WO2008025877A1

WO2008025877A1 - Method and sampling kit for assessing the condition of the gastric mucosa

Info

Publication number: WO2008025877A1
Application number: PCT/FI2007/050458
Authority: WO
Inventors: Osmo Suovaniemi
Original assignee: Biohit Oyj
Priority date: 2006-09-01
Filing date: 2007-08-28
Publication date: 2008-03-06
Also published as: FI20065542A0

Abstract

The invention concerns a method for assessing the condition of the gastric mucosa in an individual, which method comprises a) determining the concentration of the biomarkers pepsinogen I (PGI) and pepsinogen II (PGII) in a sample from the said individual, and forming the biomarker ratio PGI/PGII from the concentration values obtained for PGI and PGII, b) comparing the values obtained for PGI and PGI/PGII to the respective cut-off values or reference ranges for said biomarkers, whereby a value for at least one of the markers PGI an d PGI/PGII close to or below the lower limit of its reference range or its cut-off value is indicative of corpus atrophy in said individual. If necessary, the method can be combined with as further step of determining the Helicobacter pylori antibody and gastrin-17 (G-17) concentration in a sample from the said individual.

Description

METHOD AND SAMPLING KIT FOR ASSESSING THE CONDITION OF THE GASTRIC MUCOSA

FIELD OF THE INVENTION

The present invention is directed to a method and a sampling kit for assessing the condition of the gastric mucosa, in particular a method for detecting changes in the gastric mucosa, such as detecting atrophic gastritis and associated risk of stomach cancer in an individual. In the method, the concentration of specific biomarkers, such as the pepsinogen I (PGI) and pepsinogen II (PGII) concentration, as well as the concentration of a Helicobacter pylori antibody (Hpab) are determined. Optionally and depending on the determined concentrations for the above biomarkers, also the gastrin- 17 (G- 17) concentration, can be determined in the individual. The method can also include a step of obtaining and/or examining a gastric biopsy specimen and/or determining the vitamin B 12, homocysteine or methyl malonate concentration. Data processing means can be used to process the data obtained and to provide information, for example, in the form of a diagnosis, or suggestions for further treatment or investigations.

BACKGROUND OF THE INVENTION

Gastric cancer can be preceded by a number of different gastric diseases or conditions (so called precancerous conditions), which are chronic atrophic gastritis, pernicious anaemia, ventricular ulcer, gastric polyposis and the Menetrier disease (giant hypertrophic gastritis). Clearly identifiable changes of the mucosa are dysplasia and adenoma. The said conditions are associated with an appr. 4 to 5 fold increase of relative cancer risk, as compared to the general population. It has been established that in almost all diseases the risk is mediated over chronic atrophic gastritis.

Chronic gastritis means a prolonged inflammatory condition of the gastric mucosa. The disease can roughly be divided into the so-called superficial and the atrophic form. In superficial gastritis, the inflammatory cell infiltration is concentrated below the surface epithelium. In the case where the inflammation progresses and diffuses between the specific gastric secretory glands, one refers to chronic atrophic gastritis. In such a case, the normal glandular structures of the gastric mucosa are at least partly substituted by metaplastic changes.

The biomarkers pepsinogen I, pepsinogen II, pepsinogen I/pepsinogen II ratio and gastrin- 17, such as gastrin- 17B (fasting) and gastrin- 17S (stimulated) act as biomarkers of interest in atrophic gastritis. Also a high Hpab {Helicobacter pylori antibody) value {Helicobacter pylori infection) indicates a risk for developing atrophic gastritis. A suit- able method and kit for examining the said biomarkers is the commercially available

GastroPanel® examination (in the following GastroPanel) and software (GastroSoft®, in the following GastroSoft) supporting its use (Biohit OyJ, see also WO 02/054084 and WO2005/085871) The screening for atrophy of the corpus, mucosa of the whole stomach and antrum is described in US 6,696,262.

Helicobacter pylori is a spiral shaped, gram-negative bacterium which thrives in the mucus in the immediate vicinity of the surface epithelial cells of the gastric mucosa and in the cell interstices. After the acute stage, the inflammation is transformed into chronic gastritis. In patients suffering from chronic gastritis, in 70 to 90 % a Helicobacter pylori infection can be established (Calam, J (1994) Helicobacter pylori (Review) Eur. J. Clin Invest 24: 501-510). As Helicobacter pylori infection and chronic gastritis in the stomach are closely associated, it has been stipulated that this bacterial infection could be one etiological factor in the development of stomach cancer. It is for this reason possible that eradication of the Helicobacter pylori bacteria in the initial stages of the infection, could prevent the development of atrophy associated with chronic gastritis, and thus reduce the risk of cancer and peptic ulcers as well as the risk of vitamin B 12 deficiency and related diseases.

The GastroPanel examination measures four biomarkers in blood: pepsinogen I and II, gastrin- 17 and Helicobacter pylori antibodies. The GastroPanel examination and the Gas- troSoft software interpreting its results have been developed for use as a primary and follow-up examination in the diagnosis and treatment of patients with dyspepsia, Helicobacter pylori infection, atrophic gastritis and related risks (gastric cancer, vitamin B 12 deficiency and peptic ulcer disease). Patients screened for having values outside the reference range or cut-off values of pepsinogen I and II, gastrin- 17 values and Helicobacter pylori antibodies are indicated for having or having risk for developing atrophic gastritis (see Table 1).

If the GastroPanel examination gives a normal result, the diagnosis is either functional dyspepsia or another disease not involving the gastric mucosa. The examination diagnoses Helicobacter pylori infection, atrophic gastritis and its location (corpus, antrum or both). In addition to these diagnoses, GastroSoft software also alerts to the risks associated with atrophic gastritis of the corpus of the stomach (gastric cancer and vitamin B 12 deficiency) and to the risks associated with atrophic gastritis of the antrum (gastric cancer and peptic ulcer disease). The GastroSoft report also indicates the risk of gastroesophageal reflux disease. If necessary, the report recommends further examinations, such as gas- troscopy and biopsy specimen examination as well as vitamin B12 and homocysteine determinations (see Table 2).

Pepsinogen I and II and their ratio act as biomarkers for atrophic gastritis in the corpus of the stomach. Five out of the seven early stages of the enzyme pepsin form the pepsinogen

I group, which is produced only by the main cells in the corpus of the stomach and the mucus-secreting cells in the neck of the stomach. The remaining two form the pepsinogen

II group, which is produced in the glands of the entire stomach and to some extent also in the Brunner glands in the upper duodenum. The lower the concentration of pepsinogen I detected in the plasma sample (reference range 30 - 120 μg/1 ) and/or the pepsinogen I to II ratio (the reference value being more than 3.0), the more severe the atrophic gastritis (Zagari et al. 2002, Sipponen et al. 2001, 2002, Vaananen et al. 2003, Pasechnikov et al. 2005, Nurgalieva et al. 2005, DiMario et al. 2005). Corpus atrophy increases the risk of gastric cancer of the corpus (Varis et al. 2000, Uemura et al. 2001, Zagari et al. 2002) and may result in vitamin B 12 deficiency (Sipponen et al. 2003). An asymptomatic, progressive vitamin B12 deficiency of a few years' duration may cause permanent damage to the central and peripheral nervous system, resulting in e.g. dementia, depression and polyneuropathies. Vitamin B 12 deficiency may also increase the concentration of homocysteine in the body, which has been thought to be an independent risk factor for atherosclerosis, strokes and cardiac attacks.

One component of gastric juice is hydrochloric acid (HCl), the secretory product of the parietal, or oxyntic cell of the corpus of the stomach. It is known that the capacity of the stomach to secrete HCl is almost linearly related to parietal cell numbers (Yao et al.

2003, Samuelson et al. 2003). Acid secretion is dependent on function of the H+/K+ AT- Pase or proton pump located in the canalicular membrane of the parietal cell. Several drugs have been developed that non-competitively bind and inactivate the ATPase, resulting in strong inhibition of acid secretion. The presence of gastrin stimulates parietal cells of the stomach to secrete hydrochloric acid (HCl) / gastric acid. Gastrin and hydrochloric acid form a known feed-back controlling mechanism that is an intimate part of the normal gastric physiology (Schubert 2004; Modlin et al. 1997).

Ami dated gastrin-17, a peptide hormone, is the biomarker for atrophic gastritis of the antrum of the stomach. Physiologically, gastrin-17 is one of the most important fragments of gastrin. Gastrin (peptides consisting of 14, 17 and 34 amino acids) is formed in the G cells. G cells are found in the glandular epithelium of the antrum of the stomach and the duodenal mucosa. The blood concentration of amidated gastrin-17 produced by the G cells in the antrum continues to decrease as the atrophy of the antrum becomes more se- vere (Zagari et al. 2002, Vaananen et al. 2003, Pasechnikov et al. 2005, Sipponen et al. 2001, 2003, Nurgalieva et al. 2005, DiMario et al. 2005). If the patient has atrophic gastritis of the antrum of the stomach caused by a Helicobacter pylori infection, the fasting values of gastrin-17 are low (less than 2 pmol/1). In this case, the number of gastrin-17- secreting G cells in the mucosa of the stomach is decreased or the cells have disappeared completely (severe atrophy). The fasting value may also decrease if acid secretion in the stomach is high.

Gastric acid (HCl) inhibits the secretion of gastrin- 17 from the G cells of the antrum, resulting in a reduced concentration of gastrin- 17 in the plasma. Patients without Helicobacter pylori infection and with gastrin-17 fasting values of less than 2.0 pmol/1 may be at risk of esophageal reflux disease and its complication, Barrett's esophagus. This risk is significantly more likely if the fasting value of gastrin-17 is 1.0 pmol/1 or lower (Sippo- nen 2005).

If necessary, atrophic gastritis of the antrum can be confirmed or excluded by determining the concentration of protein-stimulated gastrin-17 (G-17S) in plasma in addition to fasting gastrin-17. It is thus possible to distinguish patients with atrophic gastritis in the antrum from patients whose low fasting concentration of gastrin-17 is entirely due to a high secretion of acid. If the antrum is not atrophied, protein stimulation increases the production of gastrin-17 in the antrum G-cells, thus increasing the amount of gastrin-17 in the blood (over 5.0 pmol/1). If the protein-stimulated gastrin-17 concentration is less than 5.0 pmol/1 and the patient has a Helicobacter pylori infection, it is very likely that the patient has atrophy of the antrum mucosa and a consequent risk of gastric cancer and peptic ulcer disease.

Table 1. Typical behavior of the relevant biomarkers in different conditions

Table 2. Summary of the data provided by the GastroPanel examination and the C- urea breath - or stool antigen test of the "test-and-treat" strategy to the doctor in charge. The GastroSoft program supplies a patient report and in consecutive examinations the graphs on the probabilities of different conditions. The reports produced by the stochastic GastroSoft are based on clinical studies comparing the results of GastroPanel examinations with results from gastroscopy and biopsy examinations.

At an early stage... 13

The GastroSoft C-urea breath or report stool antigen test report

The diagnosis for

Functional vs. organic dyspepsia. YES NO

When GastroPanel indicates the gastric mucosa is healthy, the dyspepsia complaints are often caused by functional dyspepsia or another disease not involving the gastric mucosa

H. pylori infection (gastritis) YES NOT RELIABLE (l)

Atrophic gastritis (damaged and severely dysYES NO functional gastric mucosa) and the probabilities of different conditions affecting the mucosa of the gastric corpus or antrum or both (normal, gastritis or atrophic gastritis) the risks (related to atrophic gastritis) of

Gastric cancer YES YES/NO (2)

Vitamin B 12 deficiency YES NO

Peptic ulcer disease YES YES/NO (3) the risks of the complications of

Gastroesophageal reflux disease:

Esophagitis and Barrett's esophagus YES (4) NO if necessary, a recommendation for

Gastroscopy and biopsy examination YES NO

Treatment of if. pylori infection YES YES/NO (5)

Determination of vitamin B 12 and homocysteine YES NO

Follow-up examination to monitor the incidence of atrophic gastritis YES NO the healing of the H. pylori infection YES YES the healing of atrophic gastritis YES NO

(1) The C- urea breath - and stool antigen tests give false negative results if the patient has a) atrophic gastritis (a risk of gastric cancer and peptic ulcer disease and vitamin B12 deficiency and related diseases, such as dementia, depression and polyneuropathia as well as atherosclerosis, strokes and heart attacks), b) MALT lymphoma or c) bleeding peptic ulcer disease or d) if the patient is currently re - ceiving antibiotics or PPIs (proton pump inhibitors).

(2) The risk of gastric cancer is very low without atrophic gastritis in corpus, antrum or both. But in some cases, a H. pylori infection without histologically observable atrophic gastritis may be associated with gastric cancer and peptic ulcer disease.

(3) No peptic ulcer disease with corpus atrophy (no acid, no ulcer). The risk of peptic ulcer disease is very low without antrum atrophy. (4) High pepsinogen I (over 120 μg /1) and high pepsinogen I and II ratio (over 10) and low fasting gastrin-17 (below 2 pmol /1) indicate high acid (HCl) output and risks for the complications of esophageal reflux disease.

(5) When the incidence of H. pylori -related atrophic gastritis is monitored, the pa- tient can be offered targeted, safe treatment at the right time. The need for medication and the costs and adverse effects of medication can thus be reduced. If the patient has been diagnosed with peptic ulcer disease (gastric or duodenal ulcer), the H. pylori infection has to be treated (6). It should also be treated if the patient has atrophic gastritis. The patient and the doctor may also agree on eradication treatment for other reasons for example when the patient's close relatives have been diagnosed with gastric cancer.

(6) Press Release: The 2005 Nobel Prize in Physiology or Medicine, 3 October 2005 jointly to Barry Marshall and J. Robin Warren for their discovery of "the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease": - "An indiscriminate use of antibiotics to eradicate Helicobacter pylori also from healthy carriers would lead to severe problems with bacterial resistance against these important drugs. Therefore, treatment against Helicobacter pylori should be used restrictively in patients without documented gastric or duodenal ulcer disease." http^/nobelprizc.org/mcdicine/laureates/^OOS/press.html

In the organism, acetaldehyde is formed from alcohol as a consequence of the hepatic metabolism and, locally, in the digestive tract via microbial alcohol dehydrogenase (Salaspuro et al, 1996). Carcinogenic acetaldehyde can be produced also endogenously by the oral microbes from various foodstuffs with high sugar or carbohydrate content, especially in an achlorhydric stomach. Atrophic gastritis and achlorhydria are well known risk factors of gastric cancer.

As a consequence of the microbial metabolism, acetaldehyde builds up in the stomach in the case where the stomach is free from acid or has been made acid-free by medication. For atrophic gastritis patients, microbes produce high acetaldehyde contents from ethanol and sugar in the stomach leading to an enhanced gastric cancer risk among atrophic gastritis patients (Vakevainen et al, 2002).

Our recent studies show that in an achlorhydric stomach alcohol fermentation can start very quickly by the bacteria representing normal flora of the mouth or by yeasts present in the foodstuffs, for example by common baker's or brewer's yeast. These microbes can produce significant amounts of ethanol and acetaldehyde and ethanol for example from carbohydrate containing foodstuffs, such as rice. This happens in particular, if the carbo- hydrate containing foodstuff is sweetened. For example in Asian countries the use of sweet sauces with rice is a very common practise. According to epidemiological studies the eating of rice causes a high risk for cancer in stomach.

In an acid stomach the alcohol fermentation does not occur. On the other hand Helico- bacter pylori infection and certain medicaments, such as proton pump inhibitors (PPI) raise the pH of the stomach.

About nearly 25 % of the human population in the world suffers from atrophic gastritis. From the Finnish population 8 to 12 % (depending on the age) suffers from atrophic gas- tritis and the disease is even more common among elder people. The development of achlorhydric stomach is a risk factor also for people having oesophagus reflux disease, if it is treated by PPI medications.

A problem with the prior known methods for assessment and detection of gastric mucosal changes is that gastrin- 17 is not stable in a serum or plasma sample, and must immediately be separated by centrifugation from the blood sample drawn, and a stabilizer for the gastrin- 17, such as dimethylsulfoxide, has to be added to the centrifuged sample (serum or plasma). Even though the centrifugation and stabilization in itself is rapidly carried out, this is an extra step which needs special equipment which is not always available where the blood sample is drawn. SUMMARY OF THE INVENTION

The aim of the present invention is to develop methods which solve the problem of the prior art methods by providing an easy and effective screening method by means of which it is possible to single out those individuals for which a gastrin -17 determination can be beneficial. The invention thus concerns a method for assessing the condition of the gastric mucosa, especially for assessing atrophic gastritis, and optionally an associated risk of gastric cancer in an individual.

The object of the present invention is thus a method for assessing the condition of the gastric mucosa, such as for assessing atrophic gastritis in an individual, which method comprises a) determining the concentration of the biomarkers pepsinogen I (PGI) and pepsi- nogen II (PGII) in a sample from the said individual, and forming the biomarker ratio PGI/PGII from the concentration values obtained for PGI and PGII, b) comparing the values obtained for PGI and PGI/PGII to the respective cut-off values or reference ranges for said biomarkers, whereby a value for at least one of the markers PGI and PGI/PGII close to or below the lower limit of its reference range or its cut-off value is indicative of corpus atrophy in said individual.

Typically the said method also includes a step of determining the Helicobacter pylori antibody (Hpab) concentration in said sample. This determination, which involves a comparison of the determined value for Hpab to a cut-off value or reference range, whereby a value close to or above the upper limit of the reference range or the cut-off value indicates that the tested individual has a H. pylori infection. This is valuable information in connection with the method of the invention as relating e.g. to the origin for the atrophy (H. pylori induced or autoimmune) thus being important for enabling a targeting of the treatment (e.g. eradication treatment and/or B 12 vitamin determination and treat- ment, etc) The sample for determining the biomarkers PGI, PGII and Hpab is preferably drawn as a finger prick blood sample, which can easily be obtained with a sampling kit which is also an object of the invention.

In case the above method ("the screening method") indicates, based on the fact that at least one of the markers PGI and PGI/PGII has a value which is close to or below the lower limit of its reference range or its cut-off value, that the tested individual has corpus atrophy, according to an embodiment of the invention, the method comprises the further steps of c) determining, in an individual with corpus atrophy as indicated according to step b), the concentration of the biomarker gastrin-17 (G-17) in a sample from the said individual, and d) comparing the value obtained for G- 17 to its respective cut-off value or reference range, whereby a value for G- 17 close to or below the lower limit of its ref- erence range or its cut-off value, is indicative of atrophic antrum gastritis and a consequent increased risk for gastric cancer in the antrum or the whole stomach.

More specifically, if in the above case, if the concentration of Hpab is determined as being close to or above the upper limit of its reference range or its cut-off value, then this in combination with a low value of G- 17 (both fasting G- 17 and postprandial G- 17), confirms the result and can be indicative of an increased risk of gastric cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention aims to solve the above-mentioned problem of e.g. the poor stability of gastrin-17 in a whole blood sample that can take several hours or even days to be sent for examination in a laboratory.

The invention thus contemplates performing a screening assay or examination according to which in a first step, a blood sample can be drawn, for example outside a laboratory, by a pharmacist at a pharmacy, or by any suitably trained person, for example at exhibi- tions, shopping centres or even at home. For this sampling, a kit containing the necessary means for taking the sample and forwarding it for example to a laboratory, where bio- marker concentrations can be determined, can be used. The said laboratory then carries out the determination of the PGI and PGII, normally also the Helicobacter pylori anti- body, such as IgG and IgA antibodies. According to the invention it favourable to use a IgG and IgA test combination (IgG and IgA antibodies are simultaneously measured from the same saliva, blood, serum or plasma sample), because some cases (2 to 7%) of H. pylori infection have only the IgA immune response {i.e., IgA antibody production) (Jas- kowski, T. D., T. B. Martins, Η. R. Hill, and C. M. Litwin. 1997, Kosunen, T. U., K. Seppala, S. Sarna, and P. Sipponen 1992).

The sample can easily be taken as a simple finger prick sample, which gives a sufficient volume, such as 100 - 300 μl for the said screening. In addition, a blood sample for the screening according to the invention (PGI, PGII and H. pylori IgG and IgA antibodies) can be taken at any time of the day, because there is no need for fasting and/or postprandial blood sample for G- 17 determination.

By means of the said screening biomarkers (PGI, PGII, PGI/PGII and Hpab) often asymptomatic or only mildly symptomatic atrophy of the gastric corpus (atrophic gastritis) and the associated risks (gastric cancer and vitamin B 12 deficiency) can be detected.

The reference range for pepsinogen I value is between 30 - 120 μg/1, the reference range for pepsinogen II is 3 - 10 μg/1, the reference range for PGI/PGII ratio is 3- 20, and the reference range for gastrin 17S (stimulated) value is 5 - 30 pmol/1, the reference range for gastrin 17B (fasting) is 2 - 10 pmol/1 and the reference range for Hpab is 0 - 30 EIU.

Typical cut-off values for the biomarkers are selected from the group comprising: pepsinogen I 30 μg/1, pepsinogen II 3 μg/1, PGI/PGII ratio 3, gastrin- 17S (stimulated) value 5 pmol/1, gastrin- 17B (fast) 2 pmol/1 and Hpab 30 EIU.

If the pepsinogen I value is low, it is close to the lower limit or below the reference range 30 - 120 μg/1. Close to means typically lower limit +/-5. If the pepsinogen II value is low, it is close to the lower limit or below the reference range 3 - 10 μg/1. Close to means typically lower limit +/-1. If the PGI/PGII ratio is low, it is close to the lower limit or below the reference range 3- 20. Close to means typically lower limit +/-0.5. If the gastrin 17B (fast) value is high, it is close to the upper limit or above the reference range 2 - 10 pmol/1. Close to means typically upper limit +/-0.5. If the gastrin-17S (stimulated) value is low, it is close to the lower limit or below the reference range 5 - 30 pmol/1. Close to means typically lower limit +/-1. If Hpab is high it is close to or above the upper limit of the reference value 0-30 EIU. Close to means typically upper limit +/-3.

When corpus atrophy or a risk therefore has been detected in the screening assay as described above, the person tested can be forwarded for further tests, recommendations and/or examinations. It can be recommended that the levels of vitamin B12 and homocysteine (or methylmalonate) be determined and/or that a gastroscopy and biopsy specimen examination be carried out. These will either detect precancerous lesions or early gastric cancer that can be treated, or they may show that there is no disease to be treated.

When corpus atrophy has been detected as above, the physician treating the patient may request fasting and/or postprandial (stimulated) G- 17 determinations before a gastroscopy and biopsy examination. Alternatively, an examination as described earlier (involving pepsinogens I and II, G-17 and H. pylori antibodies; i.e. the GastroPanel) may be requested in order to control and/or confirm the results of the first screening examination and to obtain additional information. The GastroPanel examination provides the physician with all the information presented above in Table 2.

In practice, the first screening examination may be carried out without a doctor's prescription. This simple, easy and affordable examination is therefore a good way to start finding out about the diseases of the gastric mucosa and associated risks. It also allows the screening and prevention of these diseases and of their risks. The kit for obtaining a sample for the screening assay is a pre-packaged sampling kit, and preferably also a mailing kit, and it can comprise typically the following components or means:

1. Finger prick device for puncturing the skin and obtaining the blood sample

2. Means for receiving and storing the sample until analyzed

3. Means for obtaining and/or maintaining hygienic or aseptic conditions in connection with sampling, such as before, during or after sampling.

4. Instructions for use of the kit, and optionally for subsequent mailing.

Especially the said kit can also contain one or more of the following components

- skin disinfectant, especially an alcohol wipe

- sticking plaster, plastic glove, gauze pad as means for maintaining hygienic or aseptic conditions after sampling

- an EDTA or other anticoagulant collection tube/device as the means for receiving and storing, and also delivering the sample for analysis

- an outer tube/container for forwarding, such as by mailing, the sample to be analyzed - a patient information request and clinical history form, and

- disposal bag,

- a Video, DVD, Compact Disk or other digital media with instructions and information relating to the test, symptoms and disease states of the gut

- an optional retest voucher and request for further information - a reply paid envelope or a means of pre -payment of postage to the laboratory such as an address label on the kit.

The form contains typically patient identification and optionally other data, assay results, instructions etc. A mailing tube or other container is usually required by postal regulations. The whole compliment of kit components can be supplied in "box ready" format. Such box may be designed to use as a "work station" in that the tubes, sampling devices and other components are held within pre-molded or cardboard formed trays. Other elements of the pre-molded or formed tray may be used for positioning, storage or collection of samples or tubes. Each kit may be identified in digital format, such as bar coding for reference purposes.

The kit for carrying out the first examination or screening is affordable and easy to obtain from a pharmacy, shopping centre or online purchase through the Internet. These factors help solve the serious medical and ethical problems (including numerous unnecessary deaths from gastric cancer) associated with the deficiencies and false negative results of the ¹³C urea breath tests and stool antigen tests used to diagnose and treat H. pylori infections according to the "test-and-treat" strategy. A breath test or stool antigen test alone is not able to detect atrophic gastritis or the associated risks. This means that, even though the H. pylori infection is treated, atrophic gastritis may be developing into gastric cancer. In addition, the patient may be unaware of the developing vitamin B 12 deficiency and the disease risks associated with it (e.g. dementia, depression and polyneuropathies, atherosclerosis, heart attacks and strokes).

The kit for the first examination makes it practically and economically possible for all persons over a risk age, e.g. over 50, to screen themselves for corpus atrophy and the associated risks (gastric cancer and vitamin B 12 deficiency). The quickly growing group of persons over 50 frequently have H. pylori infections and consequent atrophic gastritis, both of which increase with age. Atrophic gastritis is often only mildly symptomatic and, most frequently, asymptomatic. On the basis of an extensive Finnish study, it has been estimated that 250 to 300 gastric cancer deaths every year in Finland could be prevented by screening persons over 50 for H. pylori infection, the resulting atrophic gastritis and the related risks.

The screening assay easily and affordably detects H. pylori infection (present in more than half of the global population) and corpus atrophy and achlorhydria (present in al- most half the persons with H. pylori infection in the world). These conditions are estimated to affect nearly 15 % of the global population, approximately 1,000 million people (the prevalence being 5-10% e.g. in European countries, 20-30% in Asia, for example, and even more in other, still less developed areas). In people with these conditions, oral bacteria colonising the stomach produce acetaldehyde from the contents of each balanced meal.

In atrophic gastritis of corpus the concentration of pepsinogen I and the ratio of the concentrations of pepsinogen I/II decrease. In addition, since corpus does not secrete acid (HCl) due to atrophic gastritis because of the feed-back controlling mechanisms, the concentration of gastrin- 17 (G- 17) increases.

When the person has low pepsinogen I value, low pepsinogen V pepsinogen II ratio and high gastrin- 17 (in particular G-17B fast ) value, compared to the cut-off value or refer- ence range, the person is diagnosed to have atrophic gastritis in the corpus, most often due to Helicobacter pylori infection and rarely due to autoimmune disease, leading to achlorhydric or low acid stomach and production of acetaldehyde by microbes in the stomach.

An individual with an achlorhydric or low acid stomach as established by the biomarker levels above, can be treated in order to bind the acetaldehyde produced by microbes in such an environment. The treatment comprises administering to such an individual a composition comprising an effective amount of acetaldehyde -binding compound(s). An effective amount means an amount capable of binding or inactivating the amount of acet- aldehyde present in the stomach due to acetaldehyde formed from alcohol or for other reasons in stomach or in intestine and/or colon. Preferably said compound(s) comprise one or more free sulfhydryl and/or amino groups. The composition comprises a non-toxic carrier that effects, in the stomach, sustained release of said compound(s) in the stomach. Sustained or prolonged release means the release of effective substances for at least 30 minutes in the conditions of the stomach. Preferably the effective substances release for 0.5 to 8 hours, preferably 2 to 6 hours, most preferably 2 to 4 hours.

According to a preferred embodiment the compositions are taken in connection with eating, during, before or after eating. The composition can be for example mixed in the foodstuff or it can be taken before or after eating. The composition preferably releases the effective compound(s) at the time the foodstuff is in the stomach i.e. during the digestion of the food. This time is typically 2 to 4 hours. The dosage may be renewed by 4 to 10 hour intervals, preferably at 6 to 8-hour intervals.

The composition may be in the form of a preparation, for example a tablet, a capsule, a granule, powder, or a tablet or a capsule comprising powder or granules, especially a BioCyst preparation (see examples). The composition may be in a form of a monolithic or multiparticular preparation, such as tablet or capsule or granule. A single dose of the preparation may be a tablet or capsule or suitable amount of granules or a tablet or capsule comprising granules or powder.

The amount of compound(s) released in the conditions of the stomach is preferably 40-80 mg in an hour.

The task of the carrier in the composition is sustained release of the effective com- pound(s) in the conditions of the stomach.

Amino acids or other compounds or the salts thereof that suitably bind acetaldehyde and contain a free sulfhydryl (SH) and/or amino (NH2, -N, -NH- or NH3 ⁺ ) group include, for example: L-cysteine, D-cysteine, cystine, cysteic acid,cysteine glycine, threo or erythro-β-phenyl-DL-cysteine, β-tetramethylene-DL-cysteine, methionine, serine, D- penicillamine and its dipeptides with N-terminals, peptide or a protein with terminal cysteine semicarbazide, glutathione, reduced glutathione, β-mercaptoethylamine, D, L- homocysteine, D,L-homocysteic acid, N-acetylcysteine, L-cysteinyl-L-valine, β-β- tetramethylene-DL-cysteine, cysteinyl-glycine, mercaptoethylglycine, tre-(5)-β-phenyl- DL-cysteine, erythro- β-phenyl-DL-cysteine, cysteine hydrochloride, thiaminhydrochlo- ride, sodiummetabisulphite, arginine, glycine, lycine, ammonium chloride, 1,4- dithiothreitol, mercaptanes.

Cysteine and its derivatives are especially well suited to the purpose. The most suitable amino acids comprise L- and D-cysteines, compounds that are converted to cysteine or compounds which function in the same way as the L- or D-cysteines, the derivatives or salts of cysteine, especially water-soluble derivatives or salts, The most preferred com- pound)(s) are in addition to L-cysteine and D-cysteine, D-penicillamine, β- mercaptoethylamine and N-acetylcysteine, a compound converted to cysteine, or a salt or a structural analogue of these compounds capable of binding acetaldehyde. The most preferred compound is L-cysteine and the salts thereof.

A suitable amount may be a composition comprising typically 100 - 200 mg L-cysteine, preferably administered two times a day, in connection with a meal, such as before, during or after eating.

The effect of the treatment may be monitored by testing later the concentrations of pepsi- nogen I, pepsinogen I/II, and gastrin- 17. A suitable time for monitoring may be 4 weeks and 8 weeks after the treatment was started or according to the estimate of the physician.

The kit and method according to the invention provide the opportunity to obtain information about one's health at will. Most often, the kit and method according to the invention inform the person with functional dyspepsia that he does not have a H. pylori infection or atrophic gastritis, which is valuable information per se. In these cases, the complaints may be caused by a disease not related to the gastric mucosa and the related risks.

The following examples illustrate the invention.

Sampling

With a finger prick device a few drops (e.g., 100 - 300 μl) of whole blood are taken into a anticoagulant collection tube and sent to a laboratory for examination of pepsinogens I and II and H. pylori IgG and IgA antibodies. Pepsinogens I and II as well as H. pylori antibodies are stable in a whole blood sample at room temperature for several days. The screening assay may include a software (GastroSoft) interpretation for a doctor, who often with a patient may make some decisions: 1. If the stomach mucosa is healthy and the patient does not suffer from dyspepsia , there is no need for further examinations of the stomach.

2. If the mucosa of stomach is not healthy, a doctor may propose gastroscopy and biopsy sample examination or, before this examination, fasting gastrin- 17 or both fasting and postprandial gastrin- 17. 3. Or if the mucosa of the stomach is not healthy, a doctor may propose the Gastro-

Panel (pepsinogens I and II, Helicobacter pylori IgG and IgA antibodies and fasting gastrin- 17), as a confirmatory and control examination.

The data interpretation of the screening test results may contain, for example, the follow- ing advices for a doctor or/and a patient.

Example 1: The patient is Helicobacter pylori positive and has atrophic gastritis, the software (GastroSoft) recommends to visit the general practitioner (GP) or gastroen- terologist (GE) and be referred for gastroscopy, including biopsy examination as well as H. pylori and lactose intolerance biopsy quick tests. In case of severe corpus atrophy and therefore the stomach is achlorhydric, acetaldehyde binding composition (e.g. BioCyst capsules) are recommended as food additive with every meal.

Example 2: The patient is only H. pylori positive, no atrophy. Software recommends vis- iting the general practitioner or gastroenterologist.

Example 3: No H. pylori infection and no atrophic gastritis. A patient has functional dyspepsia and the problem lies outside the stomach mucosa. Is recommended to have the above mentioned gastrin- 17 examination(s) 2 above and to visit the general practitioner or gastroenterologist.

Example 4: No H. pylori infection, no atrophic gastritis and high pepsinogen I and pepsinogen I and pepsinogen II ratio. A patient may use PPFs or /and have the risk of the complications of esophageal reflux disease. Is recommended to have the above men- tioned gastrin- 17 examination(s) 2 and to visit the general practitioner or gastroenterologist.

Blood sample collection for the GastroPanel

The blood sample is taken after overnight fasting (approx. 10 hours) into an EDTA tube. The blood sample needs to be centrifuged within 30 minutes, at 2000 G for 10-15 minutes. In case the plasma is not used for assaying it needs to be frozen immediately. The samples should be mixed thoroughly after thawing. Multiple freezings and thawings should be avoided. For temporarily storage the plasma samples can be stored frozen at - 15°C, but for long-term storage should be at -20⁰C or preferably at -70⁰C. Fipemic or cloudy specimens must not be used. Alternatively, a gastrin- 17 stabilizer (such as that developed by Biohit OyJ), can be used. The addition of the stabilizer into the plasma sample immediately after separation (lOOμl / 2 ml plasma) enables the storage of the sample for 3 days in the refrigerator at 2-8°C. In case a postprandial blood sample is needed, it should be taken into an EDTA tube after 20 minutes upon the intake of the protein juice. Test kit for sample collection

Sample Collection and Testing are through a pre-packed kit, one example of this kit comprising at least the following components:

1. Finger prick device

2. Alcohol wipe

3. EDTA collection tube/device 4. Mailing tube (regulations require this)

5. Sticking plaster, plastic glove, gauze pad

6. Instructions

7. Disposal bag

8. Form 9. Prepaid response mechanism

10. DVD, video or CD or other digital medium

The components are supplied in "box ready" format. Such box may be designed to use as a "work station" in that the tubes, sampling devices and other components are held within pre-molded or cardboard formed trays. Other elements of the pre-molded or formed tray may be used for positioning, storage or collection of samples or tubes. This kit is supplied with either a pre-paid sticker on the side of the box or a prepaid envelope to mail to the laboratory after use, including disposal of gloves and wipes etc.

Comparison of the screening assay with C urea breath or stool antigen test

Table 3. Summary of the data provided by the screening (GastroView) examination and the C- urea breath - or stool antigen test of the "test-and-treat" strategy to the doctor in charge. The software (GastroView) program supplies a patient report and in consecutive examinations the graphs on the probabilities of different conditions. The reports produced by the stochastic software are based on clinical studies comparing the results of the Gas- troPanel and GastroView examinations with results from gastroscopy and biopsy examinations.

At an early stage ... The GastroSoft 13 C-urea breath or report stool antigen test report

The GastroView diagnosis for

Functional vs. organic dyspepsia. YES NO

When GastroView indicates the corpus mucosa is healthy, the dyspepsia complaints are often caused by functional dyspepsia or another disease not involving the gastric mucosa (except antrum mucosa)

H. pylori infection (gastritis) YES NOT RELIABLE (l)

Atrophic gastritis (damaged and severely dysYES NO functional gastric mucosa) and the probabilities of different conditions affecting the mucosa of the gastric corpus (normal, gastritis or atrophic gastritis)

The risks (related to atrophic gastritis) of

Gastric cancer YES YES/NO (2)

Vitamin B 12 deficiency YES NO

Peptic ulcer disease YES/NO YES/NO (3)

The risks of the complications of

Gastroesophageal reflux disease:

Esophagitis and Barrett's esophagus YES (4) NO

If necessary, a recommendation for

Gastroscopy and biopsy examination YES NO

Treatment of if. pylori infection YES YES/NO (5)

Determination of vitamin B 12 and homocysYES NO teine Follow-up examination to monitor the incidence of corpus atrophy YES NO the healing of the H. pylori infection YES YES the healing of corpus atrophy YES NO

(1) The C- urea breath - and stool antigen tests give false negative results if the patient has a) atrophic gastritis (a risk of gastric cancer and peptic ulcer disease and vitamin B 12 deficiency and related diseases, such as dementia, depression and polyneuropathia as well as atherosclerosis, strokes and heart attacks), b) MALT lymphoma or c) bleeding peptic ulcer disease or d) if the patient is currently receiving antibiotics or PPIs (proton pump inhibitors).

(2) The risk of gastric cancer is very low without atrophic gastritis in corpus, antrum or both. But in some cases, a H. pylori infection without histologically observable atrophic gastritis may be associated with gastric cancer and peptic ulcer disease. (3) No peptic ulcer disease with corpus atrophy (no acid, no ulcer). The risk of peptic ulcer disease is very low without antrum atrophy.

(4) High pepsinogen I and pepsinogen I and II ratio indicate high acid (HCl) output and risks for the complications of esophageal reflux disease (see Figure I).

(5) When the incidence of H. pylori -related atrophic gastritis is monitored, the patient can be offered targeted, safe treatment at the right time. The need for medication and the costs and adverse effects of medication can thus be reduced. If the patient has been diagnosed with peptic ulcer disease (gastric or duodenal ulcer), the H. pylori infection has to be treated (6). It should also be treated if the patient has atro- phic gastritis. The patient and the doctor may also agree on eradication treatment for other reasons for example when the patient's close relatives have been diagnosed with gastric cancer.

(6) Press Release: The 2005 Nobel Prize in Physiology or Medicine, 3 October 2005 jointly to Barry Marshall and J. Robin Warren for their discovery of "the bacte- rium Helicobacter pylori and its role in gastritis and peptic ulcer disease": - "An indiscriminate use of antibiotics to eradicate Helicobacter pylori also from healthy carriers would lead to severe problems with bacterial resistance against these important drugs. Therefore, treatment against Helicobacter pylori should be used restrictive Iy in patients without documented gastric or duodenal ulcer disease." http://nobelprize.ofg/medicinc/laureatcs/2005/prcss.html

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18, in Chigago, IL, USA

Jaskowski, T. D., T. B. Martins, H. R. Hill, and C. M. Litwin. 1997. Immunoglobulin A antibodies to Helicobacter pylori. J. Clin. Microbiol. 35:2999-3000. [PubMedl. Kosunen, T. U., K. Seppala, S. Sarna, and P. Sipponen. 1992. Diagnostic value of decreasing IgG, IgA, and IgM antibody titers after eradication of Helicobacter pylori.

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Claims

1. Method for assessing the condition of the gastric mucosa in an individual, which method comprises a) determining the concentration of the biomarkers pepsinogen I (PGI) and pepsinogen II (PGII) in a sample from the said individual, and forming the biomarker ratio PGI/PGII from the concentration values obtained for PGI and PGII, b) comparing the values obtained for PGI and PGI/PGII to the respective cut-off values or reference ranges for said biomarkers, whereby a value for at least one of the markers PGI and PGI/PGII close to or below the lower limit of its reference range or its cut-off value is indicative of corpus atrophy in said individual.

2. The method according to claim 1 comprising the further steps of c) determining, in an individual with corpus atrophy as indicated according to step b), the concentration of the biomarker gastrin-17 (G-17) in a sample from the said individual, and d) comparing the value obtained for G- 17 to its respective cut-off value or reference range, whereby a value for G-17 close to or below the lower limit of its reference range or its cut-off value is indicative of atrophic antrum gastritis and an increased risk for gastric cancer in the antrum or the whole stomach.

3. The method according to claim 1 or 2, wherein the concentration of Helicobacter pylori antibody (Hpab) is determined in the said sample, and compared to its respective cut-off value or reference range, a value close to or above the upper limit of the reference range or the cut-off value being indicative of a H. pylori infection..

4. The method according to claim 1 , wherein the sample for determining PGI, PGII and optionally Ηpab is a serum, whole blood, or plasma sample.

5. The method according to claim 4, wherein the sample is a finger prick sample.

6. The method according to claim 2 wherein the biomarker G- 17 is fasting gastrin- 17.

7. The method according to claim 2 wherein the biomarker G- 17 is stimulated gastrin- 17.

8. The method according to claim 3 wherein Hpab is a IgG and IgA antibody, preferably simultaneously measured from the sample.

9. The method according to claim 6 or 7, wherein the sample for determining G- 17 is a sample stabilized with dimethylsulfoxide.

10. The method according to claim 2, the method comprising, prior to the step c) or subsequent to the step d), a step of establishing from a gastric biopsy sample from an individual with indicated corpus atrophy according to step b), the presence of mucosal changes, including precancerous lesions and early gastric cancer.

11. The method according to any one of claims 1 - 3 comprising the additional step of determining the concentration of a biomarker selected from vitamin B 12, or of a biomarker correlating to the vitamin B 12 concentration, such as homocysteine or methylmalonate, from a sample from an individual indicated with corpus atrophy according to step b).

12. The method according to claim 1 comprising the additional steps of carrying out a confirmatory examination assaying each of the biomarkers selected from PGI, PGII and gastrin- 17, from a sample from said individual indicated with corpus atrophy by a) determining the concentration of the biomarkers pepsinogen I (PGI), pepsinogen II (PGII) and gastrin- 17 in the said sample, and forming the biomarker ratio PGI/PGII from the concentration values obtained for PGI and PGII, b) comparing the values obtained for PGI, PGI/PGII and gastrin-17 to the respec- tive cut-off value or reference ranges for said biomarkers, whereby a value for at least one of the markers PGI and PGI/PGII close to or below the lower limit of its reference range or its cut-off value in combination with a value for G- 17 close to or below the lower limit of its reference range or cut-off value, is confirmatory of atrophic gastritis and an increased risk for gastric cancer in the antrum or the whole stomach.

13. The method according to claim 12, whereby in addition the concentration of Helicobacter pylori antibody (Hpab) is determined in the said sample, and compared to its respective cut-off value or reference range, a value close to or above the upper limit of the reference range or the cut-off value being indicative of a H. pylori infection..

14. The method according to any preceding claim, wherein the reference range for pepsinogen I value is between 30 - 120 μg/1, the reference range for pepsinogen II is 3 - 10 μg/1, the reference range for PGI/PGII ratio is 3- 20, and the reference range for gastrin -17S (stimulated) value is 5 - 30 pmol/1, the reference range for gastrin- 17B (fasting) is 2 - 10 pmol/1 and the reference range for Ηpab is 0 - 30 EIU.

15. The method according to any preceding claim, wherein the cut-off values for the biomarkers are selected from the group comprising: pepsinogen 130 μg/1, pepsinogen II 3 μg/1, PGI/PGII ratio 3, gastrin- 17S (stimulated) value 5 pmol/1, gastrin- 17B (fast) 2 pmol/1 and Ηpab 30 EIU.

16. The method according to any preceding claim comprising the additional step of administering a composition for binding acetaldehyde in the stomach to the individual with indicated corpus atrophy.

17. The method according to claim 16, wherein the composition contains L- cysteine.

18. The method according to any preceding claim, comprising the additional step of entering the data values obtained for said biomarkers in a data processing means comprising an operating system, means for transceiving and processing data, said data processing means being adapted to perform the steps of comparing a measured concentration value for a biomarker to a predetermined cut-off or reference value for said biomarker, to obtain a combination of comparison results which is specific for the subject tested, and generating information in response to the said combination of comparison results, and optionally other data entered.

19. The method according to claim 18, wherein the information generated comprises suggestions for further examinations or determinations.

20. The method according to claim 19, wherein the suggestions for further examinations and determinations are selected from determination of vitamin B 12, homocysteine and methylmalonate, gastroscopy and examination of a gastric biopsy sample.

21. Sampling kit for obtaining a whole blood sample from an individual comprising

1. Finger prick device for obtaining the sample 2. Means for receiving and storing the sample

3. Means for obtaining and maintaining hygienic or aseptic conditions in connection with sampling, such as before, during and after sampling.

4. Instructions for use of the kit.

22. Kit according to claim 21 , comprising

- skin disinfectant, such as an alcohol wipe, and sticking plaster, plastic glove, gauze pad as means for obtaining and maintaining hygienic or aseptic conditions

- an anticoagulant collection tube/device as the sample receiving and storing means

- tube/container for forwarding, such as by mailing, the sample to be analyzed

- form, and

- disposal bag

and optionally an address label on the kit for the recipient of the kit.

23. Use of the sampling kit according to claim 21 for obtaining a finger prick sample for use in the method according to any preceding claim for determining the concen- tration of PGI and PGII, according to step a) and b) in claim land optionally Hpab according to claim 3 in said sample.