Comparative, Multicenter Study Estimating Association Between Germline DNA-repair Genes Mutations and PD-L1 Expression Level in Breast Cancer
Conditions: Hereditary Breast Cancer
Sponsor: Tatarstan Cancer Center

 
 
Non-Interventional Study (NIS) Protocol 
NIS Name/Code  
ESR-17-12934 
Edition Number 
1.0 
Date 
1-Jan- 2018. 
Comparative, multicenter study estimating association between germline DNA-
repair genes mutations and PD-L1 expression level in breast cancer 
 
Sponsor: 
Tatarstan Cancer Center 
The following Amendment(s) have been made to this protocol since the date of preparation: 
Amendment No. 
Date of Amendment 
Local Amendment No: 
 
Date of Local 
Amendment 
 
 

 
 
NON-INTERVENTIONAL STUDY PROTOCOL SYNOPSIS  
 
Comparative, multicenter study estimating association between 
germline DNA-repair genes mutations and PD-L1 expression level in 
breast cancer 
 
 
Co-ordinating Investigators of the Non-Interventional Study  
 
Dr. Marat G. Gordiev  
29, Sibirskiy trakt, Kazan, Russia, 420029 
 
PhD Dina D. Sakaeva  
73/1, Oktyabrya avenue, Ufa, Russia, 450002  
 
 
Study Site(s), number of subjects planned: 
It is estimated that 240 patients will be entered into the study from 2 centers (Tatarstan Cancer 
Center and Republican State Clinical Oncological Dispensary in Ufa) in approximately 1 year. 
 
Total planned Study period: 
First subject in 
January 2018 
Last subject in 
January 2019 
Study Database lock 
March 2019 
Final Study Report 
July 2019 
 
 
 
 
Medicinal Products (type, dose, mode of administration) and concomitant medication 
Not applicable. This is an observational study; therefore, patients are not assigned to a particular 
treatment by study protocol. Treatment will be according to current clinical practice. 

 
 
Rationale for this Non-Interventional Study (NIS) 
Currently there is no precise data concerning the prevalence and types DNA-repair genes 
mutations, which lead to Homologous Recombination-deficiency, and its correlation with PD-
L1 expression in BC.  
Objectives of this Non-Interventional Study 
Primary objective  
To reveal association between the presence of germline DNA-repair genes mutations and PD-
L1 expression level in tumour and immune cells in breast cancer (to reveal the differences of 
PD-L1 high tumor rate between patients with hereditary BC who have clinically significant 
germline mutations in DNA-repair genes (HR- deficiency) and patients with sporadic BC 
without such mutations).  
Secondary objectives  
- To study the frequency of germline mutations forming the familial breast cancer in Tatarstan 
and Bashkortostan republics. 
- To estimate the relation of the frequency of PD-L1 expression in tumour or/and immune cells 
with disease stage, receptor status (ER, PR, HER2neu), morphological type, Grade  
- To estimate the relation of number of tumour-infiltrating lymphocytes (TILs) with disease 
stage, receptor status (ER, PR, HER2neu), morphological type, Grade and presence of clinically 
significant mutations in genes of DNA-repair system.  
Study design 
This is a multicentre, non-interventional, prospective study to be carried out in representative 
oncology departments / institutions in order to determine the association between the presence 
of germline DNA-repair genes mutations and PD-L1 expression level in tumour and immune 
cells in breast cancer. No additional procedures besides those already used in the routine clinical 
practice will be applied to the patients. Treatment assignment will be done according to the 
current practice.  
It is planned to enroll approximately 240 subjects in Russian Federation. 
  
PD-L1 expression in tumor and immune cells in FFPE tumor tissue samples is the primary 
variable in this study.  
 
Genetic testing of germline mutations in DNA-repair system genes (NGS) in blood samples 
receiving on Screening/ Baseline Visit and PD-L1 expression testing (IHC) in FFPE tumor 
tissue samples will be carried out in Republican Clinical Oncological Dispensary, Kazan.  
 

 
 
The investigated group will consist of patients with clinical features of hereditary BC and 
revealed pathogenic germline mutations in DNA-repair genes (TP53 MLH1 MSH2 MSH6 
PMS2 EPCAM APC MUTYH CDKN2A CDK4 ATM KIT PDGFRA CDH1 CTNNA1 PRSS1 
SPINK1 BRCA1 BRCA2 FANCI FANCL PALB2 RAD51B RAD51C RAD54L RAD51D 
CHEK1 CHEK2 CDK12 BRIP1 PPP2R2A BARD1 PARP1 STK11 XRCC3). The control 
group will consist of patients with sporadic BC without mutations in these genes.  
The PD-L1 expression level in tumor and immune cells and number of TILs in FFPE tissue 
samples received prior to start of any antitumor treatment will be estimated in both groups.  
Target subject population 
Target study population will be women 18 years and older with BC diagnosed before enrolment 
into the study, consented to participate in this non-interventional study, who visit the oncology 
hospitals/departments in Kazan and Ufa.  
Study variable(s):  
Primary variable: 
PD-L1 expression in tumor and immune cells in FFPE tumor tissue samples estimated by ICH 
is the primary variable in this study. The primary variable value will be represented as “PD-L1 
high” and “PD-L1 low”. PD-L1 expression for both TC and IC in the tumor microenvironment 
will be determined by the percentage of cells expressing PD-L1 at any intensity above 
background staining. PD-L1 will be defined as high if either ≥ 25% of TC or ≥25% of IC 
expressed PD-L1, and PD-L1 was defined as low if both < 25% of TC and  <25% of IC 
expressed PD-L1.  
Secondary variables: 
Patient characteristics:  
• Age 
• ethnicity  
• Family oncological history with pointed localization of cancer (BC, OC, Prostate cancer, 
Pancreatic cancer or other localizations) and relation degree 
• Individual oncological history: BC, OC, pancreatic cancer or other localizations  
Disease information/ diagnostic procedures:  
• Disease stage and TNM classification  
• Morphological classification, Grade (1/2/3) 

 
 
• Receptor status: ER, PR, HER2neu 
• Extent of the disease 
• Profile of germline mutations in DNA-repair system genes  
• Number of TILs 
• PD-L1 expression in tumor and immune cells in FFPE tumor tissue samples estimated 
by ICH as “PD-L1 high” or “PD-L1 low” based on 50% cut-off. PD-L1 will be defined 
as high if either ≥ 50% of TC or ≥ 50% of IC expressed PD-L1, and PD-L1 was defined 
as low if both< 50% of TC and< 50% of IC expressed PD-L1.    
•  
Statistical methods 
A Non-Interventional Study is a study in which epidemiological methods including other 
methods that can be used to analyse human population health data. 
All statistical analyses will be performed by statistical software system.  
A comprehensive Statistical Analysis Plan will be prepared before database lock. 
 
 
 
 

 
 
TABLE OF CONTENTS 
PAGE 
 
TITLE PAGE ............................................. ОШИБКА! ЗАКЛАДКА НЕ ОПРЕДЕЛЕНА. 
NON-INTERVENTIONAL STUDY PROTOCOL SYNOPSIS ............................................. 2 
TABLE OF CONTENTS ......................................................................................................... 6 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................................. 9 
1. 
INTRODUCTION .................... ОШИБКА! ЗАКЛАДКА НЕ ОПРЕДЕЛЕНА. 
1.1 
Background .................................................. Ошибка! Закладка не определена. 
1.2 
Rationale for conducting this NIS ................ Ошибка! Закладка не определена. 
2. 
NIS OBJECTIVES ................................................................................................. 11 
2.1 
Primary objective ................................................................................................... 11 
2.2 
Secondary objectives ............................................................................................. 11 
3. 
STUDY PLAN AND PROCEDURES .................................................................. 11 
3.1 
Overall study design and flow chart ...................................................................... 11 
4. 
SELECTION OF SUBJECT POPULATION ........................................................ 14 
4.1 
Investigators ........................................................................................................... 14 
4.2 
Inclusion criteria .................................................................................................... 15 
4.3 
Exclusion criteria ................................................................................................... 15 
5. 
DISCONTINUATION OF SUBJECTS ................................................................ 15 
5.1 
Criteria for Discontinuation ................................................................................... 15 
5.2 
Procedures for discontinuation .............................................................................. 16 
6. 
THERAPEUTIC STRATEGY .............................................................................. 16 
6.1 
Therapeutic strategy of a Non-Interventional Study .............................................. 16 
7. 
STUDY CONDUCT .............................................................................................. 16 
7.1 
Restrictions during the study ................................................................................. 16 
8. 
MEASUREMENTS OF STUDY VARIABLES AND DEFINITIONS OF 
OUTCOME VARIABLES .................................................................................... 16 
8.1 
Primary variable ..................................................................................................... 16 
8.2 
Secondary variables ..................................... Ошибка! Закладка не определена. 
9. 
SAFETY ASSESSMENT ...................................................................................... 17 
9.1 
Definitions .............................................................................................................. 17 

 
 
9.1.1 Definition of an adverse event (AE) .............................................................................. 17 
9.1.2 Definition of serious adverse event (SAE) .................................................................... 18 
9.2 
Non-interventional studies without special safety assessment .............................. 18 
9.3 
Non-interventional studies with special safety assessment.................................... 18 
9.4 
Report on safety information ................................................................................. 19 
9.4.1 Reports on adverse events ............................................................................................. 19 
9.4.2 Reports on unexpected adverse reactions at drug administration.................................. 19 
9.4.3. Register of spontaneous reports of adverse events ....................................................... 19 
10. 
ETHICAL CONDUCT OF THE NON-INTERVENTIONAL STUDY ............... 19 
10.1 
Ethics review .......................................................................................................... 20 
10.2 
Subject Informed consent ...................................................................................... 20 
10.3 
Subject data protection ........................................................................................... 20 
11. 
STUDY MANAGEMENT .................................................................................... 20 
11.1 
Monitoring, Quality Control and Archiving .......................................................... 20 
11.2 
Training of study site personnel ............................................................................. 21 
11.3 
NIS timetable and end of study .............................................................................. 21 
12. 
DATA MANAGEMENT ....................................................................................... 22 
12.1 
Collection, monitoring, processing of data and archiving ..................................... 22 
12.2 
Reporting and publication of data .......................................................................... 22 
13. 
STATISTICAL METHODS AND SAMPLE SIZE DETERMINATION ............ 22 
13.1 
Statistical evaluation – general aspects .................................................................. 22 
13.2 
Description of outcome variables in relation to objectives and hypotheses .......... 23 
13.3 
Description of analysis sets .................................................................................... 23 
13.4          Method of statistical analysis ................................................................................ 24 
14 
Determination of sample size ................................................................................. 24 
14. 
LIST OF REFERENCES .......... ОШИБКА! ЗАКЛАДКА НЕ ОПРЕДЕЛЕНА. 
 

 
 
LIST OF TABLES 
Table 1 
Study Plan ................................................................................................. 13 
 
LIST OF FIGURES 
Figure 1 
Study Flow Chart ...................................................................................... 15 
 
LIST OF ATTACHMENTS 
Attachment 1 
7th edition of TNM classification of Breast Cancer (TNM 7) 
Attachment 2 
Staging of BC according to TNM 7 
Attachment 3  
NCCN Clinical Practice Guidelines in oncology: Genetic/Familial high risk 
assessment: Breast and Ovarian.  
Attachment 4 
AE Reporting Form 
LIST OF APPENDICES 
Appendix A 
Signatures 

 
 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS  
The following abbreviations and special terms are used in this NIS Protocol. 
Abbreviation or 
special term 
Explanation 
AE 
Adverse event  
ADR 
Adverse Drug Reaction 
Assessment 
An observation made on a variable involving a subjective judgement 
(assessment) 
CRF 
Case Report Form  
LEC 
Local Ethics Committee  
PD-L1 
Ligand of programed depth receptor  
GCP 
Good Clinical Practice 
NIS 
Non-Interventional Study 
NISA 
Non-Interventional Study Agreement 
NISP 
Non-Interventional Study Protocol 
NISR 
Non-Interventional Study Report 
BC 
Breast Cancer 
OC 
Ovarian Cancer 
PI 
Principal Investigator responsible for the conduct of a NIS at a site 
PRO 
Patient Reported Outcomes 
PS 
Performance Status 
Variable 
A characteristic of a property of a subject that may vary eg, from time 
to time or between subjects 
WHO 
World Health Organization 
TILs 
Number of tumor-infiltrating lymphocytes 
IHC 
Immunohistochemistry 
NGS 
Next generation sequencing 
ER 
Estrogen receptor 
PR 
Progesterone receptor 
 
 
 
 
 
 

 
 
1. 
INTRODUCTION 
1.1 
Background 
 
Breast cancer (BC) occupies the first place among malignancy in females (29.9% of all tumors 
in female patients in Russian Federation in 2015) [1]. 
One of the most perspective direction of the oncotherapy is anticancer immunotherapy – 
employment of inhibitors of immune checkpoints. Immune checkpoints inhibitors (such as anti-
PD-1 and anti-PD-L1 antibodies) have shown good clinical efficiency in clinical research to 
cure such malignant tumor with high mutation load, as melanoma, lung cancer, and others. 
One of the hypothesis of such effect states that, usually, more cancer neoantigens are synthetized 
in the tumors with high mutation load (driven by genome instability), causing severe lymphoid 
infiltration [2-3].  This situation is balanced by overexpression of such inhibitors of the immune 
response as PD-1 and PD-L1 [4 - 6]. 
Breast cancer – is relatively heterogenic tumor, with different genetic, morphological and 
phenotypic forms.  
Despite relatively low expression of PD-L1 in BC in general, there are reasons to believe that 
genetic instability, driven by mutations in genes involved in DNA repair, can increase the 
immunogenicity and, thus, the expression of PD-L1 in BC. 
To date, it is widely accepted that 5-10% of BC cases are represented by hereditary types, i.e. 
mediated by germline pathogenic mutations in genes of DNA reparation pathways. Hereditary 
breast cancer (HBC), as well as ovarian cancer (OC), сaused by mutations in genes BRCA1, 
BRCA2, CHEK2, TP53 и PTEN, and others. Thus, one of the promising directions here is to 
understand the inter-relation among germline pathogenic mutations associated with HBC, and 
activity of PD-L1. It would allow to optimize selection of anti-PD-L1 therapy, by forming group 
of patients (matching criteria of HBC) with high level of PD-L1 expression in cancer cells and 
tumor-infiltrating lymphocytes. 
 
1.2 Rationale for the proposed study: 
 
It is important to mention, that nearly all information, accumulated on the HBC and OC in 
Russia is based on the analysis of the females of Slavic origin [7-8]. There are reasons to assume 
that representatives of other ethnic groups (total number of nationalities in Russian Federation 
exceeds 100) in Russia, have different from Slavs spectrum if founder-mutations in BRCA1/2 
genes. Thus, generally accepted diagnostic procedures aimed on the detection of the most 
frequent in Slavs founder-mutations should result in high number of false-positive results [7]. 
For example, in the pool if patients with BC in Bashkortostan republic, mutation 
5382insC BRCA1 are found in less than 4% of cases [11].  Among, patients in Sakha republic 
(Yakutia), typical for West and East European (Slavs) populations mutations 5382insC, 
4153delA, C61G in BRCA1 was not detected at al. Such kind of discrepancy, suggests the 
domination of East-Eurasian component and low number of West-European lines of mtDNA 
and Y-chromosomes in the population of eastern regions of Russia [10, 12]. 
Also, currently there are no comprehensive studies in Russia on the frequencies of mutations in 
other than BRCA1/2 genes involved in DNA reparation. 

 
 
Here we propose complex and comprehensive research program aimed to detect relation among 
presence of germline pathogenic mutations in the DNA repair network genes (including 
BRCA1/2) and frequency of high PD-L1 expression in cancer cells and tumor-infiltrating 
lymphocytes in BC. 
Understanding of inter-relation of presence of germline pathogenic mutations in BC and 
expression of PD-L1 can become one of the important approaches to identify group of patients, 
responsive to therapy by inhibitors of immune checkpoints or to combined therapy by target 
(PARP inhibitors) and immunotherapeutic drugs. 
 
2.      NIS OBJECTIVES 
2.1 
Primary objective 
To reveal association between the presence of germline DNA-repair genes mutations and PD-
L1 expression level in tumour and immune cells in breast cancer (to reveal the difference of 
PD-L1 high tumor rate between patients with hereditary BC who have clinically significant 
germline mutations in DNA-repair genes (HR- deficiency) and patients with sporadic BC 
without such mutations). 
 
2.2 
Secondary objectives 
- To study the frequency of germline mutations forming the familial breast cancer in Tatarstan 
and Bashkortostan republics. 
- To estimate the relation of the frequency of PD-L1 expression in tumor or/and immune cells 
with disease stage, receptor status (ER, PR, HER2neu), morphological type, Grade  
- To estimate the relation of number of tumor-infiltrating lymphocytes (TILs) with disease stage, 
receptor status (ER, PR, HER2neu), morphological type, Grade and presence of clinically 
significant mutations in genes of DNA-repair system.  
3. 
STUDY PLAN AND PROCEDURES 
3.1 
Overall study design and flow chart 
This is a multicentre, non-interventional, prospective study to be carried out in representative 
oncology departments / institutions in order to determine the interrelation between the presence 
of germline DNA-repair genes mutations and PD-L1 expression level in tumour and immune 
cells in breast cancer. No additional procedures besides those already used in the routine clinical 
practice will be applied to the patients. Treatment assignment will be done according to the 
current practice.  

 
 
It is planned to enroll approximately 240 subjects in 2 centers (Tatarstan Cancer Centre and 
Republican Clinical Oncological Dispensary Ufa. 
 
PD-L1 expression in tumor and immune cells in FFPE tumor tissue samples estimated by IHC 
is the primary variable in this study. The primary variable value will be represented as “PD-L1 
high” and “PD-L1 low”.   
Genetic testing of germline mutations in DNA-repair system genes (NGS) in blood samples 
receiving on Screening/ Baseline Visit and PD-L1 expression testing (IHC) in FFPE tumor 
tissue samples will be carried out in Tatarstan Cancer Centre, Kazan.  
 
The PD-L1 expression level in tumor and immune cells and number of TILs in FFPE tissue 
samples received prior to start of any antitumor treatment will be estimated in both groups.  
The only one visit is planned for all patients in this study.   
Information regarding patient demographics, the disease characteristics, and diagnostic tests 
results will be taken from the medical records.  
Information about familial and individual oncological history will be received on the visit.  
Material and methods: 
IHC testing of PD-L1 expression level in tumor tissue samples will be conducted using 
VENTANA PD-L1 (SP263) Rabbit Monoclonal Primary Antibody. PD-L1 expression for both 
TC and IC in the tumor microenvironment will be determined by the percentage of cells 
expressing PD-L1 at any intensity above background staining. PD-L1 will be defined as high if 
either ≥ 25% of TC or ≥ 25% of IC expressed PD-L1, and PD-L1 was defined as low if both, 
<25% of TC and, <25% of IC expressed PD-L1.  
Mutations analysis: 
The germline mutations in follow panel of genes will be analysed by NGS:  
TP53 MLH1 MSH2 MSH6 PMS2 EPCAM APC MUTYH CDKN2A CDK4 ATM KIT 
PDGFRA CDH1 CTNNA1 PRSS1 SPINK1 BRCA1 BRCA2 FANCI FANCL PALB2 
RAD51B RAD51C RAD54L RAD51D CHEK1 CHEK2 CDK12 BRIP1 PPP2R2A BARD1 
PARP1 STK11 XRCC3 
DNA from tissue samples will be extracted using a QIAamp DNA Blood Mini Kit. DNA 
concentration will be measured on the spectrophotometer NanoVue Plus ( «GE Healthcare»). 
Preparing sequencing library will be performed using NimblGen SepCapEZ Choice («Roche»). 
 Sequencing will be performed on the Illumina MiSeq ( «Illumina»). 
Bioinformatic analysis will be conducted as follows: 

 
 
1. Trimming the adapter sequences and low-quality reads and using Cutadapt and Trimmomatic. 
2. Mapping reads to a reference genome sequence (GRch37/hg19) using the algorithm BWA-
MEM. 
3. Quality control of input data, alignment, concentration and coverage of target regions using 
FastQC, BAMQC and NGSrich. Expected covering target regions - by up to 400x-600x, the 
proportion of target regions covered above 100x - at least 95%, which will detect somatic 
mutation content of about 1%. 
4. Search for the nucleotide variation germinal mutations using GATK HaplotypeCaller + 
UnifiedGenotyper (to produce a combined VCF-file). 
5. Search for the nucleotide variation of somatic mutations via Mutect2 + Strelka (to produce a 
combined VCF-file). 
6. Processing consensus VCF-files using the program SnpSift (filter - read depth more than 10). 
7. Annotation using SnpEff (analysis of all transcripts), ANNOVAR (allele frequency analysis 
ExAC, 1000G and ESP6500, algorithms the functional significance SIFT, PolyPhen2, Mutation 
Taster, FATMM, CADD, DANN, Eigen) and Alamut Batch (effect on splicing, database 
dbSNP, ClinVar, HGMD Professional), BIC database. 
This pipeline has the following characteristics in the analysis our target panel of genes (exons 
+ 50 bp flanking intron regions) with sample NA12878-GIABv3.2 (Precision FDA platform): 
Precision: 100% 
Recall: 100% 
F-Measure:100% 
 
 
 
Study Flow Chart 
 
 
Day 0 Visit 
Detection of 
germline mutations 
in DNA-repair 
system genes (NGS) 
Estimation of PD-L1 
expression level and TILs 
(IHC) / Study completion 
 
Family and individual 
oncological history 

 
 
 
Table 1 
Study Plan  
Study procedures 
 
Screening/ 
Baseline Visit 
(day 0) 
Biomarker analysis 
Detection of 
presence/absence  
of germline 
mutations in DNA-
repair system 
genes (NGS) 
(1-3 months after 
day 0) 
Estimation 
of 
PD-L1 
expression level 
and TILs (IHC)  
(1 month after 
NGS results) 
Informed consent 
X 
 
 
Patient demographics 
X 
 
 
Medical history 
X 
 
 
Inclusion / Exclusion 
criteria 
X 
 
 
Receiving of blood 
samples for NGS 
Х 
 
 
Genetic testing 
 
X 
 
Request of FFPE 
tumor 
tissue 
samples for IHC  
Х 
 
 
IHC 
 
 
Х 
Disease information 
Х 
 
 
 
 
4. 
SELECTION OF SUBJECT POPULATION 
4.1 
Investigators 
Patients will be recruited by the specialists working at the Tatarstan Cancer Center in Kazan 
and at the Republican State Clinical Oncological Dispensary in Ufa. 

 
 
4.2 
Inclusion criteria 
The subject population must fulfil all of the following criteria: 
1. 
The voluntary obtained informed consent signed by both the subject and the 
investigator.  
2. 
Females 18 years age or more. 
3. 
Histologically confirmed BC with known hormone receptors and HER2neu receptors 
status, Grade of tumor, diagnosed before enrolment into the study. 
4.        Availability of FFPE tissue samples received prior to any type of antitumor treatment 
start. Tumour tissue samples must be satisfied IHC requirements for PD-L1 testing.  
5. 
Ability of blood samples receiving for NGS germline mutations testing.  
6. 
Completed medical records (stage, receptors status, demographic data) 
4.3 
Exclusion criteria  
1. 
Any evidence of uncontrolled system pathology, active infections, active bleeding 
diathesis, renal graft, including virus hepatitis B, C or HIV. 
2. 
Patients participating in clinical studies 
3. 
Any medical condition, which on the opinion of the investigator may interfere the 
patient’s participation in the study. The decision of patient exclusion can be made 
based on corresponding section in medical records (which must be completed 
prior to the visit in line with local standard) or on a visit information. It can include 
but not limited any medical conditions impeding sign informed concerns (for 
example mental disorder). 
5. 
DISCONTINUATION OF SUBJECTS 
5.1 
Criteria for Discontinuation 
Subjects may be discontinued from the NIS at any time. Specific reasons for discontinuing a 
subject from this NIS are: 
1. 
Voluntary discontinuation by the subject who is at any time free to discontinue his / 
her participation in the NIS, without prejudice to further treatment 
2. 
Incorrect enrolment e.g., the patient does not meet the required inclusion/exclusion 
criteria for the study. 

 
 
5.2 
Procedures for discontinuation 
Subjects who discontinue should be asked about the reason(s) for their discontinuation.  If 
possible, they should be seen and assessed by the investigator according to current practice. 
6. 
THERAPEUTIC STRATEGY 
6.1 
Therapeutic strategy of a Non-Interventional Study 
The assignment of a subject to a particular therapeutic strategy is not decided in advance by a 
protocol but falls within current practice. 
7. 
STUDY CONDUCT 
7.1 
Restrictions during the study 
No specific restrictions are applicable for this non-interventional study. 
 
8. 
MEASUREMENTS OF STUDY VARIABLES AND 
DEFINITIONS OF OUTCOME VARIABLES 
8.1 
Primary variable 
PD-L1 expression in tumor and immune cells in FFPE tumor tissue samples estimated by ICH 
is the primary variable in this study. The primary variable value will be represented as “PD-L1 
high” or “PD-L1 low”. PD-L1 expression for both TC and IC in the tumor microenvironment 
will be determined by the percentage of cells expressing PD-L1 at any intensity above 
background staining. PD-L1 will be defined as high if either ≥ 25% of TC or ≥ 25% of IC 
expressed PD-L1, and PD-L1 was defined as low if both< 25% of TC and< 25% of IC expressed 
PD-L1.  
 
The following data to describe the primary objective will be collected:  
The “PD-L1 high” cases rate in both patients with hereditary BC with pathogenic germline 
mutations in DNA-repair system genes and patients with sporadic BC without germline 
mutations in these genes.  
8.2.    Secondary variables 
The secondary variables for this study are defined as: 
Patient characteristics:  

 
 
• Age 
• ethnicity (Tatar, Bashkir, Slavic etc) 
• Family oncological history with pointed localization of cancer (BC, OC, Prostate cancer, 
Pancreatic cancer or other localizations) and relation degree 
• Individual oncological history: BC, OC, pancreatic cancer or other localizations  
Disease information/ diagnostic procedures:  
• Disease stage and TNM classification  
• Morphological classification, Grade (1/2/3) 
• Receptor status: ER, PR, HER2neu 
• Extent of the disease 
• Profile of germline mutations in DNA-repair system genes  
• Number of TILs 
• PD-L1 expression in tumor and immune cells in FFPE tumor tissue samples estimated 
by ICH as “PD-L1 high” or “PD-L1 low” based on 50% cut-off. PD-L1 will be defined 
as high if either ≥ 50% of TC or ≥ 50% of IC expressed PD-L1, and PD-L1 was defined 
as low if both< 50% of TC and< 50% of IC expressed PD-L1.    
9. 
SAFETY ASSESSMENT 
9.1 
Definitions 
9.1.1 Definition of an adverse event (AE) 
“Adverse Event” or “AE” means a development of any untoward medical occurrence or the 
deterioration of a preexisting medical condition in a patient or clinical trial subject and which 
does not necessarily have a causal relationship with this treatment. 
Adverse Event might be an unfavorable symptom (e.g. nausea, chest pain), factor (e.g. 
tachycardia, enlarged liver) or abnormal research result (e.g. deviation of laboratory rates, 
alteration of electrocardiogram) temporally associated with the use of a medicinal product, 
whether or not considered related to the medicinal product. 
 The following information should be reported: the use of medication during pregnancy 
(irrespectively of whether the pregnancy termination is known), and/or lactation, suicide and 
attempted suicide, suspected drug interactions.  

 
 
9.1.2 Definition of serious adverse event (SAE) 
A SAE is an AE, occurring at any dose or study phase that fulfils one or more of the 
following: 
- 
Results in death. 
- 
Is life-threatening. 
- 
Requires in-patient hospitalization or prolongation of existing hospitalization. 
- 
Results in persistent or significant disability/incapacity or substantial disruption of the 
ability to conduct normal life functions. 
- 
Is a congenital abnormality/birth defect? 
Is an important medical event that may jeopardize the subject or may require medical 
intervention to prevent one of the outcomes listed above. 
 
NOTE. 
• The Term "threat to life" with respect to SAEs means that there was an immediate risk 
of lethal outcome in the patient at the time of this event. This definition does not refer 
to event which hypothetically could result in lethal outcome, in that case, that if it had 
occurred at more severe form. 
Medical events that are important, but do not result in lethal outcome or events that are directly 
life-threatening or requiring hospitalization, can also be considered as serious adverse events in 
those cases when, in accordance with sound clinical or scientific opinions, it is hazardous for 
the patient (or patient) and when in order to prevent one of the outcomes mentioned above 
medical or surgical intervention may be required. In this case, the event is considered as serious. 
Examples of such events are common or malignant tumor, allergic bronchospasm requiring 
intensive therapy in the emergency care department or at home, hematological disorders or 
convulsions that do not result in hospitalization or the development of drug dependency or drug 
abuse. 
 
9.2 
Non-interventional studies without special safety assessment 
The active collection of any safety data will not be performed due to the non-interventional type 
of the study. Spontaneous reports of events related to safety will be reported in accordance with 
the pharmacovigilance regulatory requirements in the post-marketing period. It is imperative 
that all investigators participating in the study must be familiarized with this section of the 
Protocol. Primary investigator is responsible for training of co-investigators involved in the 
study upon the procedures of processing of spontaneously reported safety events, as well as with 
national pharmacovigilance regulatory requirements in Russia. 
9.3 
Non-interventional studies with special safety assessment 
The active collection of any safety data will not be performed due to the non-interventional type 
of the study. 

 
 
9.4 
Report on safety information  
9.4.1 Reports on adverse events 
Safety monitoring will be carried out in accordance with the local pharmacovigilance 
requirements, used in standard medical practice in the Russian Federation. 
9.4.2 Reports on unexpected adverse reactions at drug administration 
Since it is not planned to research any study drug or vaccine, and monitoring of patients 
receiving standard therapy with drugs approved for use in the Russian Federation will be carried 
out. Safety monitoring and report of adverse events, including adverse reactions, not specified 
in the package inserts, will be performed in accordance with local pharmacovigilance 
requirements, applicable for standard medical practice in the Russian Federation. 
9.4.3. Register of spontaneous reports of adverse events 
The following principles of AEs registration, reported for patients participating in the study, 
should be applied: the investigator must report AEs within the pharmacovigilance procedures 
and in accordance with local regulatory requirements: 
1. To the Federal Service on Surveillance in Healthcare: In written form to address: Russian 
Federation, 109074, Moscow, Slavyanskaya sq., 4, build. 1. Special form available at the 
moment on the following site should be used: 
http://www.roszdravnadzor.ru/i/upload/files/1308641445.19876-26263.doc. 
2. To the correspondent pharmaceutical company, i.e. marketing company owning 
registration certificate on the correspondent drug, according to the local regulatory 
requirements. 
3. In case if spontaneous AE is connected to the AstraZeneca drug, the investigator should 
submit information about AE to the Patient Safety department of AstraZeneca (in business 
and non-business hours) within 24 hours from the moment of adverse event information 
receipt via any of the following ways:  
• E-mail: AdverseEvents.ru@astrazeneca.com  
• Send a message and /or fill (Attachment 4 to the protocol) the AE Reporting Form;  
• Tel.: 8 (495) 799 56 99, Fax +7 (495) 799 56 98.  
10. 
ETHICAL CONDUCT OF THE NON-INTERVENTIONAL STUDY 
The Non-Interventional Study will be performed in accordance with ethical principles that are 
consistent with the Declaration of Helsinki, ICH GCPs. 
The Investigator will perform the NIS in accordance with the regulations and guidelines 
governing medical practice and ethics in the country of the NIS and in accordance with currently 
acceptable techniques and know-how. 

 
 
10.1 
Ethics review 
The final protocol of the Non-Interventional Study, including the final version of the Subject 
Informed Consent Form, must be approved or given a favourable opinion in writing by the Local 
Ethics Committee. 
The Local Ethics Committee must also approve any amendment to the protocol and all 
advertising used to recruit subjects for the study, according to local regulations. 
10.1 
Subject Informed consent 
The Investigator at each site will ensure that the subject is given full and adequate oral and 
written information about the nature, purpose, possible risk and benefit of the NIS.  Subjects 
must also be notified that they are free to discontinue from the NIS at any time.  The subjects 
should be given the opportunity to ask questions and allowed time to consider the information 
provided. 
The signed and dated subject informed consent must be obtained before any specific procedure 
for the NIS is performed, including: 
• 
Interview with the investigator 
• 
Fulfil the questionnaires  
• 
CRFs completion. 
The Investigator must store the original, signed Subject Informed Consent Form.  A copy of the 
signed Subject Informed Consent Form must be given to the subject.  
10.2 
Subject data protection 
The Subject Informed Consent Form will incorporate wording that complies with relevant data 
protection and privacy legislation.  Pursuant to this wording, subjects will authorise the 
collection, use and transmission of their personal data by the Investigator and by those persons 
who need that information for the purposes of the NIS. 
The Subject Informed Consent Form will explain that NIS data will be stored in a computer 
database, maintaining confidentiality in accordance with the local law for Data Protection. 
11. 
STUDY MANAGEMENT  
11.1 
Monitoring, Quality Control and Archiving 
The sponsor will carry out Monitoring and quality control in each centre to: 
• 
Provide information and support to the investigator(s) 

 
 
• 
Confirm that the research team is complying with the protocol and that data are being 
accurately recorded in the case report forms (CRFs) 
• 
Ensure that the subject informed consent forms are signed and stored at the 
investigator’s site 
• 
Ensure that the CRFs are completed properly and with adequate quality. 
  
11.1 
Training of study site personnel 
Investigators should be trained in conduction of the study. New information about the study 
should be timely given to doctors. 
Investigators should be trained in the local regulatory pharmacovigilance procedures 
requirements. If information about AE, emerged against the background of the use of any 
investigational drug or vaccine becomes known to Investigator, Investigator must collect and 
transmit such information to mentioned above contacts within the time-frames regulated by 
local pharmacovigilance requirements. 
11.1 
NIS timetable and end of study 
Before the first subject is enrolled in the NIS and any NIS related procedures are undertaken the 
following should be fulfilled 
• 
Written approval of the NIS by the Ethics Committee and/or Regulatory Authorities, 
according to local regulations 
The planned timetable for the NIS is estimated to be as follows: 
First subject in 
January 2018 
Last subject in 
January 2019 
Study Database lock 
March 2019 
Final Study Report 
July 2019 
 
 

 
 
12. 
DATA MANAGEMENT  
12.1 
Collection, monitoring, processing of data and archiving 
Each enrolled patient would be assigned with unique identification number (the least available 
from the pre-designed sequence). The individual Case Report Form (CRF), specially designed 
for this study, will be completed for each patient enrolled.  
The collection of data from out-patient medical records or disease histories for hospitalised 
patient, as well as collection of data received by a physician during the routine patient 
examination will be performed. Any special data collection procedures are not stipulated within 
the framework of this study. All patient data will be recorded by the participating physician both 
in the source documents and in the CRF. Source documents are kept by physician. 
Signed Informed Consents will be kept by the physician during the study and after the study 
completion. 
Database will have to be completed, and then passed appropriate quality check, considered to 
be full and accurate, and then will be locked. Collected data will be analyzed.  
Essential study documents must be retained by the participating physician for as long as needed 
to comply with local and international regulations. No source documents, containing patients’ 
personal data, should be taken away from the physician.  
12.2 
Reporting and publication of data 
• 
Investigators will prepare a Non-Interventional Study Report within 6 months after 
completion of the last subject. 
• 
The results from the NIS will be presented to the Investigators as a Study report at 
Q4, 2019.  
In accordance with the Declaration of Helsinki, both authors and publishers have ethical 
obligations. In publication of the results of the NIS, the authors are obliged to preserve the 
accuracy of the results.  Negative as well as positive results should be published or otherwise 
publicly available.  
13. 
STATISTICAL METHODS AND SAMPLE SIZE DETERMINATION  
13.1 
Statistical evaluation – general aspects 
This is prospective non-interventional study designed as a case-control type for exact 
assessment of influence of the mutations in DNA repair genes presence on PD-L1 expression 
in BC. Case-control studies are often used for comparison of influence a predictive factor on a 
outcome. The primary variable (PD-L1 expression) and explored risk factor (mutations in DNA 
repair genes presence) will have nominal value: high/low and yes/no respectively.  

 
 
 A Non-Interventional Study is a study in which epidemiological methods including other 
methods that can be used to analyse human population health data. 
All statistical analyses will be performed by means of the statistical software system.  
A comprehensive Statistical Analysis Plan will be prepared before database lock. 
13.2 
Description of outcome variables in relation to objectives and hypotheses 
The following measures will be analyzed: 
- 
Proportion and number of PD-L1 high cases in investigational group.  
- 
Proportion and number of PD-L1 high cases in control group.  
- 
Proportion of  PD-L1 high cases in different morphological variants patient’s subgroups.  
- 
Proportion of  PD-L1 high cases in different receptor status (ER, PR, HER2neu) patients 
subgroups.  
- 
Number of TILs in depends on germline mutation status, morphological variant, Grade, 
receptor status (ER, PR, HER2neu).  
- 
The frequency of germline mutations forming the familial breast cancer in Tatarstan and 
Bashkortostan republics. 
- 
Proportion of different BC subtypes in both investigational and base groups.  
- 
Demographic characteristics, familial and individual history for all the screened patients.  
13.3 
Description of analysis sets 
All Enrolled set will consist of all the subjects who signed informed consent to enter the study. 
Patient disposition and baseline characteristics and demography as well as analysis of the 
primary and secondary objectives will be based on the set. 
All patients will be enrolled in 2 groups 1:1. The analyses of primary objective will be conducted 
based on the results in these groups:  
• The investigated group will be consists of patients with clinical features of hereditary 
BC (according to NCCN breast and/or ovarian cancer genetic assessment guidelines 
V1.201722 ) and revealed clinically significant (pathogenic) germline mutations in DNA-
repair genes (TP53 MLH1 MSH2 MSH6 PMS2 EPCAM APC MUTYH CDKN2A 
CDK4 ATM KIT PDGFRA CDH1 CTNNA1 PRSS1 SPINK1 BRCA1 BRCA2 FANCI 
FANCL PALB2 RAD51B RAD51C RAD54L RAD51D CHEK1 CHEK2 CDK12 
BRIP1 PPP2R2A BARD1 PARP1 STK11 XRCC3) regardless morphological type, 
grade, receptor status and extent of the disease. 

 
 
• The control group will be consisting of patients with sporadic BC without germline 
mutations in DNA-repair genes (TP53 MLH1 MSH2 MSH6 PMS2 EPCAM APC 
MUTYH CDKN2A CDK4 ATM KIT PDGFRA CDH1 CTNNA1 PRSS1 SPINK1 
BRCA1 BRCA2 FANCI FANCL PALB2 RAD51B RAD51C RAD54L RAD51D 
CHEK1 CHEK2 CDK12 BRIP1 PPP2R2A BARD1 PARP1 STK11 XRCC3) regardless 
morphological type, grade, receptor status and extent of the disease. 
13.4 
Method of statistical analysis 
Considering the study objectives, the analysis strategy will be as follows:  
A descriptive analysis approach will be used to analyse the study population, baseline data and 
clinical outcomes. 
Descriptive statistics will include n, mean, median, standard deviation, minimum and maximum 
for continuous variables and n, frequency and percentage for categorical values. Proportion will 
be assessed together with 95% confidence interval, if applicable. 
The assess of differences in pd-l1 high cases frequency between two groups and association 
of germline ddr-gene mutations and pd-l1 expression level will be performed by logistic 
regression with odds ratio calculation with 95% confidence interval  
14. 
DETERMINATION OF SAMPLE SIZE 
Today the data of PD-L1 expression level in breast cancer are limited. There are a few number 
of studies, which evaluated the frequency of PD-L1 expression in different breast cancer 
variants and the data of PD-L1 expression level in these studies are very differentiated depends 
on diagnostic approach, clone of antibodies and  cut-off17-21. We have calculated 2 variants of 
sample size based on minimal and maximal literature quantity data, using the Chi-squared 
statistic (or z test). Due to the absence of exact data about PD-L1 expression in hereditary and 
sporadic breast cancer we calculated the sample size based on data from general BC population 
(mainly is represented by sporadic BC) and TNBC (significant part of which is represented by 
hereditary BC). In the articles used as basis for calculation17-21 the authors assessed PD-L1 
expression in tumor cells and the cut-off for PD-L1 positivity was 5%. In 2 studies estimated 
PD-L1 expression in total BC population as 4% and in TNBC as 11% the SP142 clone was used 
for IHC. We have found the only one study assessing concordance between SP142 and SP263 
antibodies clones for IHC (on a subset of cases including BC) and the concordance was 95%.  
We are planning to assess the PD-L1 expression in both tumor and immune cells and use the 
25% of PD-L1 positive cells as cut-off.  
Considering the listed above we assume that the sample size will be near to a mean value 
between these two calculations.  
Near 120 patients with breast cancer who have mutations in DNA-repair system genes and near 
120 patients with breast cancer without mutations will be enrolled to the study in 2 centers: 

 
 
Tatarstan Cancer Center, Kazan and Bashkortostan Clinical Oncology Center, Ufa.  We will 
need to test near 390 patients for enrolling 240 patients (expected screen failure rate 35-40%). 
This data are based on the results of own pivotal study which has shown the frequency of 
clinically significant mutations in genes of DNA-repair system in familial BC 65%.    
1. If α (two-tailed) =0,05, β =0,2, subject ratio in groups (q1 and q2)=0,5/0,5, frequency of BC 
cases with high PD-L1 expression in sporadic BC group (based on the data in general population 
of BC19) P0=0,217, frequency of BC cases with high PD-L1 expression in hereditary BC group 
(based on the data in TNBC population, significant part of the letter is represented by HBC20) 
P1=0,559, that OR 4.574, RR= 2.576 
The standard normal deviate for α = Zα = 1.960 
The standard normal deviate for β = Zβ = 0.842 
Pooled proportion = P = (q1*P1) + (q0*P0) = 0.388 
A = Zα√P(1-P)(1/q1 + 1/q0) = 1.911 
B = Zβ√P1(1-P1)(1/q1) + P0(1-P0)(1/q0) = 0.768 
C = (P1-P0)2 = 0.117 
Total group size = N = (A+B)2/C = 61 
Continuity correction (added to N for Group 0) = CC = 1/(q1 * |P1-P0|) = 6 
That sample size can be calculated as: 
Sample size (with continuity correction) 
 
N 
Outcome+ Outcome- 
Group 1: 
37 
21 
16 
Group 0: 
37 
8 
29 
Total: 
74 
29 
45 
Sample size (without continuity correction) 
 
N 
Outcome+ Outcome- 
Group 1: 
31 
17 
14 
Group 0: 
31 
7 
24 
Total: 
62 
24 
38 
 
2. If α (two-tailed) =0,05, β =0,2, subject ratio in groups (q1 and q2)=0,5/0,5, frequency of BC 
cases with high PD-L1 expression in sporadic BC group (based on the data in general population 
of BC17) P0=0,04, frequency of BC cases with high PD-L1 expression in hereditary BC group 
(based on the data in TNBC population, significant part of the letter is represented by HBC18) 
P1=0,11, that OR 2,966, RR=2,750 

 
 
The standard normal deviate for α = Zα = 1.960 
The standard normal deviate for β = Zβ = 0.842 
Pooled proportion = P = (q1*P1) + (q0*P0) = 0,075 
A = Zα√P(1-P)(1/q1 + 1/q0) = 1.032 
B = Zβ√P1(1-P1)(1/q1) + P0(1-P0)(1/q0) = 0.439 
C = (P1-P0)2 = 0.005 
Total group size = N = (A+B)2/C = 442 
Continuity correction (added to N for Group 0) = CC = 1/(q1 * |P1-P0|) = 29 
That sample size can be calculated as: 
Sample size (with continuity correction) 
 
N 
Outcome+ Outcome- 
Group 1: 
250 
28 
222 
Group 0: 
250 
10 
240 
Total: 
500 
38 
462 
Sample size (without continuity correction) 
 
 
N 
Outcome+ Outcome- 
Group 1: 
221 
24 
197 
Group 0: 
221 
9 
212 
Total: 
442 
33 
409 
 
 
 
3. Our assumption. If α (two-tailed) =0,05, β =0,2, subject ratio in groups (q1 and q2)=0,5/0,5, 
frequency of BC cases with high PD-L1 expression in sporadic BC group P0=0,14, frequency 
of BC cases with high PD-L1 expression in hereditary BC group P1=0,30, that OR 2,633, 
RR=2,114 
The standard normal deviate for α = Zα = 1.960 
The standard normal deviate for β = Zβ = 0.842 
Pooled proportion = P = (q1*P1) + (q0*P0) = 0,220 
A = Zα√P(1-P)(1/q1 + 1/q0) = 1.624 
B = Zβ√P1(1-P1)(1/q1) + P0(1-P0)(1/q0) = 0.684 
C = (P1-P0)2 = 0.026 

 
 
Total group size = N = (A+B)2/C = 208 
Continuity correction (added to N for Group 0) = CC = 1/(q1 * |P1-P0|) = 13 
That sample size can be calculated as: 
Sample size (with continuity correction) 
 
N 
Outcome+ Outcome- 
Group 1: 
117 
35 
82 
Group 0: 
117 
16 
101 
Total: 
234 
51 
183 
Sample size (without continuity correction) 
 
 
N 
Outcome+ Outcome- 
Group 1: 
104 
31 
73 
Group 0: 
104 
15 
89 
Total: 
208 
46 
162 
 
 
 
 
 
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(заболеваемость и смертность) / Под ред. А.Д. Каприна, В.В. Старинского, Г.В. Петровой. 
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01 Published 15 February 2016 
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Attachment 1.23 
TNM classification of BC  
Primary tumor (T) 
TX 
Primary tumor cannot be assessed 
T0 
No evidence of primary tumor 

 
 
Tis 
Carcinoma in situ 
Tis (DCIS) 
Ductal carcinoma in situ 
Tis (LCIS) 
Lobular carcinoma in situ 
Tis (Paget) 
Paget disease of the nipple NOT associated with 
invasive carcinoma and/or carcinoma in situ (DCIS 
and/or LCIS) in the underlying breast parenchyma. 
Carcinomas in the breast parenchyma associated with 
Paget disease are categorized based on the size and 
characteristics of the parenchymal disease, although 
the presence of Paget disease should still be noted 
T1 
Tumor ≤ 20 mm in greatest dimension 
T1mi 
Tumor ≤ 1 mm in greatest dimension 
T1a 
Tumor > 1 mm but ≤ 5 mm in greatest dimension 
T1b 
Tumor > 5 mm but ≤ 10 mm in greatest dimension 
T1c 
Tumor > 10 mm but ≤ 20 mm in greatest dimension 
T2 
Tumor > 20 mm but ≤ 50 mm in greatest dimension 
T3 
Tumor > 50 mm in greatest dimension 
T4 
Tumor of any size with direct extension to the chest 
wall and/or to the skin (ulceration or skin nodules) 
T4a 
Extension to chest wall, not including only pectoralis 
muscle adherence/invasion 
T4b 
Ulceration and/or ipsilateral satellite nodules and/or 
edema (including peau d’orange) of the skin, which do 
not meet the criteria for inflammatory carcinoma 
T4c 
Both T4a and T4b 
T4d 
Inflammatory carcinoma 
Regional lymph nodes (N) 
 
Clinical 
 
NX 
Regional lymph nodes cannot be assessed (eg, 
previously removed) 
N0 
No regional lymph node metastasis 
N1 
Metastasis to movable ipsilateral level I, II axillary 
lymph node(s) 
N2 
Metastases in ipsilateral level I, II axillary lymph nodes 
that are clinically fixed or matted or in clinically 
detected* ipsilateral internal mammary nodes in 
the absence of clinically evident axillary lymph node 
metastasis 
N2a 
Metastases in ipsilateral level I, II axillary lymph nodes 
fixed to one another (matted) or to other structures 

 
 
N2b 
Metastases only in clinically detected* ipsilateral 
internal mammary nodes and in the absenceof clinically 
evident level I, II axillary lymph node metastases 
N3 
Metastases in ipsilateral infraclavicular (level III axillary) 
lymph node(s), with or without level I, II axillary node 
involvement, or in clinically detected * ipsilateral 
internal mammary lymph node(s) and in 
the presence of clinically evident level I, II axillary 
lymph node metastasis; or metastasis in ipsilateral 
supraclavicular lymph node(s), with or without axillary 
or internal mammary lymph node involvement 
N3a 
Metastasis in ipsilateral infraclavicular lymph node(s) 
N3b 
Metastasis in ipsilateral internal mammary lymph 
node(s) and axillary lymph node(s) 
N3c 
Metastasis in ipsilateral supraclavicular lymph node(s) 
*"Clinically detected" is defined as detected 
by imaging studies (excluding 
lymphoscintigraphy) or by clinical 
examination and having characteristics 
highly suspicious for malignancy or a 
presumed pathologic macrometastasis on 
the basis of fine-needle aspiration (FNA) 
biopsy with cytologic examination. 
 
Pathologic (pN)* 
 
pNX 
Regional lymph nodes cannot be assessed (for 
example, previously removed, or not removed for 
pathologic study) 
pN0 
No regional lymph node metastasis identified 
histologically. Note: Isolated tumor cell clusters (ITCs) 
are defined as small clusters of cells ≤ 0.2 mm, or 
single tumor cells, or a cluster of < 200 cells in a single 
histologic cross-section; ITCs may be detected by 
routine histology or by immunohistochemical (IHC) 
methods; nodes containing only ITCs are excluded 
from the total positive node count for purposes of N 
classification but should be included in the total number 
of nodes evaluated 
pN0(i-) 
No regional lymph node metastases histologically, 
negative IHC 
pN0(i+) 
Malignant cells in regional lymph node(s) ≤ 0.2 mm 
(detected by hematoxylin-eosin [H&E] stain or IHC, 
including ITC) 
pN0(mol-) 
No regional lymph node metastases histologically, 
negative molecular findings (reverse transcriptase 
polymerase chain reaction [RT-PCR]) 
pN0(mol+) 
Positive molecular findings (RT-PCR) but no regional 
lymph node metastases detected by histology or IHC 

 
 
pN1 
Micrometastases; or metastases in 1-3 axillary lymph 
nodes and/or in internal mammary nodes, with 
metastases detected by sentinel lymph node biopsy but 
not clinically detected† 
pN1mi 
Micrometastases (> 0.2 mm and/or > 200 cells, but 
none > 2.0 mm) 
pN1a 
Metastases in 1-3 axillary lymph nodes (at least 1 
metastasis > 2.0 mm) 
pN1b 
Metastases in internal mammary nodes, with 
micrometastases or macrometastases detected by 
sentinel lymph node biopsy but not clinically detected† 
pN1c 
Metastases in 1-3 axillary lymph nodes and in internal 
mammary lymph nodes, with micrometastases or 
macrometastases detected by sentinel lymph node 
biopsy but not clinically detected† 
pN2 
Metastases in 4-9 axillary lymph nodes or in clinically 
detected‡ internal mammary lymph nodes in the 
absence of axillary lymph node metastases 
pN2a 
Metastases in 4-9 axillary lymph nodes (at least 1 
tumor deposit > 2.0 mm) 
pN2b 
Metastases in clinically detected‡ internal mammary 
lymph nodes in the absence of axillary lymph node 
metastases 
pN3 
Metastases in ≥ 10 axillary lymph nodes; or in 
infraclavicular (level III axillary) lymph nodes; or in 
clinically detected‡ ipsilateral internal mammary lymph 
nodes in the presence of ≥ 1 positive level I, II axillary 
lymph nodes; or in > 3 axillary lymph nodes and in 
internal mammary lymph nodes, with micrometastases 
or macrometastases detected by sentinel lymph node 
biopsy but not clinically detected†; or in ipsilateral 
supraclavicular lymph nodes 
pN3a 
Metastases in ≥ 10 axillary lymph nodes (at least 1 
tumor deposit > 2.0 mm); or metastases to the 
infraclavicular (level III axillary lymph) nodes 
pN3b 
Metastases in clinically detected‡ ipsilateral internal 
mammary lymph nodes in the presence of ≥ 1 positive 
axillary lymph nodes; or in > 3 axillary lymph nodes and 
in internal mammary lymph nodes, with 
micrometastases or macrometastases detected by 
sentinel lymph node biopsy but not clinically detected† 
pN3c 
Metastases in ipsilateral supraclavicular lymph nodes 
*Classification is based on axillary lymph 
node dissection, with or without sentinel 
lymph node biopsy. Classification based 
solely on sentinel lymph node biopsy 
without subsequent axillary lymph node 
dissection is designated (sn) for "sentinel 
 

 
 
node"—for example, pN0(sn). 
 
 
 
† "Not clinically detected" is defined as not 
detected by imaging studies (excluding 
lymphoscintigraphy) or not detected by 
clinical examination. 
 
 
 
‡ "Clinically detected" is defined as 
detected by imaging studies (excluding 
lymphoscintigraphy) or by clinical 
examination and having characteristics 
highly suspicious for malignancy or a 
presumed pathologic macrometastasis on 
the basis of FNA biopsy with cytologic 
examination. 
 
 
Distant metastasis (M) 
 
M0 
No clinical or radiographic evidence of distant 
metastasis 
cM0(i+) 
No clinical or radiographic evidence of distant 
metastases, but deposits of molecularly or 
microscopically detected tumor cells in circulating 
blood, bone marrow, or other nonregional nodal tissue 
that are no larger than 0.2 mm in a patient without 
symptoms or signs of metastases 
M1 
Distant detectable metastases as determined by 
classic clinical and radiographic means and/or 
histologically proven > 0.2 mm 
 
 
 
 
Attachment 2.23 
 
Staging of BC according to TNM  
 
Stage T 
N 
M 
0 
Tis 
N0 
M0 
IA 
T1 
N0 
M0 
IB 
T0 
N1mi M0 

 
 
  
T1 
N1mi M0 
IIA 
T0 
N1 
M0 
  
T1 
N1 
M0 
  
T2 
N0 
M0 
IIB 
T2 
N1 
M0 
  
T3 
N0 
M0 
IIIA T0 
N2 
M0 
  
T1 
N2 
M0 
  
T2 
N2 
M0 
  
T3 
N1 
M0 
  
T3 
N2 
M0 
IIIB T4 
N0 
M0 
  
T4 
N1 
M0 
  
T4 
N2 
M0 
IIIC Any T 
N3 
M0 
IV 
Any T 
Any N 
M1 
 
 
Attachment 3. 
Clinical features of hereditary BC according to NCCN breast 
and/or ovarian cancer genetic assessment guidelines V1.201722 
• 
Anyone with a family history of one or more of the following: 
o A blood relative with a known mutation in a gene that increases cancer risk 
o A blood relative with two or more primary breast cancers 
o Two or more relatives with breast cancer on the same side of the family with at least one 
diagnosed before age 50 
o A blood relative with ovarian cancer 
o A close blood relative with breast cancer before age 45 
o A blood relative with male breast cancer 
• 
Anyone of Ashkenazi Jewish ancestry with breast, ovarian, or pancreatic cancer at any age. 
• 
Anyone with a cancer diagnosis and one or more of the following: 
o A blood relative with a known mutation in a gene that increases cancer risk 

 
 
o Breast cancer at or before the age of 50 
o Triple-negative breast cancer at or before the age of 60 
o Ovarian, fallopian tube, or primary peritoneal cancer at any age 
o Male breast cancer at any age 
• 
Anyone with breast cancer at any age and one or more of the following:        
o A blood relative with a known mutation in a gene that increases cancer risk 
o An Ashkenazi Jewish ancestor 
o A close blood relative with breast cancer before age 50 
o A close blood relative with ovarian cancer 
o A second primary breast cancer 
• 
Two or more close blood relatives with breast cancer with at least one diagnosed before age 
50 
• 
Anyone with a personal or family history of three or more of the following, especially if any 
of the cases are diagnosed before age 50: 
o Pancreatic cancer 
o Prostate cancer 
o Melanoma 
o Sarcoma 
o Adrenal cancer 
o Brain tumors 
o Leukemia 
o Uterine cancer 
o Thyroid cancer 
o Kidney cancer 
o Diffuse gastric cancer 
o Colon cancer 
 
 

 
 
Attachment 4. AE Reporting Form