period, if they are believed to be related to study drugs or protocol-specified 
procedures.  
Any SAE, either related or unrelated to study treatment, must be recorded on the SAE page of the 
CRF and reported to the Pharmacovigilance Unit of the study sponsor Institution (Fondazione IRCCS 
Istituto Nazionale dei Tumori) by the Investigator or a delegate member of the investigational staff 
within 24 hours of their knowledge of the event.  
The initial report should be followed by submission of a more detailed adverse event communication 
within 5 calendar days after the Investigator first became aware of the serious event. 
A SAE report should be completed for any event where doubt exists regarding its status of 
seriousness.  If the investigators believe that a SAE not directly related to the administration of the 
investigational product(s) could be potentially caused by other conditions related to the study (such 
as withdrawal of previous therapy, or a complication of a study procedure), the relationship should 
be specified in the narrative section of the SAE page of the CRF. 
If an ongoing SAE changes in its intensity or relationship to study treatment, a follow-up SAE report 
should be completed within 24 hours. All SAEs should be followed until resolution or stabilization. 
Non-serious Adverse Events collection and reporting 
The collection of non-serious AE information should begin at initiation of the study treatment.  
If an ongoing non-serious AE worsens in its intensity, or its relationship to the study intervention 
changes, a new non-serious AE entry for the event should be reported.  
Patients who have an ongoing study treatment−related adverse event upon study completion or at 
discontinuation from the study will be followed until the event has resolved to baseline grade, the 
event is assessed as stable by the investigator, new anti-cancer treatment is initiated, the patient is lost 
to follow-up, the patient withdraws consent, or it has been determined that study treatment or 
participation is not the cause of the adverse event. 
Non-serious AEs should be reported as SAEs if they become serious.  
All identified non-serious AEs must be recorded and described on the appropriate non-serious AE 
page of the CRF.  
11.4 Laboratory test abnormalities  
Laboratory abnormalities that constitute an AE on their own (i.e., that are considered as clinically 
significant, or inducing clinical signs or symptoms, or requiring concomitant therapy or changes in 
study treatment) should be recorded on the Adverse Events CRF. Whenever possible, a diagnosis, 
rather than a symptom, should be provided (e.g., “anemia” instead of “low hemoglobin”). Laboratory 
abnormalities that meet the criteria for AEs should be followed until they have returned to normal, or 
until an adequate explanation of the abnormality is found. When an abnormal laboratory test result 
corresponds to a sign/symptom of an already reported adverse event, it is not necessary to separately 
record the lab/test result as an additional event. 
Laboratory abnormalities that do not meet the definition of an AE should not be reported as AEs. A 
Grade 3 or 4 event (severe) as per CTCAE does not automatically indicate a SAE unless it meets the 
definition of serious as previously defined and/or as per the investigator’s discretion. Any laboratory 
abnormality requiring the discontinuation or the interruption of a study drug, or requiring the subject 
to receive specific corrective therapy, represents an AE and must be reported as such. 

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12 COMPLIANCE TO THE EXPERIMENTAL TREATMENT 
12.1 Food diary during the FLA periods 
Patients randomized to arm B (experimental arm) will be given a food diary to annotate the type and 
amount of each food and beverage consumed during each of the 5-day FLA period.  
Patients’ compliance to the FLA will be evaluated through the analysis of these diaries. Quantitative 
variation within +10-50% of the allowed total daily calorie content will be considered minor 
deviations. Consuming non caloric food/beverages that are not contained in the prescribed scheme 
will be considered a minor deviation. Consuming caloric foods/beverages that are not contained in 
the prescribed scheme will be considered a major deviation, independently from the amount of the 
consumed food/beverage. Two minor deviations will define a major deviation as well. The number 
of minor and major deviations will be used to assess patients’ compliance to the prescribed FLA 
scheme. 
Patients randomized to the control arm (A) will be asked to fill the same food diaries for the recording 
of foods and beverages consumed during the same periods. The analysis of these diaries will be 
important to be sure that patients in the control arm undergo a regular diet, i.e., a normo-caloric diet 
that is based on international guidelines for cancer patients and survivors. 
 
12.2 Food diary outside the FLA periods 
All patients will be given food diaries to annotate the type and amount of food and beverage consumed 
at specific timepoints, i.e., i) during 7 consecutive days before treatment initiation ii) during one 
single day once a week (with the exception of the week in which patients receive chemotherapy plus 
Pembroluzumab plus/minus FLA) during the neoadjuvant treatment phase iii) during one single day 
every three weeks during the adjuvant treatment phase. 
This information may be useful to interpret long-term effects of the experimental treatment on 
metabolic parameters and clinical outcomes. 
Patients randomized to the control arm will be asked to fill the same food diaries for the recording of 
foods and beverages consumed during the same periods. The analysis of these diaries will be 
important to be sure that patients in the control arm undergo a regular diet, i.e., a normo-caloric diet 
that is based on international guidelines for cancer patients and survivors. 
13 DISCONTINUATION/MODIFICATION 
OF 
THE 
EXPERIMENTAL 
TREATMENT 
13.1 Discontinuation of chemo-immunotherapy 
All patients undergoing overt radiological or clinical disease progression, deterioration of clinical 
conditions or death during the study treatment will permanently discontinue all study-related 
treatments and procedures. The study treatment will be permanently discontinued in case of SAEs 

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that, according to the judgment of the physician, could compromise patient general health 
status/conditions or contraindicate/delay curative surgery.  
Patients who are not are able for any reason to maintain an adequate chemotherapy dose intensity, 
e.g., when chemotherapy administration needs to be delayed by more than three weeks for reasons 
strictly related to treatment tolerability (i.e., excluding infections, accidents or other reasons, which 
will be evaluated individually by the treating physician), will discontinue study treatments, and they 
will undergo anticipated surgery within 2-4 weeks after the last dose of chemotherapy. Patients who 
will need temporary or permanent discontinuation of Pembrolizumab for any reason (e.g., treatment 
tolerability, Pembrolizumab-related severe AEs or SAEs) will continue to receive chemotherapy, 
with or without cyclic FLA.  
During the first part of the combination therapy (PCb plus Pembroluzumab plus/minus FLA), if one 
or more than one chemotherapy drug needs to be discontinued due to toxicity, patients may 
continue treatment as follows:  
∙ If paclitaxel is permanently discontinued at any time and for any reason according to the physician 
judgment, also discontinue the other treatment of the first part of neo-adjuvant chemotherapy 
(carboplatin plus/minus FLA); then, start and complete AC or EC regimen plus Pembrolizumab  
plus/minus FLA as per protocol, then proceed with surgery.  
∙If only carboplatin is discontinued for any reason according to the physician judgment: continue with 
weekly paclitaxel (up to the completion of 12 cycles) plus triweekly Pembrolizumab plus/minus FLA 
followed by AC or EC plus Pembrolizumab plus/minus FLA as planned per protocol, then followed 
by surgery  
During the second part of the combination therapy, if doxorubicin (or epirubicin) or 
cyclophosphamide need to be permanently discontinued for any reason, proceed with anticipated 
surgery. If only Pembrolizumab is discontinued, continue AC/EC for the remaining cycles as planned 
per protocol, and  then proceed with surgery.  
 
Arm B patients, who should permanently discontinue chemotherapy for any reason, and undergoing 
anticipated surgery, will also discontinue the FLA.  
 
Note: if Pembrolizumab is permanently discontinued during the neoadjuvant treatment phase because 
of unacceptable Pembrolizumab-related toxicities, no Pembrolizumab will be administered after 
surgery. 
13.2 Modification of chemotherapy administration 
The dosage or schedule of chemotherapy can be modified in case of AEs according to clinical practice 
and physician’s judgment. The following suggestions are provided to support clinical decisions for 
adverse events of particular interest. 

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Granulocyte colony stimulating factor for primary or secondary prophylaxis of neutropenia may be 
used in selected cases, and based on the physician judgment and according to International 
Guidelines. 
 
Dose modifications for paclitaxel and carboplatin 
Recommendations for dose interruptions/modifications in case of specific treatment-emergent AEs 
are provided in the following sections. 
 
As a general rule, if dose reduction is necessary, the dose should be reduced by one dose level (see 
Table 6), and the subject should be monitored for 10 to 14 days at each dose level. If toxicity does 
not resolve during this monitoring time, drug delivery may need to be discontinued with continued 
monitoring for an additional 10-14 days. 
 
Any treatment-related non-hematological toxicity of grade higher than or equal to 2 (except for G2 
alopecia, peripheral neuropathy and fatigue, for which resolution is not necessarily needed) requires 
the treatment to be delayed until the adverse event has resolved to ≤ Grade 1. 
 
Once the dose has been reduced no re-escalation is allowed. 
Table 6:  
 
Paclitaxel Dose Levels 
Dose Level 
Paclitaxel (mg/m2) 
0 (starting dose) 
80 
-1 
75 
Carboplatin Dose Levels 
Dose Level 
Carboplatin (AUC) 
0 (starting dose) 
1.5 
-1 
1.1 
Patients experiencing any of the following toxicities during the previous cycle should have their 
chemotherapy reduced for all subsequent cycles by 1 dose level as outlined in Table 6.  
Table 7: 
 
Dose Modifications for paclitaxel alone or with carboplatin(a) 

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Toxicity 
 
Adjustment for treatment component 
believed to be associated with specific 
toxicity. Continue other treatment 
component 
as 
per 
protocol 
(see 
Paragraph 13.1). 
Neutropenia 
≥1000 x 109/L (G1/G2) 
No change to paclitaxel and/or carboplatin 
<1000 x 109/L (G3) 
Withhold paclitaxel and/or carboplatin 
until ANC≥1000 x 109/L. 
G-CSF may be used at discretion of the 
investigator.  
 
 Resume paclitaxel and/or carboplatin 
based on time of recovery: 
● ≤1 week: No change to paclitaxel 
and/or carboplatin 
● 1 but ≤3 weeks: decrease paclitaxel 
and/or carboplatin dose (-1 dose 
level) 
● ≥3 weeks: Discontinue paclitaxel 
and/or carboplatin  
 
Febrile neutropenia (≥  
38.3°C 
in 
a 
single 
temperature reading or 
a sustained temperature 
of ≥38°C for at least one 
hour associated with 
ANC < 1.0 x 109/L) 
 
Withhold paclitaxel and/or carboplatin 
until resolution. G-CSF may be used at 
discretion of the investigator.  
Resume paclitaxel and/or carboplatin 
based on number of episodes: 
● First episode: No change to 
paclitaxel and carboplatin dosages 
● Second 
episode: 
decrease 
paclitaxel and/or carboplatin dose 
(-1 dose level) 
● Third 
episode: 
Discontinue 
paclitaxel and/or carboplatin  
 

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Thrombocytope
nia 
 Grade 1  
Withhold paclitaxel and/or carboplatin until  
≥100,000/mm3. Resume paclitaxel and/or 
carboplatin based on timing of recovery:  
∙ ≤1 week: no change in paclitaxel and 
carboplatin dosage and schedule.  
∙ >1 but <3 weeks: Reduce paclitaxel and/ or 
carboplatin 
(-1 
dose 
level). 
∙ ≥3 weeks: Discontinue paclitaxel and /or 
carboplatin 
Grade 
≥2 
(<75000) 
or in the presence of 
significant bleeding or 
requiring 
blood 
transfusion  
Withhold paclitaxel and/or carboplatin until 
≥100,000/mm 
Decrease 1 dose levelb  
Discontinue paclitaxel and/or carboplatin if 
the event lasts for ≥ 3 weeks  
Anemia 
All grades 
No 
change 
to 
paclitaxel 
and 
carboplatin. 
Iron studies should be done and iron 
supplementation/ RBC transfusion can 
be given at the physician’s discretion 
Sensory 
neuropathy 
Grade 3  
Withhold paclitaxel and/or carboplatin. If 
neuropathy improves to ≤ Grade 2, 
treatment may be resumed with 1 dose 
level reduction. If neuropathy does not 
improve to ≤ Grade 2 within 3 weeks, 
discontinue treatment. 
Grade 
4 
sensory 
neuropathy 
Withhold paclitaxel and/or carboplatin. If 
neuropathy improves to ≤ Grade 2 within 
3 weeks, treatment may be resumed with 1 
dose level reduction. If neuropathy does 
not improve to ≤ Grade 2 within 3 weeks, 
discontinue treatment. 
Abnormal 
bilirubin value 
Grade 1 
Re-test bilirubin every week, continue 
study treatment 

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Grade 2 or 3 
Withhold treatment until improvement to 
Grade 1. Re- start treatment at the same 
dose level 
Grade 4 
Discontinue treatment; liver ultrasound 
should be performed immediately 
Abnormal 
AST/ALT values 
 
Grade 1 
Continue study treatment 
Grade 2 or 3 
Withhold treatment until improvement to 
Grade 1. Re- start  treatment at the same 
dose level 
Grade 4 
Discontinue treatment; liver ultrasound 
should be performed immediately 
Other Grade ≥ 3 
toxicitiesc 
 
Adjust dose or discontinue therapy as 
medically indicated. 
 
aDespite adequate/maximal medical intervention and/or prophylaxis.  
bPlatelets have to recover to ≥ 100 x 109/L (and neutrophils have to be ≥ 1000/mm3) before the start of the next cycle.  
cExcept Grade 3 fatigue, transient joint or muscle pain for which no dose modifications are required.  
Hypersensitivity Reactions to Paclitaxel or Carboplatin  
Hypersensitivity reactions rarely occur. If they do occur, minor symptoms such as flushing, skin 
reactions, dyspnea, lower back pain, hypotension, or tachycardia may require temporary 
discontinuation of the infusion. In case of mild hypersensitivity reactions, at the investigators’ 
discretion, chemotherapy can be resumed with an implementation of prophylactic drugs (i.v. 
antihistamine and 12 mg of dexamethasone), together with an infusion time prolongation. However, 
severe reactions, such as hypotension requiring treatment, dyspnea requiring bronchodilators, 
angioedema or generalized urticaria, mandate immediate discontinuation of study drug administration 
and aggressive symptomatic therapy. Patients who experience severe allergic reactions should not be 
re-challenged.  
Dose modifications for doxorubicin/epirubicin and cyclophosphamide  
Any treatment-related non-hematological toxicity of grade higher than or equal to 2 (except for G2 
alopecia and fatigue for which resolution is not needed) requires the treatment to be delayed until the 
adverse event has resolved to ≤ Grade 1. 
 
Recommendations for dose interruptions/modifications in case of specific treatment-emergent AEs 
are provided in the following sections. 
 
 
 

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Once the dose has been reduced no re-escalation is allowed.  
Table 8 
Anthracyclines and Cyclophosphamide Dose Levels 
 
Dose Level 
Doxorubicin (mg/m2) 
Epirubicin (mg/m2) 
Cyclophosphamide 
(mg/m2) 
0 (starting dose) 
60 
90 
600 
-1 
48 (-20%) 
72 (-20%) 
480 (-20%) 
Patients experiencing any of the following toxicities during the previous cycle should have 
chemotherapy dose reduced for all subsequent cycles by 1 dose level as outlined in Table 8.  
Table 9 
 
Dose Modifications for anthracyclines and cyclophosphamide (a)  
Toxicity 
 
Adjustment  
Neutropenia 
≥1000 x 109/L (G1/G2) 
No changes to AC or EC 
<1000 x 109/L (G3) 
Withhold AC or EC until ANC≥1000 x 
109/L. 
G-CSF may be used at discretion of the 
investigator.  
 
Resume treatment based on time of 
recovery: 
● ≤1 week: No change  
● 1 but ≤3 weeks: decrease -1 dose 
level 
● ≥3 weeks: Discontinue AC or EC, 
and plan surgery 
 

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Febrile neutropenia (≥  
38.3°C in a single 
temperature 
reading 
or 
a 
sustained 
temperature of ≥38°C 
for at least one hour 
associated with ANC < 
1.0 x 109/L) 
Withhold AC or EC until resolution. G-
CSF may be used at discretion of the 
investigator.  
Resume AC or EC based on number of 
episodes: 
● First episode: No change to AC or 
EC 
● Second episode: reduce AC or EC (-
1 dose level) 
● Third episode: Discontinue AC or 
EC, and plan surgery 
 
Thrombocytope
nia 
Grade 1  
Withhold AC or EC until ≥100,000/mm3, 
Resume paclitaxel and/or carboplatin based 
on timing of recovery:  
∙ ≤1 week—no change 
∙ >1 but <3 weeks: reduce 1 dose level. 
∙ ≥3 weeks: Discontinue AC or EC, and plan 
surgery 
Grade 
≥2 
(<75000) 
or in the presence of 
significant bleeding or 
requiring 
blood 
transfusion  
Withhold treatment until Plt≥100,000/mm3 
Decrease 1 levelb  
Discontinue AC or EC if treatment 
withheld for ≥ 3 weeks; plan surgery 
Anemia 
All grades 
No change to AC or EC 
Iron studies should be done and iron 
supplementation/ RBC transfusion can 
be given at the physician’s discretion 
Abnormal 
bilirubin value 
 
Grade 
1 
 
Re-test bilirubin every week, continue 
study treatment 
Grade 2 or 3 
Withhold treatment until improvement to 
Grade 1. Re- start treatment at the same 
dose level 

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Grade 3 
Repeat biochemical tests every two days; 
liver ultrasound should be performed 
immediately. Discontinue AC or EC. 
Abnormal 
AST/ALT values 
Grade 1 
Continue study treatment 
Grade 2 or 3 
Withhold treatment until improvement to 
Grade 1. Re- start treatment at a lower dose 
level 
Grade 4 
Discontinue treatment; liver ultrasound 
should 
be 
performed 
immediately. 
Discontinue AC or EC. 
Mucositis 
and 
dysphagia 
 
May 
occur 
during 
anthracycline-
cyclophosphamide 
administration. 
Temporarily withhold anthracycline if 
these side effects are moderate to severe (≥ 
grade 2), but restart full dose once they 
resolve  
Cystitis 
 
May 
occur 
with 
cyclophosphamide 
administration. 
Temporarily 
withhold 
anthracycline -cyclophosphamide if cystitis 
is moderate to severe. Encourage the 
patient to drink large amounts of water; if 
urine culture is positive, antibiotics will be 
given  
Cardiac toxicity 
 
Maximum recommended cumulative dose 
of anthracyclines is 450 mg/m2 for 
doxorubicin and 900 mg/m2 for epirubicin 
Below this cumulative dose cardiac effects 
are 
not 
expected. 
Anthracyclines will be discontinued if: 
. - congestive heart failure arises 
. - persistent arrhythmia (including sinus 
tachycardia with no demonstrable cause) 
arises  
. - asymptomatic decrease of LVEF below 
45%  

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Other Grade ≥  3 
toxicitiesc 
Adjust dose or discontinue therapy as 
medically indicated 
 
aDespite 
adequate/maximal 
medical 
intervention 
and/or 
prophylaxis. 
bPlatelets have to recover to ≥ 100 x 109/L (and neutrophils have to be ≥ 1000/mm3 before the start of the next cycle. 
cExcept Grade 3 fatigue, transient joint or muscle pain for which no dose modifications are required. 
 
13.3 Pembrolizumab discontinuation 
No dose reductions of Pembrolizumab are allowed; Pembrolizumab will be withheld or 
discontinued to manage adverse reactions as described in Table 10.  
 
Table 10 
Immune-related 
adverse 
reactions  
Severity 
Treatment modification  
Pneumonitis  
Grade 2 
 
Withhold 
until 
adverse 
reactions recover to Grades 
0-1*  
Grades 3 or 4, or recurrent 
Grade 2  
Permanently discontinue  
Colitis  
Grades 2 or 3  
Withhold 
until 
adverse 
reactions recover to Grades 
0-1*  
Grade 4 or recurrent Grade 3  
Permanently discontinue  
Nephritis  
Grade 2 with creatinine > 
1.5 
to 
≤ 3 times upper limit of 
normal (ULN) 
Withhold 
until 
adverse 
reactions recover to Grades 
0-1*  
Grade ≥ 3 with creatinine > 
3 times ULN 
Permanently discontinue  
Endocrinopathies  
Grade 
2 
adrenal 
insufficiency 
and 
hypophysitis  
Withhold treatment until 
controlled 
by 
hormone 
replacement  

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Grades 3 or 4 adrenal 
insufficiency 
or 
symptomatic hypophysitis  
Type 1 diabetes associated 
with 
Grade 
≥ 
3 
hyperglycaemia (glucose > 
250 mg/dL or > 13.9 
mmol/L) or associated with 
ketoacidosis  
Hyperthyroidism Grade ≥ 3  
Withhold 
until 
adverse 
reactions recover to Grades 
0-1*  
For patients with Grade 3 or 
Grade 4 endocrinopathies 
that improved to Grade 2 or 
lower and are controlled 
with hormone replacement, 
if indicated, continuation of 
pembrolizumab 
may 
be 
considered 
after 
corticosteroid 
taper, 
if 
needed. 
Otherwise 
treatment 
should 
be 
discontinued.  
Hypothyroidism  
Hypothyroidism may be 
managed with replacement 
therapy without treatment 
interruption.  
Hepatitis  
Grade 2 with aspartate 
aminotransferase (AST) or 
alanine 
aminotransferase 
(ALT) 
> 
3 
to 
5 times ULN or total 
bilirubin > 1.5 to 3 times 
ULN  
Withhold 
until 
adverse 
reactions recover to Grades 
0-1*  
Grade ≥ 3 with AST or ALT 
> 5 times ULN or total 
bilirubin > 3 times ULN 
Permanently discontinue  
Skin reactions  
Grade 
3 
or 
suspected 
Stevens-Johnson syndrome 
(SJS) or toxic epidermal 
necrolysis (TEN)  
Withhold 
until 
adverse 
reactions recover to Grades 
0-1*  
Grade 4 or confirmed SJS or 
TEN  
Permanently discontinue  

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Other 
immune-related 
adverse reactions  
 
Based on severity and type 
of reaction (Grade 2 or 
Grade 3)  
Withhold 
until 
adverse 
reactions recover to Grades 
0-1*  
Grades 3 or 4 myocarditis 
Grades 3 or 4 encephalitis 
Grades 3 or 4 Guillain-Barré 
syndrome  
Permanently discontinue  
Grade 4 or recurrent Grade 3  
Permanently discontinue  
Infusion-related reactions  
 
Grades 3 or 4  
Permanently discontinue  
*If treatment-related toxicity does not resolve to Grades 0-1 within 6 weeks after last dose of pembrolizumab, 
or if corticosteroid dosing cannot be reduced to ≤ 10 mg prednisone or equivalent per day within 6 weeks, 
pembrolizumab should be permanently discontinued.   
Pembrolizumab should be permanently discontinued for Grade 4 or recurrent Grade 3 immune-
related adverse reactions, unless otherwise specified in Table 10. 
Patients experiencing disease progression that precludes definitive surgery or unacceptable toxicity 
related to Pembrolizumab as neoadjuvant treatment will not receive Pembrolizumab monotherapy 
as adjuvant treatment.  
13.4 FLA discontinuation (arm B) 
Patients may permanently discontinue the FLA because of unacceptable toxicity. 
In particular, patients undergoing ≥ grade 3 hypoglycemia, or symptomatic ≥ grade 2 hypoglycemia 
during the FLA will immediately interrupt that specific FLA cycle. If the same toxicity occurs more 
than one time, the FLA will be permanently discontinued. No modifications of the dietary scheme 
are allowed on the basis of occurring toxicities. Patients for which the FLA is interrupted or 
permanently discontinued due to an AE, or because of the occurrence of clinically significant changes 
in laboratory parameters, will continue to be treated with chemoimmunotherapy. 

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Table 11 
Discontinuation guidelines for FLA-related toxicities 
Adverse event 
Toxicity (grade)  
Action 
Hypotension 
Grade 1 
Continue the FLA 
Grade 2 
Continue the FLA. If the toxicity re-occurs despite 
optimal supportive care, permanently discontinue 
study treatment 
Grade 3 or 4  
Permanently discontinue the FLA 
Hypoglycemia 
Grade 1  
(LLN - 55 mg/dL;  
LLN - 3.0 mmol/L)  
Continue the FLA 
Grade 2  
(55 - 40 mg/dL; 3.0 - 
2.2 mmol/L) without 
clinically 
relevant 
neurological 
or 
cardiac symptoms 
Continue the FLA 
Grade 2  
(55 - 40 mg/dL; 3.0 - 
2.2 mmol/L) with 
clinically 
relevant 
symptoms, including 
diplopia, 
tremor, 
convulsions, 
drop/loss 
of 
consciousness, 
arrhythmias 
requiring 
medical 
treatment, 
myocardial 
infarction.  
Interrupt that specific FLA cycle. Then the FLA can 
be re-initiated after three weeks, i.e., according to the 
pre-defined schedule. If the same toxicity occurs 
again, permanently discontinue the FLA.  
Grade 3 
Interrupt that specific FLA cycle. Then the FLA can 
be re-initiated after three weeks, i.e., according to the 

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pre-defined schedule. If the same toxicity occurs 
again, permanently discontinue the FLA.  
Grade 4 
Permanently discontinue the FLA 
Headache  
Grade 1 or Grade 2 
Continue the FLA 
Grade 3 
Permanently discontinue the FLA 
Fatigue 
Grade 1 or Grade 2 
Maintain dose level 
Grade 3 
Permanently discontinue the FLA 
 
 
 
Criteria for FLA discontinuation based on weight loss 
Mild-to-moderate weight loss (about 3-6 % of baseline body weight) is expected at the end of each 
5-day FLA cycle. After re-feeding, almost complete (>70%) restoration of pre-FLA weight is also 
expected in the majority of patients. However, some patients may fail to regain weight comparably 
to pre-FLA period, thus undergoing progressive weight loss.  
Specific guidelines for continuing/discontinuing the experimental treatment on the basis of body 
weight loss and regain are reported in Table 12. 
 
Table 12 
Guidelines for treatment discontinuation due to weight-loss 
Weight loss before initiation of a new 
FLA cycle  
Action 
Weight loss < 5% from last FLA cycle 
and   
BMI ≥ 19 kg/m2 
 Continue the FLA 
Weight loss < 5% from last FLA cycle 
and BMI < 19 kg/m2 
Withhold FLA up to the next scheduled chemotherapy 
cycle, then re-evaluate weight and BMI.   
If BMI is still < 19 Kg/m2 three weeks after the initial 
FLA delay, permanently discontinue the FLA.  
Otherwise, i.e., weight loss is <5% and BMI>19 kg/ 
m2, resume the FLA 

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Weight loss 5-10% from last FLA cycle 
and BMI ≥ 22 kg/m2 
Continue the FLA  
Weight loss 5-10% from last FLA cycle 
and BMI 19-22 kg/m2 
Withhold the next scheduled chemotherapy cycle, then 
re-evaluate weight and BMI.   
If the AE is confirmed, permanently discontinue study 
treatment.  
Otherwise, continue the FLA.  
Weight loss 5-10% from last FLA cycle 
and BMI < 19 kg/m2 
Permanently discontinue the FLA 
Weight loss > 10% from last FLA cycle 
with any BMI 
Permanently discontinue the FLA 
 
 

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14 BIOLOGICAL EVALUATIONS 
Tumor tissue and blood samples collected at prespecified timepoints (see Table 2) will be used to 
perform different biological and molecular analyses, including genomic, gene expression, proteomic, 
immunologic and metabolomic evaluations (see paragraphs below).  
Since the knowledge of biomarkers and the development of new analytic procedures is rapidly 
evolving, after the completion of the planned assessments, remaining sample materials (e.g., aliquots 
of blood or tumor samples) may be used for further biological assessments. 
14.1 Blood analyses 
Patient blood samples (total volume: approximately 50 ml) will be taken at scheduled time points 
(see Table 2) to perform immunological, metabolic and other biochemical analyses. 
With the exception of blood samples for blood cell count and standard blood chemistry, which will 
be analyzed at each participating center, blood-derived plasma and serum samples will be shipped to 
and stored at the study Sponsor Institution for the analyses detailed below. Samples will be stored for 
up to 10 years after study closure, with the additional option of further long-term storage. They will 
be stored under the Sponsor responsibility. 
Biological evaluations will be performed in the laboratories of the Department of Diagnostic 
Pathology and Laboratory at Fondazione IRCCS Istituto Nazionale dei Tumori. 
 
For sampling procedures and shipment see instructions in the appropriate sections of the Laboratory 
Manual. 
 
Immunological parameters (20 ml of peripheral blood required)  
Analyses of the impact of FLA on frequency, phenotype, function and metabolism of innate and 
adaptive antitumor immune cell populations will be performed. Our studies will include 13-color 
FACS analysis of counts and activation status of circulating inflammatory monocytes 
(CD11b+CD33+CD14+PD-L1+), polymorphonuclear myeloid-derived suppressive cells (PMN-
MDSCs: 
CD11b+CD33+CD15+HLA-DRneg), 
monocytic 
MDSCs 
(M-MDSCs: 
CD11b+CD33+CD14+ and CD11b+CD33+CD14+HLA-DRneg), dendritic cells (DCs), natural 
killer (NK: CD3negCD16+CD56dimCD25+Ki67+ perforin+), B cells, regulatory T cells 
(CD4+CD25highFoxp3+CD127+ Ki67negCTLA4+Lag3+IL- 10+) and antitumor T cells, including 
the study of cell polarization (Th1, Th2 and Th17/Th22) and the quantification of exhausted/anergic 
effector 
T 
cells 
(CD3+, 
CD8+, 
CD25dimPD-1highLag3+TIM3+Ki67neg) 
versus 
differentiated/activated T cells (CD3+, CD8+, CD25+PD-1dimPerforin+Ki67+).  
Plasma of individual patients will be analyzed to measure the concentration of pro-inflammatory (e.g. 
IL-1β, IL-2, IL-6, IL-8, IL-12, IL-17, IFN-gamma, TNF-α and chemokines MIP-1beta and MCP-1) 
and immunosuppressive cyto/chemokines (e.g. IL-4, IL-10, IL-13, TGF-beta). Serum MIF and the 
soluble part of its receptor sCD74 will be also measured. 
Global gene expression profiling will be performed to assess the transcription of metabolic genes, 
including those encoding glucose and amino acid transporters, or enzymes involved in glycolysis, 
tricarboxylic acid (TCA) cycle, fatty acid and cholesterol biosynthesis, amino acid and nucleotide 
synthesis or catabolism.  

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For each immunological parameter average measurements will be performed at scheduled time 
points, and comparisons between the experimental arms at different time points will be calculated. 
Blood cell count (8 ml of peripheral blood required) 
For each patient and each scheduled time point, the number of blood cells (red blood cells, 
neutrophils, platelets, lymphocytes) will be measured. Average values in the two treatment arms will 
be compared at different time points. 
Standard blood chemistry (12 ml of peripheral blood required) 
Patient blood will be collected at baseline and at different time points after therapy initiation. 
Measured parameters include: fasting glycemia; markers of renal (blood levels of creatinine, urea and 
uric acid) and liver (AST, ALT, total and conjugated bilirubin, alkaline phosphatase) function; 
potassium, calcium, phosphate, magnesium; lactate dehydrogenase (LDH).  
Quantification of plasma amino acids (5 ml) 
Plasma amino acids will be quantified at scheduled time points through the AccQ Tag Ultra 
Derivatization kit (Wates). Ultra-Performance Liquid Chromatography (UPLC) analysis will be 
performed to measure the concentration of all amino acids, and in particular, glutamine, glutamate, 
leucine, tryptophan, methionine, arginine. 
Lipid profile of whole blood, plasma and red blood cell membranes (3 ml) 
Blood samples (4 ml) will be collected into heparin tubes. Whole blood, purified RBC cell 
membranes and plasma will be used for lipid assessment. Cell membranes of RBCs (ghosts) will be 
prepared by lysis with hypotonic buffer and washed to eliminate hemoglobin. Phospholipids and 
neutral lipids (free, ester and oxy-cholesterol, triglycerides) of RBCs will be purified and quantified 
by HPLC-ELSD system.  
Ghost and plasma lipids will be extracted with different chloroform/methanol mixtures. Gas-
chromatographic analysis will be used to determine FA composition of whole blood, plasma, 
membrane phospholipids, as well as of each phospholipid or neutral lipid species from RBC 
membranes and plasma. The FA methyl esters will be obtained after derivatization with sodium 
methoxide in methanol 3.33% w/v and injected into gas chromatograph (Agilent Technologies 6850 
Series II). SP, SP1P and cholesterol-derived oxysterols, such as 7α-, 7β-, 7-keto-, 20-, 25-hydroxy-
cholesterol, cholesterol-5,6-epoxide and the sterol metabolite 27-hydroxy-cholesterol, will be 
measured by UPLC-MS.  
Metabolic evaluations and Growth factor analyses (3 ml) 
Plasma will be separated from patient blood samples (4 ml) collected at scheduled time points and 
frozen at -80o C. Total proteins, albumin, triglycerides; total and HDL cholesterol, prealbumin, 
transferrin, sideremia, ferritin, cholinesterase, cortisol, insulin, β-hydroxybutyrate, IGF-1; IGFBP-1, 
IGFBP-2, IGFBP-3; VEGF-A, FGF will be dosed through specific immunoradiometric or ELISA 
assays.  

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14.2 Urine analyses 
Urine samples obtained at scheduled time points will be collected to perform chemical and physical 
analyses at each participating center. In particular, urine ketone bodies will be dosed as a measure of 
patient systemic ketosis. 
14.3 Saliva and Stool analyses  
Stool samples collected at the pre-specified timepoints will be shipped to the study Sponsor 
Institution. 
Oral and Fecal Microbiota analysis 
Saliva and Fecal samples will be collected through the ORAGENE DNA Saliva and OMNIgene 
GUT (DNA Genotek, Ottawa, Canada) collection method.  
Metagenomic DNA will be extracted using the Powersoil DNA Isolation kit (Mo Bio Laboratories), 
and the variable regions of 16S ribosomal RNA gene will be amplified and then sequenced using the 
Illumina MiSeq technology platform. Sequencing data will be analyzed using the QIIME pipeline or 
Mothur program with Greengenes or Ribosomal Database Project (RDP) databases to obtain bacterial 
composition data down to the genus level.  
Bacterial metabolites in feces 
SCFAs will be extracted from fecal samples and will be analyzed by ultra-high-performance liquid 
chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). 
14.4 Biological evaluations on tumor tissue samples 
Biological evaluations on tumor tissue sample will be performed on tumor specimens obtained from 
patients’ baseline core biopsies (available as FFPE and, optionally, fresh frozen specimens), and 
surgical specimens from the same patients, depending on tissue availability.  
For all the patients enrolled in the study, a Formalin-Fixed Paraffin-Embedded (FFPE) tumor 
specimen collected before the initiation of the experimental treatment must be shipped to the study 
Sponsor Institution. Optionally, patients enrolled in the study may undergo an additional core biopsy 
of the primary tumor study Sponsor Institution for the collection of fresh-frozen tumor material for 
different types of biological analyses (see below).  
Optionally, patients will be proposed to repeat a tumor biopsy at the study Sponsor Institution 14-21 
days after the first FLA cycle completion, to collect FFPE and fresh-frozen tissue for early evaluation. 
Surgical FFPE tumor specimens will be centralized to the study Sponsor Institution. Only for patients 
enrolled at the study Sponsor Institution, surgical fresh frozen tumor material will be collected as 
well. 

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In patients undergoing disease recurrence, if a diagnostic biopsy of a metastatic lesion is indicated as 
per clinical practice (e.g., TNBC biology confirmation), an optional core biopsy will be proposed to 
the patient for the collection of fresh-frozen tumor material that will be used for subsequent biological 
evaluations.  
 
In particular, the analyses will be performed at genomic and transcriptomic level to correlate tumor 
genomic and gene expression profiles with the efficacy of the experimental arms and to evaluate the 
effect of the administered experimental treatments on gene transcription.  
Results of these analyses will be used a posteriori to select few proteins of interest whose expression 
will be analyzed on tissue samples with IHC or mass spectrometry. 
Moreover, hematoxylin and eosin (H&E) staining on tumor tissue samples will be performed to 
evaluate the proportion of tumor-infiltrating lymphocytes (TILs). 
Finally, we will perform metabolomic evaluations on tumor tissue samples. Metabolites will be 
extracted from tumor cells lysates and analyzed by mass spectrometry. 
These analyses will be performed in the Pathology Unit of Fondazione IRCCS Istituto Nazionale dei 
Tumori (INT)  
For sampling procedures and shipment see instructions in the appropriate sections of the Laboratory 
Manual. 
Gene expression analysis 
Whole-transcriptome analysis with global RNA sequencing (RNA-Seq, Illumina technology) will be 
performed in the Pathology Unit of Fondazione IRCCS Istituto Nazionale dei Tumori (INT).  
Specifically, our interest will be focused on studying expression profiles of genes involved in 
- 
Gene involved in autophagy and endosomal trafficking (e.g., ULK1, ATG5, BECN1 and 
BECN2) 
- 
Genes encoding glucose and amino acid transporters (e.g., GLUT1 and GLUT4), enzymes 
involved in glucose metabolism (e.g., hexokinase, phosphofructokinase, pyruvate kinase, 
lactate dehydrogenase), mitochondrial oxidative phosphorylation (e.g., isocitrate 
dehydrogenase, fumarate hydratase, succinate dehydrogenase), tricarboxylic acid (TCA) 
cycle, fatty acid and cholesterol metabolism, amino acid and nucleotide synthesis or 
catabolism. 
- 
Endoplasmic reticulum (ER) stress and unfolded protein response (UPR)-related genes, 
including GRP78, ATF6, IRE1a and ATF4. 
- 
DNA repair systems genes, including BRCA1 and 2, ATM, ATR, CHK1/2, TP53, PRKDC, 
RAD51, RPA, TPR, ATRIP, NBS1, MATR3, SUN1, SUN2, NESPRIN1-7, EMERIN. 
- 
Genes encoding surface and secreted proteins expressed by non-cancerous cells within the 
tumor microenvironment. In particular, we will focus on transcription of genes expressed 
by different immune cells (including CD8, CD3, IFN, perforin, granzyme, CD14, PDL1) 
in order to define specific immunological profiles. 
 
In addition, to evaluate the spatial distribution of pre-defined metabolic and immune biomarkers in 
cancer cells and in the surrounding cells in the tumor, we will perform spatial transcriptomic analyses 
through a a high-plex, high-throughput spatial profiling platform, the GeoMx®  Digital Spatial 
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In a subset of patients undergoing additional biopsy at baseline and after one treatment cycle for the 
collection of fresh material, we will also perform single cell RNA-seq (scRNA-seq) analyses to 
follow the clonal evolution of cancer cell subsets, as well as of cells in the tumor microenvironment, 
during the experimental treatment, as well as to assess the modulation of crucial metabolic pathways 
in subsets of tumor cells and cells of the immune infiltrate. ScRNA-seq analyses will be performed 
in fresh tumor material after immediate separation of single cells.  
 
Genomic assays  
We will perform next generation sequencing (NGS) analysis to evaluate the mutational status of 
around 400 genes. The panel will include, but will not be limited to, genes involved in, DNA repair 
systems, mTORC 1 (e.g., LKB1, mTORC1, PI3K, AMPK) and autophagy (e.g. BECN1) pathways. 
Peritumoral adipose tissue analysis 
In fresh breast samples collected at the time of surgery (regardless of pCR status) in patients 
undergoing surgery at the Study Sponsor Site, we will isolate peritumoral adipose tissue to perform 
transcriptomic analysis and for the isolation of peritumoral adipocytes. In patients undergoing pCR 
(i.e., for whom no residual viable tumor cells can be found at surgery), peritumoral adipose tissue 
will be identified based on the position of the magnetic seed (inserted inside the tumor before 
treatment initiation). In patients not undergoing pCR (i.e., those with residual tumor cells), adipocytes 
that are close to the residual tumor will be collected. In both cases, RNA will be extracted from 
peritumoral adipocytes to assess differences in adipose tissue transcriptomic profiles between patients 
undergoing or not undergoing pCR. In addition, living adipocytes will be separated and cultured 
under different metabolic conditions to study the possibility to reprogram adipocyte biology. 
15 STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN 
15.1 Definition of study populations 
The per-protocol (PP) population will include all enrolled patients receiving at least one full 21-day 
cycle of experimental treatment with no major violations of the eligibility criteria during study 
conduction.  Moreover, to be included in the PP population, patients should undergo breast surgery 
with curative intent in order to evaluate the achievement, or lack of achievement, of pCR. 
Therefore, patients randomized to arm A, who will have received at least one full 21-day cycle of 
chemo-immunotherapy, will be evaluated for arm A; patients randomized to arm B, who will have 
received at least one full 21-day cycle of chemo-immunotherapy plus FLA, will be evaluated for arm 
B. 
The population in which the compliance will be evaluated will include all enrolled patients with no 
major violations of the eligibility criteria during study conduction.  
The safety population will include all enrolled patients who received at least one dose of study 
treatment, whether withdrawn prematurely or not, and had at least one safety follow-up.  

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15.2 Statistical plan and sample size calculation 
Information on demographics and baseline characteristics of patients satisfying the definition of the 
study populations will be provided. The number of patients withdrawing from the study, not meeting 
the eligibility criteria, and violating the protocol will also be described. 
Patients enrolled in this study will be randomized in a 1:1 ratio to one of two experimental arms.  
Based on the assignment cohort, each enrolled patient will receive one of two experimental treatments 
as preoperative therapy before surgery of the breast and draining lymph-nodes. At randomization, 
patients will be stratified on the basis of disease clinical stage (II vs. III) and patient BMI to guarantee 
a proper balance between patients in the two treatment arms.  
 
Data from the KN522 trial indicate that the rate of pathological complete response (pCR) after neo-
adjuvant anthracycline-taxane-carboplatin based chemotherapy in combination with 
Pembrolizumab is 64.8% (95% CI, 59.9 to 69.5). In this study, we will test the hypothesis that the 
experimental treatment is able to improve the pCR from 65% to 85%.  
Specifically, by assuming a one-sided α  error of 5% and a β  error of 15% (power = 85%), 132 
patients (66 for each arm) will be needed. Finally, incorporating around 10% of patient drop-out, a 
total of 145 patients will be enrolled. Allocation of a patient to a cohort will be based on a 
randomization list. 
 
15.3 Statistical analyses 
Primary endpoint analysis  
Treatment effect will be expressed as absolute difference in pCR rates between treatments and 95% 
confidence intervals will be calculated. The analysis will be stratified by clinical disease stage 
according to TNM classification at enrollment (stage II vs. stage III), as well as by patient BMI (≥25 
kg/m2 vs <25 kg/m2). Pathological complete response is defined as absence of invasive disease in 
breast and nodes (ypT0/ypTis, ypN0). The impact of known prognostic factors (age, BMI, disease 
stage, node status, Tumor Infiltrating Lymphocytes (TILs), Grade and proliferation rate (Ki-67)) on 
the rate of pCR will be first assessed using logistic regression models. Covariates significantly 
associated with pCR will be included in a multivariate analysis to assess their independent association 
with pCR.   
 
Secondary efficacy analyses 
Secondary analyses on efficacy endpoints will be conducted on the PP population. EFS, DFS, DMFS 
and OS will be analyzed by the Kaplan-Meier (KM) method. Survival differences between the two 
treatment arms will be tested by a log-rank test. A Cox proportional hazard model will be used to 
obtain hazard ratios and 95% confidence intervals for the treatment effect on EFS, DFS, DMFS and 
OS, and stratified by disease stage at enrollment. The impact of known prognostic factors (age, BMI, 
disease stage, node status, Tumor Infiltrating Lymphocytes (TILs), Grade and proliferation rate (Ki-
67)) on patient survival (in terms of EFS, DFS, DMFS and OS) will be first assessed at univariate 
analysis. Covariates significantly associated with survival outcome will be included in a Cox 

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proportional hazard model (multivariate analysis) to assess their independent association with 
survival.  
Biologic biomarkers (including metabolic, genomic, gene expression biomarkers) modulation will be 
analyzed using generalized linear and logistic models according to the variable considered.  
 
Safety analyses and unacceptable toxicity monitoring 
Safety analyses will be conducted on the safety population. All safety parameters will be analyzed 
and presented in terms of listings and summary tables. The assessment of safety will be based mainly 
on the frequency and nature of severe (G3/G4) AEs or serious adverse events (SAEs). Number and 
percentage of patients having any severe AEs or SAEs as well as the system/organ class involved in 
the severe AE will be presented. Any other information collected (e.g., severity or suspected 
relationship to study medication) will be listed as appropriate.  
Drug-discontinuing AEs will be closely monitored throughout the study in order to promptly close 
the experimental arm in case of unacceptable toxicity. No formal safety interim analyses are planned 
for this study. Periodic safety reviews will be conducted and any outcome affecting the study conduct 
will be promptly communicated by the investigators for notification to the Ethics Committees (ECs) 
 
16 ETHICAL AND ADMINISTRATIVE CONSIDERATIONS 
16.1 Independent Ethics Committee (IEC)  
Before initiating the trial, the Investigators must obtain a written favourable opinion from the IEC for 
the study protocol, the written informed consent form, the subject recruitment procedures and any 
other written information to be provided to subjects. All the correspondence with the IEC must be 
retained in the Investigator Site File. 
Before implementing any protocol amendment, the IEC written approval must be obtained. The only 
circumstance in which an amendment may be initiated prior to IEC approval is where the change is 
necessary to eliminate apparent immediate hazards to the Patients. In that event, the IEC must be 
notified in a written form as soon as possible. 
16.2 Ethical conduct of the trial 
The study will be performed in accordance with the general principles of the International Conference 
on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 
the Declaration of Helsinki and applicable local regulatory requirements and laws. 
16.3 Informed Consent 
It is the responsibility of the Investigators to give each patient (or to the patient’s representative) full 
and adequate verbal and written information regarding the objective and procedures of the trial and 
the potential risks for taking part in the study. The patient must be informed about his/her right to 

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withdraw from the trial at any time. The patient should have time and opportunity to enquire about 
details of the trial and to decide whether to participate in the trial.   
Written Patient information must be approved by the IEC and must be given to each patient before 
any trial-related procedure is undertaken.  
It is the responsibility of the Investigators to obtain informed consent signed and dated by the patient 
or her/his legal representative or by an impartial witness, when applicable, and by the medical person 
conducting the informed consent discussion, before undertaking any trial-related procedure. One 
copy of the signed and dated Informed Consent Form should be given to the patient. The originally 
signed document should be archived in the Investigator Site File. 
The approved Patient Information sheet must not be changed without prior approval by the IEC. 
Whenever new study information becomes available during the trial conduct, the patients still on 
treatment must be informed and a new Informed Consent form or an addendum to the already signed 
Informed Consent form must be signed and dated by each patient, or her/his legal representative or 
by an impartial witness, when applicable. 
If a subject unexpectedly becomes incapable during the course of the trial, legally acceptable 
representative authorization should be obtained for the patient to continue trial participation. 
16.4 Confidentiality of information  
Information about study patients will be kept confidential and managed under the applicable laws and 
regulations, which require a signed patient authorization providing the following information: 
- 
What protected health information (PHI) will be collected from the patient enrolled in the 
study 
- 
Who will have access to that information and for what purpose 
- 
Who will use or disclose that information  
- 
The right to revoke authorization for disclosure of His/Her own PHI. 
If a patient revokes authorization to collect or use PHI, the Investigator, by regulation, retains the 
ability to use all information collected prior to the revocation of patient authorization.  
The data collection system for this study exploits built-in security features to encrypt all data, 
preventing unauthorized access to confidential participant information. 
Patient names will not be included on any forms, reports, publications, or in any other disclosure, 
except when required by laws. 
Patient names, address, birth date and other identifiable data will be replaced by a numerical code.  
All information regarding study treatment is privileged and represents confidential information. The 
Investigator agrees to keep all study results in confidence. Such information shall not be disclosed to 
third parties except to regulatory authority(ies), when requested. Individual Investigators may present 
results of the study at scientific meetings.  
 

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17 QUALITY CONTROL 
17.1 Responsibilities of the investigators 
The Investigators undertake to perform the study in accordance with Good Clinical Practice. The 
Investigators are required to ensure their compliance to the procedures required by the protocol. The 
Investigators agree to provide all information requested in the Case Report Form in an accurate and 
legible manner according to the instructions provided. The Investigators have responsibilities to take 
all reasonable steps to ensure the proper conduct of the study as regards ethics, protocol adherence, 
integrity and validity of the data recorded on the Case Report Forms.  
 
17.2 Study Monitoring  
Monitoring activities will be done in order to verify that the data are authentic, accurate, and 
complete; that the safety and rights of the subject are being protected; and that the study is conducted 
in accordance with the currently approved protocol, GCP, and all applicable regulatory requirements. 
At regular intervals during the study, each participating center will be contacted by a representative 
of the Sponsor (internal and/or external staff authorized and duly appointed by the Sponsor), through 
site visits and/or telephone calls, to review the study progress, the investigators and subjects 
adherence to protocol requirements.  
The following points will be scrutinized:  
● subject informed consent 
● subject recruitment and follow-up 
● study drug allocation  
● subject compliance to the study treatment 
● study treatment accountability  
● adverse event documentation and reporting 
According to the guidelines on ICH Good Clinical Practice, the monitor of the study will check the 
Case Report Form entries against the source documents.  
Data will be collected by eCRF and data will be systematically checked by representative of the 
Sponsor (internal and/or external staff authorized and duly appointed by the Sponsor) for consistency, 
completeness and accuracy. 
 
17.3 Audits and inspections 
Authorized representatives of a regulatory authority or Ethics Committee may perform audits or 
inspections at the study centers, including source data verification. The purpose of an audit or 
inspection is to systematically and independently examine all study-related activities and documents, 
to determine whether these activities were conducted, and data were recorded, analyzed, and 
accurately reported according to the protocol, GCP, guidelines of the ICH, and any applicable 
regulatory requirement.  

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17.4 Retention of Records 
Records and documents pertaining to the conduct of this study, including eCRFs, ICFs, Investigator 
Site File (ISF) must be retained by the Principal Investigator for at least 7 years after completion or 
discontinuation of the study, or for the length of time required by relevant national or local 
regulations, whichever is longer. After that period, the documents may be destroyed, according to 
local regulations.  
18 STUDY CONDUCTION 
18.1 Administrative Structure 
This study is sponsored by the Fondazione IRCCS Istituto Nazionale dei Tumori di Milano. 
The study Sponsor will allocate qualified personnel to the present trial. All the figures involved in the 
study design, management and conduct are qualified according to Italian laws and regulations 
concerning clinical trials, and specifically trained on the objectives, procedures and instruments of 
this trial.  
18.2 Training of study site personnel 
The Principal Investigator will ensure that appropriate training relevant to the study is given to all 
investigational staff, and that any new information relevant to the performance of this study is 
forwarded to the staff involved. 
18.3 Study timetable and end of study 
The end of the study is defined as the last visit of the last patient undergoing the study. 
The study is expected to start in January 2023 and patient enrollment should be completed by January 
2025 (expected total study duration 36 months). The study may terminate prematurely if concerns for 
safety arise. All patients will receive follow-up care in accordance with standard local clinical 
practice.  
Any SAE or non-serious AE that is ongoing at the time of this data cut-off must be followed up to 
resolution unless the event is considered unlikely to resolve by the investigator, or the patient is lost 
to follow-up.  
18.4 Data Handling 
Source documents are where data is first recorded. These include, but are not limited to, hospital 
records, clinical and office charts, laboratory and pharmacy records, diaries, microfiches, 
radiographs, and correspondence. 
Direct access to source data will be granted to authorized representatives to permit trial-related 
monitoring, audits and inspections. 

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18.5 Publication of Data 
The protocol will be registered in a publicly accessible database such as clinicaltrials.gov.  
This trial is intended for publication, even if terminated prematurely. Publication may include any or 
all of the following: posting of a synopsis online, abstract and/or presentation at a scientific 
conference, or publication of a full manuscript. In addition, after study completion and finalization 
of the study report, results of this trial will be submitted for publication and posted in a publicly 
accessible database of clinical trial results (e.g. Clinicaltrials.gov). 
 
 
 
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APPENDIX I : Quality of Life questionnaires