INCREASED RISK OF SEVERAL RELATED SEQUELS

FIELD

The present invention relates to non-invasive diagnostic tools for the risk assessment of peptic ulcer disease and diagnosis of atrophic gastritis with related risks.

BACKGROUND

The present invention relates to a method and use of gastrin- 17 as a biomarker(s) for screening of asymptomatic subjects or examining those with dyspeptic symptoms intended for disclosing healthy gastric mucosa or disease conditions with an increased risk of peptic ulcer or, atrophic gastritis with many related sequels. Optionally, the method also measures levels of

Helicobacter pylori antibodies.

Helicobacter pylori is a spiral shaped gram-negative bacterium which resides in the mucus adjacent to the surface epithelial cells of the gastric mucosa and in the cell interstices. The bacterium apparently is transferred orally from one person to the other. The effect of the bacterium on the gastric mucosa is an inflammatory reaction, mediated by wide variety of inflammation mediator substances. If not eradicated acute Helicobacter pylori infection remains persistent causing chronic gastritis. As Helicobacter pylori infection and chronic gastritis are closely associated, it has been confirmed that this bacterial infection is one of the etiological factors in the development of stomach cancer. Eradication of H. pylori infection at an early stage could prevent the development of atrophic gastritis and thus reduce the risk of gastric cancer (US 6,696,262).

It is estimated that 50% of all gastric cancer (GC) cases develop through the "Correa cascade", progressing from Helicobacter pylori (HP)-associated gastritis to mucosal atrophy, intestinal metaplasia (IM), dysplasia, to invasive adenocarcinoma. The concept on atrophic gastritis (AG) and EVI as precancerous conditions is based on long-term prospective cohort studies, demonstrating that the risk of GC is significantly increased among patients with AG, which is currently considered as the single most powerful independent risk factor of GC. Gastroscopy with biopsies is the time -honored method to diagnose and grade these gastric precancer lesions, recently re-classified by WHO as intraepithelial neoplasia (IEN), to circumvent the unsatisfactory inter-rater agreement of previous classifications. Recently an ELISA-based assay (GastroPanel®) was designed to measure the serum concentrations of four stomach- specific biomarkers: pepsinogen I (PGI) and II (PGII), gastrin- 17 (G-17) and HP IgG antibodies (IgG-HP), making it the first non-invasive diagnostic tool for detection of the subjects at risk for GC, i.e., those with AG and/or HP.

As being said, atrophic gastritis (AG) is a disease associated with a significantly increased risk of gastric cancer, being the single most important precursor condition for gastric cancer (GC) known so far. On the other hand, H. pylori -infection is the most important causative agent in the development of gastritis, and subsequent AG. H. pylori infection and AG in the antrum may lead to GC and peptic ulcer disease. It is well known that a minority of cases of AG in the corpus develop by autoimmune mechanisms. The risk of GC is 4-5 times higher among patients suffering from severe atrophy of the corpus mucosa as compared with their healthy counterparts. Among the patients with severe atrophy in the antrum, this risk is 18 fold higher than in healthy subjects, and the risk increases up to 90-fold if severe atrophy exists in both antrum and corpus (i.e., with severe panatrophy).

H. pylori infection typically starts from gastric antrum and causes first an acute, superficial HP gastritis. The infection leads to elevated level of HP antibody (HPAB) titres in the serum, and usually also up-regulated gastrin-17 levels as a result of acute inflammation. If HP-infection remains untreated, it gradually affects also the mucosa of the gastric corpus, with superficial and chronic HP-gastritis as a consequence. When untreated, said HP gastritis becomes chronic and leads to mucosal atrophy, which may gradually involve n either antrum or corpus or both. Apart from H. pylori infection, atrophic gastritis can also evolve through complex

autoimmune mechanisms. Thereby, patients withH. pylori infection or autoimmune diseases are at increased risk of atrophic gastritis, which carries an increased risk of gastric- and oesophageal cancer and several other conditions, such as the diseases related to the deficiency of vitamin B12, zinc, iron and calcium as well as malabsorption of some medicines. The prevalence of AG and GC increases with increasing age, and the risk for both diseases is highest among the subjects >45 years of age. The majority of GCs among the elderly are of the intestinal subtype, developing through the AG-to-GC sequence. Because of the high cancer risk among the elderly, the current consensus recommendations suggest endoscopy for all dyspeptic elderly people as well as for those aging above 45 (50) years.

US 201 1/104707 and EP 2557425 relate to a method for examining a person having symptomatic or asymptomatic autoimmune disease with a specifically designed biomarker panel, comprising pepsinogen I, pepsinogen II, pepsinogen I/II ratio, gastrin- 17 and H. pylori antibodies. These patent documents demonstrate how extremely important is the use of the validated combination of such biomarkers.

Zhang et al. (2007) discusses about the factors affecting human serum gastrin-17 levels and the diagnostic value of such marker in gastric precancerous lesions and gastric cancer.

However, it does not describe features of a screening method necessary for achieving the effect for detecting atrophic gastritis and related risks.

According to current clinical practice, routine procedures of stomach examination include gastroscopy, which is an invasive diagnostic tool, experienced uncomfortable by most subjects. Thus, there is a need for a simple, non-invasive diagnostic tools that are fast to perform and cost-effective tests to detect the subjects at increased risk of peptic ulcer disease, atrophic gastritis and all its possible consequences. SUMMARY OF THE INVENTION

An aim of the present invention is to solve at least some problems of the prior art. In particular, it is an aim of the present invention to provide a method and a test for screening a symptomless subject or examining a subject having the symptoms and/or biomarkers indicating an atrophic gastritis.

Furthermore, it is an aim of the present invention to provide a reliable diagnosis for revealing high gastric acid output and thereby consequently decreasing the risk of e.g. peptic ulcer disease, gastric cancer and oesophageal cancer in symptomatic or asymptomatic subjects.

More precisely the method according to the present invention is mainly characterized in that, what is stated in the characterizing part of claim 1. Considerable advantages are obtained by means of the present invention. For example, it provides a simple, non-invasive diagnostic tools that are fast to perform and cost-effective to detect subjects at increased risk of peptic ulcer disease, atrophic gastritis and related sequels. Detection of Gastrin-17 and subsequent early diagnosis of atrophic gastritis of the antrum provides possibilities to find patients at significant risk for gastric cancer in the gastric antrum, and offers tools to delineate patients at particular risk for gastric cancer, i.e. those with extended and severe atrophic gastritis in both antrum and corpus.

Next, the present technology is described more closely with reference to certain embodiments. EMBODIMENTS

The present invention relates to a method for screening symptomless subjects and examining a subject having symptoms in order to find out healthy subjects and subjects with atrophic gastritis due to Helicobacter pylori infection or autoimmune disease. H. pylori infection may be revealed by measuring the levels of H. pylori antibodies.

Gastrin-17 (P-G-17) biomarker offers a unique opportunity for diagnosing atrophic gastritis of the antrum with a simple blood test. P-G-17 is one of the most important peptide hormones of the gastrointestinal tract, playing a role in a wide variety of functions. G-17 is secreted exclusively by the gastrin-cells (G-cells) in the antrum, representing a fraction of the total gastrin concentration in the circulation. The G-17 fraction of the total gastrin can be measured with high specificity by Gastrin- 17 Advanced ELISA Test Kit (Biohit Cat. no 601 035). When dormant, the G-cells in antrum secrete only small amounts of G-l 7 hormone. The maximal secretion is achieved after physiological protein stimulation ("steak stimulus" or protein stimulation), or when the acid secretion in the stomach decreases, is low or absent. As a result of antral atrophy (i.e., loss of glands), the amount of G-cells decreases and, consequently, both the basal and post-prandial secretion of gastrin decreases.

The P-G-17 ELISA method is specific to "amidated" G-17 molecule. G-17 peptide is the most important member of the gastrin/cholecystokinin- family, which regulates the physiology of the upper gastrointestinal tract. This peptide is the biologically most active gastrin peptide, stimulating gastric acid secretion with 6 -times higher potency than the biologically next most active gastrin, G-34. The G-17-specific test allows estimation of the number and function of antral G-cells, without background noise and cross-reactivity with G-34 peptide or other gastrin fragments, which are also derived from sources other than the G-cells. The G-17 monoclonal antibody is also suitable for IHC analysis of formalin-fixed, paraffin-embedded specimens. For reference, the gastrin assays currently used in most hospital laboratories are measuring the level of total gastrin, i.e., all biologically active gastrin peptides.

As known, the H. pylori infection is the most important cause of chronic gastritis. Another well-known cause for gastritis and severe AG (atrophic gastritis) is the autoimmune mechanism, which can also be activated by H. pylori infection. The ELISA test for H. pylori is typically performed from the plasma samples. The test is based on an enzyme immunoassay technique, with purified H. pylori bacterial antigen, adsorbed on a microplate, and a detection antibody labeled with horseradish peroxidase (HRP). Gastrin-17 examination and related biomarkers

Based on the comparative studies by the gastroscopy and biopsy specimen examinations, the biomarker tests of the innovation, gastrin-17 (G-l 7) and, if necessary, H. pylori IgA & IgG antibodies have been validated to complement each other so as to form a diagnostic panel. H. pylori IgA & IgG antibodies are markers of H. pylori infection and the level of G- 17, which is usually measured in a fasting blood sample, is a marker the function and structure of antrum mucosa. The customized GastroSoft computer program is used in interpretation of the Gastrin-17 examination results. To obtain full benefit it is important to note that the gastrin- 17 biomarkers are assayed from the same blood (plasma) sample as a panel, with the optional application of GastroSoft to aid interpretation of the results.

Gastrin-17 examination is intended for safe, ethical and cost effective diagnosis and screening of dyspepsia, H. pylori infection and atrophic gastritis and related risks (10-21). These risks include gastric- and oesophageal cancer, peptic ulcer disease and the deficiency of vitamin B12, iron, zinc and calcium. Gastrin-17 also aids in the assessment of the development of gastroesophageal refiux disease (GERD) and its complications, such as erosive esophagitis and Barrett's esophagus, which may lead to esophageal cancer. Gastrin- 17 is suitable for diagnosis of atrophic gastritis as well as for indication of the cause of atrophic gastritis. If the patient diagnosed with atrophic gastritis based G-l 7 levels does not have, and has not had, H. pylori infection (H. pylori IgA & IgG antibody test and patient history), atrophic gastritis is very likely caused by autoimmune disease.

The assessment of the function and structure of antrum mucosa by the Gastrin-17 examination is extremely important because atrophic gastritis starts in most cases from the distal stomach (the antrum and its angulus), from which it may extend upwards to the corpus. Low G-l 7 and H. pylori IgA&IgG antibodies are biomarkers of atrophic gastritis of the antrum. The patients with H. pylori infection and low G-l 7 either have atrophic antral gastritis, and/or so called antral predominant gastritis characterized by high acid output and high risk of peptic ulcer disease. These patients should undergo gastroscopy and biopsy sample examinations due to an increased risk of the precancerous lesions or early cancer in the antrum. Such patients may also be at increased risk of the occurrence of GERD (gastroesophageal reflux disease) and its complications, Barrett's oesophagus and oesophageal cancer.

The term "biomarker" is herein intended to mean a measurable change taking place in a biochemical process (processes), compound(s) or cell organelle(s) of an organism. The reason for such change may be for example a disease, such as Helicobacter pylori infection or atrophic gastritis.

According to one preferred embodiment of the present invention, gastrin- 17 is (are) used as a biomarker(s) in a method for screening a symptomless subject (i.e. a person) or examining a subject having the symptoms and/or biomarkers indicating a H. pylori infection and/or an atrophic gastritis. The method preferably comprises quantitatively measuring concentrations of gastrin- 17 (basal and/or stimulated) from a biological sample, which is preferably a blood, plasma or serum sample, obtained from the said subject, and comparing obtained values to a pre-determined reference range(s) indicating a healthy gastric mucosa. It may also be beneficial to quantitatively measure the levels of H. pylori antibodies (HP AB) from the said biological sample. Furthermore, the method preferably comprises quantitatively measuring the concentrations and levels selected from options a) to e): a) gastrin-17B (basal) or/and gastrin-17S (stimulated);

b) gastrin- 17B and H. pylori IgG;

c) gastrin- 17B, H. pylori IgG and IgA;

d) gastrin-17B, gastrin-17S and H. pylori IgG;

e) gastrin- 17B, gastrin- 17S, H. pylori IgG and IgA.

Thus also a test manufactured for the purpose of screening a symptomless subject or examining a subject having the symptoms and/or biomarkers indicating a Helicobacter pylori infection and/or an atrophic gastritis according to a method disclosed herein belong to the scope of the present invention. According to further embodiment of the invention, said gastrin-17 and Helicobacter pylori antibodies are used as biomarkers for revealing high gastric acid output and a consequent risk of peptic ulcer disease. In addition, gastrin-17 andH. pylori antibodies are preferably used for decreasing a risk of peptic ulcer disease and/or gastric cancer and/or GERD (gastroesophageal reflux disease) and its complications: Barrett's oesophagus and oesophageal cancer, in symptomatic or asymptomatic subjects.

Based on the results of the present invention, if the gastrin-17 concentration in said sample is close to the lower limit or below the reference range or close to the upper limit or above the reference range, the examination is being indicative for atrophic gastritis.

Next, the diagnostic categories based on the results from Gastrin-17 examination are being described more thoroughly.

Diagnostic categories and findings based on Gastrin-17 examination and typical values thereof

Subject not having the symptoms and/or biomarkers indicating an autoimmune disease i.e. HEALTHY GASTRIC MUCOSA:

• HP antibody levels (HPAB) negative (<30 EIU)

• gastrin-17B (fast): 1 -7 pmol/1

• gastrin-17S (stimulated): 3-30 pmol/1

NON- ATROPHIC (HP) ANTRUM GASTRITIS:

• increased HPAB levels (>30 EIU)

• gastrin- 17B normal (1 -7 pmol/1) or increased (as a result of an infection)

ATROPHIC ANTRUM GASTRITIS:

• increased HPAB levels (>30 EIU)

• gastrin- 17B low (<1 pmol/1)

• gastrin-17S low, does not increase above reference value (3 pmol/1) ATROPHIC CORPUS GASTRITIS:

• increased HPAB levels or normal (<30 EIU, HP might be lost)

• gastrin- 17B increased (>7 pmol/1) (feedback from acid free corpus)

• gastrin- 17S not necessary

ATROPHIC PANGASTRITIS (BOTH ANTRUM AND CORPUS):

• increased HPAB levels or normal (<30 EIU, HP might be lost)

• gastrin- 17B low (<1 pmol/1)

• gastrin-17S does not increase above reference value (3 pmol/1)

Based on above, Gastrin- 17 examination thus cannot separate only the status between atrophic antrum gastritis and atrophic pangastritis, because in both the findings are similar based on atrophied antrum. It is known from prior art that GastroPanel® separates these two situations, because it measures the activity of the corpus directly with PGI and PGII biomarkers. Gastrin- 17, however, measures the activity of the antrum directly and the activity of the corpus indirectly (without PGI and PGII biomarkers), thus providing a cheaper test and simpler analysis.

Thus, according to one embodiment the Gastrin-17 examination according to the present invention directly measures an activity of antrum mucosa. Further, the examination gives indirect information about an activity of gastric corpus, based on a physiological positive and negative feedback mechanism between the antrum and the corpus. Increased HPAB values (above 30 EIU) are typically being indicative for H. pylori infection. Gastrin-17 examination results and their interpretation

The Gastrin-17 examination results are interpreted with customized GastroSoft program, which directly classifies the test result to one of the categories previously mentioned. In addition, the final diagnosis needs an output from a medical person, because more than one clinical diagnosis option exists for some of the results obtained by the Gastrin-17 examination. 1) HPAB level normal and G-17B normal:

• 1.1. healthy gastric mucosa

2) Increased HPAB level and normal G-17B or increased:

· 2.1. superficial HP -infection in the antrum

• 2.2. superficial HP -infection in the corpus

3) Increased or normal HPAB level and increased G-17B:

• 3.1. atrophic corpus gastritis (acid-free stomach, HP+ or HP-)

· 3.2. PPI-medication (acid-free/low acid stomach)(HP-)

4) Increased HPAB and low G-17B:

• 4.1. high acid secretion in the corpus (negative feedback)

• 4.2. atrophic antrum gastritis (absence of G-cells)

· 4.3. atrophic pangastritis (antrum and corpus both atrophic)

5) Increased HPAB levels and also low G-17S secretion:

• 5.1.atrophic antrum gastritis (absence of G-cells)

• 5.2.atrophic pangastritis (antrum and corpus both atrophic)

Diagnostic follow-up actions

Because in some of the situations (options 2.1 - 5.2 above) the result of the Gastrin-17 examination might refer to more than one actual clinical situation, few simple follow-up actions are required in order to end up to a final diagnosis:

Option 2:

• The difference is not clinically remarkable, thus HP-eradication therapy is sufficient action in both situations Option 3:

PPI medication is asked in the anamnestic information and if necessary it should be clarified, what separates situations 3.1. and 3.2.

HP eradication therapy (successful) eradicates the infection and G-17 becomes normal, thus confirming situation 2.1.

Option 4:

Low G-17B is confirmed by carrying out G-17S, whereby low value confirms situation 4.2.

Alternatively, 1 -2 weeks PPI medication is tested, and if the G-17 returns to normal, situations 4.2. and 4.3. are ruled out, whereby situation 4.1. is confirmed

Separation with only Gastrin- 17 examination is impossible

Carrying out GastroPanel-test and confirming or ruling out situation 5.2.

Carrying out gastroscopy and confirming the diagnosis with biopsies.

Antrum atrophy and high acid secretion

Gastrin- 17 examinations are typically done on a fasting plasma sample (as e.g. in 22). If a patient with H. pylori infection and low G-17 does not want to have invasive gastroscopy, atrophic gastritis of the antrum can be confirmed or excluded by assaying the concentration of protein-stimulated G-17 in plasma in addition to a fasting Gastrin- 17 examination. In case of antrum atrophy, the fasting level of G-17 is low due to the absence of the G-cells and the protein stimulation cannot increase the G-17 level. Gastric acid in turn inhibits the secretion of G-17, and in cases with high intragastric acidity alone, protein stimulation clearly raises the plasma level of G-17 (the G cell population in antrum is normal). It is thus possible to distinguish the patients with atrophic gastritis in the antrum from those whose low fasting concentration of G- 17 is entirely due to high acid secretion.

In a typical situation, if the antrum is not atrophied, protein stimulation increases the level of G-17 in the blood to over 7,0 pmol/1. If the protein-stimulated G-17 concentration is less than 3.0 pmol/1 and the patient has a H. pylori infection, it is very likely that the patient has atrophic gastritis of the antrum mucosa. The relation between low G-17, antrum atrophy and high acid secretion is explained by the well known physiological feedback mechanism between antrum and corpus. Patients without H. pylori infection and with G-17 fasting values of less than 2.0 pmol/1, who have increased G-17 levels following protein stimulation, may be at risk of the severe complications (erosive esophagitis and Barrett's oesophagus) of gastroesophageal reflux disease (GERD). This risk is significantly more likely if the fasting level of G-17 is 1.0 pmol/1 or lower. Gastrin- 17 examination directly measures an activity, and preferably structure, of an antrum mucosa and gives indirect information about an activity (and structure) of gastric corpus. This is due to the physiological positive and negative feedback mechanism between the corpus and antrum. It is known from the prior art (GastroPanel®) that the activity of the gastric corpus (pepsinogen I (PGI) and pepsinogen II (PGII) secretion) are in direct relation to the amount of the specific cells of the mucosa. Thus, the activity of the corpus also describes the structure of the corpus, consequently as in the GastroPanel® test. The difference between Gastrin- 17 examination and GastroPanel® test is that the latter measures directly H. pylori antibodies (ΗΡΑΒ), gastrin- 17, PGI, PGII and PGI/PGII ratios, whereas Gastrin- 17 examination measures only gastrin- 17 levels and optionally ΗΡΑΒ levels directly and thus provides simpler analysis, based on the biomarker levels as detected by the researchers and described earlier in the present specification.

Thus, the detection of G-17 and subsequent early diagnosis of atrophic gastritis of the antrum provides possibilities to find patients at significant risk for gastric cancer in the gastric antrum, and offers tools to delineate subjects at particular risk for peptic ulcer diseases and enables to delineate patients at highest risk for gastric cancer; i.e., those with extended and severe atrophic gastritis in both antrum and corpus. The risk of gastric cancer and peptic ulcer disease

A person with moderate or severe atrophic gastritis in the antrum (low G-17 and H. pylori IgA&IgG antibodies) has 18 times higher risk for stomach cancer than a healthy person. A person with moderate or severe atrophic gastritis in the corpus has "only" 5 times higher risk to have stomach cancer than a healthy person. If both the corpus and antrum have moderate or severe atrophic gastritis, the risk of cancer is 90 times higher (8). This information, among other things, helps to realize how extremely important (safe, ethical and cost effective; lege artis) it is to test G-17 due to the risk of gastric cancer.

G-17 is also a biomarker for the risk of peptic ulcer disease. Bleeding peptic ulcers, which are severe complications of peptic ulcer disease and increasingly due to the use of SAID medication, are killing 200 - 300 people / year in Finland (the population 5,2 million). To compare, some 400-600 people die annually from advanced gastric cancer. Proper diagnosis of people at risk of peptic ulcer disease by Gastrin- 17 examination would save people from unnecessary complications, and even from death. In addition, it is conceivable that screening of people at age over 45 in Finland with Gastrin- 17 examination would save 250 - 300 people annually from unnecessary death due to gastric cancer (patients could be found at a curable stage) (9).

Gastrin-17 examination before PPI medication

Gastrin- 17 examination is applicable and useful to be used prior to the PPI medication, to ensure that the patient does not have atrophic gastritis and hypochlorhydric or even achlorhydric stomach. In addition, PPI treatment can alleviate, and therefore mask, symptoms of serious diseases such as gastric cancer and bleeding peptic ulcer, and may thereby delay the proper diagnosis and treatment. Hypochlorhydria due to corpus atrophy and PPIs

Hypochlorhydria caused by corpus atrophy and PPIs also makes the person susceptible to the colonization of the stomach with microbes from oral cavity or from lower gut. In consumption of carbohydrates, which form part of most balanced meals, the colonized oral bacteria can elicit the production of carcinogenic acetaldehyde by fermentation in the stomach.

Hypochlorhydria of the stomach is associated with a strongly increased risk of gastric cancer (24-25). The decreased intestinal absorption of calcium due to atrophic gastritis and long-term PPI therapy predisposed to a risk for, e.g., osteoporosis and hip fractures (26). In addition, hypochlorhydric states, such as atrophic gastritis and partial gastrectomy, have long been known to be causes of iron deficiency anemia (27).

Deficiency of vitamin B12 Undiagnosed atrophic gastritis often leads to vitamin B12 deficiency, which appears to affect up to 10% of the elderly population (16). Vitamin B12 deficiency is considered to be associated with development of dementia, depression and peripheral neuropathies. In all tissues and cells, it increases in the concentrations of homocysteine that is considered an independent risk factor for atherosclerosis, heart attacks and strokes. Vitamin B12 deficiency and its causes are reversible if detected and treated early, but, unfortunately, this is rarely the case.

Summary When Gastrin-17 examination indicates that the gastric mucosa is healthy, the dyspepsia symptoms are often caused by functional dyspepsia or another disease not involving the gastric mucosa. Gastrin-17 examination can be used to differentiate the patients who really need gastroscopy from those who do not need it urgently. In this way it is possible to save and rationalize limited endoscopy resources for more important purposes. As much as 50% of dyspepsia symptoms may be of colon origin, especially in elderly population. In addition, by considering that patients with atrophic gastritis and related risks (gastric cancer, peptic ulcer disease and the deficiency of vitamin B 12, iron, zinc and calcium) are often asymptomatic, Gastrin-17 screening of the whole population over 45 years of age would help to find the individuals who require gastroscopy. This may be carried out with little or no significant change in total number of gastroscopies needed compared to the current situation, but it would result in a significant improvement in the early detection and treatment of serious disease. In addition to diagnosing H. pylori and atrophic gastritis, Gastrin-17 examination results can be used in assessment the patient's suitability and need for PPI treatment and the risk of complications of GERD.

Replacement of H. pylori ¹³ C- urea breath test and stool antigen test with Gastrin-17 examination

The above presented facts emphasize why dyspepsia andH. pylori patients should not be tested by the ¹³ C- urea breath test or stool antigen test, even though they are included in the current "test and treat" strategy. They only detect H. pylori infection but nothing else, and can

13 be unreliable even in the proper diagnosis of the on-going H. pylori detection (1-3). The C- urea breath test and stool antigen test give 40 - 50 % false negative results if the patient has atrophic gastritis; MALT lymphoma; or bleeding peptic ulcer disease; or if the patient has currently received antibiotics or PPIs (28-34). These are cases where the reliable H. pylori detection and treatment would be especially important. H. pylori IgA & IgG antibody test combination does not have these types of false negative results.

In addition, there is another reason for using the H. pylori IgA & IgG antibody test for H. pylori detection (37,38): "Nearly all infected individuals (>90%) exhibit H. /ry/on ^' -specific IgG antibodies. Most (approximately 70%) of these individuals also exhibit IgA antibodies. Approximately 7% of infected individuals are positive for IgA antibodies but negative for IgG antibodies; the reason for this aberrant response remains unclear." By referring to the studies and the scientific literature of Professor Pasechnikov et al. (14) the following is concluded:"The analysis of the literature data and results of our own research allow us to conclude that the serious medical and ethical problems of the "test and treat" strategy can be corrected simply and economically by replacing its ¹³ C- urea breath - or stool antigen test by the GastroPanel® examination. Same applies for Gastrin- 17 examination. Talley et al. (2004) indicate that in many countries, such as Sweden and the US, the "test and treat" strategy alone is not considered sufficient (37). The H. pylori tests of the "test and treat" strategy does not find atrophic gastritis and related risks, such as gastric cancer and precancerous lesions, which should be confirmed by gastroscopy and biopsy specimen examination and would be successfully treated. Consequently, Gastrin- 17 & gastroscopy and biopsy specimen examinations reveal patients with precancerous lesions and early stage gastric cancers, and, therefore, save people from unnecessary deaths because of gastric cancer." (1-3).

Gastrin-17 examination helps to avoid malpractice and its consequences The use of ¹³ C- urea breath test or stool antigen test delays correct diagnoses and treatments and may lead to malpractice and even unnecessary deaths due to, for example, misdiagnosed gastric cancer and bleeding peptic ulcers. In addition, the use of inaccurate and even misleading tests causes unnecessary costs for healthcare, social security, insurance companies, employers, and for patients themselves. Now, when the GastroPanel® and Gastrin-17

13

examinations are available, it is reasonable and ethical to give up the old tests ( C- urea breath test or stool antigen test) in the diagnosis and treatment of the patients with dyspepsia, H. pylori infection and atrophic gastritis with related risks (gastric cancer, peptic ulcer disease and the deficiency of vitamin B 12, iron, zinc and calcium). Opinion leaders, laboratories and doctors have an unquestionable authority and responsibility to recommend, offer and use the best possible examination and treatments that are available. Today, there are only three available options in proper and comprehensive, safe and ethical examination of the stomach mucosa. These are: 1) gastroscopy and histological examination of biopsy samples 2) GastroPanel® examination and 3) Gastrin-17 examination.

Gastrin-17 examination is particularly preferred in primary care and health screening especially if the endoscopic resources are insufficient. When comparing Gastrin-17 examination and gastroscopy, accurate diagnosis cannot always be made from a few biopsy specimens. In patients with atrophic gastritis, positive serology (H. pylori IgA&IgG antibodies) results may indicate an ongoing H. pylori infection in spite of negative C- urea breach test and histology results (31 -34). In addition, the histological diagnoses of two pathologists may diverge. The quality of histology is strongly dependent on experience and competence of the gastroenterologist and pathologist.

Gastrin- 17 examination is not associated with such problems since the biomarkers determined in blood give objective information on the function and structure of the stomach mucosa irrespective of the person examining them. If there are alarming changes in these biomarkers, the Gastrin-17 examination must be followed by a careful gastroscopy. In such a case, the information provided by Gastrin-17 is very helpful. The Gastrin-17 examination prevents unnecessary gastroscopies and helps target the use of sparse endoscopic resources

appropriately, particularly for the screening of colorectal cancer. The dyspeptic patients age 50 or over, whose stomach mucosa is found to be healthy should be referred for a colonoscopy, as almost half of stomach pains and disorders may be colon-related.

In medicine, it is obvious that good diagnostics go hand-in-hand with proper treatment. The business world as well as Food and Drug Administration (FDA) regulators are increasingly building this understanding into product development by supporting co -development of new drugs with development of diagnostics. This new development and the combination of the Gastrin-17 diagnostics with the PPI treatment of GERD as well as with the antibiotics and PPI treatment of H. pylori infection would promote the safe, ethical and cost effective evidence based and preventative medicine.

Gastrin- 17 examination should be required by the authorities before the reimbursement of the costs of any PPI treatment of GERD. In addition, a reliable diagnosis of H. pylori infection with related risks made by the Gastrin-17 examination or professionally performed

gastroscopy and biopsy specimen examination (gastroscopy) should be the basis for the reimbursement of the costs of the H. pylori eradication treatment. This guidance and contribution, for the development of the safe, ethical and cost effective evidence based and preventative medicine, will substantially reduce the costs of health care as well as prevent diseases, promote wellbeing and even save unnecessary deaths, for example, due to gastric cancer and bleeding peptic ulcers.

Gastroscopy has been the only method employed for the safe screening and diagnosis of dyspepsia, atrophic gastritis and gastric cancer, which is the second most common cause of cancer-related deaths worldwide. After evaluating the risk of atrophic gastritis and gastric cancer, researchers have concluded that gastrin- 17 and Helicobacter pylori antibodies can be employed effectually to screen for dyspepsia, atrophic gastritis and gastric cancer (9,11-14,19- 21 ,39,40).

Screening of atrophic gastritis and related risks (gastric cancer, peptic ulcer disease, the deficiency of vitamin B 12, iron, zinc and calcium) is now considered the standard of care for persons age 45 and older. Its omission might be a frequent source of litigations due to malpractice, what it has been considered to be in a case of the omission of colorectal cancer screening for persons age 50 or over (41).

Patent literature:

US 6696262

US 2011/104707

EP 2557425

Other references and related publications: (1) http://nobelprize.org/medicine/laureates/2005/press.html

(2) http://www.yourhealthbase.com/database/rulcer_drugs.htm

(3) http://www.gastropanel.net

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