WO2021181008A1 - Gastrin-17 test for screening and risk assesment of dyspeptic and reflux symptoms, and atrophic gastritis, with related risks - Google Patents

Gastrin-17 test for screening and risk assesment of dyspeptic and reflux symptoms, and atrophic gastritis, with related risks Download PDF

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WO2021181008A1
WO2021181008A1 PCT/FI2021/050176 FI2021050176W WO2021181008A1 WO 2021181008 A1 WO2021181008 A1 WO 2021181008A1 FI 2021050176 W FI2021050176 W FI 2021050176W WO 2021181008 A1 WO2021181008 A1 WO 2021181008A1
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gastrin
pylori
atrophic gastritis
risk
antrum
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French (fr)
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Osmo Suovaniemi
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Biohit Oyj
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/595—Gastrins; Cholecystokinins [CCK]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/062—Gastritis or peptic ulcer disease

Definitions

  • the present invention relates to diagnostic tools for screening and risk assessment of dyspeptic and reflux symptoms, and atrophic gastritis, with related risks.
  • the present invention relates to a method and use of gastrin-17 as a biomarker 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.
  • 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).
  • 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.
  • IEN intraepithelial neoplasia
  • 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.
  • atrophic gastritis 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.
  • GC gastric cancer
  • 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.
  • 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.
  • 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.
  • HPAB HP antibody
  • HP-infection remains untreated, it gradually affects also the mucosa of the gastric corpus, with superficial and chronic HP-gastritis as a consequence.
  • said HP gastritis becomes chronic and leads to mucosal atrophy, which may gradually involve n either antrum or corpus or both.
  • atrophic gastritis can also evolve through complex autoimmune mechanisms.
  • patients with H. 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, magnesium and calcium as well as malabsorption of some medicines.
  • US 2011/104707 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.
  • stomach examination includes gastroscopy, which is an invasive diagnostic tool, experienced uncomfortable by most subjects.
  • gastroscopy is an invasive diagnostic tool, experienced uncomfortable by most subjects.
  • one aim of the present invention is 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.
  • An enzyme immunoassay test according to the present invention is mainly characterized in claim 11.
  • the present technology relates to a method for screening symptomless subjects and examining a subject having dyspeptic and/or reflux symptoms in order to find healthy subjects and subjects with atrophic gastritis.
  • H. pylori infection may be revealed by measuring the levels of H. pylori antibodies.
  • Gastrin-17 is involved in the regulation mechanism of various diseases clinically. At present, the most successful application is early gastric cancer screening (which has been written into the national diagnosis and treatment guidelines). Meanwhile, drug- induced gastric damage and neurological disorders gastrointestinal stress response will be reflected by abnormal indicator of gastrin-17. It is herein suggested that gastrin- 17 be used as a routine basic follow-up index to evaluate various related diseases.
  • FIGURE 1 is a schematic picture describing that protein-rich foodstuff entering into stomach stimulates the secretion of gastrin- 17.
  • the elevated level of gastrin- 17 gives positive feedback to corpus parietal cells and as a consequence they increase the output of hydrocloridic acid.
  • FIGURE 3 is a schematic picture showing for example severe atrophic gastritis in corpus.
  • Corpus has lost its glands capable secreting e.g. gastric acid, PGI, intrinsic factor.
  • PGI gastric acid
  • Protein from food is able to stimulate the antrum G-cells and bring G-17 into circulation. But there are only few acid producing cells in corpus which G-17 can stimulate (through more complicated mechanism).
  • Gastric acid production stays low. There is going to be slow or nil negative feedback mechanism to stop the gastrin-17 production. Concentration of gastrin-17 in blood stays high also in fasting stage.
  • FIGURE 4 is a schematic picture showing for example severe atrophic gastritis in antrum there are only few or nil G-cells.
  • the stimulatory effect of a protein-rich food to antrum G-cells secreting gastrin-17 is diminished or it does not exist.
  • the blood concentration of G-17 remains low.
  • Pepsinogen I concentrations in circulation are normal or high.
  • the high acidity in stomach causes an elevated risk for peptic ulcers.
  • the present invention is thus based on gastrin-17 (P-G-17) biomarker, which 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).
  • 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.
  • steak stimulus or protein stimulation
  • the acid secretion in the stomach decreases, is low or absent.
  • 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.
  • G-l 7 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-l 7-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-l 7 monoclonal antibody is also suitable for IHC analysis of formalin-fixed, paraffin-embedded specimens.
  • the gastrin assays currently used in most hospital laboratories are measuring the level of total gastrin, i.e., all biologically active gastrin peptides.
  • 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).
  • HRP horseradish peroxidase
  • H. pylori IgA & IgG antibodies are markers of H. pylori infection and the level of G-l 7, 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. 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 reflux 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.
  • GSD gastroesophageal reflux disease
  • 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-17 and H. pylori IgA&IgG antibodies are biomarkers of atrophic gastritis of the antrum.
  • the patients with H. pylori infection and low G-17 either have atrophic antral gastritis, and/or so called antral predominant gastritis characterized by high acid output and high risk of peptic ulcer disease.
  • 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.
  • 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 dyspeptic and/or reflux symptoms and/or biomarkers indicating 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, or a saliva or urine sample, obtained from the said subject, and comparing obtained values to a pre -determined reference range(s) indicating a healthy gastric mucosa.
  • the method comprises carrying out gastric and esophaegal endoscopy preferably with biopsies, if the obtained values are indicative for i) low acid output and atrophic gastritis of the corpus, or ii) high acid output or atrophic gastritis of the antrum.
  • 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.
  • test manufactured for the purpose of screening a symptomless subject or examining a subject having the symptoms and/or biomarkers indicating high and/or low acid output and/or an atrophic gastritis according to a method disclosed herein belong to the scope of the present invention.
  • gastrin- 17 and Helicobacter pylori antibodies are used as biomarkers for revealing high gastric acid output and a consequent risk of peptic ulcer disease.
  • gastrin-17 and H. 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.
  • GERD gastroesophageal reflux disease
  • HP antibody levels HPAB negative ( ⁇ 30 EIU) gastrin- 17B (fast): 1-7 pmo 1/1 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 pmoEl) 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
  • the method for screening a symptomless subject or examining a subject having dyspeptic and/or reflux symptoms comprises: quantitatively measuring at least a concentration of gastrin- 17 biomarker from a blood, serum, plasma, saliva or urine sample obtained from the said subject, comparing obtained value to a pre -determined reference range(s), and - carrying out gastric and esophaegal endoscopy preferably with biopsies, if the obtained value is indicative for i) low acid output and atrophic gastritis of the corpus, or ii) high acid output or atrophic gastritis of the antrum.
  • 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.
  • the gastrin-17 examination 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.
  • the gastrin-17 examination results are interpreted with customized GastroSoft program, which directly classifies the test result to one of the categories previously mentioned.
  • 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.
  • 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.
  • 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.
  • Option 5 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.
  • 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.
  • G-17 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.
  • 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.
  • the fasting blood sample demonstrates that the G-17B concentration is above the reference range and/or cut-off value, it indicates low acid output and atrophic gastritis of the corpus
  • carrying out gastric and oesophageal endoscopy preferably with biopsies is relevant procedure and enables to treat pre-cancerous phase in an early stage before alarming symptoms.
  • 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.
  • 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.
  • a person with moderate or severe atrophic gastritis in the antrum (low G-17 and H. pylori IgA and 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. This information, among other things, helps to realize how extremely important (safe, ethical and cost effective; litis 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 NSAID medication, are killing 200 - 300 people / year in Finland (the population 5,5 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).
  • 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.
  • 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 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. 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. In addition, hypochlorhydric states, such as atrophic gastritis and partial gastrectomy, have long been known to be causes of iron deficiency anemia.
  • 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.
  • 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.
  • Gastrin- 17 examination is particularly preferred in primary care and health screening especially if the endoscopic resources are insufficient.
  • accurate diagnosis cannot always be made from a few biopsy specimens.
  • positive serology H. pylori IgA and IgG antibodies
  • results may indicate an ongoing H. pylori infection in spite of negative 13 C- urea breach test and histology results.
  • 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 and biopsy examination. 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.
  • GastroPanel used concomitantly with gastroscopy and biopsies in patients with dyspeptic and reflux symptoms, helps diagnosing their Hp-infection, atrophic gastritis with associated risks as well as disturbances in gastric acid output.
  • Hp-infection chronic gastritis
  • the reference values of G-17 test for healthy stomach fall within the range 1 - 7 pmol/1.
  • the reference values may be altered and may differ from the previously mentioned range due to addition of clinical data.
  • atrophic gastritis similar as early- stage gastric and esophageal cancer, is asymptomatic in most cases, the diagnosis is often delayed beyond reach of a curative therapy. In Finland, almost 1000 new cases of these cancers are found annually, and the mortality rate is more than 70%.
  • G-l 7-test helps exclude (G-17 within the cut-off range) or confirm the high acidity of the stomach contents and high acid output (G-17 below the cut-off) while suspecting an erosive reflux disease, instead of using PPI test treatment without further examination of the patient.
  • PPI-treatment can mask the symptoms and thus delay the reach of correct diagnosis. Furthermore, a long-term use of PPI-medication is associated with an increased risk of gastric and esophageal cancer as well as a risk to develop osteoporosis and dementia. In Finland, more than 10% of the population is using PPI medication occasionally or regularly.
  • G-17 levels above the cut-off values denote that the patient has atrophic gastritis with low acid output or he/she is receiving PPI-medication. In these subjects, the risk of upper gastrointestinal tract cancer is higher than on the average, and endoscopic examination is indicated.
  • G-l 7-test helps, in addition to screening of gastric disorders, detect or exclude autoimmune diseases, which might coexist in these patients as multiple manifestations: ⁇ patients with autoimmune thyroid disease (AITD) might also have an autoimmune -type atrophic gastritis (some 18%) in gastric corpus (G-17 above the cut-off values).
  • AITD autoimmune thyroid disease
  • DM1 type 1 diabetes mellitus
  • DM1 type 1 diabetes mellitus
  • RA rheumatoid arthritis
  • vitamin-B12 absorption is impaired.
  • B 12-deficiency increases the risk to develop depression, dementia, Alzheimer’s disease and polyneuropathy. Because of this, also these patients should be examined for G-17 to exclude or confirm atrophic gastritis.
  • Atrophic gastritis and acid-free stomach can change or impair the absorption of certain medications, including dipyridamol, iron- and calcium preparations, anti-fungal remedies (fluconazol, itraconazol), thyroxin and atazanovir.
  • Atrophic gastritis and prolonged PPI-medication will result in acid-free stomach, which is associated with an increased risk of gastric and esophageal cancer. This risk can be reduced by binding the carcinogenic acetaldehyde that develops in acid-free stomach by using Acetium-capsules (mvw.acetium.fi) .
  • Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

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Abstract

According to an example aspect of the present invention, gastrin-17 examination relates to the use of gastrin-17 as biomarker for screening symptomless subjects and examining subjects having dyspeptic and/or reflux symptoms in order to find out healthy subjects and subjects with atrophic gastritis. The present invention provides a simple, fast and cost effective method and test for the risk assessment of peptic ulcer disease and diagnosis of atrophic gastritis with related risks.

Description

GASTRIN- 17 TEST FOR SCREENING AND RISK ASSESMENT OF DYSPEPTIC AND REFLUX SYMPTOMS, AND ATROPHIC GASTRITIS, WITH RELATED RISKS
FIELD [0001] The present invention relates to diagnostic tools for screening and risk assessment of dyspeptic and reflux symptoms, and atrophic gastritis, with related risks.
BACKGROUND
[0002] The present invention relates to a method and use of gastrin-17 as a biomarker 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.
[0003] 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).
[0004] 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 IM 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.
[0005] 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).
[0006] 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.
[0007] Apart from H. pylori infection, atrophic gastritis can also evolve through complex autoimmune mechanisms. Thereby, patients with H. 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, magnesium and calcium as well as malabsorption of some medicines.
[0008] 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 years.
[0009] US 2011/104707 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.
[0010] 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 [0011] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.
[0012] According to an aspect of the present invention, one aim of the present invention is 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. [0013] 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. reflux disease, Barrett’s oesophagus and oesophageal cancer in symptomatic or asymptomatic subjects based on the measured gastrin-17 values and combination of gastroscopy and/or biopsy when required based on said values.
[0014] This and other aspects, together with the advantages thereof over known solutions are achieved by the present invention, as hereinafter described and claimed. [0015] The method of the present invention is mainly characterized by what is stated in the characterizing part of claim 1.
[0016] An enzyme immunoassay test according to the present invention is mainly characterized in claim 11.
[0017] Considerable advantages are obtained by means of the invention. For example, it provides simple diagnostic tools that are fast to perform and cost-effective in detecting 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.
[0018] Next, the present technology will be described more closely with reference to certain embodiments.
EMBODIMENTS [0019] The present technology relates to a method for screening symptomless subjects and examining a subject having dyspeptic and/or reflux symptoms in order to find healthy subjects and subjects with atrophic gastritis. H. pylori infection may be revealed by measuring the levels of H. pylori antibodies.
[0020] Gastrin-17 is involved in the regulation mechanism of various diseases clinically. At present, the most successful application is early gastric cancer screening (which has been written into the national diagnosis and treatment guidelines). Meanwhile, drug- induced gastric damage and neurological disorders gastrointestinal stress response will be reflected by abnormal indicator of gastrin-17. It is herein suggested that gastrin- 17 be used as a routine basic follow-up index to evaluate various related diseases.
[0021] FIGURE 1 is a schematic picture describing that protein-rich foodstuff entering into stomach stimulates the secretion of gastrin- 17. The elevated level of gastrin- 17 gives positive feedback to corpus parietal cells and as a consequence they increase the output of hydrocloridic acid.
[0022] FIGURE 2 is a schematic picture describing that when the acid output increases and pH-level of the stomach decreases below pH 2.5, it acts as a negative feedback to the antrum G-cells. This results in a lower secretion of gastrin- 17. Due to the decreased secretion of gastrin- 17 the acid output from the parietal cells is also diminished = negative feedback.
[0023] FIGURE 3 is a schematic picture showing for example severe atrophic gastritis in corpus. Corpus has lost its glands capable secreting e.g. gastric acid, PGI, intrinsic factor. Protein from food is able to stimulate the antrum G-cells and bring G-17 into circulation. But there are only few acid producing cells in corpus which G-17 can stimulate (through more complicated mechanism). Gastric acid production stays low. There is going to be slow or nil negative feedback mechanism to stop the gastrin-17 production. Concentration of gastrin-17 in blood stays high also in fasting stage.
[0024] FIGURE 4 is a schematic picture showing for example severe atrophic gastritis in antrum there are only few or nil G-cells. The stimulatory effect of a protein-rich food to antrum G-cells secreting gastrin-17 is diminished or it does not exist. The blood concentration of G-17 remains low. Pepsinogen I concentrations in circulation are normal or high. The high acidity in stomach causes an elevated risk for peptic ulcers.
[0025] The present invention is thus based on gastrin-17 (P-G-17) biomarker, which 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). [0026] 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.
[0027] The P-G-17 ELISA method is specific to “amidated” G-l 7 molecule. G-l 7 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-l 7-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-l 7 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.
[0028] 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).
[0029] Based on the comparative studies by the gastroscopy and biopsy specimen examinations, the biomarker tests of the present 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-l 7, 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.
[0030] 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. 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 reflux 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-17 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.
[0031] 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-17 and H. pylori IgA&IgG antibodies are biomarkers of atrophic gastritis of the antrum. The patients with H. pylori infection and low G-17 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.
[0032] 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.
[0033] 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 dyspeptic and/or reflux symptoms and/or biomarkers indicating 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, or a saliva or urine sample, obtained from the said subject, and comparing obtained values to a pre -determined reference range(s) indicating a healthy gastric mucosa. Furthermore, the method comprises carrying out gastric and esophaegal endoscopy preferably with biopsies, if the obtained values are indicative for i) low acid output and atrophic gastritis of the corpus, or ii) high acid output or atrophic gastritis of the antrum.
[0034] It may also be beneficial to quantitatively measure the levels of H. pylori antibodies (HPAB) 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.
[0035] Thus also a test manufactured for the purpose of screening a symptomless subject or examining a subject having the symptoms and/or biomarkers indicating high and/or low acid output and/or an atrophic gastritis according to a method disclosed herein belong to the scope of the present invention.
[0036] 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 and H. 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. [0037] 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.
[0038] Next, the diagnostic categories based on the results from gastrin- 17 examination are being described more thoroughly.
[0039] 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 pmo 1/1 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 pmoEl) 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 pmoEl) gastrin- 17S does not increase above reference value (3 pmoEl) [0040] According to one embodiment of the present invention, the method for screening a symptomless subject or examining a subject having dyspeptic and/or reflux symptoms comprises: quantitatively measuring at least a concentration of gastrin- 17 biomarker from a blood, serum, plasma, saliva or urine sample obtained from the said subject, comparing obtained value to a pre -determined reference range(s), and - carrying out gastric and esophaegal endoscopy preferably with biopsies, if the obtained value is indicative for i) low acid output and atrophic gastritis of the corpus, or ii) high acid output or atrophic gastritis of the antrum. [0041] 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.
[0042] 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.
[0043] 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)
[0044] 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.
Option 5: 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.
[0045] 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.
[0046] 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 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.
[0047] According to one embodiment of the present invention, i) if the fasting blood sample demonstrates that the G-17B concentration is above the reference range and/or cut-off value, it indicates low acid output and atrophic gastritis of the corpus, and ii) if the fasting blood sample demonstrates that the G-17B concentration is below the reference range and/or cut off value, it indicates high acid output and atrophic gastritis of the antrum. In both cases, i) and ii), carrying out gastric and oesophageal endoscopy preferably with biopsies is relevant procedure and enables to treat pre-cancerous phase in an early stage before alarming symptoms. Because atrophic gastritis and relating stomach and oesophageal cancers are usually symptomless, the diagnosis of such cancers gets delayed beyond the reach of a curing treatment. In Finland, annually 1000 people contracts these cancers with a mortality rate as high as 72%. [0048] 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 (HPAB), gastrin- 17, PGI, PGII and PGI/PGII ratios, whereas Gastrin- 17 examination measures only gastrin- 17 levels and optionally HPAB levels directly and thus provides simpler analysis, based on the biomarker levels as detected by the researchers and described earlier in the present specification.
[0049] 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.
[0050] A person with moderate or severe atrophic gastritis in the antrum (low G-17 and H. pylori IgA and 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. 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.
[0051] 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 NSAID medication, are killing 200 - 300 people / year in Finland (the population 5,5 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).
[0052] 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.
[0053] 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. 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. In addition, hypochlorhydric states, such as atrophic gastritis and partial gastrectomy, have long been known to be causes of iron deficiency anemia.
[0054] Undiagnosed atrophic gastritis often leads to vitamin B12 deficiency, which appears to affect up to 10% of the elderly population. 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. [0055] 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.
[0056] In addition, by considering that patients with atrophic gastritis and related risks (gastric cancer, peptic ulcer disease and the deficiency of vitamin B12, 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.
[0057] The above presented facts emphasize why dyspepsia and H. pylori patients should not be tested by the 13C- 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 be unreliable even in the proper diagnosis of the on-going H. pylori detection. The 13C- 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. 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.
[0058] The use of 13C- 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 examinations are available, it is reasonable and ethical to give up the old tests (13C- 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). [0059] 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.
[0060] 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 and IgG antibodies) results may indicate an ongoing H. pylori infection in spite of negative 13C- urea breach test and histology results. 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.
[0061] 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 and biopsy examination. 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.
[0062] 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. [0063] GastroPanel or 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.
[0064] 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. [0065] 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. [0066] Whenever the full GastroPanel test is not available, the G-17 component of the
GastroPanel, used concomitantly with gastroscopy and biopsies in patients with dyspeptic and reflux symptoms, helps diagnosing their Hp-infection, atrophic gastritis with associated risks as well as disturbances in gastric acid output. Hp-infection (chronic gastritis), causing peptic ulcer disease as well as Hp-related or autoimmune -type atrophic gastritis are independent risk factors of gastric cancer. In a healthy stomach, the risk of gastric cancer is very low.
[0067] According to one embodiment, the reference values of G-17 test for healthy stomach fall within the range 1 - 7 pmol/1. However, the reference values may be altered and may differ from the previously mentioned range due to addition of clinical data.
[0068] For the subjects in whom the G-17 test in the fasting blood demonstrates i) atrophic gastritis of the corpus or low acid output due to PPI-medication (G-17 above the cut off values), or ii) high acid output of the stomach or atrophic gastritis in the antrum (G-17 below the cut-off values), gastric and esophageal endoscopy (i.e. gastro-esophagoscopy) with biopsies is indicated and enables the diagnosis and treatment of cancer precursor lesions at an early stage, well before the alarming symptoms. Because atrophic gastritis, similar as early- stage gastric and esophageal cancer, is asymptomatic in most cases, the diagnosis is often delayed beyond reach of a curative therapy. In Finland, almost 1000 new cases of these cancers are found annually, and the mortality rate is more than 70%.
[0069] G-l 7-test helps exclude (G-17 within the cut-off range) or confirm the high acidity of the stomach contents and high acid output (G-17 below the cut-off) while suspecting an erosive reflux disease, instead of using PPI test treatment without further examination of the patient.
[0070] PPI-treatment can mask the symptoms and thus delay the reach of correct diagnosis. Furthermore, a long-term use of PPI-medication is associated with an increased risk of gastric and esophageal cancer as well as a risk to develop osteoporosis and dementia. In Finland, more than 10% of the population is using PPI medication occasionally or regularly.
[0071] Reflux of an acidic stomach contents (G-17 below cut-off level) into the esophagus predisposes to erosive reflux disease, Barrett's esophagitis and esophageal cancer.
[0072] G-17 levels above the cut-off values denote that the patient has atrophic gastritis with low acid output or he/she is receiving PPI-medication. In these subjects, the risk of upper gastrointestinal tract cancer is higher than on the average, and endoscopic examination is indicated.
[0073] G-l 7-test helps, in addition to screening of gastric disorders, detect or exclude autoimmune diseases, which might coexist in these patients as multiple manifestations: · patients with autoimmune thyroid disease (AITD) might also have an autoimmune -type atrophic gastritis (some 18%) in gastric corpus (G-17 above the cut-off values).
• patients with type 1 diabetes mellitus (DM1) can present with an autoimmune- type atrophic gastritis and vitamin-B12 deficiency (some 12% of DM1 patients). • similarly, the patients with celiac disease (CD), IBD, LED or rheumatoid arthritis (RA), might have a coexistent auto immune -type atrophic gastritis with its associated clinical sequels.
[0074] In the patients with atrophic gastritis, vitamin-B12 absorption is impaired.
• B 12-deficiency increases the risk to develop depression, dementia, Alzheimer’s disease and polyneuropathy. Because of this, also these patients should be examined for G-17 to exclude or confirm atrophic gastritis.
• B12-vitami deficiency results in accumulation of homocysteine into the tissues which can predispose to atheroscrelosis, cardiovascular events or stroke. G-17 test is also recommended for these patients.
[0075] Because of the fact that atrophic gastritis and acid-free stomach result in malabsorption of calcium, iron, magnesium and zinc, G-17-test is indicated to exclude atrophic gastritis among the subjects with osteoporosis and anaemia.
[0076] The risk of pneumonia, and particularly among elderly people, the risk to contract fatal intestinal infections (giardiasis, malaria, Clostridium difficile and E. coli EHEC), can be significantly increased as a result of atrophic gastritis and PPI-medication. Patients with such infections should be tested for G-17 to exclude or confirm atrophic gastritis.
[0077] Atrophic gastritis and acid-free stomach can change or impair the absorption of certain medications, including dipyridamol, iron- and calcium preparations, anti-fungal remedies (fluconazol, itraconazol), thyroxin and atazanovir.
[0078] Atrophic gastritis and prolonged PPI-medication will result in acid-free stomach, which is associated with an increased risk of gastric and esophageal cancer. This risk can be reduced by binding the carcinogenic acetaldehyde that develops in acid-free stomach by using Acetium-capsules (mvw.acetium.fi) . [0079] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term such as, for example, about or substantially, the exact numerical value is also disclosed. [0080] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. It is not intended that the invention be limited, except as by the claims set forth below. [0081] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", that is, a singular form, throughout this document does not exclude a plurality.
CITATION LIST
Patent literature
US 6696262 US 2011/104707 EP 2557425
Non-patent literature
1. Agreus L, Kuipers EJ, Kupcinskas L, Malfertheiner P, Di Mario F, Leja M, Mahachai V, Yaron N, van Oijen M, Perez Perez G, Rugge M, Ronkainen J, Salaspuro M, Sipponen P, Sugano K, Sung J. Rationale in diagnosis and screening of atrophic gastritis with stomach- specific plasma bio markers. Scand J Gastroenterol 47,136-147, 2012.
2. Aine R, Kahar E, Aitokari K, Salminen J, Eklund C, Paloheimo L, Peetsalu A, Syrjanen K. Atrophic gastritis (AG) and its clinical sequels among elderly people in Finland and Estonia. A comparative study using GastroPanel and B 12-vitamin testing of the residents in assisted-housing facilities. J. Aging Res. Clin. Pract. 5, 194-202, 2016.
3. Vohlonen I, Pukkala E, Malila N, Harkdnen M, Hakama M, Koistinen V, Sipponen P. Risk of gastric cancer in helicobacter pylori infection in a 15-year follow-up. Scand J Gastroenterol 2016;51:1159-116.
4. Sipponen P, Laxen F, Huotari K, Harkdnen M. Prevalence of low vitamin B12 and high homocysteine in serum in an elderly male population: association with atrophic gastritis and helicobacter pylori infection. Scand J Gastroenterol 2003;38:1209-121.
5. Jeong Soo Ahn, Chun-Sick Eom, Christie Y Jeon, Sang Min Park. Acid suppressive drugs and gastric cancer: A meta-analysis of observational studies. World J Gastroenterol
2013 April 28; 19(16): 2560-2568.
6. T c ram u ra- G ro n bl ad M, Bell JS, Poysti MM, Strandberg TE, Laurila JY, Tilvis RS, Soini H, Pitkala KH. Risk of death associated with use ofPPIs in three cohorts of institutionalized older people in Finland. J Am Med Dir Assoc 2012; 13:488. e9-13.
7. Sipponen P, Harkoncn M. Hypochlorhydric stomach: a risk condition for calcium malabsorption and osteoporosis? Scand J Gastroenterol 2010;45:133-138.
8. Ito T, Jensen RT. Association of long-term proton pump inhibitor therapy with bone fractures and effects on absorption of calcium, vitamin B12, iron, and magnesium.
Curr Gastroenterol Rep 2010;12:448-457.
9. Farkkila M. Miten dyspepsia tulisi hoitaa ?, Duodecim 2004; 120: 2537- 2542. - UBT-testia maahantuovan yrityksen konsulttina vilpillista kilpailua.
10. Syrjanen K, Eskelinen M, Peetsalu A, Sillakivi T, Sipponen P, Harkoncn M, Paloheimo L, Maki M, Tiusanen T, Suovaniemi O, DiMario F, Fan ZP. GastroPanel® Biomarker
Panel: The most comprehensive test for Helicobacter pylori infection and its clinical sequelae. A critical review. Anticancer Res. 2019;39:1091-1104.
11. https://www.biohithealthcare.com/wp-content/uploads/2019/11/Critisism-about-current- care-recommendations.pdf
12. https://www.biohithealthcare.com/wp-content/uploads/2019/12/Biohit-GastroPanel- Innovation.pdf

Claims

CLAIMS:
1. A method for screening a symptomless subject or examining a subject having dyspeptic and/or reflux symptoms, characterized in that the method comprises quantitatively measuring at least a concentration of gastrin- 17 biomarker from a blood, serum, plasma, saliva or urine sample obtained from the said subject, comparing obtained value to a pre -determined reference range(s), and carrying out gastric and oesophageal endoscopy preferably with biopsies, if the obtained value is indicative for i) low acid output and atrophic gastritis of the corpus, or ii) high acid output or atrophic gastritis of the antrum.
2. The method according to claim 1, characterized in that the method comprises quantitatively measuring the concentration(s) and level(s) of biomarker(s), selected from options a) to e): a) gastrin- 17B (basal) or 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.
3. The method according to claim 1 or 2, characterized in that the method directly measures an activity of antrum mucosa.
4. The method according to any of the preceding claims, characterized in that the method gives indirect information about an activity of gastric corpus, based on a physiological positive and negative feedback mechanism between the antrum and the corpus.
5. The method according to any of the preceding claims, characterized in that the reference ranges indicating the healthy gastric mucosa are for gastrin-17S 3 - 30 pmol/1, for gastrin- 17B 1 - 7 pmol/1 and for HPAB {H. pylori antibody) level below 30 EIU.
6. The method according to any of the preceding claims, characterized in that the HPAB level in said sample above 30 EIU is being indicative for Helicobacter pylori infection.
7. The method according to any of the preceding claims, characterized in that the gastrin-17 value in said sample close to the lower limit or below the reference range or close to the upper limit or above the reference range is being indicative for atrophic gastritis.
8. The method according to any of the preceding claims, characterized in that the values are indicative for non-atrophic gastritis, if the gastrin- 17B value is between 1 - 7 pmol/1 and the HPAB value is above 30 EIU.
9. The method according to any of the preceding claims, characterized in that the values are indicative for atrophic antrum gastritis or atrophic pangastritis, if the gastrin- 17B value is below 1 pmol/1, gastrin-17S value is below 3 pmol/1 and the HPAB value is below 30 EIU (when HP lost) or above 30 EIU.
10. The method according to any of the preceding claims, characterized in that the values are indicative for atrophic corpus gastritis, if the gastrin-17B value is above 7 pmoEl and the HPAB value is below 30 EIU (when HP lost) or above 30 EIU.
11. An enzyme immunoassay test for screening a symptomless subject or examining a subject having the symptoms and/or biomarkers indicating high and/or low acid output and/or an atrophic gastritis according to a method of any of the preceding claims, said test comprising means to measure concentration(s) of gastrin-17B, and/or gastrin-17S, and/or an H. pylori specific antibody or antibodies.
12. Use of means for measuring concentration(s) of gastrin-17B, and/or gastrin-17S, and/or an H. pylori specific antibody or antibodies for revealing high gastric acid output and a consequent risk of peptic ulcer disease.
13. Use of means for measuring concentration(s) of gastrin-17B, and/or gastrin-17S, and/or an H. pylori specific antibody or antibodies for diagnosing a risk of peptic ulcer disease in symptomatic or asymptomatic subjects.
14. Use of means for measuring concentration(s) of gastrin- 17B, and/or gastrin- 17S, and/or an
H. pylori specific antibody or antibodies for diagnosing a risk of gastric cancer in symptomatic or asymptomatic subjects.
15. Use of means for measuring concentration(s) of gastrin-17B, and/or gastrin-17S, and/or an H. pylori specific antibody or antibodies for diagnosing a risk of gastroesophageal reflux disease and its complications: Barrett’s oesophagus and oesophageal cancer, in symptomatic or asymptomatic subjects.
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