Prospective high-throughput genome profiling of advanced cancers: results of the PERMED-01 clinical trial

Background The benefit of precision medicine based on relatively limited gene sets and often-archived samples remains unproven. PERMED-01 (NCT02342158) was a prospective monocentric clinical trial assessing, in adults with advanced solid cancer, the feasibility and impact of extensive molecular profiling applied to newly biopsied tumor sample and based on targeted NGS (t-NGS) of the largest gene panel to date and whole-genome array-comparative genomic hybridization (aCGH) with assessment of single-gene alterations and clinically relevant genomic scores. Methods Eligible patients with refractory cancer had one tumor lesion accessible to biopsy. Extracted tumor DNA was profiled by t-NGS and aCGH. We assessed alterations of 802 “candidate cancer” genes and global genomic scores, such as homologous recombination deficiency (HRD) score and tumor mutational burden. The primary endpoint was the number of patients with actionable genetic alterations (AGAs). Secondary endpoints herein reported included a description of patients with AGA who received a “matched therapy” and their clinical outcome, and a comparison of AGA identification with t-NGS and aCGH versus whole-exome sequencing (WES). Results Between November 2014 and September 2019, we enrolled 550 patients heavily pretreated. An exploitable complete molecular profile was obtained in 441/550 patients (80%). At least one AGA, defined in real time by our molecular tumor board, was found in 393/550 patients (71%, two-sided 90%CI 68–75%). Only 94/550 patients (17%, 95%CI 14–21) received an “AGA-matched therapy” on progression. The most frequent AGAs leading to “matched therapy” included PIK3CA mutations, KRAS mutations/amplifications, PTEN deletions/mutations, ERBB2 amplifications/mutations, and BRCA1/2 mutations. Such “matched therapy” improved by at least 1.3-fold the progression-free survival on matched therapy (PFS2) compared to PFS on prior therapy (PFS1) in 36% of cases, representing 6% of the enrolled patients. Within patients with AGA treated on progression, the use of “matched therapy” was the sole variable associated with an improved PFS2/PFS1 ratio. Objective responses were observed in 19% of patients treated with “matched therapy,” and 6-month overall survival (OS) was 62% (95%CI 52–73). In a subset of 112 metastatic breast cancers, WES did not provide benefit in term of AGA identification when compared with t-NGS/aCGH. Conclusions Extensive molecular profiling of a newly biopsied tumor sample identified AGA in most of cases, leading to delivery of a “matched therapy” in 17% of screened patients, of which 36% derived clinical benefit. WES did not seem to improve these results. Trial registration ID-RCB identifier: 2014-A00966-41; ClinicalTrials.gov identifier: NCT02342158. Supplementary Information The online version contains supplementary material available at 10.1186/s13073-021-00897-9.

I -RATIONALE

Cancer biology and targeted therapies
The management of cancer patients, historically based on histological/clinical diagnostic and prognostic parameters (site, histological type, etc.) and on cytotoxic treatments (chemotherapy, radiotherapy) targeting both cancerous and healthy cells, has made significant progress over the past two decades thanks to increased knowledge of tumor biology. For example, in breast cancer, the analysis of hormone receptors (expression) and the HER2 tyrosine kinase receptor (expression and/or amplification) is now routinely carried out for not only prognosis but also the use of hormone therapy and targeted anti-HER2 therapies (1). Targeted therapies are drugs that can be injected or administered orally that target the signaling pathways that control tumor progression. The toxicity is generally limited. Efficacy is demonstrated in terms of objective response, progression-free survival, and sometimes overall survival, and is all the more important when the targeted protein represents a major and early molecular alteration in the course of the disease (e.g. imatinib that inhibits the mutated Kit receptor in gastrointestinal stromal tumors). These drugs work when the target is present in the disease. This "one target -one drug" concept has been adapted to numerous cancers both in routine clinical practice with around thirty different drugs and in clinical research with several hundred drugs in development.

High-throughput molecular characterization of tumors
At the molecular level, cancer is characterized by the accumulation and combination of multiple genetic and epigenetic abnormalities responsible for tumor origin, genomic instability, and the acquisition of an invasive and resistant phenotype that results in metastasis and death. For the past fifteen years, high-throughput molecular analyses ("omics") have revolutionized research by making it possible to develop a pan-genomic molecular portrait of a sample (2). The analyses revealed the extraordinary heterogeneity of the tumors and partly explained their histological and clinical heterogeneity. Thus, "organspecific cancer" is actually comprised of a large number of molecularly distinct diseases that merit individual treatments. Conversely, the cancers of different organs can have identical molecular alterations. This differentiation of cancers and the expansion of the catalog of alterations go hand in hand with the ever-increasing development of innovative targeted therapies. Recently, high-throughput sequencing has revealed a large number of "candidate" mutations (drivers) that, while admittedly occurring infrequently (< 5%), are potentially "targetable" by drugs already on the market or in development for the given cancer, other cancers, or other non-cancerous diseases.

Personalized medicine for advanced cancer stages
In this dynamic context of improved molecular characterization of cancers, development of new targeted therapies, and advances in terms of molecular typing, the therapeutic management must increasingly integrate the specific molecular characteristics of individual tumors. According to the American Food and Drug Administration (FDA), "personalized medicine" aims to obtain "the best medical results by selecting therapeutics according to patient's genomic profile or other patient's characteristics such as proteins, either circulating or at the cell surface". The idea being to deliver treatments based on the molecular typing of the tumor and/or metastasis, regardless of location and histological type, and is currently being applied in research and development.  (7), and Toronto in Canada (8). In these studies, typing is performed on a metastatic sample rather than the original tumor due to the appearance of new molecular alterations and resistant clones during tumor progression that may alter therapeutic indications.

The PERMED01 project
In this context, the Paoli-Calmettes Institute (IPC) plans to implement the PERMED01 project; a prospective monocentric cohort study collecting clinically annotated tissue samples from patients with advanced solid tumors in order to perform a molecular screening (portrait ) of the disease for therapeutic purposes. The IPC has all the necessary knowledge, expertise, and technological tools, particularly in Genomics (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), to properly type tumor samples. Our institute has also acquired experience in clinical genomics-based research via three prospective clinical trials, the SA02 trial based on transcriptome analysis and, more recently, the SAFIR01 and Shiva trials. This molecular portrait will complement the research of classical biomarkers (IHC, FISH, sequencing) that has been carried out for years, in addition to providing real-time information on a variety of molecular alterations of interest that could help guide patient treatment.

Primary objective
Define the number of patients whose molecular alteration(s), identified in samples in the context of this study, could lead to the delivery of "targeted" therapy.

Secondary objectives
-Define the molecular alterations of advanced malignant solid tumors.
-Compare the molecular portraits of metastatic samples with those of the primary tumor if available (heterogeneity, clonal expansion) and normal cells (somatic or constitutional mutations) from the same patient. -Determine, for the most frequent diseases (such as breast cancer; lung cancer; gynecological cancers, such as cervical, endometrial, ovarian, and fallopian tube; urological cancer, such as prostate, kidney, and bladder; and digestive cancers, such as colon, pancreatic, and liver), the relationship between these molecular alterations and the histological and clinical disease characteristics, including progression-free survival and overall survival. -Perform pan-genomic molecular analysis on metastatic samples with full exome sequencing and transcriptome analysis (expression, fusion transcripts: DNA microarray, RNA-seq). -Perform an analysis of circulating tumor DNA.
-In the case of breast cancer, validate preclinical models of therapeutic tumor response (xenografts, short-term culture, and organoids). -In cases of breast and digestive cancer, validate the isolation and characterization of circulating tumor cells.

III -ELIGIBILITY CRITERIA
The project is based on the collection and analysis of biological tumor and blood samples for any patient treated at our Institute for advanced malignant solid tumors (metastatic or locally advanced) progressive during at least one line of prior systemic therapy. All cancer types will be included whether they are common or rare cancers for which treatment options are even more limited at an advanced stage.
-Locally advanced or metastatic stage, with tumor lesion accessible for biopsy.
-Affiliated with or benefiting from social security.
-Signed informed consent form for participation in the study.

Exclusion criteria
-Person in an emergency situation, an adult who is neither a ward nor a dependent and is not under any type of guardianship or is unable to express consent. -Progressive brain metastases.
-Brain or bone metastases as the only biopsy site.
-Pregnant or breastfeeding women.

Patient registration
Patients after having been informed and accepting to participate in the study shall sign an informed consent form. Eligible patients are registered by completely filling out the inclusion form at: An inclusion number will be sent via email to the DCRD secretariat, the project head, the clinical research technician, the clinical research associate, the investigator, the biological resources center, and to the pathologist upon confirmation of inclusion.

Research methodology
Prospective, open-label, single-center trial. The sampling sites will be determined by the investigative team based on its accessibility and volume. Bone and brain metastases will not be sampled. At least 3 tumor fragments will be taken for each patient and sample site selected.
These fragments will be obtained: either via biopsy performed with radiological or endoscopic monitoring in the Radiology Department or the Medical-Surgical Exploration Unit (Unité d'Explorations Médico-Chirurgicales), respectively. The sample may be taken, depending on the location of the site selected: -by percutaneous image-guided biopsy (ultrasound or scanner), mainly using 14, 16, or 18-gauge needles via a manual, semi-automatic, or automatic mechanism. The radiologist will select the most suitable technique and type of needle for each sample.
-by biopsy at the end of an endoscopic ultrasound examination, mainly using 19 or 22 gauge needles that are 8 cm long. Biopsies are obtained by aspiration with a 20 ml syringe with a back-and-forth movement in the tumor; 1 to 3 passes are necessary to obtain a microbiopsy.
The number of biopsy fragments taken will be limited to 5 fragments per target (biopsy site) and the total number of biopsy fragments taken, all targets combined, will be limited to 10 fragments.
or via surgery performed in the Surgery Department.
The collected fragments will be transferred to the Pathology Department. These will include: -a fragment to be frozen for storage at the Biological Resources Center (BRC) for the immediate extraction of nucleic acids after verification of tumor cellularity, which will be noted in the report. Only samples with a cellularity greater than 30% will be taken into account. The CRB will be responsible for the storage and extraction of nucleic acids until recovered by the Molecular Oncology and Molecular Oncogenetics laboratories for genomic analysis. -a fragment fixed and embedded in paraffin will be stored in the Pathology Department for additional immunohistochemical or biological studies. -at least one fragment to be frozen, after verification of tumor cellularity, for storage in the CRB at -80 °C or in liquid nitrogen (depending on the mode of preservation of the tissue; in a cryotube in nitrogen or a bag at -80 °C) for subsequent proteomic and/or genomic molecular studies. -specifically, in the cases of patients with locally advanced or metastatic breast cancer and lung cancer: when an additional fragment is available, it will be sent to UMR1068 to be used in an ancillary study validating preclinical therapeutic sensitivity models (xenografts) of locally advanced or metastatic breast tumors (Appendix 3).
In the event that the tumor volume is insufficient to obtain 3 fragments, priority will be given to the fragment intended for the extraction of nucleic acids, followed by the fragment to be fixed and embedded in paraffin. If none of the samples taken allow for the characterization of the tumor's genomic abnormalities, regardless of the stage at which the failure occurs, it will be possible to propose a second biopsy to the patient as long as the inclusion criteria are still met. The decision to take a second sample is at the discretion of the investigator and his team, as is the choice of the metastatic site to sample, which may be the same as or different from the initial sample site.
In the event of a relapse during the follow-up period, the option of proposing a new sampling to the patient for an updated molecular characterization of the tumor is at the discretion of the investigator and his team.
The sample path, from its collection to the Pathology Department, the BRC, and finally the molecular laboratories will be strictly defined and traced for each patient.

-Blood samples
To definitively identify somatic alterations, it is necessary to compare the genome of cancer cells with that of normal cells (e.g. leukocytes) from the same patient. In addition, an analysis of circulating tumor DNA and circulating tumor cells may be performed as part of ancillary studies described in the appendices (Appendices 4 and 5).
For each patient there will be taken, preferably upon inclusion during a check-up, biopsy, or initial visit to an operating room (examination under anesthesia, diagnostic or therapeutic surgical procedure), 5 tubes each with 4 ml of blood in EDTA, i.e. 20 ml of blood for the extraction of DNA from normal cells and analyses of circulating tumor DNA (Appendix 4).
The tubes will be sent to the CRB within 2 hours for centrifugation (at 800G for 10 minutes at 4 °C) and aliquoting (plasma cryotubes for the study of circulating tumor DNA and pellet for DNA extraction) followed by frozen storage. In the event of a relapse during study follow-up, a new blood sample (5 tubes of 4 ml blood in EDTA) may also be taken for analysis of circulating tumor DNA.
Specifically, in cases of patients with breast cancer or cancer of the digestive tract: As part of the complementary study of circulating tumor cells described in Appendix 5, an additional sample of 5 tubes of 4 ml of blood in EDTA, i.e. an additional 20 ml, will be collected exclusively in patients suffering from locally advanced or metastatic breast cancer or metastatic digestive cancer. The total volume of blood drawn from these patients will be 40 ml.

-Clinical data
Clinical data from medical files and existing pathology databases of the IPC that were already declared to the CNIL will be recorded in the Case Report Form (CRF).
The anonymized patient data could also be used for the generation of a data warehouse model aiming to both structure clinical and genomic cancer data and set up a system allowing the data to be shared between the different members of a national group (OSIRIS: a national project model on data sharing and integration launched by the Integrated Cancer Research Sites (SIRICs, French comprehensive cancer centers) and accredited by the French National Cancer Institute).

-Molecular analyses
The molecular analyses will be carried out by the Molecular Oncology and Molecular Oncogenetics laboratories of the Paoli-Calmettes Institute. Real-time, weekly to bi-monthly analyzes will include testing for DNA copy number alterations and mutations, as well as assessing protein expression. These will concern genes of therapeutic interest that can be targeted by existing drugs or that predict the benefits of a particular systemic treatment. In our experience, it will take between 3 to a maximum of 5 weeks to obtain results (4 weeks in the SHIVA trial for the first 100 patients included). The DNA and RNA will be extracted according to a standard method and their integrity verified on gel and Agilent microarrays.

-Testing for copy number alterations: CGH-array
Gene amplifications and deletions will be identified. Tumor DNA will be hybridized on pangenome arrays for CGH-arrays (244K Agilent Technologies). The choice of microarray may vary depending on technological developments.

-Testing for mutations: targeted next-generation sequencing (NGS)
Mutations in genes of interest (around 370 selected genes, of which nearly 50 are "targetable" by existing drugs) will be identified via targeted next-generation sequencing (NGS). Two different technologies will be used: the PGM Ion Torrent system (Life Technologies) or the MiSeq platform (Illumina), with the HaloPlex kit (Agilent Technologies) including primers for the 370 genes of interest. The relevant medical literature will be monitored during the study in order to complement and update the list of genes coding for proteins of therapeutic interest.

-Validation of protein expression of relevant targets: IHC
Immunohistochemical analysis (IHC) will complement molecular analysis in certain cancers (e.g. hormone receptors and HER2 in breast cancer) and possibly validate the expression of the protein of interest identified via genomic analysis as altered (amplified, mutated). These analyses will be conducted on the fragments fixed and embedded in paraffin in the Pathology Department.

-Bioinformatics analysis of molecular data
The raw files of CGH-array and NGS results will be sent to the Institute's bioinformatics and biostatistics team. The results will then be transmitted in the form of a comprehensive standardized report, which will be recorded in the CRF. The report will be sent to the members of the multidisciplinary molecular tumor board (MTB) for scientific validation before the next meeting. All data generated (molecular analyses, IHC results, etc.) will be stored in a centralized database of the Data Management Unit of the DCRD.

-Complementary molecular analyses.
As part of the secondary objectives of the study, additional molecular analyses will be performed on the remaining tumor DNA, normal DNA, and tumor RNA, such as transcriptome analysis via DNA microarray (24)(25)(26) or by RNA-Seq on tumor RNA, or full exome analysis on tumor DNA. Concerning the blood, genomic analyses of circulating tumor DNA will also be performed.

-Patient care
Patients who are candidates for this project will be given a specialized consultation. During the consultation, the informed consent form will be given to the patient. If the patient agrees to participate in the study (signs consent form), the taking of the samples will be scheduled according to the above description (4.2-biological sampling at inclusion). Once the molecular analyses have been carried out, all the clinical and molecular data will be presented and discussed during a multidisciplinary, weekly, medical/scientific MTB.
This study does not evaluate proposed treatments. Patient care and monitoring will therefore be carried out according to the procedures and practices of the associated center or the specificities of a clinical trial in which the patient could be included, according to the recommendations of the MTB. The patients will then be followed for a period of 3 years. Follow-up data (relapse, death, etc.) will be collected annually. If a cancer susceptibility gene is identified, specific and individualized care will be offered to the patient. This care will not be carried out as part of the study and will require the signing of another specific informed consent form.

Definition
Any harmful event occurring in a person participating in biomedical research, whether or not the event is related to the research.
Any event is considered to be a serious adverse event (SAE) if it: -results in death.
-results in hospitalization of greater than 24 hours or an extension of existing hospitalization.
-causes permanent or a severe temporary disability.
-is the appearance of another cancer.
-causes a birth defect, fetal malformation, or an abortion -is medically significant.
-is a drug overdose (accidental or intentional).
-is an undesirable clinical or laboratory finding of a serious nature or considered as such by the investigator (with mention of the severity).

An event is not considered a serious adverse event if it:
-results in hospitalization of fewer than 24 hours. -resulted in a hospitalization scheduled prior to inclusion in the study, as well as any medical procedures included as part of the study (hospitalization for placement of an implant, catheter, etc.) insofar as no complications are observed.
-is an adverse event related to the treatment of the disease.
-is any hospitalization or death related to cancer progression and occurring beyond the notification period defined in the protocol.

Definition of expected serious adverse events (E-SAE)
This trial does not evaluate treatments proposed following the multidisciplinary consultation meeting. Events described as related to the sampling conducted for the purpose of this study (blood and tumor samples) will be considered as E-SAEs.
The E-SAEs below will not be reported by the sponsor:  During blood collection: mild pain, the appearance of a bruise on the arm at the site of the blood sample, "discomfort" felt when using a needle, and exceptional physical discomfort. including a low risk of perforation, which may involve in-hospital monitoring with antibiotic therapy due to the risk of infection, postoperative pain, nausea, post-sampling hemorrhage, post-sampling thrombosis, and in rare cases sepsis, pneumothorax, and pancreatitis.

Definition of a suspected unexpected adverse reaction (SUSAR)
A SUSAR is an event whose nature, intensity, and course are at odds with the actions performed in this research.

Course of action in the case of a serious adverse event
The declaration of a serious adverse event occurring within 6 days following a sample will be made within 24 hours of the event to the Department of Clinical Research and Development via the faxing or email of the serious adverse event notification form. The investigator will note for each event: its description, as clearly as possible and according to the medical terminology.
the start and end dates of the event.
the measures taken and the need or not for corrective treatment.
its course that, in the event of a non-fatal event, should be followed until recovery, a return to the previous status, or stabilization of any sequelae. -the causal relationship between this event and the samples taken as part of the study.
the causal relationship between the disease being treated and another pathology.
A follow-up report will then be sent to the sponsor to document the evolution of the event by completing the various follow-ups of the initial SAE declaration form.

Description of planned statistical methods
Qualitative data will be presented in the form of counts and percentages. The quantitative data will be summarized, with the associated 95% confidence intervals, by the mean, standard deviation, median, and extreme values.
The investigation of the relationship between the histological and clinical variables and the molecular alterations will be performed using chi-square or Fisher, Wilcoxon, or Kruskall-Wallis tests depending on the nature of the data collected.
Overall survival and recurrence-free survival will be evaluated via the Kaplan-Meier method from the date of initial diagnosis and from the date of treatment start post-biopsy. Survival comparisons of different groups of molecular alterations will be performed using log-rank analysis. Prognostic studies on overall survival and progression-free survival will be performed at the end of the study.

Expected number of research participants to be included
The primary objective is to determine the number of patients whose molecular alteration(s), identified via the samples, can lead to the utilization of targeted therapy. For this purpose, we want to be able to evaluate 300 cases over 3 years. Taking into account a technical failure rate of 35% (found in the SAFIR01 and SHIVA trials), 460 cases will need to be evaluated over 3 years. An intermediate statistical analysis will be performed after the first 100 typed cases to verify the failure rate: if the technical failure rate is greater than 40%, the study will be stopped.
As part of the Substantial Modification Request No.4 and in order to increase the numbers in the patient subpopulations for the performance of the analyzes planned as part of the secondary objectives, it is planned to include 100 additional patients during 1 year, for a total of 560 patients.
The total duration for inclusion is therefore 4 years.
The duration of patient follow-up is 3 years.
The total duration of the study is 7 years.

VII -RIGHT OF ACCESS TO DATA AND SOURCE DOCUMENTS
The investigator guarantees, for both himself and all persons involved during the progress of the trial, the confidentiality of all information provided by the Paoli-Calmettes Institute until the publication of the study's results. This confidentiality obligation will not apply to information that the investigator will be required to communicate to patients as part of their participation in the trial or to already published information.
The investigator undertakes not to publish, disclose, or use, in any way, directly or indirectly, scientific or technical information relating to the study.
However, in accordance with article R 5121-13 of the Public Health Code, the center and the investigator may provide study-related information to: the Minister of Health.
public medical health inspectors.
The trial may not be the subject of any written or oral comments without the prior approval of the sponsor; all information communicated or obtained during the trial belongs by right to the sponsor, who can freely dispose of it.

VIII -QUALITY CONTROL AND ASSURANCE
The Paoli-Calmettes Institute (IPC) is the sponsor of this project.
The sponsor is responsible for setting up a quality assurance system as described in institutional procedures so that the trial is performed in accordance with both the protocol and Good Clinical Practices.
The IPC's Department of Clinical Research and Development will coordinate the project and ensure quality control.

Regulations
The clinical trial shall be conducted in accordance with:

Ethics Committee
The promoter agrees to submit the study for prior notice to an Ethics Committee (CPP for Comité de Protection des Personnes). The information to be provided will relate to, on the one hand, the methods and nature of the research and, on the other, the guarantees provided for patients participating in this trial.

Declaration to the competent authorities
This protocol will be the subject of an authorization application to the ANSM.

Patient informed consent
Prior to carrying out research involving human subjects, the free, informed, and ongoing consent of the subjects must be obtained after they are informed, by the investigator or his representative, of the objective of the research, the conduct and duration of the study, the benefits, the potential risks and constraints of the trial as well as the nature of the product to be studied and the opinion of the Ethics Committee and authorization from ANSM.
The informed consent form will be dated and signed by both the patient and the investigator (one copy to be archived by the investigator and another given to the patient or his legal representative).
The information and informed consent form intended for the patient must be included in the same document in order to avoid any risk of a dispute regarding the content of the disseminated information.
In the case of trials whose objective is to carry out genomics or proteomics analyses, the information form must specify the type of research that will be carried out and the patient must have the possibility of either accepting or refusing the conservation of their biological samples for scientific research purposes.

Investigators' responsibilities
Investigators involved in the study agree to conduct the clinical trial in accordance with the protocol that will have been approved by the Ethics Committee and the ANSM. Investigators shall not make any modification to the protocol without the sponsor's permission.
The investigator responsible for the trial at each facility shall:  provide the sponsor with their curriculum vitae as well as those of their co-investigator.  identify the members of their team participating in the trial and define their responsibilities.  start patient recruitment after authorization from the sponsor.  try to include the required number of patients within the established recruitment period.
It is the responsibility of each investigator to:  obtain each patient's dated and signed informed consent form before any selection procedure specific to the trial.  regularly fill out the case report forms (CRFs) for every patient included in the trial and give the Clinical Research Assistant (CRA) direct access to source documents so that the latter can validate the CRF data.
 correct, sign, and date any corrections to the CRF of each patient included in the study.  notify the sponsor of serious adverse events within the required time limits.  accept regular visits from CRA and possibly from auditors appointed by the sponsor or inspectors from the supervisory authorities.
All documentation relating to the study (protocol, consent forms, CRFs, investigator files, etc.), as well as original documents (laboratory results, radiology images, consultation reports, clinical examination reports, etc.) should be kept in a safe place and treated as confidential material. The archiving of data will be the responsibility of the investigator in accordance with current legislation. The latter must retain the data as well as a patient identification list for a minimum period of 15 years after the end of the study.

Sponsor's responsibilities
The sponsor is responsible for:  acquiring insurance covering civil liabilities in the event of harmful consequences of the research for the persons involved.  communicating to investigators all information necessary for the proper conduct of the research.  requesting the approval of the Ethics Committee.  requesting authorization from the ANSM.  informing the Directors and Pharmacists of participating health care facilities.  informing the ANSM of any serious events likely to be due to the research or to its early termination the moment the sponsor has been informed.
The sponsor must archive essential documents on study conduct under conditions ensuring their safety for the minimum period stipulated in the GCPs, i.e. 15 years after the end of the research.

X -DATA PROCESSING AND RETENTION OF DOCUMENTS AND DATA RELATING TO RESEARCH
The Data Management Unit of the Paoli-Calmettes Institute will be responsible for processing the study data, which will remain the property of the Paoli-Calmettes Institute, the research's sponsor.
The data processing software used will be the ENNOV CLINICAL® software.
The Paoli-Calmettes Institute agrees to submit this project to the French Data Protection Agency.

XI -CONFIDENTIALITY AND PROPERTIES OF RESEARCH DATA
The investigators agree, for themselves and all persons involved in the trial's progress, to guarantee the confidentiality of all information provided by the Paoli-Calmettes Institute until the publication of the trial's results. This obligation of confidentiality will not apply to information that the investigator will be required to communicate to patients as part of their participation in the trial or to previously published information.
The investigators agree not to publish, divulge, or use, in any way, directly or indirectly, scientific or technical information relating to the trial.

XII -INSURANCE
The IPC agrees to acquire insurance for research involving human subjects in accordance with the current legislation, via the company SHAM (18 rue Edouard Rochet -69372 LYON CEDEX 08).

XIII -RULES RELATING TO PUBLICATION
The results of this study are the exclusive property of the Paoli-Calmettes Institute and can be used at their sole discretion. All information resulting from the trial is considered confidential, at least until the appropriate analysis and review by the sponsor and the principal investigator has been completed. Results must be published or presented in collaboration with the sponsor.
All publications, abstracts, or presentations including the results of the trial must be submitted for approval to the sponsor (IPC).
In addition, all communications, manuscripts, or presentations must include a section that mentions both the IPC and UMR 1068 as well as any organizations that contributed financially to the research.
The trial's principal investigator will be the primary signatory (1st or last author).
The investigators will be cited according to the number of patients they have or according to the depth of their involvement in the protocol and/or the pathology. The trial's statistician will be cited third, and a member of the DCRD will also be cited.
Likewise, the publication of the ancillary results (biological study) will include the names of those who carried out the ancillary work as well as the names of all the other persons involved.  b. Validation in patients included prospectively. 2-Establishment of a growth (monitoring the growth of generated organoids) and heterogeneity score: a. Rate b. Shape c. Survival and passages, presence of stem cells 3-Comparison with the existing xenograft model, taking into account the subtypes and similarity to the initial tumor. 4-Presence or not of mutations identified in the tumor. 5-Evaluation of using organoids for functional tests, study of the tumor microenvironment, testing targeted therapies (antibodies, etc.), evaluating metastatic potential (quantification of the strain's potential, migratory capacities, invasive properties, etc.).

Samples
Secondly, the creation of a breast cancer organoid bank (frozen organoids). This bank can be used to carry out functional analyses of well-characterized tumors and analyses involving targeted therapies may also be developed.

Assessment of therapeutic response
After obtaining and expanding the xenograft, the mutational status will be assessed as well as the status of the patient's tumor sample.
The tumor cells obtained via the mouse model will be cultivated in vitro for a study of therapeutic responsiveness as a "short-term culture".
These cells will then be exposed in vitro for 96 hours to a panel of drugs containing nearly 50 molecules including chemotherapies, targeted therapies, and innovative products.
The drugs considered to be "active" in vitro will be validated in vivo, in mice.
Additional analyses based more specifically on treatment effectiveness in cancer stem cells, as well as identifying anti-CSC treatments, could be carried out using this model.

for the study of invasive markers (A Badache team, CM Research Center, UMR1068)
In addition, we are implementing systematic approaches for identifying new components of invadopodia. The team involved in this project (A Badache, CRCM, UMR1068) have expertise in the study of cell migration and will study the contribution of microtubules and associated proteins, as well as the molecular components of invadopodia, to the invasive properties of CTCs. These molecules could represent new markers of invasion and metastatic progression, or even specific longer-term therapeutic targets.
The final objective of this study is to identify molecular markers involved directly or indirectly in the ability of CTCs to invade target tissues from the bloodstream. The functional data will be compared with clinical data as well as sequencing and phenotyping data from samples from these same patients.