AU721826B2 - In vitro determination for detection of tumors of the intestine - Google Patents
In vitro determination for detection of tumors of the intestine Download PDFInfo
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- AU721826B2 AU721826B2 AU67275/98A AU6727598A AU721826B2 AU 721826 B2 AU721826 B2 AU 721826B2 AU 67275/98 A AU67275/98 A AU 67275/98A AU 6727598 A AU6727598 A AU 6727598A AU 721826 B2 AU721826 B2 AU 721826B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Description
NO 98/39473 PCT/EP98/01223 In Vitro Determination for Detection of Tumors of the Intestine The invention relates to the use of an in vitro determination of intraepithelial enteric bacteria, components thereof and reaction products of the host in the latter for the detection of tumors of the intestine and precursors thereof.
Because of their poor prognosis, malignant tumors are a serious threat to human health. Possible therapies of the majority of malignant tumors are still limited at present and depend on the stage of the disease. Early detection of the disease, as well as its precursors, therefore is part of the primary target of clinical scientific research. Carcinogenesis is considered as an interaction of endogenic (genetic) and exogenic factors. While genetic alterations of the cells to date offer few possibilities for therapeutic intervention, it is hoped that understanding of the exogenic carcinogens will lead to a direct use. At the beginning, attention was focussed primarily on chemical and radioactive substances, since the action of these carcinogens is easy to trace in cell cultures and animal experiments. With the discovery of carcinogenic viruses, the infectious causes of tumor development became a primary focus of attention.
Viruses are not the sole pathogens with carcinogenic power.
A connection between certain types of carcinomas and a chronic infection with Schistosoma or liver fluke has been known for a long time.
The role of bacteria was revealed only recently, however.
Thus, Helicobacter pylori was the first bacterium to be categorized as a carcinogen of group I (WHO/IARC Convention in Lyon, 1994 and Logan RPH, Helicobacter Pylori and Gastric cancer, Lancet 1994, 334, 1078-79).
The connection between a permanent infestation of the stomach by Helicobacter pylori and stomach carcinoma is regarded as confirmed. Like stomach cancer, colorectal carcinomas are one of the most common tumor diseases worldwide. In the 1970's, the epidemiological characteristics of the propagation of colorectal carcinomas already led to the assumption that gastrointestinal bacteria are causally connected with the colorectal carcinomas (Aries, V. Crowther, J. Drasar, M. Hill, M. and Williams, R. E. Bacteria and the Etiology of Cancer of the Large Bowel, GUT 10, 334, 1969).
Despite intensive and targeted research, the postulated connection between gastrointestinal bacteria and carcinogenesis could previously be confirmed only for Helicobacter pylori. The reason for this is an extreme complexity of the intestinal flora.
In the colon content, this has bacterial densities of up to 13 /g of dry weight. Attention has thus far been paid to predominantly intraluminal bacteria, however. Whether or not a pathogen, which permanently infests the large intestine mucous membrane, plays a role in the development of colorectal carcinomas is unknown. This could not be answered with the standard known bacteriological methods.
The use of an in vitro detection of intraepithelial enteric bacteria, components thereof and reaction products of the host in the latter, for detection of tumors of the intestine and precursors of tumors of the intestine is now described.
The use is characterized in that for in vitro detection, the following steps are performed: a) Biopsy samples are shaken in an aqueous solution, and the latter are then taken up again in aqueous solution, b) Process step a) is repeated several times, and supernatants are discarded, c) the residues are converted at room temperature into an SDS-containing solution and are carefully and slowly shaken or rotated for resolution of the tissue and the DNA fraction, d) the DNA is purified with phenol and glass pearls and then resuspended in water, e) the DNA that is thus obtained is amplified with universal 16 sRNA primers, and then the intensity of the PCR product of the sample is compared to the intensity of the PCR products of standardized bacterial solutions of certain density, f) positive PCR products are cloned and sequenced and finally g) the DNA sequences that are found in the biopsy are compared to the known bacterial DNA sequences from DNA data banks.
The detection can be modified in many ways. Speciesspecific primers can be used for the PCR. Virulence factors can be detected specifically in the biopsy sample or even in stool specimens. Also, in situ hybridization can be performed. An immunohistochemical and electron microscopic detection of bacteria in the mucous membrane can also be performed. Nuclearmedicine methods such as scintigraphy or breath tests are also conceivable.
The aqueous solution that is used in process step a) of the detection is preferably a physiological common salt solution.
The SDS-containing solution that is used in process step c) of the detection is preferably a 0.5-5% SDS/physiological common salt solution.
The time for the shaking of biopsy samples in process stage a) of the detection is preferably 10-120 s, especially preferably 30-60 s.
The biopsy samples in process stage a) of the detection are preferably taken up in 100-1000 Al of physiological common salt solution, especially preferably in 500 l.
Process step a) of the detection is preferably repeated 5-20 times, especially preferably 7-10 times.
The fractions in process step c) of the detection are preferably added in a volume of 100-500 gl of 0.5-5% SDS/physiological common salt solution, whereby a 1% solution is especially preferred.
The preferred time of shaking and rotating in process stage c) of the detection is 10-24 hours, especially preferably 12 hours.
It is preferably resuspended in process stage d) of the detection in a volume of 10-50 g1 of water, especially preferably in a volume of 20 g1 of water.
The use relates in particular to the detection of E. coli bacteria in biopsies of the large intestine.
In vitro detection is also used in the examination of samples of other bodily compartments such as lymphs and bodily fluids, such as blood, fluids of the gastrointestinal tract and fluids of adjacent tissue of the stomach and the intestine.
Reaction products of the host on the attack with E. ooli or other bacteria that are characteristic of tumor invasion can be detected using commonly used analytical methods, for example an immunoassay.
The use of the in vitro detection according to the invention relates to E. coli, components of E. coli, reaction products of the host on E. coli and related bacteria in the intestinal mucous membrane or other bodily compartments outside of the gastrointestinal lumen.
The following embodiments explain the [word(s) missing] according to the invention, without the latter being limited to the examples.
Example 1 Tissues from biopsies were added to physiological common salt solution and held for two hours at 4°C for further use.
Each sample was crushed vigorously with a grinder (Vortex rod) for 1 minute in a 1.5 ml plastic tube and then added to 500 pl of fresh physiological common salt solution. This process was repeated 8 times. Then, the sample was incubated at room temperature in a 1% SDS-common salt solution and centrifuged off overnight.
DNA is extracted from the supernatant with phenol, further purified with use of a GeneClean Kit and ultimately resuspended in 30 Al of water, which is used for high-pressure liquid chromatography (HPLC).
The 5'-end of the 16S rRNA gene (600 bp) was amplified using PCR with use of a universal primer for bacteria: (c/t)(c/a)TGGCTC AG-3' (g/t)(a/g)CTGCTGGCAC-3' The 13 additional nucleotides (underlined) were bonded to the 5'-end for cloning.
To avoid false positive PCR results, before- and after-PCR stages were performed in various superstructures and parallel control experiments with omission of template DNA.
The amount of bacteria in the samples was measured using electrophoretic comparison of the PCR samples to PCR-standard solutions of bacteria with 105, 104, 103, 102 and 10 colony forming units (cfus) per il according to 25 and 30 thermocycles.
For visualization, agarose gel was treated with ethidium bromide.
Solutions with 10 cfus/gl did not provide any PCR signals.
Sample-PCR-signals with 102 cfus/Al were too weak for additional stages and were considered negative.
To determine the bacterial composition in the sample with positive PCR signals, amplified 16S rRNA sequences in a pCMV-LIC vector were cloned according to a ligation-independent cloning protocol. In a sequencer, between 40 to 200 clones were sequenced for each biopsy. The bacterial sequences were analyzed and, as described above, compared to 16S rRNA primary structures from the data banks.
Example 2 To identify the feasibility of the process according to the invention, biopsies were taken from humans.
In this connection, 82 humans underwent colonoscopies consecutively. The biopsies that were taken from the large intestines were examined for the presence of intraepithelial germs.
Three groups were formed: 1. Normal group: 42 humans with macroscopically and histologically unremarkable colonoscopic findings 2. Adenoma group: 22 humans with tubulovillous adenoma 3. Carcinoma group: 18 humans with carcinomas, of whom 11 had had a colectomy less than three years before, whereby four of the resected patients showed no sign of relapse.
None of the subjects studied had a family polyposis or had taken antibiotics two weeks before the study. One to seven biopsies were taken from each patient studied. These biopsies were studied with the process according to the invention on intraepithelial and related E. coli bacteria.
If possible, biopsies were taken both from the mucous membrane suspected of bearing tumors and from the normal mucous membrane.
Sex, average age and age bracket of the subjects studied in the three groups are as follows: Normal Adenoma Carcinoma Total number of subjects studied: 42 22 18 Average age: 45.6 67.2 Age bracket: 20-84 39-85 38-79 Women: 25 14 8 Average age: 48 68.1 64 Age bracket: 20-84 50-75 38-79 Men: 17 8 Average age: 42.2 65.5 58 Age bracket: 25-60 39-85 46-67 The quantitative determination of the bacterial concentration was carried out in 103, 56 and 47 biopsies of the subjects studied from the normal, adenoma and carcinoma groups.
In 37 of the 42 subjects studied of the normal group, no bacteria were found (0 to 100 cfu/Al). Bacterial concentrations of between 103 and 105 cfu/Al were found, however, in at least one biopsy in all of the subjects studied of the carcinoma group and in 21 of 22 of the subjects studied of the adenoma group.
Bacteria were also found in 23 of the 44 biopsies from the normal mucous membrane of the subjects studied from the adenoma group and 31 of the 37 biopsies from the carcinoma group.
PCR-positive biopsies from the normal mucous membrane and from tumor tissue of the subjects studied were determined as follows: With adenoma With carcinoma Point of removal: normal mucous Tumor normal mucous tumor membrane membrane Total number of biopsies: 44 12 37 PCR-positive biopsies: 23 11 (91) 33 9 26 and 32 PCR products from the biopsies of normal, adenoma and carcinoma groups were cloned and sequenced. In the normal group, onesample each per subject studied was sequenced.
In the adenoma/carcinoma groups, all positive PCR products from the tumor were cloned and sequenced. From the stage biopsies of normal colon sections of the tumor groups, in each case the sample with the maximum bacterial concentration was selected, cloned and sequenced.
of all sequenced clones in 17 of the 21 subjects studied from the adenoma group and 17 of the 18 subjects studied from the carcinoma group received the E. coli sequence.
The following table cites the results of the quantitative PCR and the results of the sequential analysis of cloned PCR products.
Normal Adenoma Carcinoma Number of 42 22 18 subjects studied Subjects 5 21 18 (100%) studied with a positive PCR result Number of 103 56 47 biopsies PCR-positive 8 34 40 biopsies Subjects 1 14 15 studied with dominance** of an E. coli sequence Subjects 0 3 2 (11%) studied with dominance** of an E. coli related sequence Subjects 4 4 1 studied with a mixture of different sequences Bacteroids, clostridia, pseudomonas, E. coli, none of them more than 20% of all clones *In the case of dominance, one and the same sequence is found in more than 80% of all clones 11 It is evident from the results that both in biopsies of normal-looking mucous membrane and in biopsies of tumor tissue of subjects studied with colon carcinoma and colon adenoma, intraepithelial bacteria are present.
Claims (18)
1. Use of an in vitro detection of intraepithelial enteric bacteria, components thereof and reaction products of the host in the latter for the detection of tumors of the intestine and precursors of the tumors of the intestine.
2. Use of an in vitro detection of E. coli, components of E. coli, reaction products of the host on E. coli and related bacteria in the intestinal mucous membrane or other bodily compartments outside of the gastrointestinal lumen for detection of intestinal tumors.
3. Use according to claims 1 and 2, wherein the following steps are performed for in vitro detection: a) Biopsy samples are shaken in an aqueous solution, and the latter are then taken up again in aqueous solution, b) Process step a) is repeated several times, and the supernatants are discarded, c) the residues are converted at room temperature into an SDS-containing solution and are carefully and slowly shaken or rotated for resolution of the tissue and the DNA fraction, d) the DNA is purified with phenol and glass pearls and then resuspended in water, e) the DNA that is thus obtained is amplified with universal 16 sRNA primers, and then the intensity of the PCR product of the sample is compared to the intensity of the PCR products of standardized bacterial solutions of certain density, f) positive PCR products are cloned and sequenced and finally g) the DNA sequences that are found in the biopsy are compared to the known bacterial DNA sequences from DNA data banks.
4. Use according to claim 3, characterized in that the aqueous solution that is used in process step a) is a physiological common salt solution.
Use according to claim 3, wherein the SDS-containing solution that is used in process step c) is a 0.5-5% SDS/physiological common salt solution.
6. Use according to claim 3, wherein the time for the shaking of the biopsy samples in process stage a) is 10-120 s.
7. Use according to claims 3 and 6, wherein the time for the shaking of the biopsy samples in process stage a) is 30-60 s.
8. Use according to claim 3, wherein the biopsy samples in process stage a) are taken up in 100-1000 Al of physiological common salt solution.
9. Use according to claims 3 and 8, wherein the biopsy samples are taken up in 500 gl of physiological common salt solution.
Use according to claim 3, wherein process step a) is repeated 5-20 times.
11. Use according to claims 3 and 10, wherein process step a) is repeated 7-10 times.
12. Use according to claim 3, wherein the fractions are added in process step c) in a volume of 100-500 il of 0.5-5% SDS/physiological common salt solution.
13. Use according to claims 3 and 12, wherein the fractions are added in process step c) in a volume of 100-500 Al of 1% SDS/physiological common salt solution.
14. Use according to claim 3, wherein the time of shaking and rotating in process stage c) is 10-24 hours.
Use according to claims 3 and 14, wherein the time of shaking and rotating in process stage c) is 12 hours.
16. Use according to claim 3, wherein it is resuspended in process stage d) in a volume of 10-50 gl of water.
17. Use according to claims 3 and 16, wherein it is resuspended in process stage d) in a volume of 20 Al of water.
18. Use according to claims 1-17 in samples of bodily compartments such as lymphs and bodily fluids such as blood, fluids of the gastrointestinal tract and fluids of adjacent tissue of the stomach and the intestine.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19711111A DE19711111C2 (en) | 1997-03-06 | 1997-03-06 | In vitro detection for the detection of intestinal tumors |
| DE19711111 | 1997-03-06 | ||
| PCT/EP1998/001223 WO1998039473A1 (en) | 1997-03-06 | 1998-03-04 | In vitro analysis for detection of tumours of the intestine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU6727598A AU6727598A (en) | 1998-09-22 |
| AU721826B2 true AU721826B2 (en) | 2000-07-13 |
Family
ID=7823689
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU67275/98A Ceased AU721826B2 (en) | 1997-03-06 | 1998-03-04 | In vitro determination for detection of tumors of the intestine |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0977888A1 (en) |
| AU (1) | AU721826B2 (en) |
| CA (1) | CA2293704A1 (en) |
| DE (1) | DE19711111C2 (en) |
| WO (1) | WO1998039473A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7101668B2 (en) | 2000-04-18 | 2006-09-05 | National University Of Singapore | Molecular markers |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BRPI0419002A (en) | 2004-08-13 | 2007-12-11 | Indivumed Gmbh | method for detecting and differentiating between colorectal adenoma and / or colorectal carcinoma, method for monitoring the course of colorectal adenoma, use of transthyretin as a biomarker, testing system, arrangement comprising detection molecules |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2290866B (en) * | 1994-06-29 | 1998-10-07 | Reckitt & Colmann Prod Ltd | Detection of Helicobacter pylori infection using antibodies to carbonic anhydrase |
-
1997
- 1997-03-06 DE DE19711111A patent/DE19711111C2/en not_active Expired - Lifetime
-
1998
- 1998-03-04 CA CA002293704A patent/CA2293704A1/en not_active Abandoned
- 1998-03-04 AU AU67275/98A patent/AU721826B2/en not_active Ceased
- 1998-03-04 WO PCT/EP1998/001223 patent/WO1998039473A1/en active IP Right Grant
- 1998-03-04 EP EP98912439A patent/EP0977888A1/en not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| KANAZAWA ET AL, CANCER.DIAG.TREAT.RES., 1996, 77(8), 1701-6 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7101668B2 (en) | 2000-04-18 | 2006-09-05 | National University Of Singapore | Molecular markers |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2293704A1 (en) | 1998-09-11 |
| EP0977888A1 (en) | 2000-02-09 |
| WO1998039473A1 (en) | 1998-09-11 |
| DE19711111C2 (en) | 1999-09-02 |
| AU6727598A (en) | 1998-09-22 |
| DE19711111A1 (en) | 1998-09-17 |
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| FGA | Letters patent sealed or granted (standard patent) | ||
| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |