Endogenous Mechanisms of Inactivation of the Endothelium Tumor

Study Overview
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Brief Summary
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The role of immunity in the development of cancers, and the associated escape mechanisms, have attracted renewed interest since the publication of tests testing immunological checkpoint inhibitors. One of the steps in the probably least studied immunological response is the penetration of immunocompetent cells within the tumor across the vascular barrier. This infiltration is suggested as a prognostic and predictive marker of treatment response, particularly in triple negative HER2 (Human Epidermal Growth Factor Receptor-2) overexpressing breast cancers. The methods of evaluating these infiltrates are complex and have been the subject of recommendations. A better understanding of the mechanisms of infiltration of immunity cells within tumors will certainly help to better understand the impact of cancer treatments and develop new therapeutic strategies. It is this issue of vascular endothelium that Dr. Soncin's team is developing as part of an INCa (Institut National du cancer) project. The egfl7 / VE-statin (vascular endothelial-statin) gene is thought to be involved in transendothelial passage of immune cells from vascular lumen to tumor. Its expression has already been studied in a series of breast cancers. Other markers of endothelial activation are currently being identified. The main objective of this project will be to better understand the behavior of the endothelium in a population of breast cancer where the infiltrate in immune cells is precisely likely to play a leading role. This retrospective cohort of 250 to 300 cases treated with adjuvant and neoadjuvant will be immunologically characterized using the recommendations of Salgado et al. that a multicentric team of pathologists will take ownership. This evaluation will be counter-appraised. Once our cohort is immunologically characterized, our project will focus on better understanding the endothelial mechanisms involved: which cells? immunophenotyping of immunity cells. By which vessels? (measurement of densities in blood and lymphatic vessels, density in HEV). By what mechanisms? Do the actors identified in vitro within the Inca project have an in vivo translation

Detailed Description
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The main objective of this project will be to better understand the behavior of the endothelium in a population of breast cancer where the infiltrate in immune cells is precisely likely to play a leading role. This retrospective cohort of 250 to 300 cases treated with adjuvant and neoadjuvant will be immunologically characterized using the recommendations of Salgado et al., that a multicentric team of pathologists will take ownership. This evaluation will be counter-appraised. Once our cohort is immunologically characterized, our project will focus on better understanding the endothelial mechanisms involved: which cells? immunophenotyping of immunity cells. By which vessels? (measurement of densities in blood and lymphatic vessels, density in HEV). By what mechanisms? Do the actors identified in vitro within the Inca project have an in vivo translation

Official Title
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Endogenous Mechanisms of Inactivation of the Endothelium Tumor

Conditions
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Breast Cancer, Colon Cancer

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Men and women aged 18 and over Confirmed histological diagnosis of breast carcinoma at localized or metastatic stage or colorectal cancer Treated at the Oscar Lambret Center or the Henri Becquerel Center between 1/1/2005 and 31/12/2007 Having undergone surgery for excision of the primary tumor and / or a metastasis resected specimen available Patients who gave their consent Exclusion Criteria: History of other cancers Breast or colic tumors with other histological profiles Patient treated for breast or colonic recurrence

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Patients with breast cancer<br>Patients clinical data collection Selection of patients tumor samples and centralization at the Oscar Lambret and the Henri Becquerel centres Realization of tumor samples series of cuts and paraffin shavings for: Quantitative RT-PCR (Reverse Transcription PCR): Assessment of the expression of the genes regulating the endothelium activation: egfl7, SetD5 (SET Domain Containing 5), other genes and microRNAs identified in the high-throughput screen realized by the Dr. F Soncin Semi-quantitative evaluation of the same genes expression by in situ hybridization techniques (if probes available) Semi-quantitative evaluation of the expression of the corresponding proteins by immunohistochemistry techniques (If antibodies available) Characterization of lymphocyte populations Measurement of the endothelial cell density, the lymphatic endothelium and the High Endothelial Venules | |
| Patients with colorectal cancer<br>Patients clinical data collection Selection of patients tumor samples and centralization at the Oscar Lambret Center and the Henri Becquerel Center by the teams of Dr. Yves-Marie Robin and Jean-Michel Picquenot, Head of the Anatomy and Cytopathology Departments Realization of series of cuts and paraffin shavings of the tumor samples for: Quantitative RT-PCR: Assessment of the expression of the genes regulating the endothelium activation: egfl7, SetD5, other genes and microRNAs identified in the high-throughput screen realized by the Dr. F Soncin Semi-quantitative evaluation of the same genes expression by in situ hybridization techniques (if probes are available) Semi-quantitative evaluation of the expression of the corresponding proteins by immunohistochemistry techniques (If antibodies are available) Characterization of lymphocyte populations Measurement of the endothelial cell density, the lymphatic endothelium and the High Endothelial Venules | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| correlation between the expression levels of the genes involved in the regulation of endothelial activation and the degree of tumor-infiltrating lymphocytes | The expression of the genes (egfl7, SetD5, other genes and microRNAs) will be evaluated quantitatively by RT-PCR. The amount of RNA obtained corresponds to a relative amount of RNA relative to the amount of RNA measured in a sample used as a reference. A semi-quantitative evaluation will also be carried out by in situ hybridization techniques and immunohistochemistry of endothelial expression of endothelial activation regulation markers: 0+ no labeled vessels, 1+ <30% of labeled vessels, 2+ between 30 and 60% of marked vessels, 3+> 60% of marked vessels The lymphocyte infiltration will be evaluated by measuring the ratio between the surface infiltrated by mononuclear cells and the tumor surface (for intratumoral TILS) or stromal (for total stromal TILS). The analysis will be done on H & E slides and the data will be expressed as a percentage. | 24 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| reproducibility of lymphocyte infiltration assessment on H & E slides |  | 24 months | 
| study of the subgroups of the cohort with a predominant lymphocytes infiltration cancer | description of subpopulations of immunocompetent cells by specific complementary immunolabeling of CD4 + T lymphocytes (mature T helper lymphocytes expressing the surface protein CD4), cytotoxic CD8 (T lymphocytes expressing the surface protein CD8), T-regulatory (CD25 / FoxP3), B-cell lymphocytes , macrophages and NK (Natural Killer) cells | 24 months | 
| Cell density description | Description of density in vascular endothelial (CD31 / 34 labeled), lymphatic (LYVE / podoplanin-labeled) and HEV (High endothelial venom MECA79 + (Rat Monoclonal Anti-Peripheral Node Addressin Antibody )) cells and correlation with lymphocyte infiltrate levels | 24 months | 
| Prognostic value assessment | Evaluate the prognostic value of biomarkers and lymphocytic infiltrate in terms of Global Survival and Survival Without Recurrence | 24 months | 
| Expression levels of genes involved in the regulation of endothelial activation | In subpopulations of patients treated in neo-adjuvant or metastatic situations: assess the expression levels of genes involved in the regulation of endothelial activation | 24 months | 
| treatment response data | In subpopulations of patients treated in neo-adjuvant or metastatic situations: assess the treatment response data (chemotherapy in patients with breast cancer, chemotherapy and antiangiogenic in colon cancers) | 24 months | 
| lymphocytic infiltrate data | For patients treated for breast cancer: study lymphocytic infiltrate data | 24 months | 
| the existence of a BRCA1 and or BRCA 2 mutation (which are tumour suppressor genes) | For patients treated for breast cancer: assess the existence of a BRCA1 and or BRCA 2 mutation | 24 months | 
| association between lymphocytic infiltrate data and the RER phenotype (the replication error phenotype) | For patients treated for colon cancer: to study the association between lymphocytic infiltrate data and the RER phenotype | 24 months | 
| The RER phenotype (the replication error phenotype) | For patients treated for colon cancer: to study the RER phenotype | 24 months |