Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-01
2002-04-16
Ungar, Susan (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007200, C435S007230
Reexamination Certificate
active
06372439
ABSTRACT:
BACKGROUND OF THE INVENTION
The United States government has certain rights in this invention by virtue of a grant from the National Institutes of Health NIDDKD to James R. Goldenring.
Gastric cancer worldwide is the second highest cause of death from cancer. While rates of gastric cancer have been decreasing in the United States, gastric cancer prevalence remains high in other parts of the world, especially in Asia. Presently, patients in endemic regions for gastric cancer, especially Japan and China, undergo screening by upper endoscopy. There is no alternative at present for discovery of early cancer other than by endoscopy. Grading of these endoscopies is based solely on hematoxylin and eosin staining. There are no prognostic markers that could indicate those at risk for future cancer development. Therefore, while biopsies can reveal early gastric cancers, patients in high risk regions who are initially negative by endoscopy must be followed with endoscopies to rule out future developments.
The epidemiological association between chronic
H. pylori
infection and gastric carcinoma is now well established, such that the Working Group Meeting of the International Agency for Research on Cancer with the World Health Organization recently classified
Helicobacter pylori
as a group 1 human gastric carcinogen (Peura, D. A.
Gastroenterology
113, S4-S8 (1997)). Parsonnet and colleagues reported that 84% of patients with gastric cancer had serum antibodies against
H. pylori
, as opposed to 66% of uninfected matched controls (Parsonnet, et al.
New Eng. J. Med
. 325, 1127-1131(1991)).
H. pylori
infection induces a chronic gastritis progressing to atrophic gastritis, foveolar hyperplasia and intestinal metaplasia (Mobley, H. T. L.
Gastroenterology
113, S21-S28 (1997)). Recent studies have indicated that chronic gastritis associated with
Helicobacter pylori
contributes to the development of gastric adenocarcinoma (Peura, D. A.
Gastroenterology
113, S4-S8 (1997); Parsonnet, et al.
New Eng. J. Med
. 325, 1127-1131 (1991); Forman et al.
Br. J. Med
. 302, 1302-1305 (1991); Normura, et al.
New Eng. J. Med
. 325 1132-1136 (1991)). Nevertheless, the mechanism of progression from chronic gastritis to malignant disease remains unclear, and the relationship of intestinal metaplasia,
H. pylori
infection and the development of cancer continues to be controversial. Moreover, while an association between gastric cancer and infection with
H. pylori
has recently been established, the cell of origin for gastric adenocarcinoma is controversial. This does not establish a mechanism between the bacteria and the cancer, and provides little or no guidance for correlating treatment of, or risk associated with,
H. pylori
as it relates to development of gastric cancer.
It is therefore an object of the present invention to provide screening methods for early gastric cancer.
It is a further object of the present invention to provide means for assessing the degree of early gastric cancer and for screening and following patients at risk for gastric cancer.
It is a still further object of the present invention to provide means for serological testing for patients at risk of gastric cancer.
SUMMARY OF THE INVENTION
It has been determined that a specific metaplastic lineage that contains immunoreactivity for a trefoil polypeptide, spasmolytic peptide, is associated with and gives rise to the vast majority of human adenocarcinomas. The identification of this Spasmolytic Polypeptide Expressing Metaplasia (SPEM) is a major factor for grading of biopsies of the stomach to assess risk for gastric cancer. It also forms the basis of a method for serological screening for those at risk for gastric cancer. In a preferred embodiment, antibodies to spasmolytic peptide (hSP) are used in immunostaining of biopsies of gastric tissue obtained by endoscopy for grading biopsies. Those patients having these cells, characterized by a morphology more typical of a type of cell present normally in the intestine and not stomach, Brunner's gland cells, are at risk of developing adenocacinoma. Since these cells express hSP, antibodies or nucleic acid probes hybridizing to mRNA encoding hSP, can be used for rapid detection of the cells in tissue biopsies. The antibodies can also be used in serological tests for screening and following patients at risk for gastric cancer. In combination with evidence of previous or present invention with
H. pylori
, the tests are predictive of the likelihood of developing adenocarcinoma.
DETAILED DESCRIPTION OF THE INVENTION
Metaplastic cell lineages arising in response to chronic injury are precursors for the evolution of dysplasia and adenocarcinoma. This sequence is well characterized in the case of the Barrett's epithelium, a columnar specialized intestinal metaplasia in the distal esophagus of patients with esophageal reflux (Haggitt, R. C.
Hum. Pathol
. 25, 982-993 (1994)). While a subtype of intestinal metaplasia has been associated with gastric adenocarcinoma (Dixon, et al.
Am. J. Surg. Pathol
. 20, 1161-1181 (1996); Filipe et al.
Int. J. Cancer
. 57, 324-329 (1994)); Correa, P.
Cancer Res
. 48, 3554-3560 (1988)), the link between these lineages and the evolution of gastric adenocarcinoma has not been clear. It has now been determined that there is an association between SPEM, detectable in biopsies based on the presence of cells having a morphology similar to Brunner's gland cells, and adenocarcinoma. Although normal cells in the stomach, such as mucus neck cells, express hSP, these cells are not predictive of adenocarcinoma.
I. Methods and Reagents for Screening
A. Histology of the Gastric Tissues
Presently, gastric mucosal biopsies are fixed in formalin and embedded in paraffin. Microtome sections of tissues are then stained with hematoxylin and eosin. These stained slides are examined for loss of parietal cells (oxyntic atrophy), ulceration, inflammatory infiltrates, and alterations in cell lineages including increased numbers of surface mucous cells (foveolar hyperplasia), the presence of goblet cells (intestinal metaplasia), as well as dysplasia and adenocarcinoma. Dysplasia and adenocarcinoma are judged by changes in nuclear morphology, loss of cytoplasmic space, loss of polarity and invasion of submucosa or vasculature. Brunner's glands are not present in the normal stomach but can be observed in the duodenum.
B. Markers of SPEM
Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a group of peptides, including BCEI, expressed from the pS2 gene; hITF, expressed from the TFF3 gene; and hSP, expressed from the SML1 gene. These peptides are abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenance of surface integrity. (Schmitt, et al.,
Cytogenet. Cell Genet
. 72(4), 299-302 (1996)). Human spasmolytic peptide (hSP) was identified by Romasetto, et al.,
EMBO J
., 9(2), 407-414 (1990), based on homology to pancreatic spasmolytic polypeptide, sequenced and determined to be separately encoded on the genome from pS2. The gene sequence and amino acid sequences of hSP can be obtained from GenBank, accession number 1477545. Both are present in normal stomach epithelium. The patterns and timing of the expression of the trefoil peptides are different from each other. It is thought that S2 plays an important role in the proliferation of intestinal epithelial cells during the acute phase of mucosal ulceration, whereas ITF may be involved in differentiation of the cells, particularly to form goblet cells, during the recovery phase of acute colitis. (Itoh, et al.,
Biochem. J
. 318(Pt 3), 939-944 (1996)). Immunostaining for SP in the intact mucosa has been determined to be confined to the mucous neck cells, but following exposure to stress it was significantly enhanced and occurred also in the cells of the basal region of gastric glands, as reported by Konturek, et al.,
Regul. Pept
. 68(1), 71-79 (1997). Konturek, et al. (1997) proposed that SP plays an important role in healing of stre
Goldenring James R.
Lee Jeffrey R.
Schmidt P. Henry
Calkins, Esq. Charles W.
Kilpatrick & Stockton LLP
Rothchild, Esq. Cyntiha B.
Ungar Susan
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