RNA and Heat Shock Protein Biomarkers in Radiation-induced Fibrosis in Breast Cancer

Study Overview
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Brief Summary
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The purpose of this study is to seeking a molecular signature of pathological radiation induced fibrosis based on the response of skin fibroblasts after irradiation, comparing two groups of patients distinguished by their individual radiosensitivity. The signature will integrate recent insights in terms of alternative splicing of mRNAs and level of expression of non-coding RNAs, particularly long non-coding RNAs, snRNAs, snoRNAs and microRNAs. In each group each expression patterns of candidate HSP proteins potentially predictive of pathological radiation induced fibrosis (HSP27, HSP70, αβ crystalline) in the serum and on cell culture will be characterized.

Official Title
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Study of RNA and Heat Shock Protein (HSP) Derived Biomarkers in Radiation-induced Fibrosis in Patients Treated for Breast Cancer.

Conditions
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Breast Carcinoma, Fibrosis

Intervention / Treatment
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* Other: skin biopsies
* Other: blood samples


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: women age ≥ 18 and <70 years old non metastatic disease ECOG performance status 0 or 1 chest size ≤ 110 cm et bra size <D absence of reconstructive breast surgery patient able to undergo blood samples (haematological conditions allowing blood sample) non-evolving carcinological disease absence of systemic inflammatory disease (other than scleroderma) or diabetes no inflammatory ou infectious flare on biopsy site at the time of inclusion invasive or in situ breast carcinoma ability to provide an informed written consent form affiliation to a social security system Then stratification into two groups : group 1 : radio-sensitive patients Post-operative radiotherapy completed at least 6 months ago AND radiation induced dermal and/or soft tissue toxicity (dermatitis, fibrosis, atrophy) rated > 2 (CTCAE v4.0 scale) group 2 : radio-tolerant (control) patients Post-operative radiotherapy completed more than 4 years ago AND radiation induced dermal and/or soft tissue toxicity (dermatitis, fibrosis, atrophy) rated ≤1 (CTCAE v4.0 scale) . Exclusion Criteria: age <18 or > 70 years old evolutive cancer / metastatic disease chest size > 110 cm et bra size ≥ D previous reconstructive breast surgery ECOG performance status > 1 systemic inflammatory disease or diabetes inflammatory ou infectious flare on biopsy site at the time of inclusion, very significant ulceration in the treated breast anemic patients use of oral anticoagulants pregnant or likely to be in 6 months patients deprived of liberty or under supervision non-affiliation to a social security system

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 70 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Biomarkers<br> | Other: skin biopsies<br>* Biopsies (12 G) will be performed : in non-irradiated breast skin in irradiated breast skin<br>Other: blood samples<br>* blood samples are collected: 10 ml in EDTA tube 2,5 ml in PAXgene Blood RNA tube 4 ml in EDTA tube<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Global mRNA alternative splicing and expression of non-coding RNAs profiles in healthy dermal fibroblasts | frequency of inclusion of individual exons within the set of mRNA isoforms (overall splicing profile) and variation in expression of non-coding RNAs | 6 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Transcriptomic signature of pathological induced fibrosis when comparing the primary outcome between the two populations on cultured fibroblasts |  | 6 months | 
| Transcriptomic signature of pathological induced fibrosis when comparing the primary outcome between the two populations on serum |  | 6 months | 
| Individual radiosensitivity on healthy dermal fibroblasts | The micronuclei will be counted 24 hours after ex vivo irradiation with an indirect immunofluorescence assay (53BP1 + pATM antibodies) | 6 months | 
| Comparison of the overall mRNA splicing and non-coding RNA expression profiles between non irradiated and irradiated dermal fibroblasts in the same individual |  | 6 months | 
| Changes in cellular distribution of the main non-coding RNAs whose expression varies significantly within the pre-identified signature between the 2 groups of patients | The cellular distribution is defined as the compartment (nucleoplasm, nucleolus, intra-nuclear corpuscles, cytosol, RE, mitochondria ...) marked by the fluorescent probe labeled to the non-coding RNAs of interest (RNA-FISH) | 6 months | 
| seric HSP proteins potentially predictive of pathological induced fibrosis | HSP27, HSP70 and αB crystalline measured in serum with ELISA assay | 6 months | 
| Cellular distribution of specific HSP on fibroblast culture in each group of patients | immunolabeling of HSPs and spatial mapping and sub-nuclear distributions | 6 months | 
| Potential interactions between DNA damage response proteins and candidate HSP | Collocation of HSPs with pATM and 53-BP1 (confocal microscopy / FLIM) | 6 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Skin biopsy, Radiotherapy, RNA biomarker, HSP biomarker, Exon junction array, Gene expression, Radiosensitivity prediction