WO2015049372A1

WO2015049372A1 - Method for in vitro investigating mitochondrial dna levels in a biological sample, kits and uses thereof

Info

Publication number: WO2015049372A1
Application number: PCT/EP2014/071239
Authority: WO
Inventors: Eliette Camille Touati; Julien Fernandes; Valérie Michel; Javier Torres Lopez
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut Pasteur; Instituto Mexicano del Seguro Social
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut Pasteur; Instituto Mexicano del Seguro Social
Priority date: 2013-10-03
Filing date: 2014-10-03
Publication date: 2015-04-09
Anticipated expiration: 2016-04-03
Also published as: EP2857520A1

Abstract

The invention relates to an in vitro method for investigating the level of mtDNA in a biological sample removed from a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition by pooling said biological sample into categories, upon determination of the level of mtDNA in said biological sample. The invention also relates to a method for in vitro monitoring or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, comprising a step of measuring, in parallel to the investigation of the level of mtDNA, the level of any one of the following parameters: cytokine IL-8, platelet count (PLT) and/or mean platelet volume (MPV) and/or percentage of large platelets (LPLT), inflammatory markers such as IL-6 and/or IL-23, or any combination of these parameters, and concluding about the health status of the tested patient. The invention also relates to kit(s) suitable for carrying out the method(s) of the invention and their use.

Description

METHOD FOR IN VITRO INVESTIGATING MITOCHONDRIAL DNA LEVELS IN A BIOLOGICAL SAMPLE, KITS AND USES THEREOF.

The present invention relates to the field of in vitro testing methods based on the investigation of the level of mitochondrial DNA (mtDNA) in biological samples collected from individuals, animals or humans, and in particular relates to methods that can be applied to the monitoring and/or diagnosing of the health status of a patient susceptible of suffering from a gastric cancer or susceptible of suffering from condition(s) that may evolve in a gastric cancer, especially methods suitable for the monitoring or the detection of a risk of gastric carcinogenesis. The invention also relates to kits for performing the methods of the invention and their uses.

The present invention further relates to methods and means for the investigation or detection of a risk of development of precancerous lesions or early gastric cancer, in particular methods and means involving biomarkers enabling the further evaluation of the necessity to perform further clinical investigations.

Gastric cancer (GC) is the second leading cause of cancer-related death in developing countries and the fourth in industrialized countries. It affects about one-million people per year.¹ Although the incidence of GC and mortality rates have gradually decreased in the last decades, it remains an important public health problem worldwide with approximately 800 000 deaths per year.

GC is often asymptomatic or causes only nonspecific symptoms in its early stages. By the time heavy symptoms occur, the cancer has often reached an advanced stage and may have also metastasized.

Actually and as a consequence, GC is often diagnosed at an advanced stage, e.g. at stage IV for 79% of the tumors. These tumors generate early hematogenic metastasis to the liver, lung, brain and skeleton. Consequently, gastric cancer carries a poor prognosis with an overall 5- years survival rate around 15%.² However if it is diagnosed at an early stage, it can be a curable disease.³ These data highlight the importance and the need for characterization and validation of early GC biomarkers to reduce the morbidity and mortality associated to gastric adenocarcinoma. GC arises from the complex interplay of various factors including environmental factors and host genetic factors.^{4, 5} However, the major risk factor occurring for 60 to 80% of gastric cancer cases is Helicobacter pylori infection. The prevalence of H. pylori infection is high, with 80 to 95% of the population infected in developing countries and up to 30% to 40% of adults in industrialized countries.⁶ The infection mostly persists life-long if not treated. All infected individuals develop a gastritis which can evolve to peptic ulcer diseases in 10% of the cases, while gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma will develop for 3% and 0.3% of infected subjects, respectively.^{6, 7}

Gastritis, in particular atrophic gastritis, may be characterized by a chronic inflammation of the gastric mucosa, associated by high production of oxidative species known to damage DNA, further rendering mtDNA highly susceptible to damage and depletion.

Gastric adenocarcinoma is a malignant epithelial tumor, originating from glandular epithelium of the gastric mucosa. A major proportion of GC, i.e. more than 90%, are adenocarcinomas. Based on an histological distinction, there are two major types of gastric adenocarcinoma: intestinal type or diffuse type. Adenocarcinomas tend to aggressively invade the gastric wall, infiltrating the gastric mucosa, and present several degrees of differentiation, from well to poorly differentiated.

MALT lymphoma (or MALToma) is a form of lymphoma involving the mucosa-associated lymphoid tissue (MALT), frequently found in the stomach. This disease originates from B cells in the marginal zone of the MALT, and is also called extranodal marginal zone B cell lymphoma. Gastric MALT lymphoma is frequently associated (but not in all cases) with a chronic inflammation resulting from the presence of H. pylori, or linked with the presence of H. pylori. In the context of the invention, GC encompasses gastric adenocarcinoma and MALT lymphoma.

GC of intestinal-type is mostly induced by H. pylori infection. It develops through a sequence of precursor lesions from atrophic gastritis to intestinal metaplasia (IM) then dysplasia and cancer.⁸ IM are recognized as preneoplastic lesions. It is important to notice that the eradication of the infection at an early stage (IM) can reverse and more importantly prevent the development of precancerous lesions.⁵ Accordingly, in humans several studies showed regression of precancerous lesions after H. pylori eradication,^{9 10} also confirmed in animal models. ^{11 , 12}

In intestinal-type of GC, the sequential steps in the precancerous process can be considered as (in evolving order): gastritis (superficial or, at a later stage, chronic atrophic gastritis, for example), metaplasia (small intestinal or colonic metaplasia), and finally, dysplasia.

The cancerous (or neoplasic) stage may then be entered, depending on the evolution of the disease. The clinical stages of GC can be classified using known classifications systems, e.g. the TNM Classification of Malignant Tumours staging system that describes the extent of a patient's cancer. Using this type of classification, one can distinguish between Stages 0, I, II, III or IV. Other types of staging systems can be used.

In Stage 0, the gastric cancer is limited to the inner lining of the gastric mucosa and may be treatable by surgery when found very early, without need for chemotherapy or radiation treatments.

In Stages I and II, the disease has penetrated the deeper layers of the gastric mucosa, and may be treated by surgery, sometimes associated with chemotherapy and/or radiation treatments.

In Stage III, the disease may have penetrated other nearby tissues distant lymph nodes. Treated as for Stage II, a cure is still possible in some cases.

In Stage IV, the disease has spread to nearby tissues and more distant lymph nodes, or has metastasized to other organs. A cure is very rarely possible at this stage. Some techniques are used to prolong life or improve symptoms, including laser treatment, surgery, and/or stents to keep the digestive tract open, and chemotherapy.

Mitochondria are essential organelles of eukaryotic cells and possess their own genome. Defects in mitochondrial functions are associated with various human diseases including cancer.¹³ In humans, mitochondrial DNA (mtDNA) is a 16.6Kb circular DNA molecule present at hundreds to thousands copies per cell.¹⁴ Mutations in mtDNA have been reported in all cancer examined to date. As example in colorectal cancer, presence of mtDNA mutation is associated with a poor prognosis.¹⁵ The presence of mtDNA mutations has also been described at early stages of gastric carcinogenesis.^{16, 17} In H. pylori chronic gastritis patients, mtDNA mutations are significantly more frequent in gastric cancer patients than in cancer free patients.¹⁸ According to the inventors previous studies, mtDNA mutations are induced in vitro in H. pylori infected gastric epithelial cell¹⁹ and in the gastric mucosa of mice chronically infected.²⁰ Tumor specific changes in mtDNA copy number have also been reported in a broad range of primary human cancers.^{21 , 22}. Xia et al, reported mtDNA depletion in the peripheral blood of patients strongly associated with stage I breast tumors.²⁸ A decrease in mtDNA content has been described in most tumor tissues of advanced GC, compared with nearby non-tumorous control tissue.²³ Variation of mtDNA level in cancer patients compared to healthy subjects has also been described for circulating cell-free mtDNA in the plasma and serum, leading to postulate that level of circulating mtDNA can constitute a potential preventive/diagnosis cancer marker detectable by a non-invasive method.¹⁹ Indeed, changes in mtDNA content in circulating blood samples have been described in patients with various types of tumors. As example, levels of circulating mtDNA were significantly higher in patients with urologic malignancies compared to healthy volunteers,²⁴ as also reported in several case-control studies in breast,²⁵ colorectal²⁶ and lung cancer.²⁷ These studies indicate that increased mtDNA content in peripheral blood should be associated with elevated cancer risk. In a recent case-control study conducted within a large prospective cohort of women residing in Shangai, no association was shown between leukocyte mtDNA copy number and presence of gastric tumor.²⁹ However a positive association between low mtDNA copy number in blood drawn within the two years prior to cancer diagnosis and risk of developing GC was observed, suggesting that circulating blood mtDNA levels could be an indicator of the presence of GC lesions at an early stage.

So far, variation of mtDNA levels in circulating blood can therefore be considered as a potential marker for the development of serological diagnosis test for a detection of the presence of gastric preneoplasia or cancer lesions.

However, the literature is not consistent when it comes to determining what variation(s) in mtDNA levels may be correlated with the presence of gastric preneoplasia or cancer lesions associated with a risk of gastric cancer, let alone what particular mtDNA levels may be associated with such a risk.

The present invention proposes a solution to this uncertainty, and further provides tools for investigating the level of mtDNA in a biological sample, in particular in a blood sample, said sample being obtained from a patient susceptible of suffering from a gastric cancer condition or condition(s) susceptible to evolve in a gastric cancer, in particular for monitoring or detecting a risk of an ongoing gastric carcinogenesis process. The method of the invention accordingly enables to conclude that a risk of an ongoing gastric carcinogenesis process is present and/or enables the determination of the relevancy to perform further clinical investigations.

As used herein, the term "gastric carcinogenesis" encompasses both the precancerous manifestations described above including gastritis, preneoplastic lesions such as intestinal metaplasia (small intestinal or colonic metaplasia), neoplastic lesions such as dysplasia, that may evolve in gastric cancer, and the different cancerous stages of gastric cancer, in particular, use of the expression "gastric carcinogenesis" covers the development of gastric cancer since detection of the presence of a gastritis or associated lesions in a patient. Accordingly, the investigation methods described herein enable at least the determination of a risk of gastric carcinogenesis in a patient, a sample of which is assayed according to the methods described herein. In some cases, as described herein, said risk may appear stronger.

The method of the invention can suitably be used for detecting or monitoring the biological parameters of a patient, and/or providing information about, in particular, but not exclusively, diagnosing, the health status of such a patient.

The present invention relies on studies in which the peripheral leucocytes mtDNA levels have been measured in patients, and a posteriori correlated to various steps of the gastric carcinogenesis process. Relevant variations of mtDNA levels have been surprisingly found during the progression from gastric inflammatory lesions in gastritis patients until the development of malignant lesions, supporting the notion that circulating mtDNA level can be considered as a potential biomarker for detection of gastric carcinogenesis, in particular at early steps during gastric carcinogenesis.

Accordingly, the present invention relates to an in vitro method for investigating the level of mtDNA in a biological sample removed from a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, by pooling said biological sample removed from such a patient into categories, said method comprising the steps of:

a. Determining the level of mtDNA in said biological sample, and b. Comparing the level of mtDNA determined in step a. with a normal threshold value determined for healthy subjects, and c. From the comparison made in step b., assigning the tested biological sample to one of the following categories: Group I: if the level of mtDNA determined in step a. is decreased with respect to the normal threshold value introduced in step b. by less than 2 folds, i.e., the ratio of the level of mtDNA determined in step a. over the normal threshold value determined for healthy subjects of step b. is inferior to 2,

Group II: if the level of mtDNA determined in step a. is from 2 and 20 folds with respect to the normal threshold value introduced in step b., i.e., the ratio of the level of mtDNA determined in step a. over the normal threshold value determined for healthy subjects of step b. is equal or superior to 2, but equal or inferior to 20,

Group III: if the level of mtDNA determined in step a. is increased with respect to the normal threshold value introduced in step b. by more than 20 folds, i.e., the ratio of the level of mtDNA determined in step a. over the normal threshold value determined for a pool of healthy subjects of step b. is superior to 20.

Criteria to define healthy individuals for the purpose of determining normal threshold value(s) are further defined below, and include a negative serology for H. pylori, no chronic inflammation and any pathology detected over 6 months before the analysis, as well as no antibiotic or antiinflammatory treatment.

The biological sample obtained from a patient may be a tissue sample, in particular from a biopsy or may be a blood sample or a fraction of a blood sample such as a cellular fraction. The disclosure and examples provided herewith in relation to blood sample would similarly apply to other biological samples except when technically irrelevant.

According to a particular embodiment, the assignment of the tested biological sample to one of the categories that are Group I, II or III is achieved by determining the value of mtDNA level change, which therefore constitutes, in this particular embodiment, step c.

According to a particular embodiment, the biological sample removed from such a patient is a blood sample. According to a more particular embodiment, such a blood sample is beforehand treated to isolate leukocytes from which total DNA is prepared and purified. The measure of mtDNA level is performed on the preparation of total DNA of the leukocytes.

According to a particular embodiment, a level of mtDNA as determined in step a., which enables the assignment of the tested biological sample to Group I or Group III, is a strong indicator for the presence, or strongly indicates the possibility of presence, of gastritis lesions (in Group I) or gastric neoplasia (in Group III), leading to further clinical investigation for the subject(s) assigned to these two groups, including endoscopy and anatomic-pathology analysis.

According to another particular embodiment however, assignment of the tested biological sample to Group II may still be an indicator for the presence of an ongoing carcinogenic process, albeit not detectable by the sole assignment of the tested biological sample to Group II. Further clinical investigation(s) may still be required. According to a particular embodiment, investigation of further physiological parameter(s), such as, investigation of the level of IL-8, as described herein, may be envisioned.

According to another approach, generally applicable within the context of the present invention, it will be understood that assignment of the tested biological sample to one of the categories described in step c. provides quantification of the change in the level of mtDNA following the comparison made in step b., since the categories listed in step c. do not overlap one another.

Thus, according to a particular approach of the invention, aimed at providing a simple and non-invasive method for investigating physiological parameters of subjects, in particular in order to conclude about the health status of a patient, according to the definition provided herein, the present invention provides an in vitro method for investigating the level of mtDNA and IL-8 level in a biological sample removed from a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, and/or in vitro monitoring and/or diagnosing the health status of such a patient, said method comprising the steps of:

a. determining the level of mtDNA in said biological sample, and b. comparing the level of mtDNA determined in step a) with a normal threshold value determined for healthy subject(s), and c. from the comparison made in step b), determining if the level of mtDNA determined in step a) is increased with respect to the normal threshold value introduced in step b) by more than 20 folds, and

d. measuring, in parallel, according to the definition provided herein, to the investigation of the level of mtDNA of step a), the level of cytokine IL-8 in a biological sample removed from the patient tested in step a), and

e. Concluding about the health status of the patient on the basis of the results obtained from step c. and d.

According to a particular embodiment, conclusion is made in step e) of a suspicion of a risk of precancerous or cancerous lesions for the assayed patient if the level of mtDNA determined in step a) is increased with respect to the normal threshold value introduced in step b) by more than 20 folds (Group III defined herein) and the level of cytokine IL-8 for said patient is superior to 100 pg/mL. Further clinical investigations as defined herein should be carried out for said patient. Such a method may enable an early detection of patients susceptible of developing gastric cancer, by a simple blood sampling.

According to this particular embodiment, the detection of patients with high level of mtDNA change (>20 folds, group III) and IL-8>100pg/ml would be a strong indication of the presence of gastric inflammation and probable pre-cancer or cancer lesions by a simple blood sampling.

By "patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition", it is meant a patient presenting a suspicion for developing gastric cancer, for example because of physical clues, family history or complaint(s) indicating to the practitioner that an etiology of gastric cancer may be present or may become present. It also includes patients with gastroesophageal reflux, with chronic gastric pain as well as H. pylori seropositive subjects or H. pylori seronegative subjects, which have been beforehand eradicated for H. pylori infection. This definition also encompasses individuals having a declared gastric cancer condition or condition(s) susceptible to evolve in a gastric cancer condition, under treatment or not, which should be monitored. A particular group of patients eligible for the performance of the method of the invention is a group of patients with chronic inflammation associated with gastritis, or patient(s) with gastroesophageal reflux, with chronic gastric pain as well as H. pylori seropositive subjects. As mentioned above, a "patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition" may also be H. pylori negative at the time of the sampling and/or testing because of a successful eradication of the infection. The above definition also includes patients previously diagnosed for chronic atrophic gastritis or other gastric lesion that need a clinical follow-up.

In a particular embodiment, the method of the invention may be carried out on sample isolated (collected, removed) from an individual who has been previously diagnosed for a gastric cancer or for lesions that may lead to a gastric cancer (pre-neoplasic condition) and who, optionally, may have been subjected to a treatment, such as surgery and/or chemotherapy and/or radiations treatment, in particular an individual pertaining to the above-mentioned particular group(s) of patient(s). In a more particular embodiment, the method of the invention is carried out on total DNA preparation purified from leukocytes previously isolated from a blood sample isolated (collected, removed) from an individual as defined in the paragraph above.

Under "gastric cancer" are encompassed: gastric adenocarcinoma, both diffuse or intestinal types, and MALT lymphoma.

By "investigating the level of mtDNA" it is meant detecting, in particular quantifying a "level of mtDNA", e.g. a quantity of mtDNA in an assayed biological sample collected from an individual, animal or human, with respect to a quantity considered as a standard value, said level reflecting the abundance of mtDNA copies in said sample.

By "level of mtDNA", it is meant a result qualifying the change in mtDNA with respect to a standard or normal level and in particular a value aimed at quantifying or representing the amount, in particular the number of copies of mtDNA, in particular the absolute number of copies of mtDNA, in the tested biological sample, or a relative amount of mtDNA in the tested biological sample, in particular when normalized with respect to a quantity of nuclear DNA (nDNA) or another suitable reference also present in said biological sample, or with respect to normalizing genes or DNA sequences pertaining to nDNA. In a particular embodiment, the investigation according to the invention may thus be achieved without determining the absolute quantity of mtDNA in the sample but by evaluating the variation (increase of the level or the decrease of the level) of said mtDNA and assigning said variation to a range as defined above.

To obtain a "level of mtDNA" according to the present invention, use is made of techniques enabling nucleic acids quantification from a biological sample. Such technique enables the determination of the average concentration (or amount) of nucleic acids, i.e., DNA or RNA, especially mtDNA within the context of the present invention, present in a sample. Several methods can be used to establish such concentrations (or amounts), including (1 ) spectrophotometric analysis of nucleic acids and their further quantification and (2) quantification using the measurement of the fluorescence intensity of dyes that bind to nucleic acids and selectively fluoresce when bound, as well as (3) quantification after specific nucleic acids amplification, such as in the real time PCR technique, which also relies on the detection of a fluorescent dye bound to said nucleic acids to be detected and quantified. All these techniques may be used with their corresponding appropriate containers for the nucleic acids to quantify, such as cuvettes or microplates or arrays, as well as combined in more integrated systems such as chips, lab-on-a-chip and microfluidic supports (including microfluidic cartridge or chip). Depending on the technique used, prior isolation of mtDNA to be quantified may be required, according to the common knowledge in the art of nucleic acids analysis.

By "a normal threshold value determined for healthy subjects", it is meant a value found by assaying one biological sample from an healthy subject or alternatively found by assaying several biological samples from several distinct healthy subjects, the resulting normal threshold value being then determined as the mathematical mean of the levels values of all the assayed healthy subjects biological samples, or alternatively found by assaying a pool of biological samples from several distinct healthy subjects.

According to a particular embodiment, such a normal threshold value is determined for a pool of healthy subjects.

By "healthy subject(s)" it is meant subjects that would have no symptoms of gastric disorders or patients referred to for gastroscopy with gastric biopsies corresponding to a normal phenotype, and in both cases not infected with H. pylori or that received an eradication therapy followed by a confirmed negative H. pylori serology. In a particular embodiment, the biological sample removed from such healthy subject(s) is a blood sample.

More particularly, by "normal threshold value determined for healthy subjects or for a pool of healthy subjects", it is meant a value, corresponding to a level of mtDNA, which may be predetermined according to the knowledge of the skilled in the art because it is known to correspond to a normal mtDNA value, or, according to a preferred embodiment, a value determined from assays made on subjects, in particular individuals, known to be healthy, i.e., not presenting a gastric cancer condition or suffering of condition(s) susceptible to evolve in a gastric cancer, or as explained above, said patients providing samples which either are individually tested and the results obtained from said samples treated to provide a mean val ue or a median value or said samples are tested as pooled samples to provide a so-called normal value. The thus obtained value provides a threshold that determines the reference value for normal samples. Healthy individuals for the determination of mtDNA normal values correspond to subjects with a negative serology for H. pylori, with no symptoms of gastric pathologies susceptible to evolve in gastric cancer or MALT lymphoma, and no other pathologies and inflammatory diseases detected and which have not been under antibiotic or anti-inflammatory treatment for the last 6 months.

By "fold", it is meant the expression of a change, in particular a number, describing how much a given quantity changes from a normal to a tested value, the normal value being in particular the "normal threshold" and the tested value being the mtDNA copy number or a representative value thereof, in the tested sample. For example, a normal value of 30 and a tested value of 60 correspond to a fold change of 2, or in common terms, a two-fold increase. To the contrary, a normal value of 60 and a tested value of 30 correspond to a fold change of 0.5, or in common terms, a 0.5 fold decrease, also referred to as a "minus" two-folds decrease (expressed in negative terms, with a "minus" sign before the number). Fold changes therefore correspond to a ratio of the tested value to the normal value. In other words, the fold change results from the determination of a ratio of the tested value against the normal value.

The invention accordingly also relates to a method of determining a "normal threshold" value for the mtDNA which value is associated with an absence of gastric cancer condition or an absence of a condition susceptible to evolve in a gastric cancer condition as disclosed herein. The determination of "normal threshold" value may be regarded as involving a pooling method to the extent that it results either from pooling the results or from pooling the samples said samples being obtained from healthy subjects. Healthy subjects refer to a subject with no diagnosed chronic inflammation either gastric or other and with a negative H. pylori serology. It can also be a subject that is not treated by antibiotherapy or Non-Steroidal Anti-Inflammatory Drug(s) (NSAID), with a normal blood count and in the absence of any medical treatment at the time of the sampling and/or testing. According to another particular embodiment the normal values providing the threshold to determine the level of change in mtDNA of tested biological sample are the result of the above disclosed pooling method when performed on a pre-determined relevant population, as also detailed above.

The method of the invention can subsequently be used for monitoring or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, such a method comprising steps a., b. and c. as disclosed above and further comprising the following step:

d. from the assignment to a Group I, II or III made in step c. as described above, concluding about the health status of a patient from which the tested biological sample has been removed.

By "concluding about the health status" it is meant concluding that the individual, the biological sample of which is tested, may present a risk of suffering of a gastric cancer or from condition(s) that may evolve in a gastric cancer, i.e., condition(s) indicating a risk that a gastric carcinogenesis is present in the tested individual. Accordingly, further clinical investigations may be ordered.

According to a particular embodiment, the expression "to conclude about the health status of a patient from which the tested biological sample has been removed" means that conclusion is made to proceed with further clinical investigation(s) for said patient.

According to a particular embodiment, the expression "concluding about the health status" and/or the expression "proceed with further clinical investigation (s) for said patient' encompasses enrolment of said patient in a procedure of closer therapeutic monitoring, i.e., said patient is recommended with or directly incorporated in a therapeutic follow-up comprising a regular monitoring of his/her condition or health status over time, and optionally further clinical investigations regarding its health status.

In particular and according to a finding related to the present invention, assignment of a mtDNA level value in Group I or III in step c. as described above, provides the information that the patient from whom the tested biological sample has been removed is susceptible of suffering from gastric lesions, which also suggests performance of further clinical investigations.

Accordingly, conclusion "about the existence of a risk of gastric carcinogenesis at an early stage" means that the method of the invention enables detecting at an early stage the presence of lesions associated with gastric carcinogenesis and/or the evolution of such a gastric carcinogenesis and/or may encompass the performance of further clinical investigations.

As non-limitative examples, other investigations methods aimed at confirming or excluding the presence of as gastric carcinogenesis process may be optical gastroscopic examination, computed tomography (or CT) scanning of the abdomen, biopsies for histological examination, various blood tests, e.g., Complete Blood Count (CBC) to check for anemia.

Accordingly, it is another object of the invention to provide a method for monitoring or detecting a risk of gastric carcinogenesis in a patient, wherein said method comprises identifying whether the level of mtDNA of the assayed sample has a value that pertains to Group I or Group III, a risk of gastric carcinogenesis being present if the biological sample removed from said patient has such a value. By "a risk of gastric carcinogenesis" it is meant a risk of presenting a gastric cancer condition or a risk of presenting condition(s) susceptible to evolve in a gastric cancer condition, which encompass:

- precancerous manifestations of gastric cancer, that may further evolve in gastric cancer, such as: gastritis or gastritis lesions (superficial or, at a later stage, chronic atrophic gastritis, for example), metaplasia (small intestinal or colonic metaplasia), and finally, dysplasia, and

- the different cancerous (or neoplasic) stages of gastric cancer, as defined above.

According to a particular finding, the method of the invention enables concluding to the existence of a risk of gastric carcinogenesis, in particular at an early stage, when the tested biological sample pertains to Group I and the mtDNA level change measured with respect to normal value is less than 0.5 fold.

In a particular embodiment, by "early stage of gastric carcinogenesis" it is meant a pre-neoplasic stage, as defined above, for example a gastritis stage.

According to another particular finding, the method of the invention enables to conclude to the existence of a risk of a gastric cancer, when the tested biological sample pertains to Group III and the mtDNA level change measured with respect to normal value is more than 30 folds.

According to a particular embodiment of the method of the invention, if the tested biological sample pertains to Group I and determination is made that the mtDNA level change is less than 0.5 fold with respect to normal threshold value, conclusion is made about the existence of a risk of gastric carcinogenesis at an early stage, in particular a pre-neoplasic stage, for example at a gastritis stage.

According to a particular embodiment of the method of the invention, if the tested biological sample pertains to Group III and determination is made that the mtDNA level change is more than 20 folds with respect to normal threshold value, conclusion is made about the existence of a gastric pre- neoplasia or gastric cancer.

According to a particular embodiment of the method of the invention, if the tested biological sample pertains to Group III and determination is made that the mtDNA level change is more than 30 folds with respect to normal threshold value, conclusion is made about the existence of a gastric cancer.

According to a particular embodiment, the gastric carcinogenesis referred to herein is associated with an Helicobacter pylori infection or is an Helicobacter py/or/^'-induced gastric carcinogenesis.

Accordingly, the invention also comprises a method of investigating the level of mtDNA in a sample removed from a patient, where the patient has been previously or is simultaneously or in parallel tested for infection by Helicobacter pylori. The detection of H. pylori may be performed on a fraction of the sample removed from the patient, in particular on the serum fraction of a blood sample by carrying out a step of detection of antigens specific for H. pylori infection. People infected by H. pylori have specific IgA and IgG antibodies that can be easily detectable. In addition, the search for the presence of CagA antigens can also confirm the presence of H. pylori. Another method to detect H. pylori is the ¹³C urea breath test, a non- invasive test with high sensitivity widely used in human medicine (Graham et al, 1987, Lancet, 1 : 1 174-1 177). This respiratory test allows an indirect measure of the H. py/or/^'-associated urease activity. Presence of H. pylori can also be detected in stools by immunoassay indicating the presence of H. pylori antigens or by amplification of H. pylori DNA in particular by polymerase chain reaction (PCR) using specific primers for H. pylori genes sequences, which are available in the literature to one skilled in the art, and detection of the amplified DNA.

For detection of H. pylori, the blood sample may be prepared on the one hand to purify the cellular fraction of the blood sample, in particular the mononuclear cells or leukocytes containing the mtDNA to be assayed and on the other hand to collect the serum enabling the detection of H. pylori infection.

In a particular embodiment, the tested biological sample is obtained from a patient diagnosed with gastric carcinogenesis and under treatment for this condition or not, and/or a patient having an ongoing, treated or not, Helicobacter pylori infection, and/or a patient having antecedents of Helicobacter pylori infection(s), eradicated by prior or ongoing treatment or not, and/or an individual having gastric pain and/or a family history of gastric cancer.

The method for investigating the level of mtDNA in a biological sample according to the invention is performed using any relevant process for the quantification of nucleic acids, as in particular introduced above. It especially involves treating the sample to gain access to mononuclear cells, in particular leukocytes, and furthermore to provide access to nucleic acid, especially by extracting the nucleic acid, and optionally by separating the mtDNA from nuclear DNA. The method also involves using polynucleotides, in particular probes and/or oligonucleotides when they are complementary to a region of interest in the mtDNA, said polynucleotides optionally including a detectable label or marker (such as a fluorochrome or a radioelement) in order to allow quantitative detection of the mtDNA.

According to an embodiment of the invention, the method for investigating the level of mtDNA in a biological sample and optionally the method of determining a "normal threshold" value involve(s) a step of amplification of the mtDNA, possibly a step of differential amplification of the mtDNA with respect to the nuclear DNA.

According to a specific embodiment, the "level of mtDNA" is determined by quantitative polymerase chain reaction (q-PCR). Primers specific for the 12sRNA mitochondrial gene may be used, although one skilled in the art can suitably choose other genes or sequences of the mitochondrial genome for implementing such a technique, following guidance available in the literature of this field with respect to this technique. PCR (polymerase chain reaction) is a common method for amplifying DNA. In order to amplify small amounts of DNA, a DNA template, at least one pair of specific oligonucleotide primers, nucleotides (dATP, dCTP, dGTP, dUTP), a suitable buffer solution and a thermo stable DNA polymerase are required. A substance marked with a fluorophore is generally added to one reagent of this mixture in a thermal cycler that contains sensors for measuring the fluorescence of the fluorophore after it has been excited at the required wavelength allowing the generation rate to be measured for one or more specific products. Real-time PCR (q-PCR) is generally applied to the detection and quantification of DNA in samples to determine the presence and/or abundance of a particular DNA sequence in these samples. A measurement is made after each amplification cycle, which enables the quantification of the amplified product in real time.

Real-time PCR is performed by using a real-time PCR apparatus, and after each cycle, the levels of fluorescence are measured with a detector. Used dyed generally only fluoresce when bound to the DNA amplified through PCR, and the increase of fluorescence is detected, corresponding to increasing presence of the amplified products, at each amplification cycle.

Real-time PCR can be used to quantify nucleic acids by either relative quantification or absolute quantification . Absolute quantification gives the exact number of target DNA molecules by comparison with DNA standards using a calibration curve. By this method, it is possible to determine the number of mtDNA copies in patients suspected for the presence of gastric pre-neoplasia or neoplasia and compare this number to the number of mtDNA copies defined in healthy subjects. (Ref: von Wurmb-Schwark et al, 2002, Forensic Science International, 126: 34-39). Relative quantification enables determining fold-differences between a target sequence, the quantity of which is to be determined, and a "housekeeping sequence".

In order to quantify the presence of a specific DNA target sequence, representative of the copy number of the mtDNA, it is indeed convenient to express its relative level in relation to another DNA sequence called a "normalizing sequence" or "housekeeping sequence", which is selected for its almost constant rate of expression. Housekeeping sequences are usually found in genes involved in the functions related to basic cellular survival, which normally implies constitutive gene expression. This enables the provision of a ratio expressing the presence of the amplified sequence of interest over the presence of the amplified selected normalizer. This method allows obtaining a value evaluating the relative presence of the amplified sequence of interest actually knowing its absolute quantity within the tested sample.

Commonly used normalizing sequences are those found in genes coding for the following proteins. As a non-limitative list, tubulin, glyceraldehyde-3-phosphate dehydrogenase, albumin, cyclophilin, ribosomal RNAs sequences can be used.

Real time PCR allows quantification of the desired product at any point in the amplification process by measuring fluorescence. Measurement is expressed using a Cycle Threshold (d) value (d; PCR cycle at which the fluorescence of the sequence of interest is detected; the lowest is the CT value, the more abundant is the target sequence). To quantify the presence of the target sequence when a normalization sequence is used, a normalization procedure such as the AAC-r-method can be used, said AAC-r-method being used for analyzing a relative gene expression.

According to a more specific embodiment of the invention, the level of mtDNA" is determined by quantitative polymerase chain reaction (q-PCR) through reference to a selected normalizer gene or nDNA sequence, the level of mtDNA being calculated according to the formula 2^ACt, wherein ACt = CtnDNA - CtmtDNA, as described in reference publications Fan et al, 2009 , J Cancer Res Clin Oncol, 135 ; 983-989 and/or Chatre and Richetti, 2013, J of Cell Sciences, 126 : 914-926. With this calculation method, the level of mtDNA can be calculated using the ACT of average Cj of mtDNA and nDNA (ACT = CtnDNA - CtmtDNA,) as 2^ACt. The primers used for amplification can be chosen by one skilled in the art according to the common knowledge in the field of this technique, as indicated in particular in the above-mentioned reference publications.

The invention thus concerns a method wherein the level of mtDNA is determined by quantitative polymerase chain reaction (q-PCR) following the steps of:

- preparing the biological sample to provide access to the

nucleic acid, especially mitochondrial nucleic acid of cells;

- contacting the prepared sample with oligonucleotide primers targeting the mtDNA;

- performing amplification cycles,

- simultaneously running amplification of a normalizer nDNA,

- quantitatively detecting the mtDNA and the normalizer nDNA.

- determining the level of mtDNA through reference to a selected normalizer nDNA sequence, the level of mtDNA being calculated according to the formula 2^ACt, wherein ACt =

CtnDNA - CtmtDNA-

The invention also concerns a method for investigating the level of mtDNA in a biological sample wherein the biological sample is from a patient diagnosed with gastric carcinogenesis and under treatment for this condition or not, and/or a patient having an ongoing, treated or not, Helicobacter pylori infection, and/or a patient having antecedents of Helicobacter pylori infection(s), eradicated or not, and/or an individual having gastric pain and/or a family history of gastric cancer.

According to a particular embodiment, the mtDNA of the tested sample is detected or quantified by using a region in the 12sRNA gene of the mitochondrial genome, and the nDNA of the tested sample is detected or quantified by using a region in the 18sRNA gene of the nuclear genome. Other couples of mtDNA nDNA primers (probes) can be chosen and used by the person skilled in the art, as reminded above, for example mtDNA encoded ATPase 8 gene and nDNA glyceraldehyde-3- phosphodehydrogenase (GAPDH) or Beta 2 microglobuline (B2M).

Accordingly, in a particular embodiment, oligonucleotide primers and/or DNA probes suitable for carrying out the invention may advantageously be selected for their capacity to hybridize to a strand of the 12sRNA gene or to a strand of the 18sRNA strand. Other combination of couples of mtDNA and nDNA genes specific primers can be also used as primers and/or DNA probes able to hybridize to a sequence of the mtDNA encoding genes: NADH dehydrogenase subunit 1 (ND1 ) or subunit 5 ( ND5) or COX1 coupled with primers for glyceraldehyde-3- phosphodehydrogenase (GAPDH), Beta 2 microglobuline (B2M) or β actin for nDNA encoding genes. As long as the target sequence is known, which is the case of mtDNA, obtaining primers and/or DNA probes such as disclosed above is a matter of routine experimentation. The oligonucleotide (forward and reverse) primers have a length adapted to specifically priming the targeted mtDNA or nDNA and in particular have a length within a range of 10 to 30 nucleotides. Examples of oligonucleotide primers are polynucleotides comprising or consisting of the sequences disclosed in the experiments provided herein. The probes may have a length of 50 to several thousand of nucleotides, in particular may have a length of 50 to 500 nucleotides.

Oligonucleotides and probes to carry out the detection of mtDNA are selected for their ability to hybridize specifically with the targeted mtDNA and accordingly would not hybridize significantly in PCR operating conditions with nuclear DNA. Preferably, the chosen mtDNA sequences are highly specific to mitochondrial genome and nonexistent in nuclear genome.

The invention thus also relates to the use of the pairs of oligonucleotides either as unique pair or combined primer pairs having the following sequences hybridizing to the 12SRNA: 12S (forward): 5'- GCT CGC CAG AAC ACT ACG AG; (reverse): 5'- CAG GGT TTG CTG AAG ATG GCG for the detection of mtDNA. It may further involve primers as the following hybridizing with a nuclear encoded 18SRNA: 18S (forward): 5'- GAG AAA CGG CTA CCA CAT CC; (reverse): 5'- GCC TCG AAA GAG TCC TGT AT.

In a particular embodiment, the method of the invention is performed on the basis of a biological sample removed from a patient, that is a blood sample or, in particular when the patient already present gastric mucosa lesions, a biopsy sample, i.e. a sample containing cells obtained from tissues, in particular a biopsy sample of gastric mucosa. All layers of the gastric mucosa may be used for sampling. In this context, mtDNA and/or DNA extraction and isolation can be performed according to commonly known methods after isolation of cells of interest in the biological sample removed from a patient.

According to another particular embodiment, the level of mtDNA is determined by testing circulating blood mtDNA, in particular is determined by testing the mtDNA of leukocyte(s), in particular leukocyte(s) isolated from a blood sample, especially when circulating blood samples are used for performing the method of the invention. Leukocytes are of interest to measure mtDNA level(s) in the context of gastric cancer detection as they respond to pro-inflammatory mediators present in the systemic circulation through mechanisms involving mitochondrial alteration and dysfunction. Leukocytes act as a sensor of metabolic stress associated to cancer leading to propose mtDNA level in circulating leukocytes as valuable biomarkers for cancer prevention/detection.

In a more particular embodiment, the method of the invention is performed using a preparation of DNA isolated from a biological sample constituted of peripheral leukocytes isolated from sample removed from a patient, that is a blood sample or, in particular when the patient already present gastric mucosa lesions, previously detected by a biopsy sample such as a biopsy sample of gastric mucosa. All layers of the gastric mucosa may be used for sampling and preliminary detection of the lesions. MtDNA and/or DNA extraction and isolation can be performed according to commonly known methods.

According to another aspect, the invention also relates to a method for preparing a biological sample in order to further perform investigation of the level of the mtDNA in a patient according to the embodiments described in the present disclosure. Accordingly, such a method for preparing a biological sample may comprise the steps of:

- removing a blood sample from a patient;

- optionally, separating the blood sample removed from the patient in one or more (in particular 2 or 3), fractions;

- optionally, conducting a separation step by centrifugation on the blood sample removed from the patient or the fractions obtained in the previous step, thereby obtaining on one hand a pellet comprising in particular cells and in the second hand a liquid fraction;

- optionally, isolating peripheral leukocytes (PBMC) directly from the blood sample removed from the patient or from fractions obtained from said blood sample; means such as column-based cell isolation devices, automated cell isolation devices, or appropriate separation systems may be used for this purpose;

- optionally, obtaining a plasma or serum sample directly from the blood sample removed from the patient or from fractions obtained from said blood sample;

Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot. The clot is removed by centrifugation and the resulting supernatant, designated serum, is removed . Plasma is produced when whole blood is collected in tubes that are treated with an anticoagulant. The blood does not clot in the plasma tube. The cells are removed by centrifugation. The supernatant, designated plasma is removed from the cell pellet. Preparation of plasma or serum samples through common method described in the art is known.

According to a particular embodiment, the method for preparing a biological sample comprises a step of isolation of DNA, in particular mtDNA, from cells, in particular from PBMC, obtained through any one of the steps described above.

According to a particular embodiment, the method for preparing a biological sample results in a preparation of DNA isolated from a biological sample constituted of peripheral leukocytes isolated from sample removed from a patient. DNA isolation through common method described in the art is known.

The method for preparing a biological sample described above may be coupled with a method for the investigation of the level of the mtDNA in a patient or other parameter(s), as described and/or exemplified herein.

In this context and according to a particular embodiment, the biological sample removed from a patient is a blood sample. According to a more particular embodiment, such a blood sample is beforehand treated to isolate leukocytes from which total DNA is prepared and purified. The measure of mtDNA level may be performed on the preparation of total DNA of the leukocytes.

In a particular embodiment of the invention, the investigation of the level of the mtDNA in a patient is associated with the determination of a different biological parameter measured in order to improve the significance or the interpretation of the results obtained in relation with the determination of mtDNA level with a view to ascertain the patient's condition. Other biological parameters in this regard include the parameters which are known to provide indication as to a condition susceptible to evolve toward gastric carcinogenesis, such as measurement of cytokine, especially interleukin expression in particular pro-inflammatory interleukin, including IL-8 expression. According to a particular embodiment, the determination of these particular pro-inflammatory interleukin levels, including IL-8 level is performed on plasma obtained during the centrifugation of the blood sampling, or serum obtained during processing of the result of the blood sampling, made for the determination of the levels of mtDNA described above, from the same individual.

According to a particular embodiment, leukocytes and plasma are collected on the same blood sample. during a centrifugation step or leukocytes and serum come from a same single blood sample collected on a patient.

Another object of the invention is accordingly a method for detecting physiological parameter(s) of a patient, and/or monitoring said physiological parameter(s) and/or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, said method comprising the following steps:

a. Performing a method of the invention for investigating the level of mtDNA in the tested patient as described above, and b. Measuring, in parallel to the investigation of the level of mtDNA of step a., the level of cytokine IL-8 in a biological sample removed from the patient tested in step a., and

c. Concluding about the health status of a patient on the basis of the results obtained from step a. and b.

By "measuring, in parallel to the investigation of the level of mtDNA", it is meant that the measurement of the level of cytokine IL-8 is made, by using samplings of biological material(s) in a same individual, collected within a close interval of time, i.e. avoiding an interval of time that may render both parameters not representative of the physiological status of the individual at a given time, in particular for evaluating both parameters at a same given time t. According to a particular embodiment, the measurement of the level of cytokine IL-8 is made in the plasma isolated in parallel with leukocytes during a centrifugation step, by using samplings of biological material(s) in a same individual, collected within a close interval of time as defined above, in particular collected at the same time from a single blood sample later separated in fractions for measuring 1 ) mtDNA level(s), 2) the level of cytokine IL-8 in the plasma isolated in parallel with leukocytes from said single blood sample from a patient.

The inventors have indeed assessed the pertinence of measuring the level of cytokine IL-8 for a same tested individual, in particular for providing a better evaluation of the health status of said tested individual. A strong association between cancer and inflammation has long been observed. Association between circulating cytokine level(s) and gastric cancer risk has also been reported, highlighting in particular that an high H. pylori prevalence and increased circulating IL-8 level may be associated with increased risk of gastric cancer (Epplein et al, 2013, Cancer Causes Control, DOI: 10.107/s10552-013-0284-z).

Levels of cytokine IL-8 can be measured using commonly known methods in the art such as, as a non limitative example, through enzyme- linked immunosorbent assay(s) (ELISA) (Engvall E and Perlman P, 1971 , Immunochemistry, 8: 871 -874). For example, levels of cytokine IL-8 can be measured using the ELISA kit commercialized by BD Biosciences: BDOptEIA Set Human IL-8. Cat. No. 555244.

According to a particular embodiment, if one determines that the level of mtDNA pertains to Group I or Group II and the level of cytokine IL-8 is above 100 pg/mL, conclusion can be made of a risk of presence of a gastric cancer.

According to another particular embodiment, if one determines that the level of mtDNA pertains to Group III and the level of cytokine IL-8 is above 100 pg/mL, conclusion can also be made of a risk of presence of a gastric cancer. The fact that both the level of mtDNA pertains to Group III and the level of cytokine IL-8 is above 100 pg/mL strongly suggest the presence of an established gastric cancer.

According to a particular embodiment, the method of the invention for detecting a risk of presence of a gastric cancer in a tested patient comprises determining whether the level of mtDNA pertains to Group III and determining whether the level of cytokine IL-8 is above 100 pg/mL.

In another aspect, the invention also relates to a method for detecting physiological parameter(s) of a patient, and/or monitoring physiological parameter(s) and/or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, said method comprising the following steps:

a. Performing a method of the invention for investigating the level of mtDNA in the tested patient as described above, and b. Measuring, in parallel to the investigation of the level of mtDNA of step a., the level of any one of the following parameters: cytokine IL-8, platelet count (PLT) and/or mean platelet volume (MPV) and/or percentage of large platelets (LPLT), inflammatory markers such as IL-6 and/or IL-23, or any combination of these parameters in a biological sample removed from the patient tested in step a., and

Matowicka-Karna J et al (Volume 2013, article ID 401623, Clinical and Developmental Immunology) have evidenced the contribution of platelets and inflammatory markers in gastric cancer. Generally cancer cells are a source of inflammatory cytokines and growth factors such as IL-6 or IL-23. IL-6 and IL-23 are actively involved in the pathogenesis and development of tumors. They facilitate tumor growth by inhibiting apoptosis of tumor cells and by angiogenesis induction within the tumor. IL-6 is a pleiotropic cytokine involved in immune responses, inflammatory reactions, and haematopoiesis. A direct association between increasing plasma levels of IL-6 and gastric cancer risk has been reported in Wong et al, 201 1 , Cancer Sciences, 102: 191 1 -1915. IL-23 strongly induces the secretion of IFN-y, affects the haematopoiesis by stimulating thrombocytopoiesis, and the production of neutrophils, and stimulates the production of acute phase proteins. Platelets take part in inflammation and in cancerous diseases. They play an active role in the inflammatory process due to secreted proinflammatory factors, chemokines, and growth factors. An excessive number of platelets is the cause of a significant increase in the risk of metastases in each stage of cancer and an indicator of poor prognosis, for example, in gastric, lung, and kidney cancer. In cancer disease, an increase in the percentage of large platelets (LPLT) is observed, and because young, metabolically active platelets appear in the circulation, this may lead to an increase in MPV (mean platelet volume).

"Platelet count" (PLT) can be expressed as platelet count per unit volume of blood, generally ranging between 150.10⁹ and 350.10⁹ per liter of blood.

"Mean platelet volume" (MPV) can be calculated on the basis of distribution volume, generally ranging from 7,8 to 1 1 ,5 femtoliter (fl).

"Large platelets" are defined as platelets having a volume above 20 femtoliter (fl).

PLT, MPV and LPLT parameters can be collected through an hematology analyzer instrument, available in the art.

Levels (or concentrations) of IL-6 and/or IL-23 parameters can be measured using commonly known methods in the art, in particular through ELISA testing.

The above-mentioned parameters may also enable distinguishing between different gastric cancer stages. Reference is made to Matowicka- Karna J et al (Volume 2013, article ID 401623, Clinical and Developmental Immunology). In particular, high levels of IL-23 and IL-6 have been observed in patients with early gastric cancer. In the experiments described by Matowicka-Karna et al, a control level of IL-23 in a group of normal subjects was found to be 5.21 ±3.65 pg/ml compared to 20.42±1 .44 pg/ml in a group of early gastric cancer patients. Control level for IL-6 was found to be 2.45±1 .44 pg/ml in a group of normal subjects compared to 10.80±8.24 pg/ml in a group of early gastric cancer patients.

According to a particular embodiment, the method for determining physiological parameter(s) of a patient, and/or monitoring said physiological parameter(s) and/or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition of the invention encompasses a determination of any combination of the above-mentioned parameters, in particular a combination of determination of the level of mtDNA and the level of at least one of the parameters selected amongst: IL-8, PLT, MVP, LPLT, IL-6, IL- 23, or a combination of determination of the level of mtDNA and the level of IL-8 and the level of at least one of the parameters selected amongst: PLT, MVP, LPLT, IL-6, IL-23, in particular a combination of determination of the level of mtDNA and the level of IL-8 and the level of any one of either IL-6 or IL-23.

The invention therefore also relates to a method for in vitro monitoring physiological parameter(s) of a patient, and/or or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, said method comprising the following steps:

a. Performing a method of the invention as described herein for investigating the level of mtDNA in the tested patient, and b. Measuring, in parallel to the investigation of the level of mtDNA of step a., the level of any one of the following parameters: cytokine IL-8, platelet count (PLT) and/or mean platelet volume (MPV) and/or percentage of large platelets (LPLT), inflammatory markers such as IL-6 and/or IL-23, or any combination of these parameters in a biological sample removed from the patient tested in step a., and

According to a particular embodiment, such a step c. of conclusion about the health status of a patient takes into account the value(s) of a level of IL-23 and/or of a level of IL-6 for detecting a risk of presence of a gastric cancer condition or the presence of a gastric cancer condition, in particular a gastric cancer condition at an early stage, in a tested patient, or takes into account a relevant increase of any one or all of such value(s) with respect to a normal threshold value determined for healthy subjects, as defined herein and according to the data provided herein and/or by Matowicka- Karna et al. An increase may be calculated in fold(s) for quantifying said increase.

According to a specific embodiment, conclusion is made of a risk of presence of a gastric cancer condition or conclusion is made of the presence of a gastric cancer condition, in particular a gastric cancer condition at an early stage, when the IL-23 level found in the tested sample(s) is superior to about 18 or about 19 or about 20 pg/ml, in particular superior to 20,5 pg/ml, and/or when the IL-6 level is superior to about 8 or about 9 or about 10 pg/ml, in particular superior to 10,8 pg/ml.

According to another specific embodiment, such a conclusion is made in combination with deduction(s) made with respect to another parameter, as described herein.

According to a particular embodiment, conclusion of the presence of a risk of a gastric cancer condition requires performing further clinical investigation(s), as described herein.

Another object of the invention is to provide a kit suitable for carrying out a method of the invention as defined herein, comprising: - At least one pair of specific oligonucleotide primers specific for hybridization with mtDNA and, optionally, at least one pair of specific oligonucleotide primers specific for hybridization with H. pylori nucleic acid(s) sequence(s), and, optionally, one or several of the following reagents,

- nucleotides (e.g. dATP, dCTP, dGTP, dUTP),

- a DNA polymerase, in particular a thermostable DNA polymerase, such as a Taq DNA Polymerase,

- at least one dye for staining nucleic acids, in particular a dye detectable in a real-time PCT equipment,

- optionally, a buffer solution,

- optionally, reagents necessary for the hybridation of the primers to their targets,

- optionally, a reference dye and,

- optionally a notice providing instructions for use and expected values for interpretation of results.

According to a particular embodiment, a kit of the invention comprises primers suitable for amplifying 12S and optionally 18S gene sequences or fragments thereof, such as 5'- GCT CGC CAG AAC ACT ACG AG and 5'- CAG GGT TTG CTG AAG ATG GCG nucleotide sequences (12S), and/or 5'- GAG AAA CGG CTA CCA CAT CC and 5'- GCC TCG AAA GAG TCC TGT AT sequences (18S).

Other primers may be used within such a kit, as detailed hereabove. Another object of the invention is to provide a kit suitable for carrying out a method of the invention as defined herein, comprising:

- at least one antibody specific for a protein selected amongst:

IL-8, IL-6, IL-23 or a combination of several antibodies specific for IL-8, IL-6, IL-23, and, optionally, at least one antibody specific for H. pylori antigen(s), such as CagA antigens, and, optionally, one or several of the following reagents, - a secondary antibody or reagent to reveal a complex between specific antibody(ies) recited above and its(their) target,

- optionally, a buffer solution,

- optionally, an assay plate, and

Another object of the invention is to provide a kit suitable for carrying out a method of the invention as defined herein, comprising a combination of some of the agents, or all the agents, mentioned in the above-described kits, i.e., tubes and/or means allowing the separation of leukocytes and plasma from blood samples, and reagents necessary to isolate DNA from leucocytes and to perform both mtDNA detection and quantification including couples of primers specific to mtDNA and nDNA genes as described above also including Taq DNA polymerase, deoxynucleotides mix, buffer and dye needed for qPCR reaction. This kit may also include all or some reagents for the detection of a protein selected amongst: IL-8, IL-6, IL-23 by enzyme like immunoassay (ELISA), specific antibodies allowing to quantify these proteins also including the necessary positive and negative controls to perform the assays and, optionally, at least one marker specific for H. pylori antigen(s).

The invention also relates to the use of kit(s) according to the invention for investigating the level of mtDNA or of the other parameter(s) described herein in a biological sample, and/or monitoring said parameter(s) or the health status of a patient and/or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition and/or monitoring or detecting a risk of gastric carcinogenesis, or suffering from a gastric cancer condition, including the health status of said patient with respect to an H. pylori infection, according to the description provided herein, in all its detailed or encompassed embodiments. The invention also relates to the use of agents, as described herein, in particular when the kits suitable for implementing the invention are described, for the manufacture of a kit suitable for or aimed at performing the method of the invention as described herein. Instructions for use or guidance for implementing the method of the invention and/or instructions for use or guidance in order to obtain a suitable kit may advantageously be provided.

The present invention is a basis for a non-invasive test for measuring mtDNA levels, in particular in circulating blood. By non-invasive test, it is meant that the method of the invention is in particular an in vitro method. Said method does not need the presence of a medical practitioner for its implementation. According to the invention mtDNA levels may be used as an efficient biomarker for an early detection of gastric carcinogenesis, in particular an early detection of the presence of gastric lesions involved or at the basis of a gastric carcinogenesis process. The present invention may be especially suitable for prevention purposes, but also for monitoring the progression of a gastric carcinogenesis process, subject to an on -going treatment or not, monitoring a shift from a pre-neoplasic condition to a neoplasic condition, or as a follow-up after a cure to screen for a recurrence of the disease. For this purpose, the method of the invention simply relies on the detection and/or monitoring of physiological parameter(s) of a patient.

The method of the invention may be used on patients under treatment, such as chemotherapy treatment and/or radiations treatment, as an indicator of treatment efficiency, disease stage, and disease development.

The method of the invention may be used on patients presenting a HER2 overexpressing gastric adenocarcinoma, treated or not with trastuzumab (Herceptin®, Genentech). Other known drugs used in gastric cancer chemotherapy are (non-limitative examples): 5-fluorouracil, cisplatin, epirubicin, etoposide, docetaxel, oxaliplatin, capecitabine, or irinotecan. As a result of performing the methods of the invention on patients, other investigations methods aimed at confirming or excluding the presence of as gastric carcinogenesis process may be indicated, such as an optical gastroscopic examination, computed tomography or CT scanning of the abdomen, biopsies for histological examination, various blood tests may also be done, such as Complete Blood Count (CBC) to check for anemia.

The invention will be further described herein, referring to the following figures and experimental section.

Two studies have been carried out. The first study was based on two cohorts of Mexican adult patients (Cohort 1 and Cohort 2 herein). The profile of these patients is summarized in Table 1 below. Conclusions on the basis of this first study can also be found in Fernandes et al., 2014, Cancer Epidemiology, Biomarkers and Prevention, DO I: 10.1 158/1055- 9965.EPI-14-0471 , which is, by reference, incorporated herein in its entirety. For the 74 gastric cancer patients in the Cohort 1 in this published study all the criteria previously determined to be taken in consideration including age, gender and smoking habits have been documented. Patients for whom one or several data were missing with respect to these criteria have been dismissed from the statistical analysis in Fernandes et al., 2014, compared to the data provided herein. This does however not impact the conclusions to be drawn from the data presented herein, which are the same as in Fernandes et al., 2014. A second study has been carried out on European patients (Cohort 3 herein). The profile of these patients is summarized in the Results section below. The Material and Methods section and the Results section below refer to the first study as the study done on "Mexican adult patients" and refer to the second study as the study done on "European adult patients". The group corresponding to the "Mexican adult patients" is a relatively isolated population compared to the "European adult patients" . Accordingly, the "European adult patients" are part of a group that is more heterogenous, in particular, such a group is constituted of individuals who have more genetic variations between themselves.

Legends of figures

Figure 1. Circulating mtDNA content in white blood cells of NAG and GC patients compared to healthy blood donors / Mexican adult patients. A) MtDNA was quantified by quantitative real-time PCR from DNA isolated from peripheral leukocytes isolated from healthy blood donors H. py/or/^'-negative (open triangles) and from non atrophic gastritis (NAG) (open circles) and GC patients (closed triangle) from cohort 1 . MtDNA relative values was measured as previously described according to nuclear DNA (nDNA) (see Material and methods).³⁵ Each symbol corresponds to a single patient. Bars and numbers indicate geometric means. Data are obtained from measures done in triplicate in three independent experiments. *P<0.05 ; **P<0.001 . B) Peripheral leukocytes mtDNA level in NAG and GC patients according to the type of cancer either diffuse (n=31 ) or intestinal (n=20) or the presence of hyperplasia (n=19), metastasis (n=19) or late-stage cancer judged inoperable by surgeons (n=17) C) Distribution of the samples according to their mtDNA peripheral blood content in three different groups, Group I : mtDNA<2, Group II : 2<mtDNA<20 and Group III : mtDNA>20. No healthy samples are found in Group I. Samples with mtDNA level > 30 include only H. pylori infected gastric cancer patients. D) Samples with mtDNA<1 can subdivided in 2 sub- groups : Group la : mtDNA<0.5 corresponding exclusively to NAG samples and Group lb : mtDNA<1 including 94% of NAG samples (16/17) and 6% of GC samples (1 /17).

Figure 2. Comparison of IL-8 levels in sera of NAG and GC patients / Mexican adult patients. A) IL-8 was quantified by using the ELISA kit BDOptEIA Set Human IL-8. Cat. No. 555244 commercialized by BD Biosciences according to the recommendations of the fabricant, in the sera from NAG and GC patients from cohort 1 . The mean IL-8 level is higher in GC (median= 3.95) than in NAG (median= 16.65) patients. B) Analysis of a correlation between IL-8 serological levels and peripheral leukocytes mtDNA amount in NAG and GC patients from cohort 1 . A positive significant correlation between IL-8 and mtDNA was detected for NAG samples (r=0.586 ; p=0.0041 ) but not in the case of GC patients.

Figure 3. Comparison of mtDNA level at various steps of the gastric carcinogenesis process / Mexican adult patients. Quantification of peripheral leukocytes mtDNA level in NAG, IM and GC patients from cohort 2. MtDNA was measured as previously reported³⁵ and described in material and methods. Figure 4 : Circulating mtDNA content in white blood cells of NAG and GC patients compared to healthy blood donors according to their H. pylori serology / Mexican adult patients. MtDNA was quantified by quantitative real-time PCR from DNA isolated from peripheral leukocytes isolated from healthy blood donors H. py/or/^'-negative (open circles) and from non atrophic gastritis (NAG) H. pylori negative (grey circles) and H. pylori positive (closed circles) and GC patients H. pylori negative (grey diamond) and H. pylori positive (closed diamonds) from cohort 1 . MtDNA relative values was measured as previously described according to nuclear DNA (nDNA) (see Material and methods).³⁵ Each symbol corresponds to a single patient. Bars and numbers indicate geometric means. Data are obtained from measures done in triplicate in three independent experiments. ^*P<0.05 ; ^**P<0.001 .

Figure 5. Scheme summarizing possible paths for testing a blood sample according to the present disclosure. Figure 6. Circulating mtDNA content in leukocytes of patients with gastritis and gastric cancer compared with healthy subjects / European adult patients. Intervals I, II, III correspond to a range of mtDNA level : <2 ; 2-20 and >20 as defined with the Cohorts 1 and 2 study and in Fernandes et al, 2014. In line with the previous data collected through Cohorts 1 and 2, 98% of the healthy samples are found in the group II. 22% and 24% of, respectively, of gastritis and gastric cancer samples are found in group III, compared to 7% and 28% respectively with the Mexican patients. Each symbol corresponds to a single patient. Bars, geometric mean. Samples were tested in triplicate in three independent experiments. ^***p<0.0001 .

Figure 7. Circulating mtDNA content in leukocytes from gastritis patients compared to healthy subjects / European adult patients. A) H. pylori negative and positive patients, B) in Non atrophic and atrophic gastritis patients, C) In the absence or presence of metaplasia. Mean mtDNA values have the tendency to increase with the severity of gastritis lesions. Each symbol corresponds to a single patient. Bars, geometric mean. Samples were tested in triplicate in three independent experiments. ^* p<0.05, ^*** p<0.0001 .

Figure 8. Comparison of circulating mtDNA levels among gastric cancer patients / European adult patients. No differences are observed between cancer samples at stade III and stade IV as well as between cancer of intestinal type and diffuse type. Mean mtDNA values have the tendency to increase with the severity of gastritis lesions. Each symbol corresponds to a single patient. Bars, geometric mean. Samples were tested in triplicate in three independent experiments. ^*** p<0.0001 . Figure 9. IL-8 plasmatic level in patients with gastritis and gastric cancer / European adult patients. 65% and 92% of samples with IL- 8>100pg/ml correspond to gastritis and gastric cancer patients, respectively. In contrast, samples with IL-8<100pg/ml concern the majority of healthy cases. Mean mtDNA values have the tendency to increase with the severity of gastritis lesions. Each symbol corresponds to a single patient. Bars, geometric mean. Samples were tested in triplicate in three independent experiments. *** p<0.0001 .

Figure 10. Quantification of mtDNA in gastric biopsies of patients from the mexican cohort 2. Mitochondrial DNA was measured by qPCR on DNA isolated from gastric tissues samples from patients with gastritis, intestinal metaplasia and gastric cancer. Compared to gastritis and patients with intestinal metaplasia, mean mtDNA value in gastric biopsies from gastric cancer patients is 3.9 and 5.3 fold lower in gastritis samples and samples with intestinal metaplasia. Each symbol correspond to one patient. *** pvalue<0.001 .

Material and methods

Mexican adult patients - Study population.

Two cohorts (Cohort 1 and Cohort 2) of Mexican adult patients were studied. The profile of these patients is summarized in Table 1 below. Cohort 1 included 48 healthy blood-donors as the reference group, 28 patients with non-atrophic gastritis (NAG), and 78 with gastric cancer (GC) for a total of 154 adults recruited during the period 2009-201 1 . The healthy individuals were blood donors recruited at the blood bank of the Instituto Mexicano del Seguro Social (IMSS), Medical Center SXXI. Cohort 2 included 48 patients with NAG, 34 patients with intestinal metaplasia (IM), and 49 with gastric cancer (GC) for a total of 131 patients recruited during the period 1999-2002. Patients from both cohorts were adults who were attended for gastroduodenal diseases at the Instituto Mexicano del Seguro Social (IMSS) Medical Center SXXI, in Mexico City. The inventors selected patients who were not under treatment for cancer and who had not been treated with antibiotics, bismuth compounds, proton pump inhibitors and non-steroidal anti-inflammatory drugs for at least two weeks prior to the study. Diagnosis was based on endoscopic examination and histopathology studies. ³⁰ All patients and controls were informed about the study and asked to sign a consent letter. The study was approved by the ethical committee from the National Council for Research on Health, IMSS.

European adult patients - Study population.

A cohort of patients (Cohort 3) from 2 AP-HP units, Unit of Gastroenterology at A. Pare hospital in Boulogne-Billancourt headed by Pr. D. Lamarque and Unit of Digestive Oncology at the European G. Pompidou hospital in Paris, headed by Pr J. Taieb, was constituted. It included 29 gastritis patients (A. Pare hospital, Boulogne-Billancourt, France) and 30 gastric cancer patients (European G. Pompidou hospital, Paris, France). Mean age for gastritis patients is 60 years-old and 60.2 for gastric cancer patients. The sex ratio (males/females) was 0.52 and 2.33 for gastritis and gastric cancer patients respectively. Among gastritis samples, 48% have a positive serology for H. pylori. The H. pylori serology status for gastric cancer patients was not available. A control group of healthy volunteers with a negative H. pylori serology (n=50), collected at the clinical investigation and biological resources access platform (ICAREB) Institut Pasteur, France was also included. All patients and volunteers were adults who have not been treated with antibiotics, bismuth compounds, proton pump inhibitors or NSAIDs during the 6 preceding months. They have been informed of the study and signed a consent document. All samples were identified by a reference number and analysed blindly.

Mexican adult patients - Collection of clinical samples, histological analysis and DNA extraction

For patients with gastritis or precancerous lesions, gastric biopsies collected from both the antrum and the corpus were taken. Biopsies were processed either for histology or immediately frozen at -70 °C. For histology, biopsies were immersed in formalin and processed for H&E staining for diagnosis of the lesions. Presence of non-atrophic gastritis, atrophic gastritis, intestinal metaplasia, or dysplasia was documented in each biopsy. The presence of H. pylori bacteria was confirmed by Giemsa staining. For patients with gastric cancer one fraction from the tumor lesion and one from the adjacent tissue were collected during surgery and divided for either histology or DNA extraction. For histology, tissues were immersed in formalin and processed for staining with H&E and analysed for H. pylori and for type of gastric tumor.

Circulating DNA was prepared from a sample of 10 ml of peripheral blood from each patient, and mononuclear cells were purified by centrifugation through a Ficoll-Hypaque density gradient. DNA was isolated from these cells using the salting-out microtechnique and frozen at -70°C until tested for mtDNA. The serum fraction was frozen at -20°C until tested for serology to H. pylori antigens. Mexican adult patients - ELISA for IgG against H. pylori antigens.

IgG antibodies against H. pylori whole-cell antigens and against CagA H. pylori protein were tested in sera, using enzyme-linked immunoabsorbent assays which were previously validated by the inventors. ³¹ European adult patients - Determination of H. pylori serology

H. pylori serology was determined with IgG antibodies against H. pylori whole-cells antigens using an Enzyme-like Immunosorbant assay (ELISA) kit (Serion Elisa Classic) according to the recommendations of the fabricant.

Mexican adult patients - Quantification of mitochondrial DNA by quantitative Polymerase Chain Reaction (q-PCR)

The quantification of mtDNA was performed as previously described ^{33, 35}, on total DNA isolated from circulating leucocytes using the StepOne™ Plus Real-Time PCR system and FastStart Universal SYBR Green Master (Applied Biosystems) following the manufacturer's instruction. MtDNA was quantified using a region in the 12SRNA gene. The nuclear encoded 18SRNA gene was used as an endogenous reference. Primers used to amplify the 12S and 18S gene sequences were: 12S (forward): 5'- GCT CGC CAG AAC ACT ACG AG; (reverse): 5'- CAG GGT TTG CTG AAG ATG GCG and 18S (forward): 5'- GAG AAA CGG CTA CCA CAT CC; (reverse): 5'- GCC TCG AAA GAG TCC TGT AT.³⁴ The qPCR was carried out in 20μΙ of total reaction volume containing 5μΙ of DNA (200 pg), 10μΙ of FastStart Universal SYBR Green Master, 0,2μΙ_ of primers (10μΜ) using an initial denature step at 95°C for 10 minutes and 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. All samples were analyzed in triplicate. To determine the quantities of mtDNA and nDNA present in blood samples, the average threshold cycle number values (Ct) of the nDNA and mtDNA were obtained from each case. The level of mtDNA was calculated using the delta Ct (ACt) of average Ct of nDNA and mtDNA (ACt = CtnDNA-Ct_mtDNA) as 2^ACt as previously described. ³⁵

European adult patients - Isolation of DNA and quantification of mtDNA

For each patient of Cohort 3, 15ml of blood were collected and peripheral blood mononuclear cells (PBMC) and plasma were separated through centrifugation on Leucosep ready to use tubes with PANCOLL. PBMC were frozen at -80°C until preparation of DNA and plasma were stored at -20°C.

Samples from healthy volunteers from the ICAREB platform were received as PBMC and plasma and stored as described above.

DNA was isolated from PBMC fraction using a DNeasy blood and tissue kit (Qiagen) and mitochondrial DNA (mtDNA) was quantified by quantitative polymerase chain reaction (qPCR) through reference to a selected nuclear gene (nDNA). The mtDNA was quantified using a region in the 12SRNA gene of the mitochondrial genome. A sequence in the 18SrRNA was selected as the nuclear reference gene.

The following couples of primers have been used in the qPCR reactions : 12SRNA : 12S forward : 5'-GCT CGC CAG AAC ACT ACG AG-3' and 12S reverse : 5'-CAG GGT TTG CTG AAG ATG GCG for the detection of mtDNA. 18SRNA : 18S forward : 5'-GAG AAA CGG CTA CCA CAT CC-3' and 18S reverse : 5'-GCC TCG AAA GAG TCC TGT AT-3' for the detection of nDNA. The level of mtDNA was calculated according to the formula 2^ACt , with ACt= CtnDNA-CtmtDNA as previously described [Fan et al, 2009, J. Cancer Res Clin Oncol, 135 : 983-989 ; Chatre and Ricchetti, 2013, J. of Cell Sciences, 126 : 914-926].

Mexican adult patients - Quantification of Interleukin 8 (IL-8) level in the serum

IL-8 levels were quantified in the serum of patients by enzyme-linked immunosorbent assay (ELISA) by using the ELISA kit BDOptEIA Set Human IL-8. Cat. No. 555244 commercialized by BD Biosciences according to the recommendations of the fabricant.

European adult patients - Determination of IL-8 levels

Plasmatic levels of IL-8 were measured by the ELISA kit : Duo Set Human

CXCL8/IL-8 (RetD Systems) according to the recommendations of the fabricant.

Mexican adult patients - Data analysis

The Mann-Whitney U test was used to compare mtDNA level in peripheral blood between healthy subjects and patients at various stages of the gastric pathologies, as well as to compare mtDNA level isolated from gastric biopsies. Differences were considered significant when P<0.05. For Odd Ratio (OR) determination the 95% confidence intervals were determined according to the Woolf s method (Woolf B, Ann Hum Genet, 1955, 19: 251 - 253).

European adult patients - Statistical analysis

The Mann-Whitney U test was used to compare mtDNA and IL-8 levels in peripheral blood between healthy subjects and patients at various stages of the gastric pathologies. Differences were considered significant when P<0.05.

Results

Table 1. Characteristics of the study population / Mexican adult patients

Number of patients Mean age Sex ratio H. pylori serology¹

(range) (MZF)

Healthy subjects 48 32 (18-62) 1.58 <1 : 100%

Non-atrophic gastritis 28 53 (17-82) 0.68 <1 : 50% >1 : 50%

Gastric cancer 78 62 (38-89) 0.8 <1 : 39% >1 : 61%

Total of patients with gastric diseases 106

Non-atrophic gastritis 46 50 (30-78) 0.37 <1 : 31% >1 : 69%

Intestinal metaplasia 31 61 (33-80) 0.48 <1 : 21% >1 : 79%

Gastric cancer 49 62 (31-86) <1 : 35% >1 : 65%

Total of patients with gastric diseases 131

Two different groups of patients are analyzed in the study. In each groups, samples were collected around the same period of time, from 2009-2011 for cohort 1 and 1999-2002 for cohort 2.

a Patients with H. pylori serology <1 are considered non-infected and > 1 are H. pylori positive.

b Healthy subjects are blood donors among which are included in the study only H. pylori negative samples.

Characteristics of the studied cohorts / Mexican adult patients

Table 1 describes the general characteristics of the patients included in the study led on Mexican adult patients. In the cohort 1 , half of the non-atrophic gastritis (NAG) patients were H. py/or/^'-positive compared to 61 % in the gastric cancer (GC) group. In the control group, samples are from blood donors, all H. pylori negative. Among GC patients, 28 were diagnosed as diffuse type with 9 cases inoperable and 6 with metastasis. Twenty GC were of intestinal type with 8 cases inoperable, 7 with hyperplasia and 7 with metastasis. In the cohort 2, 69% of the NAG patients are H. pylori positive compared to 75% and 65% in IM and GC groups. Overall, IM and GC patients were older with a mean age of 61 and 62 respectively, compared to 50 for NAG patients as also observed for cohort 1 .

Levels of MtDNA in peripheral leucocytes from patients with gastritis and gastric cancer / Mexican adult patients

In cohort 1 , mtDNA was quantified in DNA isolated from leucocytes of patients with non-atrophic gastritis (NAG) (n=28) and gastric cancer (n=78) and compared with healthy H. pylori negative subjects (n=48). The mean mtDNA value in the circulating blood white cells was 3-fold lower in patients with NAG (p=0.02) and slightly higher 1 .4-fold (p=0.0013) in GC patients compared to healthy subjects (Figure 1 A). The comparison between NAG and GC patients showed a mean mtDNA level 4-fold higher in GC samples (P=0.0009). Among GC samples, 31 and 20 were diffuse and intestinal type, respectively. In both cases, mtDNA level was comprised between 1 and more than 30, compared to NAG samples found between 0 and less than 30 (Figure 1 B). Also when comparing GC samples of all types, according to the presence of hyperplasia, metastasis or judged as inoperable by surgeons, no significant differences were observed on the variations of mtDNA level between the different GC groups. As reported in the table 1 , the H. pylori serology was positive for half of the NAG and 61 % of GC patients. No significant difference was observed comparing circulating blood white cells mtDNA level in H. py/or/^'-positive and negative patients either both groups of patients (Figure 4). According to their mtDNA level, samples can be classified in 3 main groups corresponding to mtDNA<2 (Group I), 2<mtDNA<20 (Group II) and mtDNA>20 (Group III) (Figure 1 C). The distribution of samples among these groups was not influenced by age or gender of the studied-population (data not shown), and showed important differences according to the gastric pathologies. In the Group I with mtDNA<2 no healthy subjects were detected compared to 46% NAG and 14% GC samples. Indeed, for all healthy subjects mtDNA levels were found in Group II comprised between a value of 2 to 20. Whereas for NAG and GC patients, the distribution of mtDNA level was broader with samples in the Groups I and II for NAG and I to III for GC. Furthermore, samples with mtDNA<1 corresponded mainly to NAG 39% (OR= 4.28 ; 95% Cl=2.1 1 -8.69) with only 2% GC (OR= 0.14; 95% Cl=0.03- 0.64) (Figure 1 D). MtDNA values lowest than 0.5 concerned only NAG samples (Figure 1 D). In addition, mtDNA levels >10 were more associated with NAG (OR=2.33 ; 95% Cl=0.69-7.81 ) and GC (OR=5.13; 95% Cl=1 .95- 13.48) than healthy subjects. Highest levels of mtDNA>30 were only observed in GC patients (Figure 1 A).

Comparison of serological levels of IL-8 in NAG and GC patients / Mexican adult patients

Chronic inflammation is associated with atrophic gastritis and plays an important role at early steps during the promotion of carcinogenesis³⁶. In order to further improve the significance of the measure of circulating mtDNA regarding the inflammatory background of the patient, the level of the pro-inflammatory cytokine IL-8 in the serum was measured in 22 NAG and 77 GC patients from cohort 1 . As reported in Figure 2A, the mean IL-8 value was 4-fold lower in NAG compared to GC patients. All NAG patients showed IL-8 levels lower than 50 pg/ml . A significant correlation was observed between progressive increase of IL-8 and peripheral leucocytes mtDNA levels for NAG patients (r=0.586 ; P=0.0041). However this group concerned only 22 samples (Figure 2B). In GC patients, two groups of samples according to IL-8 levels can be distinguished with IL-8<50 and IL- 8>100 pg/ml corresponding to 79% and 21 % of patients respectively. It is to be noticed that for 93% of GC patients, IL-8 higher than 100pg/ml is associated with mtDNA level of Group I or I I. No significant correlation was observed between IL-8 and peripheral leucocytes mtDNA levels in GC patients. In addition, there were no significant differences in IL-8 according to the different groups defined for mtDNA level (data not shown).

Levels of circulating mtDNA in white blood cells of patients with preneoplastic lesions (IM) or GC / Mexican adult patients.

In order to analyse if the levels of mtDNA varied according to the suggested clinical history of GC, mtDNA was quantified in NAG (n=46), IM (n=31 ) and GC (n=49) patients in a second series of samples (Cohort 2) (Table 1 ). Despite the fact that overall the mean mtDNA levels measured for samples from cohort 2 were lower than for cohort 1 was more recent, no significant differences were observed when comparing circulating mtDNA level between patients with NAG and patients with IM (Figure 3A). As observed in the cohort 1 , levels of mtDNA in GC patients were 3-fold higher that those observed in patients with NAG (P=0.02) and IM (P=0.002). When distribution of mtDNA levels were analyzed according to the gastric pathology, the inventors found that values higher than 2 were observed in 35% of GC patients, compared to 4% in patients with NAG (OR=1 1 .69; 95% CI=2.52-54.22) and 10% in patients with IM samples (OR=4.96; 95% Cl=1 .31 -18.72). Thus, peripheral leucocytes mtDNA levels were higher in GC than IM patients. According to the data obtained from the two cohorts, inventors' results showed that peripheral leucocytes mtDNA level shifted to higher values between gastric pre-neoplastic (NAG and IM) and neoplastic stages (GC), establishing a signature assessing the evolution of the disease, that may be used when monitoring the health status of monitored patients or individuals. According to data from cohorts 1 and 2, the combination of mtDNA level related to group III (>20) and IL-8 levels >100pg/ml corresponds to a signature for the presence of gastric preneoplasia and/or gastric cancer lesions and/or a signature assessing the evolution of the disease, as detailed above.

Levels of mtDNA in peripheral leukocytes from patients with gastritis and gastric cancer / European adult patients.

In the healthy group, the relative mean mtDNA level was 4.72 with values ranging from 1 .8 to 20, corresponding to 98% of the samples (49/50) in the mtDNA group II with 2<mtDNA<20 as previously defined by studying

Cohorts 1 and 2 and summarized in Fernandes et al, 2014, Cancer

Epidemiology Biomarkers and Prevention (Figure 6).

Higher mean mtDNA levels are observed in gastritis (2.7-fold ; p<0.0001 ) and gastric cancer (2.6-fold ; p<0.0001 ) patients compared to healthy subjects. MtDNA levels ranged between 4.85 to 40 in the gastritis group and 4.36 to 32.6 in the gastric cancer group.

Among gastritis samples 80% are found in the mtDNA group II and 20% in the mtDNA group III (mtDNA>20). The presence of H. pylori infection had no effect on the peripheral leukocytes mtDNA values (Figure 7A). It is important to notice that mtDNA values increased with the severity of the lesions, as indicated when comparing non atrophic gastritis and atrophic gastritis samples (Figure 7B), and gastritis samples without and with metaplasia (Figure 7C), indicating that higher level of mtDNA could also reflect the severity of lesions among gastritis cases.

According to the results obtained with the Cohorts 1 and 2 and summarized in Fernandes et al, 2014, Cancer Epidemiology Biomarkers and Prevention, 27% of gastric cancer samples presented higher mtDNA levels (>20) and classified in mtDNA group III. No significant differences were observed when comparing mtDNA values for cancer samples at stades III to IV or between cancer of diffuse and intestinal type (Figure 8). Plasmatic levels of IL-8 in gastritis and gastric cancer patients / European adult patients.

Chronic inflammation is associated with atrophic gastritis and plays an important role at early steps during the promotion of carcinogenesis. In order to further improve the significance of the measure of circulating mtDNA regarding the inflammatory background of the patient, the plasmatic levels of the pro-inflammatory cytokine IL-8 were measured. Mean IL-8 values were 3-fold higher in gastritis and gastric cancer patients compared to healthy subjects (p<0.0001 ) (Figure 9). Importantly, for 65% of gastritis and 92% of gastric cancer cases, IL-8 was higher than 100pg/ml compared to 4% of the cases in the healthy group. Importantly, among gastric cancer patients, 4 of them with the highest plasmatic IL-8 levels, from 175 to 342pg/ml, died since the beginning of the study. These data suggest that higher IL-8 levels are associated with the severity of gastric lesions.

Discussion

As reminded in the introductory section, gastric cancer represents a major health burden worldwide. Gastric cancer is often diagnosed at an advanced stage and consequently carries a poor prognosis with an overall 5-years survival rate around 15%. Importantly, if it is diagnosed at an early asymptomatic stage, it can be a curable disease. Two types of gastric cancer can be distinguished, the intestinal and diffuse type. The intestinal type develops through progressive changes in the gastric mucosa from non-atrophic gastritis, atrophic gastritis, intestinal metaplasia, dysplasia and gastric cancer.

The first study carried out on two cohorts of Mexican adult patients (Cohort 1 and Cohort 2 herein), which is also documented in Fernandes et al., 2014, Cancer Epidemiology, Biomarkers and Prevention, DOI: 10.1 158/1055-9965. EPI-14-0471 , which is, by reference, incorporated herein in its entirety, enabled characterization of biomarker(s), i.e., physiological parameter(s) to be detected, applicable in the context of a method of detection of said biomarker(s). Conclusions may be drawn from such a detection of said physiological parameter(s) in order to evaluate a risk of presence of a carcinogenic process, and/or detection of gastric cancer or lesions possibly associated with a gastric cancer or the possible later occurrence of a gastric cancer. These tools are valuable in a context of gastric cancer prevention,

In the first study, the inventors have in particular measured the levels of mtDNA by real-time PCR on DNA isolated from leucocytes and the levels of IL-8 in serum of non-atrophic gastritis and gastric cancer patients, compared to asymptomatic controls. The inventors identified three ranges of changes in mtDNA values, <2.0 (group I), 2.0 to 20 (group II) and >20 (group III). Group I included mainly non-atrophic gastritis and few gastric cancer cases. Group III corresponded almost exclusively to gastric cancer patients. All controls felt in interval II, together with some non-atrophic gastritis and gastric cancer cases; IL-8 levels >50 pg/ml were observed exclusively in GC patients, including those within mtDNA group II.

As a result, and within the context of the present invention, conclusion can be made of the presence of a risk of presence of precancerous lesions or early gastric cancer, regarding the health status of the analysed patient, with the recommendations set forth in the present disclosure. In particular, conclusion can be made of a risk of presence of non-atrophic gastritis or gastritis when the level of mtDNA pertains to group I, and a risk of presence of gastric cancer when the level of mtDNA pertains to group III. The classification of an individual in groups I or III may be an indication (a first indication) of the presence of gastric inflammation and probable gastric cancer, respectively.

In these results, it has also been found that a level of mtDNA superior to 8.46 was significantly associated with the presence of a gastric cancer. ROC analysis performed to determine a cutoff value of mtDNA that will differentiate gastric cancer cases from asymptomatic H. pylori negative controls found a value of 8.46, with a specificity of 80% but a sensitivity of 47%.

These results also enabled concluding that in most of the cases, higher concentration of IL-8 (in particular, concentrations above 50 pg/mL, but also, by extension, concentrations above 100 pg/mL), differentiated patients with gastric cancer with "normal" mtDNA levels (group II), despite of the lack of correlation between mtDNA and IL-8 levels. With respect to the level of 100pg/mL set forth herein as relevant in the context of the present invention, IL-8 levels superior to 100pg/ml are exclusively found in patients with gastric preneoplasia and gastric cancer lesions and except 2 healthy samples, 48-50 healthy patients are under this IL-8 level value.

According to the data of Fernandes et al., 2014, amongst samples pertaining to group II regarding mtDNA level, 70% of finally acknowledged gastric cancer cases presented IL-8 levels superior to 100 pg/mL. However, amongst samples pertaining to group III regarding mtDNA level, 90% of finally acknowledged gastric cancer cases presented IL-8 levels superior to 100 pg/mL in this European cohort. They represent only 16% of samples in the published study (Fernandes et al, 2014), which is based on the analysis of more homogenous group of patients (Mexican patients).

As a result of these experiments, the inventors have designed possible paths for testing a blood sample according to their discoveries, summarized in the scheme of Figure 5. Accordingly, patients with samples pertaining to group I or group III according to their mtDNA level should be considered as suspect for pre-neoplasia or gastric cancer lesions, and further clinical investigations should be performed. In the particular context of group I, further monitoring of the status of the patient should at least be performed, in order to establish or not the absence of change of the status of its mtDNA level over time. Patients with samples pertaining to group II according to their mtDNA level may be further investigated with respect to other physiological parameters, such as IL-8 levels. Differently, samples pertaining to group II may be further investigated according to other techniques, but IL-8 testing may improve, amongst patients whose mtDNA level change pertains to group II, those to be presumed to have a gastric cancer.

In their first study, the inventors validated their results regarding mtDNA levels group assignment in a second cohort of patients, confirming that mtDNA was significantly higher in gastric cancer than in patients with preneoplastic lesions. These data led to propose, in particular, that circulating levels of both, mtDNA and IL-8 can be potential and effective biomarkers for the early detection of patients at risk of gastric cancer.

A second study has been carried out on European patients (Cohort 3 herein). This second study enabled confirming the results of the quantification of mtDNA level changes to another cohort of patients (Cohort 3) from different geographic origin. This second study also confirmed the interest of coupling the quantification of mtDNA level changes with the measure of plasmatic IL-8, and further validated the approach designed by the inventors, in particular, but not only, with respect to the pertinence of mtDNA level changes and IL-8 levels as biomarkers to reveal the presence of gastric cancer lesions at an early stage.

The approach of the inventors is therefore independent from the geographic origin of the assayed individuals, and confirmed with a second study extending the amount of patients to which the approach of the inventors has been applied to.

In line with the results obtained in the first study, almost all healthy individuals have been found to have mtDNA level changes enabling them to be classified in group II. Group III has been associated with a risk of presence of gastric pre-neoplastic or cancer lesions.

Based on the results obtained in the first study on cohorts of patients from Hospital in Mexico (Cohorts 1 and 2) and in the second study using another cohort from Parisian Paris hospitals (AP-HP) (Cohort 3), the inventors confirmed that for all cohorts : - Circulating leukocytes mtDNA levels in healthy subjects (Hp^" ICAREB healthy subjects and Hp^" blood donors in Mexico) match with mtDNA group II (2<mtDNA<20);

- Higher levels of mtDNA corresponding to group III (mtDNA>20) are associated with gastric pre-neoplastic or cancer lesions (see, in particular, Figure 6 for the results in Cohort 3); as indicated in the table 2;

- MtDNA levels has a tendency to increase with the severity of the gastric lesions (Figure 7), at the gastritis stage; However no difference was observed between mtDNA level in gastric cancer

Stage III and Stage IV (Figure 8).

- Plasmatic IL-8 levels are higher (>100pg/ml) in gastric cancer and gastritis patients. The highest levels of IL-8 are found associated with the most serious cases as indicated in the table 3.

According to data obtained in both studies and as highlighted in bold fonts in the tables 2 and 3, both mtDNA level associated to group III (>20) and IL-8 level superior to 100pg/ml reflect high risk for the presence of gastric pre-neoplasia or gastric cancer lesions for the tested individual.

Generally, the detection methods described herein constitute a first indication for the patients, upstream endoscopic investigation and specific follow-up. The findings of the inventors indicate that testing for circulating mtDNA, and optionally IL-8 amongst other biomarkers, might offer reliable minimally invasive biomarkers, to screen populations at risk for gastric cancer. They pave the way to the development of circulating mtDNA measure, and optionally other biomarkers measure as described herein, as a predictive/early-diagnostic biomarkers in a context of gastric carcinogenesis, including when the severity of the possible carcinogenic lesions has to be evaluated (increase in the change levels of mtDNA at the gastritis stage). Table 2: Comparison of the distribution of samples from Mexican adults patients and European adult patients according to mtDNA level group I, II and III and gastric pathology.

Table 3: Comparison of the distribution of samples from Mexican adults patients and European adult patients according to IL-8 levels inferior or superior to 100pg/ml

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Claims

1 . An in vitro method for investigating the level of mtDNA in a biological sample removed from a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition by pooling said biological sample into categories, said method comprising the steps of: determining the level of mtDNA in said biological sample, and comparing the level of mtDNA determined in step a) with a normal threshold value determined for healthy subject(s), and from the comparison made in step b), assigning the tested biological sample to one of the following categories:

Group I: if the level of mtDNA determined in step a) is decreased with respect to the normal threshold value introduced in step b) by less than 2 folds,

Group II: if the level of mtDNA determined in step a) is increased from 2 to 20 folds with respect to the normal threshold value introduced in step b),

Group III: if the level of mtDNA determined in step a) is increased with respect to the normal threshold value introduced in step b) by more than 20 folds.

The method of claim 1 , for monitoring or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, said method further comprising the following step:

d. From the assignment to a Group I, II or III, made in step c), concluding about the health status of a patient from which the tested biological sample has been removed.

3. The method of any one of claim 1 or 2, for monitoring or detecting a risk of gastric carcinogenesis in a patient, wherein a risk of gastric carcinogenesis exists if the biological sample removed from said patient pertains to Group I or Group III.

4. The method of claims 2 or 3, wherein if the tested biological sample pertains to Group I determining whether the mtDNA level is less than 0.5 fold with respect to normal threshold value, and, if so, concluding about the existence of a risk of gastric carcinogenesis at an early stage, in particular a pre-neoplasic stage, for example at a gastritis stage.

5. The method of any one of claims 2 to 4, which comprises a previous, simultaneous or parallel step a detection of an Helicobacter pylori infection, in particular through detection of antigen(s) specific for H. pylori infection, or through an assay involving DNA amplification and subsequent detection of said DNA, or detection of the presence of specific H. pylori IgA and IgG antibodies in a biological sample removed from the tested patient, or through an ¹³C urea breath test performed on the tested patient

6. The method of any one of claims 1 to 5, wherein the biological sample is from a patient diagnosed with gastric carcinogenesis and under treatment for this condition or not, and/or a patient having an ongoing, treated or not, Helicobacter pylori infection, and/or a patient having antecedents of Helicobacter pylori infection(s), eradicated or not, and/or an individual having gastric pain and/or a family history of gastric cancer. The method of any one of claims 1 to 6, wherein the level of mtDNA is determined by quantitative polymerase chain reaction (q-PCR) following the steps of:

- preparing the biological sample to provide access to the

nucleic acid, especially mitochondrial nucleic acid of cells;

- performing amplification cycles,

- simultaneously running amplification of a normalizer nDNA,

- quantitatively detecting the mtDNA and the normalizer nDNA.

CtnDNA - CtmtDNA-

The method of any one of claims 1 to 7, wherein the biological sample removed from the patient is a blood sample or a biopsy sample, in particular a biopsy sample of gastric mucosa.

The method of any one of claims 1 to 8, wherein the level of mtDNA is determined by testing circulating blood mtDNA, in particular is determined by testing the mtDNA of leukocyte(s).

A method for in vitro monitoring or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, said method comprising the following steps on a biological sample removed from said patient:

a. Performing a method according to any one of claims 1 to 9 for investigating the level of mtDNA in the tested patient, and b. Measuring, in parallel to the investigation of the level of mtDNA of step a., the level of cytokine IL-8 in a biological sample removed from the patient tested in step a, and

The method of claim 10, for detecting a risk of presence of a gastric cancer in a tested patient comprising determining whether the level of mtDNA pertains to Group I or Group II and determining whether the level of cytokine IL-8 is above 100 pg/mL

The method of claim 10, for detecting a risk of presence of a gastric cancer in a tested patient comprising determining whether the level of mtDNA pertains to Group III and determining whether the level of cytokine IL-8 is above 100 pg/mL

A method for in vitro monitoring or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition, said method comprising the following steps:

a. Performing a method according to any one of claims 1 to 9 for investigating the level of mtDNA in the tested patient, and b. Measuring, in parallel to the investigation of the level of mtDNA of step a., the level of any one of the following parameters: cytokine IL-8, platelet count (PLT) and/or mean platelet volume (MPV) and/or percentage of large platelets (LPLT), inflammatory markers such as IL-6 and/or IL-23, or any combination of these parameters in a biological sample removed from the patient tested in step a., and c. Concluding about the health status of a patient on the basis of the results obtained from step a. and b.

14. Kit suitable for carrying out a method as defined in any one of claims 1 to 13, comprising:

- At least one pair of specific oligonucleotide primers specific for hybridization with mtDNA and, optionally, at least one pair of specific oligonucleotide primers specific for hybridization with H. pylori nucleic acid(s) sequence(s), and, optionally, one or several of the following reagents,

- nucleotides (e.g. dATP, dCTP, dGTP, dUTP),

- optionally, a buffer solution,

- optionally, a reference dye and,

- a notice providing instructions for use and expected values for interpretation of results.

15. Kit suitable for carrying out a method as defined in any one of claims 1 to 13, comprising:

- at least one antibody specific for a protein selected amongst:

IL-8, IL-6, IL-23 or a combination of several antibodies specific for IL-8, IL-6, IL-23, and, optionally, at least one antibody specific for H. pylori antigen(s), such as CagA antigens, and, optionally one or several of the following reagents, - a secondary antibody or reagent to reveal a complex between specific antibody(ies) recited above and its(their) target,

- optionally, a buffer solution,

- optionally, an assay plate, and

- optionally a notice providing instructions for use and expected values for interpretation of results. itable for carrying out a method as defined in any one of claims 1, comprising:

- nucleotides (e.g. dATP, dCTP, dGTP, dUTP),

- optionally, at least one buffer solution,

- optionally, a reference dye,

- at least one antibody specific for a protein selected amongst:

IL-8, IL-6, IL-23 or a combination of several antibodies specific for IL-8, IL-6, IL-23, and, optionally, at least one antibody specific for H. pylori antigen(s), such as CagA antigens, and, optionally one or several of the following reagents,

- a secondary antibody or reagent to reveal a complex between specific antibody(ies) recited above and its(their) target, - optionally, an assay plate, and

- optionally, a notice providing instructions for use and expected values for interpretation of results. 17. Use of a kit according to any one of claims 14 to 16 for investigating the level of mtDNA in a biological sample and/or of the level(s) of any one of the following parameters: IL-8, IL-6, IL-23 or combination(s) thereof, and/or monitoring or diagnosing the health status of a patient susceptible of suffering from a gastric cancer condition or susceptible of suffering of condition(s) susceptible to evolve in a gastric cancer condition and/or monitoring or detecting a risk of gastric carcinogenesis, or suffering from a gastric cancer condition, including the health status of said patient with respect to an H. pylori infection.