WO2002087630A2

WO2002087630A2 - Determination of gastric and oesophageal tumours

Info

Publication number: WO2002087630A2
Application number: PCT/EP2002/004242
Authority: WO
Inventors: Alfredo Di Leo; Michelle Linsalata; Francesco Russo
Original assignee: Eurospital SpA
Current assignee: Eurospital SpA
Priority date: 2001-04-27
Filing date: 2002-04-17
Publication date: 2002-11-07
Anticipated expiration: 2003-10-27
Also published as: AU2002302542A1; ITMI20010885A0; WO2002087630A3; ITMI20010885A1

Abstract

A method for the diagnosis of gastric and oesophageal tumors in human patients which comprises 1) administration of 13C ornithine or a metabolically active precursor thereof, and 2) quantitation of the ¿13?CO2 in the breath.

Description

A METHOD FOR THE DETERMINATION OF GASTRIC AND OESOPHAGEAL TUMOURS

The present invention relates to a method of early, non-invasive diagnosis of neoplastic transformation of the gastric and oesophageal mucosa.

The method is based on determination of the ornithine decarboxylase activity of the gastric and oesophageal tissues, using a breath test with ¹³C labelled ornithine.

BACKGROUND OF THE INVENTION

Tumours of the upper digestive tract (oesophagus and stomach) are of considerable health and social importance. Oesophageal tumours are universally distributed, and are considered to be the second most widespread form of cancer in the world after liver carcinoma. In Italy, the incidence of this tumour is approximately 3-5 cases per 100,000 inhabitants. Half of all tumours of the gastroenteric tract are located in the stomach. This means that out of every 100 deaths from tumours, over 16 are caused by gastric tumours.

Great importance is attributed to pre-cancerous lesions, which are characterised by variations in proliferation and precede the onset of the full- scale neoplastic process. Among the most important modifications affecting the oesophagus is the so-called "Barrett oesophagus" (BE), a condition in which the normal multi-layer squamous epithelium of the oesophagus is replaced by columnar epithelium. Barrett oesophagus predisposes the patient to the development of adenocarcinoma at the same site of the metaplasia. The risk of adenocarcinoma is 30-35 times higher than among the general population.

As far as the stomach is concerned, the intestinal type of gastric cancer, prevalent in men and the elderly, seems to be closely correlated with type B gastritis of the antrum, an inflammatory process often associated with intestinal metaplasia. The latter is often associated with dysplasia, an anatomopathological alteration which in itself is an indicator of increased risk of gastric cancer. In fact, in both metaplasia and dysplasia, the various detectable parameters of cell proliferation are similar to those found in gastric cancer (1 , 2).

Cell proliferation represents a critical stage in the carcinogenesis of the gastro-oesophageal mucosa. Indeed, increased cell proliferation in microscopically normal tissue distant from the carcinoma seems to be one of the first abnormalities which occurs during the development of gastric carcinoma. The polyamines, including spermine, spermidine and putrescine, play a crucial role in cell proliferation and differentiation in normal and neoplastic gastric tissue. In man, higher levels of polyamines have been found in the tissue, serum and urine of patients with stomach and oesophageal cancer than in healthy patients. Ornithine decarboxylase (ODC) is a key enzyme in polyamine synthesis. Impaired regulation of ODC activity is involved not only in invasive processes and tumour metastasis, but also in neoplastic transformation and tumour growth. Therefore, ODC is now considered to be a real oncogene (3, 4). Recent publications demonstrate that cancerous gastrointestinal mucosa presents significantly higher polyamine levels and ODC activity than the healthy mucosa surrounding the tumour. Other studies showed higher ODC mRNA levels in oesophageal carcinoma than in the normal surrounding mucosa; these levels are correlated with the presence of tumour metastasis, the histology of the tumour and its prognosis.

A key role seems to be played by polyamines and ODC also in conditions that predispose the patient to the development of carcinoma of the gastrointestinal tract. Increased ODC activity has been found in Barrett oesophagus. Moreover, ODC activity is considerably increased in the antral biopsies of patients with intestinal metaplasia. A decreasing range of ODC activity has been found in the colon from adenocarcinoma to benign adenomatous polyps and the normal mucosa surrounding the tumour.

Therefore ODC activity represents a marker for cell hyperproliferation, and consequently plays an important role in the early diagnosis of neoplastic transformation of the gastro-oesophageal mucosa. Nowadays, the assay of the enzyme is performed on surgical samples, biopsy tissues or cultured cells, still using a radiometric method that evaluates the CO₂ released by ornithine labelled with the radioactive isotope ¹⁴C (5-7).

For detection of malignant transformations of the gastrointestinal mucosa, a biochemical marker which can be used non-invasively, ie. without the need for tissue samples (biopsy tissue or surgical samples), has not yet been identified. DISCLOSURE OF THE INVENTION

It has now been found that the proliferative activity of gastro- oesophageal mucosa which is cancerous or at risk of neoplastic transformation can be evaluated in vivo by means of a non-invasive diagnostic protocol based on the administration of ¹³C ornithine, and subsequent detection of labelled C0₂ in the breath. In particular, the increase in C levels in the exhaled air is correlated with increased ornithine decarboxylase activity, which, as previously described, is a marker of cell hyperproliferation and neoplastic transformation of the gastro-oesophageal mucosa.

Therefore, the present invention relates to a method of diagnosing gastric and oesophageal tumours which comprises 1) administration of C ornithine or a metabolically active precursor thereof, and 2) determination of the ¹³CO₂ in the breath. This method is known as breath test and has been applied, for example, to diagnosis of Helicobacter pilori infection or bacterial infection in general (US 6067989 and US 5944670), of the liver function (US 61 10122 and US 5961470), of intestinal disorders (US 4830010) and various other conditions.

Breath tests with ¹³CO₂ are based on the administration of a substrate containing a functional group labelled with the stable isotope ¹³C. The functional group is enzymatically detached during the passage through the gastrointestinal tract, during its absorption or during subsequent metabolic steps. Then the detached portion containing the labelled substrate undergoes further metabolic transformations that lead to the production of C0₂, which mixes with the pool of circulating bicarbonates in the bloodstream and is eventually exhaled with the breath. Breath samples are usually collected by having the patient blow into test tubes through a straw. After chromatographic separation, the CO₂ is introduced into a mass spectrometer (IRMS: isotope ratio mass spectrometer) for measurement of the C0₂/ CO₂ ratio. The results of this differential measurement are expressed as delta per thousand [δ C_PDB] (isotopic abundance expressed as the relative difference per thousand from the reference standard, PDB).

Unlike breath tests based on the use of substrates labelled with the radioactive isotope ¹⁴C, the use of ¹³C does not present the disadvantages associated with exposure to radiation which, though low, prevents 14 C from being used in children, pregnant women or repeatedly in the same patient. The breath test does not require the presence of medical personnel, but can be run by skilled technicians trained in the use of the necessary apparatus.

Moreover, the method obviously does not involve any direct action on the human body.

The labelled substrate can be administered orally in the form of a meal or drink to be taken on an empty stomach before the test. In addition to ornithine C, one of its metabolically active precursors (ie. a substance which can generate ornithine after metabolic transformation) such as ornithine alphaketoglutarate can be used. The ¹³CO₂ value detected in the breath, expressed as indicated above (δ¹³Cp_DB)_. will generally be compared with a reference value. The reference value can be obtained by comparing a statistically significant sample of healthy volunteers and patients with a confirmed gastro-oesophageal tumour or pre-cancerous state, and will be determined as the "cut-off value which discriminates between the two groups.

The period after administration of the labelled compound during which the test must be performed to give the best result can be established individually, depending on the response of the patient under examination, and will generally range between a minimum of 15-20 minutes and a maximum of

150-180 minutes.

The data shown in the following examples, which relate to experiments carried out in vivo and in vitro, demonstrate the efficacy of the method and its validation with a conventional ¹⁴C radiometric assay. EXAMPLE 1 - in vitro tests (¹³ C vs. ¹⁴C)

The study was conducted according to the following steps:

1. Development of a method able to detect tissue ODC activity by releasing CO₂ to be analysed by mass spectrometry.

2. Comparison between the radiometric method and the IRMS method, and evaluation of the possibility of detecting quantitative differences between healthy and pathological tissue using C.

For this purpose, samples of gastric tissue, both cancerous and normal tissue surrounding the tumour, were taken from 1 1 patients who underwent surgery to remove stomach tumours and incubated with ornithine labelled with ' C and ¹⁴C; the signal deriving from the catabolism of the labelled ornithine by ODC was determined by mass spectrometry (¹³C) and the radiometric method (¹⁴C). Readings of two assays carried out on samples of normal (N) and pathological (P) gastric tissue Sample ¹³C-N ¹³C-P ¹⁴C-N ¹⁴C-P

1 9.130 6.240 136.0 107.0

2 8.970 32.580 134.0 620.0

3 3.000 3.440 55.0 52.0

4 6.700 11.620 186.0 231.0

5 2.500 10.910 36.0 166.0

6 0.420 22.350 11.0 760.0

7 0.730 5.460 23.0 73.0

8 4.640 1.450 144.0 59.0

9 5.730 8.290 97.0 175.0

10 1.200 14.480 23.0 272.0

11 3.730 15.420 73.0 208.0

¹³C-N ¹³C-P ¹⁴C-N ¹⁴C-P

Mean 4.250 12.02 83.45 247.5

Std. deviation 3.093 9.078 59.51 232.3

Std. error 0.9327 2.737 17.94 70.04 Data are expressed as delta C_PDB for the mass spectrometry assay ( C) and as pmol CO₂/h/mg prot for the radiometric assay (¹⁴C).

The graphs in Fig. 1 show the correlation between the results obtained with the two methods in normal and pathological tissue. EXAMPLE 2 - In vivo tests In vivo experiments were conducted on 4 healthy volunteers to whom

C labelled ornithine was administered. Main features of the ¹³C-Ornithine Breath Test

The patient fasts for at least 6 hours, and is subsequently given an oral dose of citric acid before the test, and a dose of ¹³C labelled ornithine with (1 mg/kg of body weight) dissolved in water; the test requires a single administration; quantitative variations in the exhaled CO₂ are detected by mass spectrometry on serial breath samples taken from time 0 (time of administration of the labelled meal) to the end of the fourth hour.

The curve relating to the four volunteers (Fig. 2) shows a release of

CO₂ due to the reaction catalysed by ODC. This release is demonstrated by the gradual increase of ¹³C_PBB values, which peaks around 60-80 minutes after administration of the labelled meal, and then decreases at the subsequent serial tests until the end of the 240th minute.

REFERENCES

1. Conti EMS and Romagnoli L. "Epidemiologia e fattori di rischio dei tumori dell'esofago e dello stomaco." From "I tumori delle prime vie digestive". Battelli T, Crespi M, Lopez M, Manocchi P, Mattioli R and Pilone A (eds.), Monduzzi publishers, Bologna 1992.

2. Jass JR. "Role of intestinal metaplasia in the histogenesis of gastric carcinoma." J Clin Pathol 1980;33:801-810.

3. Tabor CW, Tabor H. Polyamines. Annu Rev Biochem 1982;53:749-790. 4. Auvinen M, Paasinen A, Andersson LC, Holtta E. "Ornithine decarboxylase activity is critical for cell transformation." Nature 1992; 360:355-358.

5. M. Linsalata, F. Russo, A. Cavallini, P. Berloco, A. Di Leo. "Polyamines, Diaminooxidase (DAO) and Ornithine Decarboxylase (ODC) Activity in Colorectal Cancer and in Normal Surrounding Mucosa." Dis Colon Rectum, 1993; 36: 662-667.

6. F. Russo, M. Linsalata, I. Giorgio, M.L. Caruso, R. Armentano, A. Di Leo. "Polyamine levels and ODC activity in intestinal-type and diffuse-type gastric carcinoma." Digest Dis Sci 1997; 42: 576-579. 7. M. Linsalata, F. Russo, M Notarnicola, P. Berlocco, A. Di Leo. "Polyamine profile in human gastric mucosa infected by Helicobacter pylori." Ital J Gastroenterol Hepatol 1998; 30: 484-489.

Claims

1. Method for the diagnosis of gastric and oesophageal tumours in a human patient, which comprises: 1) administration of ¹³C ornithine or a metabolically active precursor thereof, and 2) quantitation of the ¹³CO₂ in the breath.

2. Method as claimed in claim 1, in which the quantity of CO₂ is determined as the ¹³CO₂/¹²CO₂ ratio by chromatographic separation of the exhaled mixture and subsequent mass spectrometry analysis of the CO₂ containing fraction .

3. Method as claimed in claim 2, including comparison of the CO₂/ CO₂ ratio with a reference value.

4. Method as claimed in claims 1-3, in which the quantitation of CO₂ in the breath is performed between 15 and 180 minutes after the administration of C ornithine.

5. Method as claimed in any of the preceding claims, for diagnosis of "Barrett oesophagus" and pre-cancerous stomach lesions.