WO2020089428A1

WO2020089428A1 - New prognostic method of pancreatic cancer

Info

Publication number: WO2020089428A1
Application number: PCT/EP2019/079904
Authority: WO
Inventors: Daniel Olive; Juan Iovanna; Jean-Charles Dagorn
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université D'aix Marseille; Centre National De La Recherche Scientifique (Cnrs); Institut Jean Paoli & Irene Calmettes
Priority date: 2018-11-02
Filing date: 2019-10-31
Publication date: 2020-05-07

Abstract

The present invention relates to a prognostic method of pancreatic cancer. In the present study, the inventors use specific antibodies to monitor the concentrations of soluble forms of BTLA in plasma of advanced PD AC patients, which are not eligible for resection, by ad hoc developed ELIS As. They showed that high plasma levels of these immune checkpoints correlate with poor outcome and can be used as prognostic factors in non-resectable PDAC. Thus the present invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising determining in a sample obtained from the patient the expression level of BTLA.

Description

NEW PROGNOSTIC METHOD OF PANCREATIC CANCER

FIELD OF THE INVENTION:

The present invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTLA ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

BACKGROUND OF THE INVENTION:

Pancreatic Ductal Adenocarcinoma (PD AC) is a major public health issue. It is predicted to reach the second leading cause of cancer-related death in Western countries by 2030 (1). Currently, surgery is the only chance for a prolonged survival but, unfortunately, only less than 20% of the PDAC patients are eligible. Despite the improvement in surgical techniques, there has been no major breakthrough in the therapeutic arsenal. In addition, PDAC is one of the most heterogeneous cancers with low chemotherapeutic sensitivity due to a dense desmoplastic stroma, a weak vasculature and significant biological aggressivity. Still today, the prognosis remains extremely dark with a 5-year survival rate around 7% (2). Inflammation and immune evasion are two major hallmarks of cancer and are crucial steps for PDAC progression (3). The immune checkpoint family, belonging to the immunoglobulin superfamily refers to transmembrane receptors and co-receptors involved in immune homeostasis to potentiate inflammation, autoimmunity but also tumor immunity (4, 5). However, in cancer, suppressive immune checkpoints are often hyper-activated to ensure an effective evasion of tumor cells from immune surveillance 6. These immune checkpoints include in part, the B7/butyrophilin- like receptors such as butyrophilin sub-family 3A/CD277 receptors (BTN3A) (7, 8), the B and T lymphocyte attenuator (BTLA) belonging to the B7-like receptors (9, 10) and the programmed death protein (PD-l) with its ligands PD-L1 and PD-L2. PD-1/PD-L1 pathways blockade (e.g. Nivolumab, Pembrolizumab) has been widely studied in solids tumors and lymphomas and shows promising therapeutic effect particularly in melanomas and non-small cell lung cancer (11, 12). However, their efficacy for other solid tumors such as PDAC remains controversial (13). Other immune checkpoints such as the BTN3A sub-family, who play an important role in VY9V52 T-cells activation and regulation, have been described as therapeutic targets in cancer. BTN3A members have been recently shown to be prognostic factors in solid cancers (8). On the other hand, the B and T lymphocyte attenuator (BTLA) have been shown to inhibit tumor specific CD8+ T cell proliferation and their high expression correlates with a shorter survival in some solid tumors mainly of the gastro-intestinal track including gastric cancer (9, 10). Despite considerable efforts, the complete role of these molecules in cancer immunotherapy and the specific role of soluble ectodomains of these receptors remain to be elucidated. Recently, the plasmatic soluble forms of sPD-l and sPD-Ll have been shown to negatively correlate with survival in myeloma (14).

SUMMARY OF THE INVENTION:

In the present study, the inventors use specific antibodies to monitor the concentrations of soluble forms of BTLA in plasma of advanced PD AC patients, which are not eligible for resection, by ad hoc developed ELISAs. They showed that high plasma levels of these immune checkpoints correlate with poor outcome and can be used as prognostic factors in non-resectable PDAC.

Thus, the present invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTLA ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value. Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

A first aspect of the invention relates to a method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTLA ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value. According to these particular embodiment, the sample can be a pancreatic tumor sample that is to say a sample obtained from the pancreatic tumor or a biopsy obtained from a pancreatic tumor.

In a particular embodiment, BTLA is sBTLA-l that is to say soluble BTLA.

A second aspect of the invention relates to a method for predicting the invasiveness of a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTLA ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

In one embodiment, the pancreatic cancer is a stage Tl pancreatic cancer, a stage T2 pancreatic cancer, a stage T3 pancreatic cancer or a stage T4 pancreatic cancer according to the UICC-TNM classification.

In another embodiment, the pancreatic cancer is a pancreatic ductal adenocarcinoma (PD AC), a pancreatic adenocarcinoma, a pancreatic serous cystadenomas (SCNs), a pancreatic intraepithelial neoplasia, pancreatic mucinous cystic neoplasms (MCNs), a non-resectable pancreatic ductal adenocarcinoma (PD AC) or a non-resectable pancreatic adenocarcinoma.

As used herein, the term“patient” denotes a human with a pancreatic cancer according to the invention.

As used herein, the term“BTLA” for“B- and T-lymphocyte attenuator” denotes a protein that in humans is encoded by the BTLA gene. BTLA has also been designated as CD272. BTLA expression is induced during activation of T cells, and BTLA remains expressed on Thl cells but not Th2 cells. Like PD1 and CTLA4, BTLA interacts with a B7 homo log, B7H4. However, unlike PD-l and CTLA-4, BTLA displays T-Cell inhibition via interaction with tumor necrosis family receptors (TNF-R), not just the B7 family of cell surface receptors. BTLA is a ligand for tumour necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immune responses.

As used herein, the term“survival time” denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as pancreatic cancer (according to the invention). The survival time rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.

As used herein and according to the invention, the term“survival time” can regroup the term OS.

As used herein, the term“Overall survival (OS)” denotes the time from diagnosis of a disease such as pancreatic cancer (according to the invention) until death from any cause. The overall survival rate is often stated as a two-year survival rate, which is the percentage of people in a study or treatment group who are alive two years after their diagnosis or the start of treatment.

As used herein and according to all aspects of the invention, the term“sample” denotes, blood, peripheral-blood, serum, plasma or cancer biopsy and particularly pancreatic cancer biopsy.

Measuring the expression level of BTLA can be done by measuring the gene expression level of BTLA or by measuring the level of the protein BTLA and can be performed by a variety of techniques well known in the art.

Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A“detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et ak, such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-l -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2’-disulfonic acid; 4,4’-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDamino fluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitro tyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N’-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphtho fluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20l320l6, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).

Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.

Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hyhridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et ah, Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et ah, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929. Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 nm) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 nm). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are“specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR (or q RT-PCR).

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.

According to the invention, the level of BTLA proteins may also be measured and can be performed by a variety of techniques well known in the art. For measuring the expression level of BTFA, techniques like EFISA (see below) allowing to measure the level of the soluble proteins are particularly suitable.

In the present application, the“level of protein” or the“protein level expression” or the “protein concentration” means the quantity or concentration of said protein. In another embodiment, the“level of protein” means the level of BTFA proteins fragments. In still another embodiment, the“level of protein” means the quantitative measurement of BTFA proteins expression relative to a negative control.

According to the invention, the protein level of BTFA may be measured at the surface of the tumor cells or in an extracellular context (for example in blood or plasma).

Typically protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or EFISA performed on the sample.

Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as EFISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS). etc. The reactions generally include revealing labels such as fluorescent, chemio luminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins BTLA. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the protein BTLA. Thus, in a particular embodiment, fragment of BTLA protein may also be measured.

In a particular embodiment, the detection of the level of BTLA can be performed by flow cytometry. When this method is used, the method consists of determining the amount of BTLA expressed on tumor cells. According to the invention and the flow cytometry method, when the florescence intensity is high or bright, the level of BTLA express on tumor cells is high and thus the expression level of BTLA is high and when the florescence intensity is low or dull, the level of BTLA express on tumor cells is low and thus the expression level of BTLA is low.

In another embodiment, the extracellular part of the BTLA protein is detected. Predetermined reference values used for comparison of the expression levels may comprise“cut-off’ or“threshold” values that may be determined as described herein. Each reference (“cut-off’) value for BTLA level may be predetermined by carrying out a method comprising the steps of

a) providing a collection of samples from patients suffering of a pancreatic cancer; b) determining the level of BTLA for each sample contained in the collection provided at step a);

c) ranking the tumor tissue samples according to said level

d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level,

e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient;

f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve;

g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets

h) selecting as reference value for the level, the value of level for which the p value is the smallest.

For example the expression level of BTLA has been assessed for 100 pancreatic cancer samples of 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the best expression level and sample 100 has the worst expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding pancreatic cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.

The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels. In routine work, the reference value (cut-off value) may be used in the present method to discriminate pancreatic cancer samples and therefore the corresponding patients.

Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.

The man skilled in the art also understands that the same technique of assessment of the expression level of a protein should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a protein of a patient subjected to the method of the invention.

Such predetermined reference values of expression level may be determined for any protein defined above.

According to the invention, the reference values for BTLA may be 1.91 ng/ml.

A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of BTLA in the sample obtained from the patient.

The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively the kit of the invention may comprise amplification primers that may be pre- labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

The present invention also relates to BTLA as biomarker for outcome of pancreatic cancer patients.

The present invention also relates to sBTLA as biomarker for outcome of pancreatic cancer patients.

The present invention also relates to BTLA as biomarker of invasiveness of pancreatic cancer and more particularly for PD AC.

Thus, in another aspect, the invention relates to an anti-BTLA antibody, for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above. In one embodiment, the anti-BTLA antibody of the present invention is an isolated anti- PD-l antibody (mAb BTLA 8.2) which is obtainable from the hybridoma accessible under CNCM deposit number 1-4123 or the anti-BTLA antibody 629.3 as described in the patent application WO2017144668.

In another embodiment, the anti-BTLA antibody comprises the 6 CDRs of the antibody obtainable from the hybridoma accessible under CNCM deposit number 1-4123 and derivatives thereof or of the antibody 629.3 described above.

In one embodiment, the antibodies can comprises the variable domains of the antibodies identified above or can be a monoclonal or a chimeric antibody of the antibodies identified above.

The invention also relates to a method for treating a pancreatic cancer in a patient with a bad prognosis as described above comprising the administration to said patient of an anti- BTLA antibody.

Another aspect of the invention relates to a therapeutic composition comprising an anti- BTLA antibody for use in the treatment of pancreatic cancer in a patient with a bad prognosis as described above.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intrathecal or subcutaneous administration and the like.

Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Compounds used to already treat pancreatic cancer can be used in combination with an anti-BTLA according to the invention. These compounds can be selected in the group consisting in Gemcitabine, 5-fluorouracil (5-FU), Irinotecan, Oxaliplatin, Albumin-bound paclitaxel, Capecitabine, Cisplatin, Paclitaxel, Docetaxel and Irinotecan liposome.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Correlation between plasma level of immune checkpoints sBTLA) and non- resectable PD AC patient overall survival. A significant inverse correlation was found between , sBTLA and patient overall survival. The correlation was established using the parametric Pearson correlation coefficient (r).

Figure 2: Receiver operating characteristics (ROC) curve analysis of plasma level for sBTLA. For each marker, ROC curves were plotted for sensitivity and specificity of survival classification (left panels). The withe circle shows the optimal values of specificity and sensitivity for optimal threshold values (Youden index associated criteria). The plasma levels of each marker were plotted for STS and LTS patients (right panels). The dashed lines represent the optimal thresholds obtained by ROC analysis. (AUC: area under the curve).

Figure 3: Kaplan Meier analysis of overall survival in patients with high and low plasma level of sBTLA.

EXAMPLE:

Material & Methods

Patients Three expert clinical centers (Paoli-Calmettes Institute, Hospital Nord and Hospital La Timone) from Marseille participated in this study after receiving ethics review board approval. Thirty-two diagnosed patients with non-resectable PD AC (locally advanced n=l2 and already metastatic at diagnosis n=20) were included in this project led by the Paoli-Calmettes Institute (clinical trial NCT01692873). Consent forms of informed patients were collected and registered in a central database. All patients were followed from diagnosis until their death or the last follow-up date. Full annotated clinical characteristics were listed in a central database. We confirm that the present study was performed in accordance with good clinical practice guidelines and the Declaration of Helsinki.

Determination of soluble BTLA concentrations in plasma

We decided to have ELIS As of the markers BTLA produced by DYNABIO S.A. (Parc de Luminy, Marseille France) according to our specifications. These specifications included i/ verification by tandem mass spectrometry of the antigen sequence ii/ optimization of the assay by testing all combinations of available monoclonal antibodies in capture and detection, targeting maximal signal/background ratio and sensitivity. Combinations of two or more antibodies in coating and/or detection were also tested to improve performances iii / checking sample compatibility (serum vs plasma, interference of the matrix), iv/ ensure that assay can be run at room temperature for easy handling and robustness.

The ELISA follow the same schedule: All steps are run at room temperature. Plates are coated overnight with the antibody selected for capture then washed. Remaining binding sites are blocked to minimize background. All next steps end with plate washing. For PD-L1 assay, all steps are conducted under shaking. Samples to be tested are incubated for 3 h. Then, the biotinylated antibody selected for detection is incubated for 30 min, followed by incubation for 15 min with the avidin-peroxidase conjugate. Finally, the substrate TMB is incubated for 15 min, the reaction stopped with H2S04 and the O.D. read at 450 nm. Concentrations are established by comparison with a range obtained with known concentrations of the recombinant antigen. The recombinant antigen was home-synthesized.

Studies comparing concentrations of all the marker measured in serum and plasma from the same blood collection showed that apparent concentrations in serum were at least ten times less than in plasma. This observation shows that clotting results in the apparent loss of a large part of the assayed proteins. Because the mechanism of such loss is unknown, determination of protein concentrations in serum might be affected by factors other than the clinical status of the patient. As consequence, use of serum samples could be misleading and should be avoided. All samples assayed in this study were plasmas. We also observed in the EFISA an interference of the plasma matrix, which becomes negligible when plasma samples are diluted at least 1/5. In the present study, all plasma samples were at least diluted 1/5 before assay.

Blood samples

The samples were obtained, under consent, at the time of the EUS-FNA biopsy procedure. According to inclusion criterias, all patients were naive of any chemotherapeutic treatment during blood sampling. Total blood fractions were processes within 4 hours from the sampling and centrifuged at 2,200 g during 15 min at 4°C in presence of EDTA. The supernatants (plasma fraction) were aliquoted in cryotubes and stored a -80°C until processing.

Statistical analysis

Correlation analysis was performed using the Pearson correlation test according to the Gaussian distribution of data. Overall survival analysis was performed using the Kaplan-Meier method and log-rank test. Univariate analysis of survival was performed using the Cox- regression model. The receiver operating characteristic (ROC) curves were generated for each marker. The areas under the curves (AUC) were assessed to evaluate each marker performance for discriminating short from long term survivors. SPSS PASW 23.0 (SPSS Inc. Chicago, IL, USA) software was used for regression analysis. ROC curves were generated using the MedCalc software for Windows, version 18.2.1 (MedCalc Software, Ostend, Belgium). GraphPad Prism 5.01 (GraphPad software Inc. La Jolla, CA, USA) was used for correlation and Kaplan-Meier survival analysis. P values <0.05 were considerate significant.

Results

Non resectable PD AC patients

Between 2012 and 2016, EUS-FNA tumor biopsies and blood samples of 32 non resectable PD AC patients were collected. All patients were recruited under the Paoli-Calmette Insitute clinical trial NCT01692873 (https://clinicaltrials.gov/show/NCT0l692873) exclusively in case of pancreatic ductal adenocarcinoma diagnosis. The overall survival median of this cohort is 6.9 months (95% Cl: (4.4-10.19)) that is very close to the worldwide reference OS median of between 6-8 months for this type of non-operable patients under palliative chemotherapy 15. We split the cohort in two subgroups according to the 6 months OS cut-off. Patients who died of disease before six months were named short term survival patients (STS, n=l6) and those who died after six months were named long term survival patients (LTS, n=l6). All the patient’s characteristics are provided in Table 1. Ninety percent of patients (29/32) received palliative treatments consisting of gemcitabine (n=8), gemcitabine-capecitabine (GEMZAR®/XELODA®) combination treatments (n=2), FOLFIRINOX combination (n=l9). The levels of sBTLA is highly correlated in non-operable PD AC patients

In this study, a specific ELISA tests was utilized to measure plasma level of sBTLA. Median values was 2.22 ng/ml for sBTLA (range 0 to 11.68 ng/ml). Note that 10/32 patients presented values below the limit of detection for sBTLA.

Plasma levels of sBTLA negatively correlate with overall survival in non resectable PD AC patients

High expression of BTLA (rp=-0.38, pval=0.03, respectively) is associated with poor prognosis in advanced pancreatic cancer (see figure 1).

Determination of the optimal cut-off for BTLA marker to classify short term versus long term PD AC survivors

We used ROC curve analysis 20 to determine for each marker the optimal cut-off that discriminates short-versus long-term survivors. As shown in Ligure 2 (left panels), the optimal cut-off is 1.91 ng/ml for sBTLA (AUC = 0.78, pval=0.00l). The plasma level of the marker is plotted in the right panels for long term and short term survivors, the dashed line indicate the threshold level of the marker.

Clinical characteristics of patients with high plasma concentrations of immune checkpoints.

We classified, for the marker tested, the patients with low and high plasma levels for each immune checkpoint, using cut-offs determined beforehand with ROC curves. We plotted the overall survival for these patients by Kaplan Meier curves (Ligure 3). Lor the biomarker analyzed, we observe strong significant differences in overall survival medians between patients with plasma concentrations above and under thresholds. Patients with high level (>1.91 ng/ml) have an overall survival median of 3.4 months versus 17.4 months for patients with low level of sBTLA (log rank pval=0.0035).

Conclusion

In conclusion, our study reveals that monitoring the concentration of soluble forms of inhibitory immune checkpoints in plasma like sBTLA can help predict survival in advanced and unresectable PD AC patients.

REFERENCES:

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INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 bis)

Form PCT/RO/134 (July 1998; reprint Ja

Claims

CLAIMS:

1. A method for predicting the survival time of a patient suffering from a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTLA ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

2. A method according to claim 1 wherein BTLA is sBTLA.

3. A method for predicting the invasiveness of a pancreatic cancer comprising i) determining in a sample obtained from the patient the expression level of BTLA ii) comparing the expression level determined at step i) with its predetermined reference value and iii) providing a good prognosis when the expression level determined at step i) is lower than its predetermined reference value, or providing a bad prognosis when the expression level determined at step i) is higher than its predetermined reference value.

4. A method according to claims 1 to 3 wherein the pancreatic cancer is a pancreatic ductal adenocarcinoma (PDAC), a pancreatic adenocarcinoma, a pancreatic serous cystadenomas (SCNs), a pancreatic intraepithelial neoplasia, pancreatic mucinous cystic neoplasms (MCNs) or a non resectable pancreatic adenocarcinoma..

5. An anti-BTLA antibody for use in the treatment of pancreatic cancer in a patient with a bad prognosis according to claims 1 to 4.

6. A method for treating a pancreatic cancer in a patient with a bad prognosis according to claim 1 to 4 comprising the administration to said patient of an anti-BTLA antibody.