WO2000070348A1

WO2000070348A1 - Methods for predicting and/or diagnosing the risk of gastric cancer

Info

Publication number: WO2000070348A1
Application number: PCT/AU2000/000441
Authority: WO
Inventors: Robert Llewellyn Clancy; Gerald Pang
Original assignee: Onco Alert Pty Ltd
Priority date: 1999-05-14
Filing date: 2000-05-15
Publication date: 2000-11-23
Also published as: CA2372761A1; KR20020033093A; AUPQ037799A0; EP1183540A1; BR0010558A; HK1044988A1; JP2002544520A; MXPA01011721A; EP1183540A4; CN1355885A

Abstract

The present invention relates to methods of predicting the risk of developing cancer and in particular to a method for diagnosing, and/or predicting the risk of developing gastric cancer in a subject infected with Helicobacter.

Description

METHODS FOR PREDICTING AND/OR DIAGNOSING THE RISK OF

GASTRIC CANCER

TECHNICAL FIELD

The present invention relates to methods of predicting the risk of developing

cancer and in particular to a method for diagnosing, and/or predicting the risk of

developing, gastric cancer in a subject infected with Helicohacter pylori.

BACKGROUND

Helicohacter pylori infection is now recognised as an essential pre-requisite for the

development of gastric cancer. About 30% of the population become infected with this

bacterium and commonly present with chronic gastritis. This may be complicated by

gastric or duodenal ulceration, or may present as non-ulcer dyspepsia. A sizeable

number of carriers are asymptomatic. However, in a small number of patients with H.

pylori, their condition evolves through stages (including epithelial cell metaplasia and

dysplasia) into neoplasia. The factors responsible for this evolution are complicated, but

involve geographical, environmental and genetic parameters. Of particular importance is

the host response. Current evidence supports the theory that a particular T cell response

known as Thl (characterised by production of γinterferon (γlFN) but not interleukin-4

(IL-4)) as promoting mucosal damage. Alternatively, a ThO response can occur which

includes balanced production of these cytokines (γlFN and IL-4) and which favours

protection from mucosal damage. Patterns of mucosal cytokine response associated with

neoplastic transformation and tumour progression have not been described.

Current Management Practice of H. pylori Infection

H. pylori is an essential component of the chain of events leading to chronic

gastritis and peptic ulceration. Eradication of infection with antibiotics induces an 80- 90% cure rate of peptic ulceration. A widely accepted treatment paradigm is based on

detection of infection using antibody assays, followed by combination antibiotic therapy

without prior endoscopic diagnosis. Endoscopy, before eradication therapy is generally

accepted when 'danger' symptoms (eg, severe pain, bleeding) occur, or a significant risk

of gastric cancer is present. However, endoscopy is a procedure which is associated with

its own risks and is to be avoided if possible.

At present, no non-invasive test exists which would allow for prediction or

diagnosis of gastric cancer in patients which Helicohacter infection. Such a test would

be particularly valuable for patients who present with relatively mild symptoms but who

are identified as being in a "high risk" category and who would otherwise automatically

be required to undergo an endoscopy - with its attendant risks. Even in patients who

present with "danger symptoms" and who may still require an endoscopy, such a non-

invasive test could be used as a complementary tool in diagnosis. This change in

practice could have a significant impact on health economics.

It is an object of the present invention to overcome or ameliorate at least one of the

disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

It has surprisingly been found that mucosal IgG2 anti-H. pylori antibody and γlFN

levels are decreased and IL-4 levels are elevated in patients having Helicohacter

infection when gastric cancer or precancer lesions (metaplasia and dysplasia) are present.

These changes are also reflected in the blood of such patients. However, the changes

are not seen in other disorders in which Helicohacter pylori is colonising the gastric

mucosa. According to a first aspect, the present invention provides a method of diagnosing

and/or determining the risk of developing gastric cancer in a subject with a Helicohacter

infection, including:

a) determination of IgG2 anti-H. pylori antibody level in the subject;

b) comparison of the IgG2 anti-H. pylori antibody level with a predetermined

control IgG2 anti-H. pylori antibody level, wherein a reduction in the level of IgG2 anti-

H. pylori antibody in the subject compared to the control indicates the presence and/or

increased risk of developing gastric cancer.

According to a second aspect, the present invention provides a method of

diagnosing and/or determining the risk of developing gastric cancer in a subject with a

Helicohacter infection, including:

a) determination of γlFN level in the subject;

b) comparison of the γlFN level with a predetermined control γlFN level, wherein

a reduction in the level of γlFN in the subject compared to the control indicates the

presence and/or increased risk of developing gastric cancer.

According to a third aspect, the present invention provides a method of diagnosing

infection, including:

a) determination of IL-4 level in the subject;

b) comparison of the IL-4 level with a predetermined control IL-4 level, wherein

an elevation in the level of IL-4 in the subject compared to the control indicates the

presence and/or increased risk of developing gastric cancer. According to a fourth aspect, the present invention provides a method of

Helicohacter infection, including a combination of a method according to the first aspect

and/or a method according to claim second aspect and/or a method according to the third

aspect.

According to a fifth aspect, the present invention provides a method of diagnosing

infection, including a combination of a method according to the second aspect and a

method according to the third aspect.

Preferably, the Helicohacter infection is a Helicohacter pylori infection.

Preferably, the IgG2 anti-H. pylori antibody, γlFN and/or IL-4 levels are

determined by detection in a sample of biological fluid such as for example blood,

saliva, gastric fluid and the like.

Preferably, the measurement of IgG2 anti-H. pylori antibody, γINF and/or IL-4

either simultaneously provides, or can be performed simultaneously with, a method

which provides an indication of H. pylori status.

Preferably, the IgG2 anti-H. pylori antibody and/or γlFN and/or IL-4 are detected

by a near-subject assay. The assay may, however, also be a laboratory-based test.

Preferably, the assay is an antibody assay although it will be understood that other

known methods of measurement can also be effectively used. Most preferably, the assay

is an ELISA . According to a sixth aspect, the present invention provides a method of predicting

the risk of, and/or diagnosing, gastric cancer in a subject having a Helicohacter infection

by

a) determining the frequency of IgG2 anti-H.pylori antibody- and/or γlFN- and/or

IL-4-producing cells in the subject's blood; and

b) comparison of the frequency of IgG2 anti-H.pylori antibody- and/or γlFN-

and/or IL-4-producing cells in the subject's blood with a predetermined control level,

wherein a reduction in the level of IgG2 anti-H.pylori antibody- and/or γlFN-producing

cells and/or an elevation in IL-4-producing cells in the subject's blood indicates the

presence and/or increased risk of developing gastric cancer.

It will be clear to the skilled addressee that the blood may be purified to provide an

enriched white blood cell population and the white blood cell population may be further

fractionated to obtain specific cell populations.

Preferably, the IgG2 anti-H.pylori antibody- and/or γlFN- and/or IL-4-producing

cells are stimulated with H. pylori antigen prior to measurement of IgG2 anti-H.pylori

antibody and/or γlFN and/or IL-4.

According to a seventh aspect, the present invention provides a method of

predicting the risk of, and/or diagnosing, gastric cancer in a subject having a

Helicohacter infection by

a) determining the frequency of IgG2 anti-H.pylori antibody and/or γlFN and/or

IL-4-producing cells in the subject's gastric mucosa; and

b) comparison of the frequency of IgG2 anti-H.pylori antibody and/or γlFN and/or

IL-4-producing cells in the subject's gastric mucosa with a predetermined control level, wherein a reduction in the level of IgG2 anti-H.pylori antibody- and/or γlFN-producing

cells and/or an elevation in IL-4-producing cells in the subject's gastric mucosa indicates

the presence and/or increased risk of developing gastric cancer.

Preferably, the cells are derived from a biopsy sample.

cells are detected by flow cytometry.

Control levels of IgG2 anti-H. pylori antibody, IL-4 and/or γlFN can be established

in samples of biological fluids obtained from normal individuals, ie. those not having an

established H pylori infection, or they can be established in samples from subjects with

H. pylori infection who have uncomplicated chronic gastritis or asymptomatic infection

or the like. In certain cases, in which subjects are followed prospectively, control levels

may be internal levels, i.e. the subject's own control levels.

The method of the present invention can also be used to diagnose and/or determine

the risk of developing pre-cancer lesions such as metaplasia or dysplasia by way of

measurement of IgG2 anti-H. pylori antibody, γlFN and/or IL-4.

It will be clear to the skilled addressee that ratios of IgG2 anti-H. pylori antibody,

γlFN or IL-4 to other parameters such as, for example total IgG anti-H. pylori antibody

may be useful as a predictor of, or in the diagnosis of, gastric precancerous or cancerous

conditions, including situations in whch dysplasia and metaplasia are present.

Refinement of the prediction and/or diagnosis of precancerous or cancerous conditions

may require that specific ratios be utilised, such as the ratio of IL-4:γIFN, IgG2:total IgG

or IgG2:IgGl. However, other ratios may also be useful. In the context of the present invention, the abbreviations "γlFN" and "IFNγ" have

been used interchangably in the specification to refer to the cytokine γ interferon.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Detection of IL-4 in supematants of gastric mucosal cultures from subjects

with gastric cancer or pre-cancer lesions (metaplasia or dysplasia). In uncomplicated H

pylori infection (or in benign peptic ulcers) a Thl pattern of cytokine (eg, γlNF) is

found.

Figure 2. This figure illustrates a high level of correlation between secretion of IL-4

from mucosal biopsies, and H pylori antigen stimulated blood T cells. IL-4 was not

secreted from antigen stimulated T cells in untreated subjects with uncomplicated

chronic gastritis and H pylori infection.

Figure 3. Cytokine (IL-8, IL-4 and γlFN) production in the gastric mucosa of subjects

infected with H. pylori..

Figure 4. IgGl and IgG2 anti-H. pylori antibody levels in serum of H. rμ/or.-infected

subjects having various gastrointestinal disorders.

Figure 5. IgGl and IgG2 anti-H. pylori antibody levels in serum of H. j-rμ/ rz^'-infected

subjects having various gastrointestinal disorders.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention will now be described in more detail with reference to non-limiting

examples. It was previously known that total IgG anti-H. pylori antibody levels in blood and

gastric mucosa can be used as an indicator of H. pylori status. In the following examples,

therefore, it will be understood that, while IgG anti-H. pylori can be utilised as a general

indicator of H. pylori status, the invention also relates to the measurement of the IgG2

subclass which can be used as a predictor of, or in the diagnosis of, gastric cancer.

Techniques for measurement of cytokines and antibodies in human samples are

well-known in the art and protocols and reagents are readily available. Examples of

some of the techniques used are indicated below as an illustration of how some

measurements may be performed.

Unless indicated otherwise, standard techniques which can be ascertained from

standard texts and laboratory manuals may be employed.

Example 1 Determination of cytokine and antibody levels in a blood sample

The standard assay involves coating microwells of a 96- well microtitre plate with

monclonal anti-IL-4 ( MoAb ). After removal of antibody and washing with

PBS/Tween 20, 100 uL of whole blood is added to each well containing an equal

volume of AIM-V medium. After incubation for 24 hrs at 37°C, the plasma

supernatant is removed for measurement of γlFN by ELISA (Figure 1). The amount of

IL-4 captured by IL-4 MoAb in each well is measured by ELISA (Figures 1 and 2).

IgGl and IgG2 subclass anti-H. pylori levels or IgG2/IgG ratios in serum from clotted

blood or plasma supernatant (above) are measured by ELISA (Figures 4, 5). All samples

are stored at -80°C until assay. Assay svstem for measurement of IL-4 alone or IL-4 and anti-H pylori IgG antibodies at

the same time.

Wells of a 96-well flat-bottomed microtitre plate are coated with 2 μg/mL of

monoclonal anti-IL-4 capture antibody in sodium bicarbonate buffer pΗ 8.5. After

removal of antibody solution, an equal volume of freshly collected whole blood is added

to each well. After incubation for 24 hrs at 37°C, the plasma supernatant as removed and

IL-4 bound is detected by reaction with biotinylated anti-IL-4 antibody and

strepavidin-peroxidase conjugate. The amount of IL-4 is measured by colour

development read in a plate reader with the appropriate standards.

On the same plate, IgG anti-H. pylori antibody is detected by adding the plasma

supernatant to wells coated with 4 ug/mL of H. pylori antigens using an ELISA assay.

The results are shown in Table 1.

Table 1 IL-4 production and anti-H. pylori IgG antibody in whole blood

Subject IL-4 production (pg/mL) H. pylori IgG (ELISA Index) SΪ 42.77 0.696

52 9.4 1.61

53 13.49 1.86

54 108.25 0.95

55 9.4 1.83 S6 18.1 0.67

57 9.4 4.32

58 19.41 3.22

59 56.64 3.48 S10 15.1 3.42 Sll 9.4 0.12 Example 2 Frequency of IL-4 and γlFN producing cells in gastric mucosa

Gastric T cells are isolated from biopsy tissues obtained at endoscopy. The tissues

is rinsed in ImM dithiothreitol and ImM EDTA to remove epithelial cells and

intraepithelial cells before extraction of lamina propria T cells in serum-free AIM-V

medium containing 40 U collagenase (Worthington Biochemical) for 2-3 hrs. The

viability of the mononuclear cells after removal of undigested materials was >90% by

trypan blue exclusion. Isolated gastric mononuclear cells from individual biopsies are

usually too low (about 0.503 x 10 cells per biopsy) for antigen-mediated re-stimulation

in bulk cultures. Therefore, IL-4 and γlFN producer frequencies in each cell isolate are

determined by intracellular staining and then analysed on the FACS Vantage using 3-

colour flow cytometry. Isolated gastric cells were activated with PMA and ionomycin

and PMA, stained with PerCP-CD3 monoclonal antibody (Becton Dickinson) and then

processed for intracellular staining with FITC-γlFN and PE-IL-4 monoclonal antibody as

described above.

Unless indicated otherwise above, standard techniques which can be ascertained

from standard laboratory texts were used.

Table 2 provides an example of the predictions/diagnoses which can be made on

the basis of the above tests.

Table 2

Example 3 Frequency of IL-4 and γlFN producing cells in peripheral blood

Intracellular cytokine staining and detection by flow cytometry is used to estimate

cytokine-producer frequencies of IL-4 and γlFN amongst different subjects. This allows

comparison of results obtained from gastric biopsy tissue where analysis by limiting

dilution culture following antigen re-stimulation is not possible due to low numbers of

cells isolated per biopsy. Peripheral blood mononuclear cells or whole blood is activated

with phorbol myristate acetate (PMA, 50 ng/mL) and 1 μM ionomycin for 4-5 hrs in the

presence of 2 μM monensin, fixed, permeabilised and stained with FITC/PE labelled

γIFN/IL-4 (Bectin-Dickinson). γlFN and IL-4 frequencies are then analysed by flow

cytometry with matched isotype IgG control and gated for lymphocytes.

The frequencies of IL-4 and γlFN producing cells in peripheral blood from

subjects with or without H. pylori infection are shown in Tables 3 and 4. The ratios of

γIFN:IL-4 producing cells were higher in subjects infected with H. pylori than in

non-infected subjects.

Limiting dilution analysis was used to determine quantitative estimates of the

frequency of circulating IL-4 and γlFN-secreting cells in blood using short-term cultures

stimulated with Ηp recombinant antigen (citrate synthase of Ηp 0310). A non-protective

recombinant antigen Ηp-0162 was used as a negative control. Cells are seeded in V-

bottomed 96-well microplate using twofold dilution from 10³ to 2.5 x 10 cells at 24

replicates per cell concentration. Cultures were stimulated with a predetermined

concentration of citrate synthase or Hp 0310 antigen in the presence of rIL-2 (5 U/mL)

for 3 days. Controls contained no responder cells or responder cells in medium and rlL-

2 without antigen. As IL-4 is unstable an antibody capture method is used with bound IL-4 measured by ELISA using a matched antibody pair (Endogen/CSL). γlFN

production is measured in the supernatant by standard methods. Frequencies of

peripheral blood mononuclear cells producing IL-4 and γlFN are calculated by

maximum likelihood method using appropriately validated computer software.

Table 3 Cytokine producing cells in H. pylori antibody POSITIVE subjects

Subjects γIFN(%) IL-4 (%) γIFN:IL-4 ratio

SI 18.3 2.5 7.3 S2 25.4 3.3 7.6

S3 26.0 11.5 2.3

S4 9.6 2.6 3.7

S5 14.8 5.6 2.6

Mean ± SE 4.7 ± 1.15*

Table 4 Cytokine producing cells in H. pylori antibody NEGATIVE subjects

Subjects γlFN (%) IL-4 (% γIFN:IL-4 ratio

S6 24.8 28.3 0.9

S7 8.9 3.0 3.0

S8 8.1 2.6 3.1

S9 29.9 29.1 1.0

S10 25.9 17.2 1.5

Mean ± SE 1.9 ± 0.48*

* p=0.054

Figures 1 to 5 provide results obtained utilising the tests exemplified below in studies of

subjects having various gastrointestinal conditions i.e. reflux, gastritis, duodenal ulcer,

gastric ulcer and gastric cancer. The Figures are self-explanatory and show that levels of IgG2, γlFN and IL-4 can be used as predictors of, and in the diagnosis of, gastric cancer

in patients having H. pylori infection.

Although the invention has been described with reference to specific examples, it

will be appreciated by those skilled in the art that the invention may be embodied in

many other forms without departing from the spirit or intent of the inventive concept.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of diagnosing and/or determining the risk of developing gastric cancer

in a subject with a Helicohacter infection, including:

a) determination of IgG2 anti-H. pylori antibody level in the subject;

b) comparison of the IgG2 anti-H. pylori antibody level with a predetermined

increased risk of developing gastric cancer.

2. A method of diagnosing and/or determining the risk of developing gastric cancer

in a subject with a Helicohacter infection, including:

a) determination of γlFN level in the subject;

presence and/or increased risk of developing gastric cancer.

3. A method of diagnosing and/or determining the risk of developing gastric cancer

in a subject with a Helicohacter infection, including:

a) determination of IL-4 level in the subject;

presence and/or increased risk of developing gastric cancer.

4. A method of diagnosing and/or determining the risk of developing gastric cancer

in a subject with a Helicohacter infection, including a combination of a method according to claim 1 and/or a method according to claim 2 and/or a method according to

claim 3.

5. A method of diagnosing and/or determining the risk of developing gastric cancer

in a subject with a Helicohacter infection, including a combination of a method

according to claim 2 and a method according to claim 3.

6. A method according to any one of claims 1 to 6 wherein the Helicohacter infection

is a Helicohacter pylori infection.

7. A method according to any one of claims 1 to 7 wherein the IgG2 anti-H. pylori

antibody, γlFN and/or IL-4 levels are determined by detection of the levels in a sample

of biological fluid.

8. A method according to claim 7 wherein the biological fluid is blood.

9. A method according to claim 7 wherein the biological fluid is saliva.

10. A method according to claim 7 wherein the biological fluid is gastric fluid.

11. A method according to any one of claims 1 to 10 wherein the measurement of

IgG2 anti-H. pylori antibody, γINF and/or IL-4 either simultaneously provides, or can be

performed simultaneously with, a method which provides an indication of H. pylori

status.

12. A method according to any one of claims 1 to 11 wherein the IgG2 anti-H. pylori

antibody, γlFN and/or IL-4 are detected by a near-subject assay.

13. A method according to any one of claims 1 to 11 wherein the assay is a laboratory-

based test.

14. A method according to claim 12 or claim 13 wherein the assay is an antibody

assay.

15. A method according to claim 14 wherein the antibody assay is an ELISA.

16. A method of predicting the risk of, and/or diagnosing, gastric cancer in a subject having a Helicohacter infection by

IL-4-producing cells in the subject's blood; and

b) comparison of the frequency of IgG2 anti-H.pylori antibody- and/or γlFN-

presence and/or increased risk of developing gastric cancer.

17. A method according to claim 16 wherein the blood is purified to provide an

enriched white blood cell population.

18. A method according to claim 17 wherein the white blood cell population is further

fractionated to obtain specific cell populations.

19. A method according to any one of claims 16 to 18 wherein the IgG2 anti-H.pylori

antibody- and/or γlFN- and/or IL-4-producing cells are stimulated with H pylori antigen

prior to measurement of IgG2 anti-H.pylori antibody and/or γlFN and/or IL-4.

20. A method of predicting the risk of, and/or diagnosing, gastric cancer in a subject

having a Helicohacter infection by

a) determining the frequency of IgG2 anti-H.pylori antibody and/or γlFN and/or

IL-4-producing cells in the subject's gastric mucosa; and

the presence and/or increased risk of developing gastric cancer.

21. A method according to claim 20 wherein the cells are derived from a biopsy

sample.

22. A method according to claim 20 or claim 21 wherein of IgG2 anti-H.pylori

antibody and/or γlFN and/or IL-4-producing cells are detected by flow cytometry.