Liquid Biopsy and Pancreas Cancer: Detection of AXL(+) CTCs (CTC-AXL-PANC)

In solid cancers, some more aggressive tumor cells actively detach from the primary lesion and then travel through the circulating compartment to reach distant organs and form micro-metastases. These circulating tumor cells (CTCs) that have become disseminated tumor cells (DTCs) flourish in their new environments and may remain dormant for many years after the complete resection of the primary tumor. Detecting CTCs in the blood is also relevant for assessing tumor progression, prognosis and therapeutic follow-up. The non-invasive, highly sensitive for CTCs analysis is called "liquid biopsy". Pancreatic adenocarcinoma and breast cancer remain among cancers of very poor prognosis and thus represent a major therapeutic challenge. In recent years, the Axl membrane tyrosine kinase receptor has been the target of growing interest. Activation of the Gas6/Axl signaling pathway is associated with, among other things, tumor cell growth and survival, epithelial to mesenchymal transition (EMT) or drug resistances. In addition, Axl overexpression is frequently identified in patients with pancreatic adenocarcinoma and is associated with a poor prognosis. For example, the Laboratoire des Cellules Circulantes Rares Humaines (LCCRH) at the CHU and the University of Montpellier has developed two new "CTC-AXL" tests to detect CTCs expressing Axl: one using the CellSearch® (gold standard and FDA-approved) system and the other using the EPIDROP technique. The purpose of this research project is to assess the concordance of the "CTC-AXL" measurement by the innovative EPIDROP technique and the CellSearch® technique in patients with metastatic pancreatic or breast cancer.

In solid cancers, after the formation and growth of the primary tumor, some more aggressive tumor cells actively detach from it and then travel through the circulating compartment to reach distant organs (bone marrow - liver - lung - brain...) and constitute new foci or micro-metastases. These circulating tumor cells (CTCs) that have become disseminated tumor cells (DTCs) flourish in their new environments and may remain dormant for many years after the complete resection of the primary tumor. Due to events not fully elucidated, DTCs can develop on site giving rise to macroscopic metastases but also join again the circulating compartment in the form of CTCs, swarm, colonize other organs and cause secondary metastases.

Detecting CTCs in the blood is very relevant for assessing tumor progression but also promising in terms of cancer disease prognosis and therapeutic follow-up. This new approach, published for the first time in 2010 under the term liquid biopsy, is therefore defined as a non-invasive blood test, extremely sensitive, achievable in real time and that allows the analysis of CTCs.

Currently, the choice of targeted therapies for a given patient is made after analyzing the primary tumor for expression and/or genomic status of specific molecular targets.

Many studies show that metastatic cells have phenotypic and genotypic characteristics distinct from those of most of the primary tumor. This can be explained either because metastatic cells acquire new genomic skills over time, or because a subset of metastatic sub-clone pre-exists within the primary tumor but has escaped detection by standard tissue biopsy techniques.

A direct analysis of CTCs could provide important additional information to prevent patients from inappropriate, costly treatments and harmful side effects.

For several years, the AXL protein, a tyrosine kinase receptor, has emerged as a new strategic target in oncology. Over-expression of AXL has been frequently identified in patients with pancreatic adenocarcinoma. AXL is a member of the Tyro3-Axl-Mer family, like its ligand protein Gas-6 (growth arrest-specific). An activation of the Gas6/AXL signaling pathway results in the activation of several effector pathways such as RAS/RAF/MEK/ERK or PI3K/AKT and is associated with, among other things, tumor cell growth and survival, metastatic formation and dissemination, Epithelial-to-mesenchymal transition (EMT) or drug resistances. It has been shown clinically that the AXL protein is a factor of poor prognosis and resistance to reference treatments (radiotherapy, chemotherapy or targeted therapy). Thus, many therapeutic strategies have been proposed and developed to inhibit the AXL pathway, ranging from chemical molecules, blocking its kinase activity and therefore the underlying signaling pathways, to nucleotide aptamers, AXL fusion proteins, and monoclonal antibodies.

Pancreatic adenocarcinoma, the 4th leading of cancer related deaths, remains among cancers of very poor prognosis and thus represents a major therapeutic challenge. The median overall survival is 11.1 months after optimal treatment (FOLFIRINOX). The clinical relevance and oncogenic potential of AXL in the progression of different types of tumors have been largely evidenced. Indeed, 50% to 75% of pancreatic adenocarcinoma samples have overexpression of AXL and the level of expression of AXL is correlated with clinical parameters indicating tumor aggressiveness and poor prognosis such as frequency of distant metastases or the survival.

In this context, the LCCRH lab, which has specialized in the detection and analysis of CTCs for 20 years, has developed a CTC-AXL detection test using the CellSearch® system. The CellSearch® system is the only method approved by the Food and Drug Administration (FDA) for the detection of CTC in colorectal, breast and metastatic prostate cancers. In addition, the LCCRH holds a patent for another technology for the detection and characterization of live and functional CTCs, called EPIDROP. The implementation of the AXL research is already done for AXL labelling on CTCs in EPIDROP as well as the visualization of the AXL cleavage by live CTC.

Thanks to this unique functional test of CTCs, it is easy to imagine offering an oncology 'oncogram' by testing in real time the effectiveness of drugs on CTCs and personalized medicine to patients.

This real-time liquid biopsy proposal on functional CTCs is quite innovative in Oncology.

To date, there are no studies on the study of functional CTCs related to AXL.

Primary objective:

- Evaluate the concordance of the CTC-AXL measurement (inclusive) by the innovative EPIDROP technique and the CellSearch technique®

Secondary objectives:

* Evaluate the accuracy of the CTC-AXL measurement (inclusive) between EPIDROP and CellSearch®
* Assess the degree of agreement between CTC-AXL measurement (inclusive) by EPIDROP and CellSearch®
* Evaluate the overall survival of patients with metastatic pancreatic cancer based on the number of circulating tumour cells carrying the AXL marker measured by EPIDROP or CellSearch® at inclusion
* Evaluate the progression-free survival of patients with metastatic pancreatic cancer based on the number of circulating tumour cells carrying the AXL marker measured by EPIDROP or CellSearch® at inclusion
* Culture CTCs from the blood sample (EDTA 10mL)
* Creation of a single liquid biopsy bio-bank for pancreatic cancer involving only plasma storage

  * Study the expression of PD-L1 (at inclusion) and detect the CTC subgroup expressing PD-L1 using EPIDROP and CellSearch® techniques;
  * Study the circulating immune system in this cohort of patients with metastatic pancreatic cancer by performing immunological analysis on blood mononuclear cells;
  * Determine if there is a significant correlation between the detection of CTC and CTC-PD-L1 and the circulating immune system (T cells, NK cells, B cells, macrophages, immune checkpoints, platelets);
  * To assess the correlation between CTC, CTC-PD-L1 and immune cells and the clinical outcomes (progression-free survival, overall survival) of these patients with metastatic pancreatic cancer.

Eligibility Criteria:
Inclusion Criteria:

* The patient is at least 18 years old;
* Patients with pancreatic cancer with remote metastases, naïve of any treatment, that is, eligible for a first line of treatment;
* Patients with oral consent

Exclusion Criteria:

* Non-affiliation or non-beneficiary of a Social Security regimen;
* Frailty persons according to Article L1121-6 of the CSP;
* Adult protected or unable to give consent as per Article L1121-8 of the CPMP;
* Pregnant or lactating women as per MSC L1121-5.
* Not included for monitoring difficulties (mutation, insufficient motivation, predictable poor compliance, priority associated pathology in care, etc.)