BMS_PD-L1_onco : Assessment of the PD-L1 Protein as a Biomarker in Oncology and Hematology

Diffuse large B-cell lymphomas (DLBCLs) represent 25 to 30% of adult non-Hodgkin lymphomas in western countries. DLBCLs are aggressive cancer but potentially curable with multi-agent chemotherapy. Whereas R-CHOP regimen has led to a marked improvement in survival, this disease remains a biologically heterogeneous entity. New therapeutic strategies are required including identification of patients' subgroups with different prognostic.

This project is based on BMS_LyTrans and Goelams 075 clinical trial. A study of whole blood transcriptome in 75 DLBCL patients and in 87 controls showed that PD-L1 (CD274) gene was overexpressed in DLBCL patients. Preliminary results demonstrated that PD-L1 is detected in plasma of DLBCL patients with a significantly higher concentration than in controls. This protein was selected as a potential biomarker because of its established role in anti-tumoral immunity. Interaction between PD-L1 and its receptor PD-1 is known to inhibit activation of immune responses by inducing T-lymphocytes anergy and/or apoptosis. Moreover, a direct involvement of PD-L1 in the protection of cancer cells from lysis by activated T lymphocytes has been demonstrated. PD-L1 expression has been described in several solid tumours, including ovary cancer, breast cancer, colon cancer, renal cell carcinoma, non-small cell lung carcinoma and in hematological malignancies such as T-NHL, MM and Hodgkin's lymphoma. Furthermore the expression of PD-L1 by tumour cells is associated with poor prognosis. The blockade of PD-L1/PD-1 axis may represent a novel therapeutic approach in aggressive cancers. These first results incite to identify the cells releasing soluble PD-L1 and to investigate its role in the anti-tumoral immunity in DLBCL patients.

The aim of this study is to identify cells producing soluble PD-L1 in DLBCL patients at diagnosis in comparison to others tumours known to express PD-L1 (metastatic breast cancer, Hodgkin's lymphoma, non-small cell lung cancer).

Secondary purposes are :

* To confirm the presence of plasma soluble form of PD-L1 in others malignancies
* To study surface expression of PD-L1 on circulating tumour cells with multiparameter fow cytometry and Veridex® technology in DLBCL and metastatic breast cancer patients
* To study surface expression of PD-L1 on circulating endothelial cells in DLBCL, Hodgkin lymphoma and metastatic breast cancer patients (subpart ended in late 2012)
* To study surface expression of PD-L1 on different types of leukocytes (monocytes, B and T lymphocytes)
* To separate circulating tumour cells expressing PD-L1 by immunomagnetic or Cell-sorting method
* To develop ELISPOT technique to study the release of soluble PD-L1 in culture supernatants of selected cells (subpart ended mid 2013)
* to evaluate the correlation between the expression of PD-L1 in the plasma and \*) the expression of PD-L1 in the tumor, \*\*) the expression of PD-L1 and other molecules in the bronchoalveolar liquid (whenever available from routine) in non-small cell lung cancer
* to evaluate the response to treatment according to plasma PD-L1 expression in non-small cell lung cancer
* to evaluate the susceptibility to develop a disease according to the single nucleotide polymorphisms of the PD-L1 gene in DLBCL and non-small cell lung cancer
* Constitution of the different cohorts and collection of samples Main cohort : de novo DLBCL at diagnosis Secondary cohorts: Hodgkin's lymphoma, metastatic breast cancer, non small cell lung cancer Control cohorts : healthy volunteers (blood donors), patients with immune thrombocytopenia (ITP)
* Quantification of plasma soluble PD-L1 in the different cohorts

Eligibility Criteria:
Inclusion Criteria:

General inclusion criteria :

* Age ≥ 18 years and ≤ 75 years,
* Life expectancy more than 4 months
* Signed informed consent obtained
* Social security affiliation is mandatory
* Non previously treated (even by corticotherapy),
* HIV negative, HBs negative, HCV negative

Inclusion criteria for DLBCL patients :

* A biopsy proven diagnosis of de novo DLBCL according to the current WHO criteria,
* Immunohistochemistry for GCB/nonGC classification according to Hans' algorithm
* Patients with advanced-stage disease defined as Ann Arbor stages III or IV, or stages I or II with bulky disease (\>7cm)

Inclusion criteria for non-small cell lung cancer patients :

* A biopsy proven diagnosis of de novo non-small cell lung cancer (all stages) according to the current WHO criteria

Inclusion criteria for Hodgkin's lymphoma patients :

* A biopsy proven diagnosis of Hodgkin's lymphoma according to the current WHO criteria

Inclusion criteria for metastatic breast cancer or with lymph node involvement :

* A biopsy proven diagnosis infiltrating lobular or ductal breast carcinoma
* with lymph node involvement or metastasis

Inclusion criteria for patients with immune thrombocytopenia (ITP) :

* Primary ITP was defined by the IWG as a platelet count less than 100 G/L in the absence of other causes or disorders that may be associated with thrombocytopenia.
* Bone marrow examination excluding a central aetiology of thrombocytopenia

Inclusion criteria for healthy volunteers :

- Inclusion criteria for blood donation according to the Etablissement Français du Sang (EFS) criteria

Exclusion Criteria:

General non-inclusion criteria :

* Age \< 18 years et \> 75 years,
* Pregnant women,
* Person legally involved in a case
* No social security affiliation
* Signed informed consent not obtained,
* Preliminary treatment (even corticoid treatment).
* HIV positive, HBs positive, HCV positive

Non-inclusion criteria for DLBCL patients :

* Lymphoma other than DLBCL,
* Transformation of a low grade lymphoma to a high grade lymphoma (DLBCL),
* Extranodal marginal zone lymphoma of MALT lymphoma,
* Post-transplant lymphoproliferative disorders,
* Lymphoblastic lymphoma,
* Burkitt's lymphoma,
* Carcinoma or history of carcinoma except in situ cervical carcinoma.

Non-inclusion criteria for non-small cell lung cancer patients : None

Non-inclusion criteria for Hodgkin patients :

- Non Hodgkin's lymphoma

Non-inclusion criteria for metastatic breast cancer or with lymph node involvement :

* Carcinoma other than infiltrating lobular or ductal breast carcinoma
* Chemotherapy in 30 days preceding the inclusion
* Hormonotherapy in 7 days preceding the inclusion
* Carcinoma or history of carcinoma except in situ cervical carcinoma.
* Hemoglobin level \< 10g/dl

Non-inclusion criteria for patients with immune thrombocytopenia (ITP) :

- Central aetiology of the thrombocytopenia

Non-inclusion criteria for healthy volunteers :

- Exclusion criteria for blood donation according to the Etablissement Français du Sang (EFS) criteria