Classifications

• • 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
  • G01N33/56922—Campylobacter
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/50—Determining the risk of developing a disease

Definitions

• 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.
• 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.
• the method also measures levels of
• 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).
• GC gastric cancer
• 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. 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.
• 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 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.
• 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.
• An aim of the present invention is to solve at least some problems of the prior art.
• the method according to the present invention is mainly characterized in that, what is stated in the characterizing part of claim 1.
• the present invention 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.
• 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).
• 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 amount of G-cells decreases and, consequently, both the basal and post-prandial secretion of gastrin decreases.
• 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.
• 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). Gastrin-17 examination and related biomarkers
• 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.
• GDD gastroesophageal refiux 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-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.
• 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 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.
• 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);
• 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 belong to the scope of the present 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.
• gastrin-17 andH are used as biomarkers for revealing high gastric acid output and a consequent risk of peptic ulcer disease.
• 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
• the examination is being indicative for atrophic gastritis.
• HEALTHY GASTRIC MUCOSA Subject not having the symptoms and/or biomarkers indicating an autoimmune disease i.e. HEALTHY GASTRIC MUCOSA:
• 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. Gastrin-17 examination results and their interpretation
• pangastritis • 5.2.atrophic pangastritis (antrum and corpus both atrophic)
• PPI medication is asked in the anamnestic information and if necessary it should be clarified, what separates situations 3.1. and 3.2.
• 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.
• 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.
• Gastrin- 17 examination measures only gastrin- 17 levels and optionally ⁇ levels directly and thus provides simpler analysis, based on the biomarker levels as detected by the researchers and described earlier in the present specification.
• 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&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; litis) 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 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 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.
• 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).
• hypochlorhydric states such as atrophic gastritis and partial gastrectomy, have long been known to be causes of iron deficiency anemia (27).
• 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 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.
• 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.
• 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.
• 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.”
• 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.
• the use of inaccurate and even misleading tests causes unnecessary costs for healthcare, social security, insurance companies, employers, and for patients themselves.
• 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&IgG antibodies
• results may indicate an ongoing H. pylori infection in spite of negative C- urea breach test and histology results (31 -34).
• 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
• 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 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.
• researchers 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).
• Patent literature
• Pepsinogen A to Pepsinogen C A sensitive Test for Atrophic Gastritis. Scan J Gastroenterol 1989: 24: 870-876.
• Goldkorn I et al Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component. J Biol Chem.l989;264:18768-74.
• hypoglycaemia and meals in duodenal ulcer with and without acid hypersecretion to pentagastrin are hypoglycaemia and meals in duodenal ulcer with and without acid hypersecretion to pentagastrin.
• Helicobacter pylori infection does auto-immuity affect progression to atrophic gastritis? Eur J Gastroenterol Hepatol. 5(Suppl 2):S27-S29.

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• 2014
  • 2014-10-16 CN CN201410548436.4A patent/CN105242045A/zh active Pending
  • 2014-10-16 CN CN202110776404.XA patent/CN113514647A/zh active Pending
• 2015
  • 2015-06-15 WO PCT/FI2015/050431 patent/WO2015189480A1/en active Application Filing
• 2016
  • 2016-06-02 HK HK16106253.1A patent/HK1218159A1/zh unknown

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