WO2015158722A1

WO2015158722A1 - Method for diagnosing or monitoring a cancer

Info

Publication number: WO2015158722A1
Application number: PCT/EP2015/058086
Authority: WO
Inventors: Diane MÈGE; Medhi OUAISSI; Françoise DIGNAT GEORGES; Laurence Panicot-Dubois; Christophe Dubois
Original assignee: Université D'aix-Marseille; INSERM (Institut National de la Santé et de la Recherche Médicale); Assistance Publique Hopitaux De Marseille
Priority date: 2014-04-15
Filing date: 2015-04-14
Publication date: 2015-10-22

Abstract

The present invention relates to a method for monitoring the outcome of a given cancer in a blood sample of a patient. It also relates to a method for diagnosing a given cancer in a blood sample of a patient, comprising the following steps: a) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in said sample, b) comparing the respective concentrations obtained in step a) to the respective ranges of reference values, and c) if all the concentrations obtained in step a) are included in said respective ranges of reference values, establishing that said patient suffers from a given cancer.

Description

METHOD FOR DIAGNOSING OR MONITORING A CANCER

FIELD OF THE INVENTION

The present invention relates to an in vitro method for monitoring the outcome of a given cancer in a blood sample of a patient. It also relates to a method for the diagnosis of a given cancer. Particularly said method relies on the establishment of a signature of specific microparticles concentrations, which may be called a "microparticulosome".

BACKGROUND OF THE INVENTION

Microparticles (MPs) are heterogeneous plasma membranes vesicles (0.1- Ιμιη) bearing proteins and biomarkers of their cells of origin. MPs are released from different cell types, such as platelets, endothelial cells, leukocytes, and erythrocytes, via the budding of the outer cell membrane during cell activation or apoptosis (Piccin et al., 2007; Zahra et al., 2011). Platelet MPs were originally studied because of their procoagulant activity (Chargaff et al, 1946) and have been found to play a role in numerous diseases, including infectious, autoimmune, inflammatory and cardio-vascular diseases, thromboembolic events, and different cancers (Barteneva et al., 2013). In addition to serving as biomarkers, MPs are involved in different activities, including procoagulant and fibrinolytic activities, vascular remodeling, or neoangiogenesis, through such effectors on their surface as tissue factor (TF), plasminogen activators (Lacroix and Dignat-George, 2012), inflammatory cytokines, or vascular endothelial growth factor, respectively. The roles of MPs in cancer is also increasingly being recognized, and MPs have been reported to be involved in tumor growth, immune evasion, chemoresistance, initiation of the tumor stem cell niche, neoangiogenesis, and extracellular matrix degradation (Rak, 2010; D'Souza-Schorey et al, 2012; Van Doormaal et al., 2009). Moreover, circulating plasma MPs appear to support the increased procoagulant activity associated with cancer. This increased risk of thromboembolic events, particularly in pancreatic cancer (20%; Chew et al., 2006), is due to increased levels of soluble activated TF (Khorana et al., 2008), and increased MP TF activity in cancer patients with venous thromboembolism has been described (Manly et al., 2010). Indeed, many studies have reported an increase in the procoagulant activity of MPs associated with different cancers, such as pancreatic (PC; Tesselaar et al., 2007; Zwicker et al., 2009; Manly et al., 2010; Thaler et al., 2012), colorectal (CRC; Hron et al., 2007; Zwicker et al., 2009; Manly et al, 2010; Thaler et al, 2012), gastric (Thaler et al, 2012), lung (Manly et al., 2010), breast (Tesselaar et al., 2007), testicular (Van den Hengel et al., 2013), brain (Thaler et al., 2012), and hematopoietic cancers (Auwerda et al., 2011). Nonetheless, few studies have analyzed the signature of MPs in cancer based on the levels of total, subpopulation, procoagulant, and tumor MPs. Some authors have observed an increased level of total MPs in gastric (Baran et al., 2010), lung (Fleitas et al., 2012), ovarian (Graves et al., 2004), and breast (Liebhardt et al., 2010) cancers compared to healthy subjects, and Kim et al. reported an elevated level of platelets MPs in patients with gastric cancer compared to healthy subjects (Kim et al., 2003).

There is thus a need to improve the fine diagnosis of cancer. Particularly, there is a need for a reliable method for diagnosing a given cancer, using said circulating MPs. Said method has to be quick and easy to perform, has to be reliable, sensitive and specific, without any laborious experimentation. The method has to be as less invasive as possible.

SUMMARY OF THE INVENTION

The inventors have established a method for diagnosing a cancer, and a method for monitoring the outcome of a given cancer, using concentrations of different subpopulations of microparticles (MPs). This approach is reliable, sensitive and specific for a type of cancer, and can be easily performed. Thus, it allows diagnosing a given cancer in a patient, or monitoring the outcome of a given cancer in a patient, thanks to a blood sample thereof and to multiple comparisons of concentrations of MPs subpopulations. Said concentrations of MPs subpopulations indeed constitute, for each type of cancer, a specific signature. This signature may be called a ' 'microparticulo s ome' ' .

The invention thus relates to a method for diagnosing a given cancer in a blood sample of a patient, comprising the following steps:

a) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in said sample, b) comparing the respective concentrations obtained in step a) to the respective ranges of reference values, and

c) if all the concentrations obtained in step a) are included in said respective ranges of reference values, establishing that said patient suffers from a given cancer.

Said method is an in vitro method, performed with a simple blood sample of a patient.

The invention also relates to a method for monitoring the outcome of a given cancer of a patient, comprising the following steps:

a) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in a blood sample of said patient, before treatment of said patient, said treatment being chosen among chemotherapy, radiotherapy and surgery,

b) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in a blood sample of said patient, after said treatment of said patient,

c) comparing the respective concentrations obtained in step a) to the respective concentrations of step b), and

d) increases of at least 17%, preferably of at least 30%, preferably at least 35%, more preferably 40%, between the concentrations of platelet microparticles, fibrin microparticles and leukocyte microparticles, obtained in step b) and the ones obtained in step a) respectively, and a decrease of at least 30%, preferably at least 35%, more preferably 40%, between the concentration of erythrocyte positive microparticles obtained in step b) and the one obtained in step a), are indicative that the patient is in remission.

According to this method, the measures performed on a patient before treatment in step a) constitute the references.

The invention also relates to a method for monitoring the outcome of a given cancer in a blood sample of a patient, comprising the following steps: a) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in said sample,

b) comparing the respective concentrations obtained in step a) to the respective ranges of reference values, and

c) if all the concentrations obtained in step a) are included in said respective ranges of reference values, establishing that said patient is in remission.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term "patient" refers to an individual with symptoms of and/or suspected of having a cancer. It denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human. As used herein, the term "diagnostic" or "diagnosing" refers to the correlation between the results (MPs subpopulations concentrations) obtained thanks to a blood sample of a given patient, and the given cancer from which said patient suffers. Indeed, the method of diagnostic according to the invention allows determining, in view of the MPs subpopulations concentrations in a blood sample of a patient, which given cancer afflicts said patient.

As used herein, the blood sample used in the methods of the invention may be chosen from whole blood, plasma and serum. Preferably, the blood sample is plasma, and more preferably platelet-poor plasma (PPP). Said PPP is plasma with very low number of platelets (< 10* 10 I^L). As explained in the examples, typically said PPP may be obtained by the following process:

centrifuging the blood sample previously citrated at a RCF (Relative Centrifugal Force) of 150-250 x g for 10 to 20 minutes, preferably 15 minutes, to obtain the platelet-rich plasma (PRP) (corresponding to the supernatant);

- centrifuging PRP at a RCF of 1000- 1500 x g for 10 to 20 minutes, preferably 15 minutes, optionally in the presence of an inhibitor of platelet activation (like prostacyclin), to obtain PPP (corresponding to the supernatant); PPP is then centrifuged at 5000-6000 x g for 2 to 5 minutes, preferably 3 minutes; and optionally

freezing the aliquots, which are stored, preferably at liquid nitrogen and then at - 80°C, until the measurements are performed.

Diagnosing methods

Thus, accordingly, in a first aspect, the invention relates to a method for diagnosing a given cancer in a blood sample of a patient, comprising the following steps:

a) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in said sample,

Preferably, without any mention of the contrary, the cancer is chosen from malignant solid tumors, preferably from colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, gastric cancer and gallbladder cancer, most preferably from colorectal cancer and pancreatic cancer.

Indeed, each type of cancer has its own profile of MPs subpopulations concentrations. Said MPs subpopulations concentrations are easily detected, and in a sensitive way.

Preferably, step a) of the method of the invention further comprises measuring, in said sample, at least one concentration of MPs, preferably all the concentrations of MPs, said MPs being chosen from endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles.

Step a) comprises measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in the blood sample of the patient. Preferably, step a) of the method of the invention further comprises measuring, in said sample, at least one concentration of MPs, preferably all the concentrations of MPs, said MPs being chosen from endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles.

As described in the whole present application, and without any mention of the contrary, the measure of the concentration of a given subpopulation of MPs is performed by mixing the sample of the patient, preferably PPP, with Annexin V preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies, preferably specific antibodies against CD41, CD235a, CD lib, CD31, tissue factor, fibrin, MUC1, CEA and/or CA19-9. Said antibodies depend on the targeted microparticles, as explained in the following paragraph.

The fluorescent label is well-known in the art and may be chosen from fluorescein isothiocyanate (FITC), phycoerythrin and its conjugates like phycoerythrin-Cy7, phycocyanins and allophycocyanin, and Alexa Fluor compounds.

The enzymatic label is also well-known in the art and may be chosen from horseradish peroxidase, alkaline phosphatase, glucose oxidase and beta-galactosidase. As described in the whole present application (i.e. for the different methods), typically, for measuring the concentrations of the different subpopulations of MPs, the following protocol may be used:

- The blood sample of the patient, preferably PPP, is incubated with Annexin V, preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies, preferably specific antibodies against CD41, CD235a, CDllb and fibrin, and optionally a control antibody. Indeed, said appropriate specific antibodies are targeted against:

CD41, for platelet MPs which are positive, and/or for endothelial MPs which are negative,

- CD235a, for erythrocyte MPs which are positive, and/or for leukocyte MPs which are negative,

CD31 for endothelial MPs which are positive, CD lib for leukocyte MPs which are positive, and

tissue factor, fibrin, MUC1, CEA and CA19-9 for the tissue factor positive microparticles, fibrin positive microparticles, MUC1 positive microparticles, CEA positive microparticles and CA19-9 positive microparticles, respectively Said appropriate specific antibodies are commercially available and well-known, as notably explained in the examples.

- The resulting mixture is incubated, and preferably paraformaldehyde is added to fix the sample after incubation.

- Then, polystyrene fluorospheres (like CytoCount beads from Dako) are added, in a volume which is equal to the volume of the patient's sample.

- Then the samples and controls are measured using a flow cytometer, typically a Gallios flow cytometer.

The concentrations are expressed as the number of MPs/μΕ of blood sample, preferably PPP.

Preferably, in the first step of the protocol, the blood sample of the patient, preferably PPP, is incubated with Annexin V and at least all of the specific antibodies against CD41, CD235a, CD lib and fibrin.

Preferably, the specific antibody targeted against fibrin is 59D8, which binds to fibrin but not to fibrinogen. Said antibody is described in "Application of "ATTEMPTS" for drug delivery", of Naik et al, Journal of Controlled Release 101, 20025, 35-45. A control antibody may also be used, so as to measure the background. Said control antibody is typically added at the beginning of the protocol, with all the above-mentioned appropriate specific antibodies, in the same sample. Alternatively, the blood sample of the patient is divided into 2 subsamples, one of them being mixed with the appropriate specific antibodies as described above, and the other one being mixed with the control antibody.

A very detailed protocol is typically described in the examples.

Therefore, at the end of step a), the concentrations of the different subpopulations of MPs are determined. Then, the method of the invention comprises a step b) of comparing the respective concentrations obtained in step a) to the respective ranges of reference values. In other words, the concentrations of the different subpopulations of MPs obtained in step a) are compared to the respective ranges of concentrations considered as reference values. For example, the concentration of platelet MPs obtained in step a) is compared to the range of platelet MPs reference values.

For a given MPs subpopulation, said range of reference values is preferably the range from statistical minimal to statistical maximal concentrations of said MPs subpopulation of patients who are known to be afflicted by a given cancer (for example colorectal or pancreatic cancer). In any case, all the concentrations are obtained thanks to the method of the invention, and according to the same protocol (so as to be comparative).

Finally, the method of the invention comprises a step c): if all the concentrations obtained in step a) are included in the respective ranges of reference values, then establishing that said patient suffers from a given cancer.

For example, if the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, are all included in their respective ranges of reference values, then this means that the patient suffers from a given cancer.

Specifically, and preferably, the method according to the invention is for diagnosing colorectal cancer or pancreatic cancer.

Most preferably, the method according to the invention is for diagnosing colorectal cancer in a blood sample of a patient, and comprises the following steps:

b) comparing:

- the concentration of platelet microparticles obtained in step a) to the range of 2000 to 8000, preferably of 2200 to 7500, more preferably of 2300 to 7130, - the concentration of erythrocyte microparticles obtained in step a) to the range of 80 to 700, preferably of 90 to 500, more preferably of 100 to 480,

- the concentration of leukocyte microparticles obtained in step a) to the range of 1 to 25, preferably of 1 to 22, more preferably of 1 to 20, and

- the concentration of fibrin positive microparticles obtained in step a) to the range of 5 to 100, preferably of 7 to 95, more preferably of 8 to 90,

said concentrations being expressed in microparticles^L,

c) if all the concentrations obtained in step a) are included in said ranges of step b), establishing that said patient suffers from colorectal cancer.

More preferably, the method for diagnosing colorectal cancer described in the previous paragraph further comprises in step a) the measures of the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles in said sample, and further comprises in step b) the comparisons of:

- the concentration of endothelial microparticles obtained in step a) to the range of 1 to 35, preferably of 1 to 30, more preferably of 1 to 27,

- the concentration of tissue factor positive microparticles obtained in step a) to the range of 0.2 to 500, preferably of 0.3 to 450, preferably of 0.4 to 430,

- the concentration of MUC1 positive microparticles obtained in step a) to the range of 0 to 60, preferably of 0 to 50, more preferably of 0 to 47,

- the concentration of CEA positive microparticles obtained in step a) to the range of 0 to 60, preferably of 0 to 50, more preferably of 0 to 45,

- the concentration of CA19-9 positive microparticles obtained in step a) to the range of 0 to 200, preferably of 0 to 150, more preferably of 0 to 130, and

- the concentration of total microparticles obtained in step a) to the range of 3500 to 10000, preferably of 3700 to 9500, more preferably of 3770 to 9100,

said concentrations being expressed in microparticles^L. Alternatively, more preferably, the method of the invention is for diagnosing pancreatic cancer in a blood sample of a patient, and comprises the following steps:

b) comparing:

- the concentration of platelet microparticles obtained in step a) to the range of 1400 to 10000, preferably of 1500 to 9900, more preferably of 1500 to 9850,

- the concentration of erythrocyte microparticles obtained in step a) to the range of 40 to 1200, preferably of 50 to 1150, more preferably of 55 to 1100,

- the concentration of leukocyte microparticles obtained in step a) to the range of

4 to 50, preferably of 5 to 45, more preferably of 6 to 42, and

- the concentration of fibrin positive microparticles obtained in step a) to the range of 90 to 500, preferably of 100 to 450, more preferably of 110 to 420,

said concentrations being expressed in microparticles^L,

c) if all the concentrations obtained in step a) are included in said ranges of step b), establishing that said patient suffers from pancreatic cancer.

More preferably, the method for diagnosing pancreatic cancer described in the previous paragraph further comprises in step a) the measures of the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles in said sample, and further comprises in step b) the comparisons of:

- the concentration of endothelial microparticles obtained in step a) to the range of 50 to 300, preferably of 55 to 250, more preferably of 57 to 230,

- the concentration of tissue factor positive microparticles obtained in step a) to the range of 5 to 200, preferably of 7 to 180, more preferably of 10 to 170,

- the concentration of MUC1 positive microparticles obtained in step a) to the range of 0 to 50, preferably of 0 to 30, more preferably of 0 to 20,

- the concentration of CEA positive microparticles obtained in step a) to the range of 0 to 200, preferably of 0 to 170, more preferably of 0 to 150, - the concentration of CA19-9 positive microparticles obtained in step a) to the range of 5 to 700, preferably of 7 to 600, more preferably of 9 to 500, and

- the concentration of total microparticles obtained in step a) to the range of 5000 to 22000, preferably of 5100 to 21500, preferably of 5110 to 21200,

said concentrations being expressed in microparticles^L.

Lactadherin embodiment

According to a specific embodiment ("lactadherin embodiment"), the invention relates to a method for diagnosing a given cancer in a blood sample of a patient, comprising the following steps:

a) measuring the concentrations of platelet microparticles, endothelial microparticles and fibrin positive microparticles in said sample, by mixing said sample with lactadherin preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies,

As shown in Example 2, lactadherin is very specific for the microparticles listed in step a). Lactadherin is a protein also called milk fat globule-EGF factor 8 protein (Mfge8), encoded under Q08431 for the human version in Uniprot. Thus, the invention also aims the use of lactadherin for measuring a concentration of microparticles.

Fluorescent labels, enzymatic labels and appropriate specific antibodies are as described above.

Preferably, step a) of the method of the lactadherin embodiment further comprises measuring, in said sample, at least one concentration of MPs, preferably all the concentrations of MPs, said MPs being chosen from erythrocyte microparticles, leukocyte microparticles, MUC1 positive microparticles, CEA positive microparticles, podoplanin positive microparticles and total microparticles. Typically, for this embodiment, the following protocol may be used for measuring the concentrations of the different subpopulations of MPs:

- The blood sample of the patient, preferably PPP, is incubated with lactadherin, preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies, preferably specific antibodies against CD41, CD235a, CDllb and fibrin, and optionally a control antibody

The concentrations are expressed as the number of MPs^L of blood sample, preferably PPP.

A very detailed protocol is typically described in example 2.

Most preferably, the method of the lactadherin embodiment is for diagnosing a cancer in a blood sample of a patient, and comprises the following steps:

b) comparing:

- the concentration of platelet microparticles obtained in step a) to the range of 500 to 14000, preferably of 525 to 13500,

- the concentration of endothelial microparticles obtained in step a) to the range of 0 to 1500, preferably of 0 to 1300, and

- the concentration of fibrin positive microparticles obtained in step a) to the range of 0 to 1110, preferably of 0 to 1100,

said concentrations being expressed in microparticles^L, c) if all the concentrations obtained in step a) are included in said ranges of step b), establishing that said patient suffers from a cancer.

More preferably, the method for diagnosing a cancer described in the previous paragraph further comprises in step a) the measures of the concentrations of erythrocyte microparticles, leukocyte microparticles, MUCl positive microparticles, CEA positive microparticles, podoplanin positive microparticles and total microparticles in said sample, and further comprises in step b) the comparisons of:

- the concentration of erythrocyte microparticles obtained in step a) to the range of 1 to 3500, preferably of 5 to 3300,

0 to 1200, preferably of 0 to 1100,

- the concentration of MUCl positive microparticles obtained in step a) to the range of 0 to 2100, preferably of 0 to 2000,

- the concentration of CEA positive microparticles obtained in step a) to the range of 0 to 19000, preferably of 0 to 18500,

- the concentration of podoplanin positive microparticles obtained in step a) to the range of 0 to 7000, preferably of 0 to 6500, and

- the concentration of total microparticles obtained in step a) to the range of 5000 to 80000, preferably of 6000 to 78500,

said concentrations being expressed in microparticles^L.

Monitoring methods with reference values

The invention also relates to a method for monitoring the outcome of a given cancer in a blood sample of a patient, comprising the following steps:

c) if all the concentrations obtained in step a) are included in said respective ranges of reference values, establishing that said patient is in remission. All the technical features disclosed for the method for diagnosing a given cancer described above are applicable. The only difference resides in the definition of the range of reference values. In this case, for a given MPs subpopulation, said range of reference values is preferably the range from statistical minimal to statistical maximal concentrations of said MPs subpopulation of patients who are known to be in remission of said given cancer. In any case, all the concentrations are obtained thanks to the method of the invention, and according to the same protocol (so as to be comparative). Preferably, step a) of said method for monitoring the outcome of a given cancer according to the invention further comprises measuring the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles in said sample.

Lactadherin embodiment

According to a specific embodiment, the invention relates to a method for monitoring the outcome of a given cancer in a blood sample of a patient, comprising the following steps:

Monitoring methods on a given patient

The invention also relates to a method for monitoring the outcome of a given cancer of a patient, comprising the following steps: a) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in a blood sample of said patient, before treatment of said patient, said treatment being chosen among chemotherapy, radiotherapy and surgery,

d) increases of at least 17%, preferably of at least 30%, preferably at least 35%, more preferably 40% between the concentrations of platelet microparticles, fibrin microparticles and leukocyte microparticles, obtained in step b) and the ones obtained in step a) respectively, and a decrease of at least 30%, preferably at least 35%, more preferably 40%, between the concentration of erythrocyte positive microparticles obtained in step b) and the one obtained in step a), are indicative that the patient is in remission.

In some embodiments, preferably, in step d), an increase of at least 50%, preferably at least 60%, between the concentrations of fibrin microparticles obtained in step b) and the ones obtained in step a), is observed.

Preferably, the given cancer is chosen from malignant solid tumors, preferably from colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, gastric cancer and gallbladder cancer. Most preferably, it is chosen from pancreatic and colorectal cancer.

Preferably, the treatment is defined below in the "Methods of treatment" section. Indeed, as shown in the examples, particularly in Table 3, it is possible to monitor a patient afflicted of a given cancer, like colorectal cancer, over time, and determining if he is in remission or not, by measuring different microparticles concentrations.

The time interval between step a) and step b) may be variable, for example from 24h to several weeks, several months or several years. Of course, the blood samples of the patient used in steps a) and b) are different.

Preferably, the time interval between step a) and step b) is of 24h or 48h, when the treatment is a surgery.

Preferably, the time interval between step a) and step b) is one week, when the treatment is chemotherapy.

Preferably, according to said method, step a) and step b) further comprise measuring the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles, in a blood sample of said patient, before and after treatment of said patient, respectively, and wherein, in step d):

an increase of at least 90%, preferably of at least 95%, more preferably of at least 100%, between the concentration of endothelial microparticles obtained in step b) and the one obtained in step a),

an increase of at least 10%, preferably of at least 15%, more preferably of at least 20% between the concentration of total microparticles obtained in step b) and the one obtained in step a),

and decreases of at least 30%, preferably of at least 40%, more preferably of at least 50% between the concentrations of tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles and CA19-9 positive microparticles obtained in step b) and the ones obtained in step a), respectively, are indicative that the patient is in remission. Lactadherin embodiment According to a specific embodiment, the invention relates to a method for monitoring the outcome of a given cancer of a patient, comprising the following steps:

a) measuring the concentrations of platelet microparticles, endothelial microparticles and fibrin positive microparticles, by mixing a blood sample of said patient with lactadherin preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies, before treatment of said patient, said treatment being chosen among chemotherapy, radiotherapy and surgery,

b) measuring the concentrations of platelet microparticles, endothelial microparticles and fibrin positive microparticles, by mixing a blood sample of said patient with lactadherin preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies, after said treatment of said patient,

d) increases of at least 20%, preferably at least 25%, more preferably 27% between the concentrations of platelet microparticles and endothelial microparticles, obtained in step b) and the ones obtained in step a) respectively, and a decrease of at least 50%, preferably at least 60%, between the concentration of fibrin positive microparticles obtained in step b) and the one obtained in step a), are indicative that the patient is in remission.

Methods of treatment

The invention finally relates to a method for treating a specific cancer in a patient, comprising the following steps:

a) measuring the concentrations of platelet microparticles, erythrocyte microparticles, leukocyte microparticles and fibrin positive microparticles, in a blood sample of said patient,

b) comparing the respective concentrations obtained in step a) to the respective ranges of reference values, c) if all the concentrations obtained in step a) are included in said respective ranges of reference values, establishing that said patient suffers from a specific cancer, and d) treating said patient in consequence for said specific cancer. In the context of the invention, the term "treatment" or "treating", as used herein, means curing the disease, but also alleviating or decreasing the progress of the disorder or condition to which such term applies, or alleviating, decreasing the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

Said treatment may comprise chemotherapy, radiotherapy, and/or surgery. Preferably, it comprises one or more chemotherapeutic agents. As used herein, the expression "anticancer agent" or "chemotherapeutic agent" refers to compounds which are used in the treatment of cancers.

Anti-cancer agents include but are not limited to temozolomide, fotemustine, dacarbazine, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L- asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan (CPT-11), SN-38, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustine and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, monoclonal antibodies against EGF receptor or VEGF, such as bevacizumab, cetuximab and panitumumab, imatimb mesylate, hexamethyhnelamine, topotecan, genistein, erbstatin, lavendustin and also bortezomib (also called PS341, and sold by Millenium Pharmaceuticals under the name Velcade). The invention will now be illustrated thanks to the examples and figures below. FIGURE LEGENDS

Figure 1: Characterization of the microparticle subpopulations in colorectal diseases. Concentrations of total -(A), platelet- (B), leukocyte- (C), erythrocyte- (D) and endothelial- (E) derived MPs in plasma between patients presenting with colorectal cancer (CRC) or benign colorectal diseases (Benign CR) and healthy subjects. Statistically significant for p-value < 0.05.

Figure 2: Characterization of procoagulant and tumor-derived microparticles in colorectal diseases. Concentrations of TF- (A), Fibrin- (B), CEA- (C), CA19-9- (D) and MUC1- (E) positive MPs in plasma between patients presenting with colorectal cancer (CRC) or benign colorectal diseases (Benign CR) and healthy subjects. Statistically significant for p-value < 0.05.

Figure 3: Characterization of the microparticle subpopulations in colorectal and pancreatic diseases. Concentrations of total- (A), platelet- (B), leucocyte-(C), erythrocyte- (D) and endothelial- (E) derived MPs in plasma between patients presenting with colorectal (CRC) or pancreatic cancer (PC) and benign pancreatic diseases (Benign P). Statistically significant for p-value < 0.05.

Figure 4: Characterization of procoagulant and tumor-derived microparticles in colorectal and pancreatic diseases. Concentrations of Fibrin- (A), TF- (B), Podoplanine- (C), CEA- (D), CA19-9- (E) and MUC1- (F) positive MPs in plasma between patients presenting with colorectal (CRC) or pancreatic cancer (PC) and benign pancreatic diseases (Benign P). Statistically significant for p-value < 0.05.

Figure 5: Detection of MPs issued from the cancer cell line Bx PC-3.

AnV : MPs detected using Annexin V;

LD: MPs detected using lactadherin.

Figure 6: Platelet derived MPs (PMPs) detected with lactadherin/CD41+ and Annexin/CD41+.

A) PMP LD: PMPs detected using lactadherin. B) PMP AnV : PMPs detected using Annexin V.

CCR: Colorectal Cancer

Turn Benigne CR: benign colorectal disease

Crohn: Crohn's disease

CP: chronic pancreatitis

Turn Benigne P: benign pancreatic disease

C estomac: gastric cancer

C Vesicule: gallbladder cancer. Figure 7: Erythrocyte derived MPs (EryMPs) detected with lactadherin/CD235a+ and Annexin/CD235a+.

A) EryMP LD: EryMPs detected using lactadherin.

B) EryMP AnV : EryMPs detected using Annexin V.

CCR: Colorectal Cancer

Turn Benigne CR: benign colorectal disease

Crohn: Crohn's disease

CP: chronic pancreatitis

Turn Benigne P: benign pancreatic disease

C estomac: gastric cancer

C Vesicule: gallbladder cancer.

Figure 8: Endothelial derived MPs (EMPs) detected with lactadherin/CD31+/CD41- and Annexin/CD31+/CD41-.

A) EMP LD: EMPs detected using lactadherin.

B) EMP AnV : EMPs detected using Annexin V.

CCR: Colorectal Cancer

Turn Benigne CR: benign colorectal disease Crohn: Crohn's disease

CP: chronic pancreatitis

Turn Benigne P: benign pancreatic disease

C estomac: gastric cancer

C Vesicule: gallbladder cancer.

Figure 9: Fibrin derived MPs (MP Fib) detected with lactadherin and Annexin.

A) MP Fib + LD: MP Fib detected using lactadherin.

B) MP Fib + AnV : MP Fib detected using Annexin V.

CCR: Colorectal Cancer

Turn Benigne CR: benign colorectal disease

Crohn: Crohn's disease

CP: chronic pancreatitis

Turn Benigne P: benign pancreatic disease

C estomac: gastric cancer

C Vesicule: gallbladder cancer.

Example 1

MATERIALS AND METHODS

Study population

The inventors prospectively included all the patients who underwent surgery for colorectal cancer (CRC) and pancreatic cancer (PC) in the Digestive Surgical Department of Timone's Hospital (Marseille, France) since 2011 and 2009, respectively. Patients undergoing surgery for benign colorectal diseases, i.e., Crohn's disease or infectious diverticulitis, and benign pancreatic disease, i.e., chronic pancreatitis, were also included. Healthy subjects without surgical history were included. All the individuals signed an institutional review board-approved consent form.

The data collected for each patient were as follows: demographic data, including age and sex of the patient; clinical data, including medical history, surgical history, history of venous thromboembolism, and anticoagulant therapy within the 3 months prior to surgery; and biological data, including hemogram, levels of carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9)). The CRC and PC staging was defined according to Tumor Node Metastasis (TNM) classification, i.e., stage I for located tumor, stages II and IIA for parietal extension, stages IIB and III for lymph extension, and stage IV for metastatic extension (AJCC Cancer staging Manual, 2002). Any preoperative chemotherapy and/or radiotherapy were recorded.

Platelet-poor plasma (PPP) containing microparticles (MPs) in human blood

Venous fasted blood samples were collected in citrated tubes and prepared within two hours following the drawing, as described by Lacroix et al (Lacroix et al., 2012). Citrated blood was centrifuged at 200 x g for 15 minutes to obtain the platelet-rich plasma (PRP). PPP was obtained after the centrifugation of PRP at 1100 x g for 15 minutes, with prostacyclin (PGI2) added to prevent platelet activation. PPP was then centrifuged at 7000 x g for 3 minutes. Aliquots were snap-frozen in liquid nitrogen and stored at -80°C until the measurements were performed.

Flow cytometric analysis of PPP samples

The circulating MPs contained in the PPP samples were analyzed using a Gallios flow cytometer (Beckman Coulter), as previously described by Robert et al. (Robert et al., 2012). The flow cytometry instrument settings and MP gating were performed with Megamix beads (BioCytex).

The MP cellular origin was detected with a combination of Annexin V and specific antibodies against cells of origin. Briefly, fluorescein isothiocyanate (FITC, Tau Technologies)-conjugated Annexin V was used to label phosphatidylserine in the MP membrane. Phycoerythrin-Cy7 (PC7)-conjugated anti-CD41 (Beckman Coulter), phycoerythrin (PE)-conjugated anti-CD31 (Beckman Coulter), allophycocyanin (APC)- conjugated anti-CDllb (Beckman Coulter), and APC-Alexa Fluor 750 (APC-A-Fluor 750)-conjugated anti-CD235a (Beckman Coulter) were used to identify platelet (Annexin V+/CD41+), endothelial (Annexin V+/CD31+/CD41-), leukocyte (Annexin V+/CDllb+/CD235a-), and erythrocyte (Annexin V+/ CD235a+) MPs. Matched isotype control antibodies labelled with an appropriate fluorescent dye were purchased from Beckman Coulter.

Procoagulant MPs were determined using a combination of brilliant blue-conjugated Annexin V purchased from Biolegend and antibodies identifying procoagulant activity. These procoagulant MPs were determined by the detection of MPs decorated with TF on their surface (defined as Annexin V+/TF+) using an Alexa 488-conjugated anti-TF antibody obtained from Abeam (abl7375). The inventors also detected MPs decorated with fibrin, the final product of the coagulation cascade (define as Annexin V+/Fib+), with a specific Alexa 488-conjugated anti-fibrin antibody, called 59D8; this antibody was a generous gift from Pr B Furie (Harvard Medical School, Boston). These antibodies and their isotype controls were labeled in-house with an Alexa Fluor 488 protein labeling kit following the manufacturer's instructions (Life Technologies).

Tumor MPs were characterized by the detection of MPs expressing tumor markers on their surface using Alexa 647-conjugated anti-Mucinel (Mucl, Novocastra), PE- conjugated anti-Podoplanin (BioLegend), PE-conjugated anti-CEA (Abeam), and Alexa 647-conjugated anti-CA19-9 (Novocastra). Matched isotype control antibodies were labeled in-house with an Alexa 647 protein labeling kit following the manufacturer's instructions (Life Technologies) or were purchased from BioLegend and Abeam.

The MP labeling was based on a 30-minute incubation of patient PPP (30 μί) with Annexin V and appropriate antibodies; 300 μΐ_^ of calcium buffer was then added to improve the binding of Annexin V to phosphatidylserine. After a 3-minute incubation, 100 μΐ_^ of paraformaldehyde (16%) was added to fix the sample, and 30 μΐ_^ of CytoCount beads (Dako) was added; these beads were necessary to determine the concentration of MPs in PPP. The concentrations are expressed as the number of MPs^L of PPP. The samples and associated controls were measured within an hour using a Gallios flow cytometer. The sample analyses were performed with Kaluza software (Beckman Coulter), as already described by Lacroix et al. (Lacroix et al., 2012). Statistical analysis

The qualitative and quantitative variables are expressed as percentages and the median with a range. The MPs levels are expressed as MPS/ J L and were compared between groups and tumor stages using the Student t or nonparametric Mann-Whitney tests, as appropriate. A one-way ANOVA was used in cases of more than two groups. All the tests were two-tailed, and differences were considered significant at p<0.05. All the analyses were performed using the GraphPad software.

RESULTS

Study population

The inventors included 85, 36, 15, and 18 patients presenting with CRC, PC, benign colorectal and benign pancreatic diseases, respectively. Twenty healthy subjects were also included. Table 1 below summarizes the demographic, clinical, and biological data of the different groups. The healthy subjects were significantly younger and included more females than other groups. No significant difference regarding the median age and sex ratio were observed between the two types of cancers or between the two types of benign diseases.

CRC PC BENI GN "CR" BENI GN "P" HEALTHY P n 85 36 15 18 20

Sex M F 59 (69) / 26 (31) 21 (58) / 15 (42) 7 (47) / 8 (53) ₁₄ (78) / 4 (22) 5 (25) / 15 (75) 0.002

Median age (years) 71.5 [28.7-93.3] 69 [55-80] 57.4 [32.1-86.6] 52,5 [25-76] 38 [20-63] <0.0001

Medical history

• Uiabetus 15 (18) 6 (17) 4 (27) 0 0 NS

• Arteriopathy 14 (16) 5 (14) 0 0 0 NS

• Active smoker 23 (27) 7 (19) 3 (20) 3 (17) 5 (25) NS

• Thromboembolic 4 (5) 3 (8) 2 (13) 1 (6) 0 NS events 17 (20) 1 (3) 1 (7) 0 0 0.005

• Cancer

Digestive surgical history 31 (36) 5 (14) 9 (60) 0 2 (10) <0.0001

Anticoagulant/antiagregant

22 (26) 9 (25) 2 (13) 1 (6) 0 0.03 treatment

Biology (median) *

• Haemoglobin (g/dL) 12.1 [7-16.5] 12.7 [9-14.7] 11.1 [9.6-14.6] 13 [10-16] NS

• Leukocytes (G/L) 7.8 [2.4-18] 7.1 [6.2-20] 9 [3.8-13] 7.8 [4.2-14] NS

• Platelets (G/L) 260.5 [95-610] 271 [117-469] 276 [141-726] 285 [138-456] NS

TP (%) 90 [17-126] 87 [55-126] 93 [69-120] 95 [65-109] NS

• ratio TCA 1 [0.8-2.2] 1.03 [0.8-1.7] 1 [0.8-1.8] 1 [0.8-1,3] NS

CEA (ng/mL) 3.6 [0-1837] 3.15 [1.4-53.4] 3.4 [1.3-3.7] NS

CA19-9 (U/mL) 21.3 [1-749] 84.3 [21.5-2069] 9.85 [2.5-326] 0.0003

Stages 0.001

• In situ 10 (12) 0

• Located 16 (19) 0

• Parietal extension 23 (27) 7 (19)

• Node extension 22 (26) 18 (50)

• Metastasis 14 (16) 11 (31)

Table 1: Demographic data of different groups (CRC = colorectal cancer, PC = pancreatic cancer, BENIGN "CR" = benign colorectal diseases, BENIGN "P" = benign pancreatic diseases) * data not available for the patients.

5

No significant difference was noted with regard to the biological data, except that CA19-9 was higher in PC. Some 71% of the patients presenting with PC had advanced disease, whereas the CRC stages were rather similar. 0 Characterization of the concentration and origin of circulating MPs in CRC, benign colorectal disease, and healthy patients

The inventors first determined the concentration of MPs in CRC and benign colorectal disease groups compared to healthy subjects (table 2). Surprisingly, the concentration of total MPs, as measured using Annexin V, was significantly decreased in the CRC and benign colorectal diseases compared to the healthy subjects, whereas there was no significant difference between the CRC and benign colorectal diseases (figure 1A).

BENIGN

CRC PC BENIGN « P » HEALTHY

« CR »

Subpopulations

Procoa ulant MPs

Tumor MPs

Table 2: Microparticulosome signature based on a four code system :

low to high levels of MPs (MPsfaL): white < horizontal lines < underlined numbers < diagonal lines

The same results were obtained using another marker of total circulating MPs (lactadherin, data not shown), confirming a decreased production or an increased "consummation" of total MPs in CRC and benign colorectal diseases in comparison to healthy subjects. The concentration of circulating MPs was next determined according their cellular origin, as previously described (Lacroix et al., 2010; Zahra et al., 2011). Again, no significant difference was observed between the CRC and benign colorectal disease groups (Figure 1A to IE). However, concentrations of platelet-derived and endothelial-derived MPs were significantly decreased in the two groups compared to healthy subjects (figures IB and IE). The inventors observed that the differences in the concentrations of the MP subpopulations were not influenced by the concentrations of the cells of origin, as no difference was noted between the groups. Previous reports have demonstrated an active participation of platelet- and endothelial-derived MPs in the inflammation, angiogenesis, and growth of a tumor (Van Doormaal et al., 2009; Rak, 2010; D'Souza-Schorey et al, 2012); circulating MPs may fuse with endothelial or cancer cells (Ratajczak et al., 2006; Mause et al, 2010), aggregate platelets, or interact with inflamed epithelium (Thomas et al., 2009). All these processes may explain the consummation of circulating MPs, leading to a decrease in their plasma concentrations under pathological conditions.

Characterization of procoagulant and tumor MPs in CRC, benign colorectal disease, and healthy patients

It has been previously described that MP-related procoagulant activities are increased in different cancers, including CRC and PC (Hron et al., 2007; Tesselaar et al., 2007; Zwicker et al., 2009; Manly et al., 2010; Thaler et al., 2012). Therefore, to compare the concentrations of circulating procoagulant MPs in the different groups, the inventors determined both the presence of TF on the surface of MPs and the generation of fibrin. Indeed, fibrin is the final product of the blood coagulation cascade and thus represents a pertinent indicator of the procoagulant activity expressed by circulating plasma MPs. A significant decrease in the concentration of TF-bearing MPs was observed in the CRC group compared to the healthy subjects (figure 2A), though the concentration of fibrin- bearing MPs was not significantly different between the CRC and benign colorectal disease groups (figure 2B). These results are in accordance with the slightly increased risk of venous thromboembolism in patients suffering from CRC in comparison to cancers associated with thrombosis, such as pancreatic or lung cancers (Chew et al., 2006). The inventors next determined the concentration of three glycoprotein tumor markers, CEA, CA.19.9, and Mucl, on the surface of circulating MPs present in the plasma of colorectal benign or tumor patients and healthy controls. No significant difference was observed in the concentration of MPs bearing these three tumor- associated antigens in the different group (figures 2C to 2E). This lack of difference between the groups may be predominantly due to the low concentration of Annexin V- positive MPs in the CRC group in comparison to the healthy controls. Furthermore, although commonly detected in the serum of patients suffering from CRC, CEA and CA.19.9 are not specific for cancer, and CEA 19.9 and Mucl have never been specifically detected in association with CRC. Altogether, these results indicate that a single analysis of the concentrations of total, procoagulant, or tumor MPs present in plasma may not be sufficient to distinguish between benign and cancer pathologies. However, based on the different results obtained in the different groups, the inventors propose a hallmark of microparticles; this "microparticulosome signature" was based on the concentration and not the percentage of MPs detected in the plasma (table 2). A four code system was depicted corresponding to the plasma concentration of MPs detected from high (diagonal lines) to low (white) levels of MPs. Based on this table, the inventors observed that the concentrations of Annexin V, platelets, erythrocytes, and endothelial-derived MPs were high in the healthy group, "intermediate" in the benign colorectal diseases, and low in the CRC subpopulation. Although the concentration of Annexin V-positive MPs was slightly decreased in the benign colorectal disease group, the levels of platelet- and erythrocyte-derived and Mucl- and CEA-bearing MPs were increased in this group compared to the CRC group.

Comparison of the microparticulosome generated in CRC, PC, and inflammatory- associated diseases

To determine whether the signatures observed in the CRC and benign colorectal disease groups were specific for these pathologies, the inventors next examined another cancer, PC, and its associated benign disease, chronic pancreatitis. The concentrations of Annexin V-positive MPs (figure 3A), leukocyte-derived (figure 3B), and endothelial- derived MPs (figure 3C) were significantly increased in the PC group compared to the CRC group. In contrast, no significant difference was observed in the concentration of platelet-MPs (figure 3D) and erythrocyte-derived MPs (figure 3E) between the cancers. Interestingly, the concentration of endothelial MPs was also significantly increased in PC in comparison to the benign pancreatic disease group (figure 3C). Moreover, the inventors observed that the differences in the concentrations of MP subpopulations were not influenced by the concentrations of the cells of origin, as no differences were noted between the groups. PC is associated with an increased risk of thrombosis, and different reports have demonstrated an increase in the number of MPs bearing active TF on the surface (Tesselaar et al., 2007; Zwicker et al., 2009; Manly et al., 2010; Thaler et al., 2012). Accordingly, the inventors observed a significant increase in fibrin-bearing MPs in the PC group compared to the benign pancreatic disease and CRC groups (figure 4A) and also the benign colorectal disease and healthy groups (data not shown). Interestingly, the levels of TF expressed in the activated and inactivated forms on the surface of MPs were not different between the studied subpopulations (figure 4B). Podoplanin is a glycoprotein that is overexpressed in different types of cancer (Lowe et al., 2012), and recent studies have highlighted the role of its ligand CLEC-2 in the activation and aggregation of blood platelets (Wicki and Christofori, 2007). The inventors hypothesized that PC cell-derived MPs may aggregate platelets (Thomas et al., 2009) via this pathway of activation, suggesting that concentrations of podoplanin- positive MPs may increase in patients suffering from PC. Indeed, the levels of podoplanin-positive MPs were significantly higher in the patients suffering from PC in comparison to the other pathological groups (Figure 4C), though these levels were comparable to the observations in the healthy group (table 2). Again, this finding is mainly because the concentration of total plasma MPs was twice as high in healthy subjects in comparison to PC patients. Regarding two other tumor markers usually used in digestive cancer diagnostic, no significant difference was noted between CRC and PC for CEA-positive MPs (Figure 4D) whereas CA19-9-positive MPs were significantly increased in PC and inflammatory pancreatic diseases compared to CRC (Figure 4E). These results reflect the biological data of these patients, in which no significant difference was observed for serum CEA, contrary to serum CA19-9. However, CRC was associated with a significantly increased Mucl-positive MPs level (Figure 4F). Mucl is an ubiquitous marker of glandular epithelium. It has been already described on the surface of MPs associated with CRC, PC, gastric, ovarian and breast cancers. These results highlight the need to establish a complete plasma microparticulosome that is specific for a particular cancer or inflammatory disease instead of following a unique plasma biomarker. Evolution of the microparticulosome signature with the evolution of CRC

To determine whether the plasma microparticulosome could change with the evolution of a cancer, the inventors compared the MP signature in 11 patients presenting with CRC, both preoperatively and after remission. The concentrations of Annexin V-positive MPs, all the subpopulations of MPs, TF- and fibrin-positive MPs, and tumor-MPs were determined as described above. The results were compiled using the same four code signature (table 3).

PREOPERATIVE

REMISSION CRC CRC P

Total MPs 5289 0.1

Subpopulations

PMP Ϊ 4667 0.4

EMP 1.5 52 0.003

EryMP 342.5 138 0.08

LeuMP 6.5 11 0.3

Procoagulant MPs

TF positive 1 24 0.2

Fibrin positive 22 0.1

'* j

Tumor MPs

MUC1 positive 0.1

PODO positive 24 67 0.1

CEA positive 0.002

CA19-9 positive 0 0 0.1

Table 3: Microparticulosome signature preoperatively and after remission from CRC (n = 11), based on a four code system :

low to high levels of MPs (MPsfaL): white < horizontal lines < underlined numbers

Interestingly, after remission, the concentrations of Annexin V-positive MPs returned to the levels observed in the benign colorectal disease group, with an increase in the plasma concentrations of platelet-, erythrocyte-, and leucocyte-derived MPs and a decrease in TF-bearing MPs, even though the fibrin-positive MPs were slightly increased following remission. Interestingly, plasma Mucl-, CEA-, and CA19-9- MPs concentrations were not detectable, and the plasma podoplanin-MPs concentration was that observed in the benign colorectal disease group (data not shown). These results indicate that the microparticulosome signature is different before and after CRC remission, indicating that it could be used to characterize the evolution of the disease. Although different studies have previously demonstrated an increase in Annexin V- positive platelets or CEA- or mucine-1 -positive MPs in different pathologies (Kim et al., 2003;Graves et al., 2004; Tesselaar et al., 2007; Zwicker et al., 2009; Baran et al., 2010; Liebhardt et al., 2010; Fleitas et al., 2012), there are no data thus far comparing the concentration and origin of plasma MPs according to the type of cancer or its corresponding benign inflammatory disease. Here, the inventors demonstrate that a single analysis of MP concentration might be insufficient to characterize a cancer and its evolution. However, the results obtained by establishing a signature based on all the MPs present in the bloodstream may more accurately reflect the presence and evolution of a cancer. Altogether, these results demonstrate the importance of characterizing different proteins present on the surface of circulating MPs. This MP signature may, for instance, be characterized for a single individual during diagnosis, and its evolution may constitute a pertinent indicator of the evolution of the disease.

Example 2

MATERIALS AND METHODS

Study population

The inventors prospectively included all the patients who underwent surgery for colorectal cancer (CRC) and pancreatic cancer (PC) in the Digestive Surgical Department of Timone's Hospital (Marseille, France) since 2011 and 2009, respectively. Patients undergoing surgery for benign colorectal diseases, i.e., Crohn's disease or infectious diverticulitis, and benign pancreatic disease, i.e., chronic pancreatitis, were also included. Healthy subjects without surgical history were included. All the individuals signed an institutional review board-approved consent form. Agreement reference of UMR-1076 tissue collection was DC- 2013-1815. Collected data were demographic (age, sex), clinical (medical and surgical history, history of venous thromboembolism, recent anticoagulant therapy, TNM stages of CRC and PC and preoperative chemoradiotherapy), biological (hemogram, levels of carcinoembryonic antigen -CEA-, and carbohydrate antigen 19-9 -CA19-9-).

Platelet-poor plasma (PPP) containing microparticles (MPs) in human blood

Venous fasted blood samples were collected in citrated tubes and prepared within two hours following the drawing, as described by Lacroix et al (Lacroix et al., 2012). Citrated blood was centrifuged at 200 x g for 15 minutes to obtain the platelet-rich plasma (PRP). PPP was obtained after the centrifugation of PRP at 1100 x g for 15 minutes, with prostacyclin (PGI2) added to prevent platelet activation. PPP was then centrifuged at 7000 x g for 3 minutes. Aliquots were snap-frozen in liquid nitrogen and stored at -80°C until the measurements were performed. Cell lines and culture conditions

BxPC3 (ATCC- CRL1687) and HT29 (ATCC-HTB39) cell lines were respectively grown in RPMI 1640 medium or McCoy medium. Media were supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1% fungizone. Cells were grown at 37°C in a humidified atmosphere with 5% C02.

Purification of MPs issued from cancer cell lines

The isolation of MPs was adapted from Berckmans et al. (2001). In brief, cells at 80% of confluency were incubated for 15 h in OptiMEM medium. MP-rich medium was centrifuged at 1,500 g to eliminate cellular debris and ultracentrifuged at 20,000 g to isolate MPs. The final pellet containing MPs was resuspended in 0.5 ml PBS. Aliquots were snap-frozen in liquid nitrogen and stored at -80°C until the measurements were performed.

Flow cytometric analysis of PPP samples

The circulating MPs contained in the PPP samples were analyzed using a Gallios flow cytometer (Beckman Coulter), as previously described by Robert et al. (Robert et al., 2012). The flow cytometry instrument settings and MP gating were performed with Megamix beads (BioCytex). The MP cellular origin was detected with a combination of Annexin V or lactadherin and specific antibodies against cells of origin. Procoagulant MPs were determined using a combination of Annexin V or lactadherin and antibodies directed against fibrin, the final product of the coagulation cascade. The fibrin antibody was a generous gift from Pr Furie (Harvard Medical School, Boston). These antibodies and their isotype controls were labeled in-house with an Alexa Fluor labeling kit. Tumor MPs were characterized by the detection of MPs (Annexin V or lactadherin positive) expressing tumor markers on their surface, such as Mucinel (Mucl), Podoplanin, CEA and CA19-9. Matched isotype control antibodies were labeled in-house with an Alexa fluor labeling kit. Each specific antibody was matched with an isotype control antibody labeled with an appropriate fluorescent dye.

Annexin Positives MPs

The MP labeling was based on a 30-minute incubation of patient PPP (30 ^L) with Annexin V and appropriate antibodies; 300 of calcium buffer was then added to improve the binding of Annexin V to phosphatidylserine. After a 3-minute incubation, 100 of paraformaldehyde (16%) was added to fix the sample, and 30 of CytoCount beads was added; theses beads were necessary to determine the concentration of MPs in PPP. The concentrations are expressed as the number of MPs/microliter of PPP or PBS. The samples and associated controls were measured within an hour using a Gallios flow cytometer. The sample analyses were performed with Kaluza software, as already described by Lacroix et al.

Lactadherin Positives MPs

The MP labeling was based on a 30-minute incubation of patient PPP (10 ^L) with lactadherin and appropriate antibodies; 300 of PBS -/- was then added to dilute the sample. After a 3-minute incubation, 100 of paraformaldehyde (16%) was added to fix the sample, and 10 μί of CytoCount beads was added; theses beads were necessary to determine the concentration of MPs in PPP. The concentrations are expressed as the number of MPs/microliter of PPP or PBS. The samples and associated controls were measured within an hour using a Gallios flow cytometer. The sample analyses were performed with Kaluza software, as already described by Lacroix et al. RESULTS Lactadherin MPs positives versus Annexin MPs positives

To demonstrate that lactadherin recognizes better the MPs produced by a cancer, the inventors first compared the number of MPs produced by a cancer cell line (Bx PC-3) in vitro using lactadherin and Annexin V.

As illustrated in Figure 5, the use of lactadherin instead of Annexin V increases by about 3 times the number of MPs detected.

The inventors next compared in the samples from patients the number and the origin of MPs using Annexin V or lactadherin.

As illustrated in Figure 6, the number of platelets derived MPs (PMPs) detected in plasma from patients was identical using lactadherin or Annexin V (and using antibodies against CD41). The same result was observed when erythrocyte-derived microparticles (EryMP) were detected using antibodies against CD235a (Figure 7).

However, a significant difference was observed when comparing the number of endothelial-derived (Figure 8, using antibodies against CD31) and fibrin positive MPs (Figure 9). In both cases the use of lactadherin increases the number of detected MPs.

Based on these results, the inventors conclude that lactadherin is more sensitive to detect cancer-cell derived MPs, fibrin derived MPs and endothelial derived MPs than Annexin V.

The inventors finally compared the lactadherin positive MPs in the same patients suffering from a cancer before and after the surgery. The results, showed in the table below, confirm that lactadherin is much more sensitive that Annexin V. Table showing MPs in the same patients suffering from cancer before and after surgery: N = 10

Low to high levels of MPs (MPsfaL): white < horizontal lines < underlined numbers < diagonal lines Before surgery

LACTADHE IN ANNEXIN P LACTADHERIN ANNEXIN P

Total MPs 5339 <0.0001 6690 <0.0001

PMPs 4490 0.8 4370 0.4

EMPs 183 1.5 0.007 0.05

EryMPs 265 0.8 130 0.5

Leu MPs 5.7 0.1 10.6 12.6 0.8

Fibrin 0.8 0 Z6 0.06 positive

MUC1 0.02 0 0 0.5 positive

PODO 19 0.6 117 0.97 positive

CEA 0.9 0.4 1.4 0 0.006 positive

CA19-9 0 0 0.01 0 0 0.1 positive

REFERENCES

In the present application, references are the following:

Auwerda, J.J.A., Y. Yuana, S. Osanto, M.P.M. de Maat, P. Sonneveld, R.M. Bertina, and F.W.G. Leebeek. 2011. Microparticle-associated tissue factor activity and venous thrombosis in multiple myeloma. Thromb. Haemost. 105: 14-20. doi: 10.1160/TH10-03- 0187.

Baran, J., M. Baj-Krzyworzeka, K. Weglarczyk, R. Szatanek, M. Zembala, J. Barbasz, A.

Czupryna, A. Szczepanik, and M. Zembala. 2010. Circulating tumour-derived microvesicles in plasma of gastric cancer patients. Cancer Immunol. Immunother. 59:841-850. doi: 10.1007/s00262-009-0808-2.

Barteneva, N.S., E. Fasler-Kan, M. Bernimoulin, J.N.H. Stern, E.D. Ponomarev, L. Duckett, and I.A. Vorobjev. 2013. Circulating microparticles: square the circle. BMC Cell Biol. 14:23. doi: 10.1186/1471-2121-14-23.

Chamouard, P., D. Desprez, B. Hugel, C. Kunzelmann, C. Gidon-Jeangirard, M. Lessard, R.

Baumann, J.-M. Freyssinet, and L. Grunebaum. 2005. Circulating cell-derived microparticles in Crohn's disease. Dig. Dis. Sci. 50:574-580.

Chargaff, E., and R. West. 1946. The biological significance of the thromboplastic protein of blood. . Biol. Chem. 166: 189-197.

Chew, H.K., T. Wun, D. Harvey, H. Zhou, and R.H. White. 2006. Incidence of venous thromboembolism and its effect on survival among patients with common cancers. Arch. Intern. Med. 166:458-464. doi: 10.1001/.458.

D' Souza-Schorey, C, and J.W. Clancy. 2012. Tumor-derived microvesicles: shedding light on novel microenvironment modulators and prospective cancer biomarkers. Genes Dev. 26: 1287-1299. doi: 10.1101/gad.192351.112.

Van Doormaal, F.F., A. Kleinjan, M. Di Nisio, H.R. Biiller, and R. Nieuwland. 2009. Cell- derived microvesicles and cancer. Neth J Med. 67:266-273.

Fleitas, T., V. Martinez-Sales, V. Vila, E. Reganon, D. Mesado, M. Martin, J. Gomez-Codina, J.

Montalar, and G. Reynes. 2012. Circulating endothelial cells and microparticles as prognostic markers in advanced non-small cell lung cancer. PLoS ONE. 7:e47365. doi: 10.1371/journal.pone.0047365.

Graves, L.E., E.V. Ariztia, J.R. Navari, H.J. Matzel, M.S. Stack, and D.A. Fishman. 2004.

Proinvasive properties of ovarian cancer ascites-derived membrane vesicles. Cancer Res. 64:7045-7049. doi: 10.1158/0008-5472.CAN-04-1800.

Van den Hengel, L.G., A.Q.M.J. van Steijn-van Tol, R.M. Bertina, H.H. Versteeg, and S.

Osanto. 2013. Microparticle-associated tissue factor activity in plasma is unaffected by cytolytic chemotherapy treatment in metastatic testicular cancer patients. Thromb. Res. 131 : 187-189. doi: 10.1016/j.thromres.2012.11.026.

Hron, G., M. Kollars, H. Weber, V. Sagaster, P. Quehenberger, S. Eichinger, P.A. Kyrle, and A.

Weltermann. 2007. Tissue factor-positive microparticles: cellular origin and association with coagulation activation in patients with colorectal cancer. Thromb. Haemost. 97: 119-123.

Khorana, A.A., C.W. Francis, K.E. Menzies, J.-G. Wang, O. Hyrien, J. Hathcock, N. Mackman, and M.B. Taubman. 2008. Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer. . Thromb. Haemost. 6: 1983-1985. doi: 10.1111/j.1538-7836.2008.03156.X.

Kim, H.K., K.S. Song, Y.S. Park, Y.H. Kang, Y.J. Lee, K.R. Lee, H.K. Kim, K.W. Ryu, J.M.

Bae, and S. Kim. 2003. Elevated levels of circulating platelet microparticles, VEGF, IL- 6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor. Eur. J. Cancer. 39: 184-191.

Lacroix, R., and F. Dignat-George. 2012. Microparticles as a circulating source of procoagulant and fibrinolytic activities in the circulation. Thromb. Res. 129 Suppl 2:S27-29. doi:10.1016/j.thromres.2012.02.025.

Lacroix, R., C. Judicone, P. Poncelet, S. Robert, L. Arnaud, J. Sampol, and F. Dignat-George.

2012. Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. . Thromb. Haemost. 10:437-446. doi:10.1111/j. l538-7836.2011.04610.x.

Lacroix, R., L. Plawinski, S. Robert, L. Doeuvre, F. Sabatier, S. Martinez de Lizarrondo, A.

Mezzapesa, F. Anfosso, A.S. Leroyer, P. Poullin, N. Jourde, M.-S. Njock, C.M. Boulanger, E. Angles-Cano, and F. Dignat-George. 2012. Leukocyte- and endothelial- derived microparticles: a circulating source for fibrinolysis. Haematologica. 97: 1864- 1872. doi: 10.3324/haematol.2012.066167.

Lacroix, R., S. Robert, P. Poncelet, R.S. Kasthuri, N.S. Key, F. Dignat-George, and ISTH SSC Workshop. 2010. Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. . Thromb. Haemost. 8:2571-2574. doi:10.1111/j.1538-7836.2010.04047.X.

Liebhardt, S., N. Ditsch, R. Nieuwland, A. Rank, U. Jeschke, F. Von Koch, K. Friese, and B.

Toth. 2010. CEA-, Her2/neu-, BCRP- and Hsp27-positive microparticles in breast cancer patients. Anticancer Res. 30: 1707-1712.

Lowe, K.L., L. Navarro-Nunez, and S.P. Watson. 2012. Platelet CLEC-2 and podoplanin in cancer metastasis. Thromb. Res. 129 Suppl LS30-37. doi: 10.1016/S0049- 3848(12)70013-0.

Manly, D.A., J. Wang, S.L. Glover, R. Kasthuri, H.A. Liebman, N.S. Key, and N. Mackman.

2010. Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism. Thromb. Res. 125:511-512. doi: 10.1016/j.thromres.2009.09.019.

Mause, S.F., and C. Weber. 2010. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ. Res. 107:1047-1057. doi: 10.1161/CIRCRES AHA.110.226456.

Piccin, A., W.G. Murphy, and O.P. Smith. 2007. Circulating microparticles: pathophysiology and clinical implications. Blood Rev. 21 : 157-171. doi: 10.1016/j.blre.2006.09.001. Rak, J. 2010. Microparticles in cancer. Semin. Thromb. Hemost. 36:888-906. doi: 10.1055/s- 0030-1267043.

Ratajczak, J., M. Wysoczynski, F. Hayek, A. Janowska-Wieczorek, and M.Z. Ratajczak. 2006.

Membrane-derived micro vesicles: important and underappreciated mediators of cell-to- cell communication. Leukemia. 20: 1487-1495. doi: 10.1038/sj.leu.2404296.

Robert, S., R. Lacroix, P. Poncelet, K. Harhouri, T. Bouriche, C. Judicone, J. Wischhusen, L.

Arnaud, and F. Dignat-George. 2012. High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles— brief report. Arterioscler. Thromb. Vase. Biol. 32: 1054-1058. doi: 10.1161/ATVBAHA.111.244616.

Tesselaar, M.E.T., F.P.H.T.M. Romijn, I.K. Van Der Linden, F.A. Prins, R.M. Bertina, and S.

Osanto. 2007. Microparticle-associated tissue factor activity: a link between cancer and thrombosis? /. Thromb. Haemost. 5:520-527. doi: 10.1111/j. l538-7836.2007.02369.x.

Thaler, J., C. Ay, N. Mackman, R.M. Bertina, A. Kaider, C. Marosi, N.S. Key, D.A. Barcel, W.

Scheithauer, G. Kornek, C. Zielinski, and I. Pabinger. 2012. Microparticle-associated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients. . Thromb. Haemost. 10: 1363-1370. doi: 10.1111/j.1538-7836.2012.04754.X.

Thomas, G.M., L. Panicot-Dubois, R. Lacroix, F. Dignat-George, D. Lombardo, and C. Dubois.

2009. Cancer cell-derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo. . Exp. Med. 206: 1913-1927. doi: 10.1084/jem.20082297.

Wicki, A., and G. Christofori. 2007. The potential role of podoplanin in tumour invasion. Br. J.

Cancer. 96: 1-5. doi: 10.1038/sj.bjc.6603518.

Zahra, S., J. A.M. Anderson, D. Stirling, and C.A. Ludlam. 2011. Microparticles, malignancy and thrombosis. Br. J. Haematol. 152:688-700. doi: 10.1111/j. l365-2141.2010.08452.x.

Zwicker, J.I., H.A. Liebman, D. Neuberg, R. Lacroix, K.A. Bauer, B.C. Furie, and B. Furie.

2009. Tumor-derived tissue factor-bearing microparticles are associated with venous thromboembolic events in malignancy. Clin. Cancer Res. 15:6830-6840. doi: 10.1158/1078-0432.CCR-09-0371.

Claims

1. Method for monitoring the outcome of a given cancer of a patient, comprising the following steps:

d) increases of at least 17%, preferably of at least 30%, preferably at least 35%, more preferably 40% between the concentrations of platelet microparticles, fibrin microparticles and leukocyte microparticles, obtained in step b) and the ones obtained in step a) respectively, and a decrease of at least 30%, preferably at least 35%, more preferably 40% between the concentration of erythrocyte positive microparticles obtained in step b) and the one obtained in step a), are indicative that the patient is in remission.

2. Method according to claim 1, characterized in that step a) and step b) further comprise measuring the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles, in a blood sample of said patient, before and after treatment of said patient, respectively, and wherein, in step d): an increase of at least 90%, preferably of at least 95%, more preferably of at least 100%, between the concentration of endothelial microparticles obtained in step b) and the one obtained in step a), an increase of at least 10%, preferably of at least 15%, more preferably of at least 20% between the concentration of total microparticles obtained in step b) and the one obtained in step a),

and decreases of at least 30%, preferably of at least 40%, more preferably of at least 50% between the concentrations of tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles and CA19-9 positive microparticles obtained in step b) and the ones obtained in step a), are indicative that the patient is in remission.

3. Method according to any one of claims 1 to 2, characterized in that the given cancer is chosen from malignant solid tumors, preferably from colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, gastric cancer and gallbladder cancer.

4. Method for diagnosing a given cancer in a blood sample of a patient, comprising the following steps:

5. Method according to claim 4, characterized in that step a) further comprises measuring at least one concentration, preferably all the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles, in said sample.

6. Method according to claim 4 or 5, characterized in that the given cancer is chosen from malignant solid tumors, preferably from colorectal cancer, pancreatic cancer, breast cancer, prostate cancer and lung cancer.

7. Method according to any one of claims 4 to 6, characterized in that it is for diagnosing colorectal cancer in a blood sample of a patient, and it comprises the following steps:

b) comparing:

- the concentration of platelet microparticles obtained in step a) to the range of 2000 to 8000, preferably of 2200 to 7500, more preferably of 2300 to 7130,

- the concentration of erythrocyte microparticles obtained in step a) to the range of 80 to 700, preferably of 90 to 500, more preferably of 100 to 480,

said concentrations being expressed in microparticles^L,

8. Method according to claim 7, characterized in that step a) further comprises measuring the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles in said sample, and that step b) further comprises comparing:

- the concentration of MUC1 positive microparticles obtained in step a) to the range of 0 to 60, preferably of 0 to 50, more preferably of 0 to 47, - the concentration of CEA positive microparticles obtained in step a) to the range of 0 to 60, preferably of 0 to 50, more preferably of 0 to 45,

said concentrations being expressed in microparticles^L.

9. Method according to any one of claims 4 to 6, characterized in that it is for diagnosing pancreatic cancer in a blood sample of a patient, and it comprises the following steps:

b) comparing:

- the concentration of leukocyte microparticles obtained in step a) to the range of 4 to 50, preferably of 5 to 45, more preferably of 6 to 42, and

said concentrations being expressed in microparticles^L,

10. Method according to claim 9, characterized in that step a) further comprises measuring the concentrations of endothelial microparticles, tissue factor positive microparticles, MUC1 positive microparticles, CEA positive microparticles, CA19-9 positive microparticles and total microparticles in said sample, and that step b) further comprises comparing:

- the concentration of CEA positive microparticles obtained in step a) to the range of 0 to 200, preferably of 0 to 170, more preferably of 0 to 150,

- the concentration of CA19-9 positive microparticles obtained in step a) to the range of 5 to 700, preferably of 7 to 600, more preferably of 9 to 500, and

said concentrations being expressed in microparticles^L.

11. Method according to any one of preceding claims, characterized in that the blood sample is chosen from whole blood, plasma and serum, preferably is platelet-poor plasma.

12. Method according to any one of the preceding claims, characterized in that the measure of the concentration of a subpopulation of microparticles in the sample is performed by mixing said sample with Annexin V preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies.

13. Method for diagnosing a given cancer in a blood sample of a patient, comprising the following steps:

a) measuring the concentrations of platelet microparticles, endothelial microparticles and fibrin positive microparticles in said sample, by mixing said sample with lactadherin preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies, b) comparing the respective concentrations obtained in step a) to the respective ranges of reference values, and

14. Method according to claim 13, characterized in that it comprises the following steps: a) measuring the concentrations of platelet microparticles, endothelial microparticles and fibrin positive microparticles in said sample, by mixing said sample with lactadherin preferably conjugated to a fluorescent or enzymatic label, and appropriate specific antibodies,

b) comparing:

said concentrations being expressed in microparticles^L,

c) if all the concentrations obtained in step a) are included in said ranges of step b), establishing that said patient suffers from a cancer.

15. Method according to claim 13 or 14, characterized in that it further comprises in step a) the measures of the concentrations of erythrocyte microparticles, leukocyte microparticles, MUC1 positive microparticles, CEA positive microparticles, podoplanin positive microparticles and total microparticles in said sample, and further comprises in step b) the comparisons of:

- the concentration of leukocyte microparticles obtained in step a) to the range of 0 to 1200, preferably of 0 to 1100, - the concentration of MUC1 positive microparticles obtained in step a) to the range of 0 to 2100, preferably of 0 to 2000,

said concentrations being expressed in microparticles^L.

16. Method for monitoring the outcome of a given cancer of a patient, comprising the following steps:

17. Method for monitoring the outcome of a given cancer in a blood sample of a patient, comprising the following steps:

18. Use of lactadherin for measuring a concentration of microparticles.