Diagnosis of endometrial precancers

Details Diagnosis of endometrial precancers Diagnosis of endometrial precancers

2001-05-31

2003-11-18

Huff, Sheela (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007100

Reexamination Certificate

active

06649359

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to immunological methods for diagnosis of conditions characterized by abnormal levels of PTEN protein.
BACKGROUND OF THE INVENTION
PTEN tumor suppressor gene mutations are the most frequent genetic lesion in the highest incidence female genital tract tumor, endometrioid endometrial adenocarcinoma. The role of PTEN in mediating risk conferred by an abnormal hormonal environment and endometrial hyperplasias is uncertain, because of inadequate reagents for protein expression studies in situ and controversy in precancer diagnosis.
Somatic mutation or deletion of the PTEN tumor suppressor gene has been reported in approximately 40% (1;2) and 40 (3)-76% (4), respectively, of endometrial adenocarcinomas. Further evidence for PTEN function within the female reproductive tract is evident in Pten knockout mice that develop complex proliferative endometrial lesions (5). In humans, familial inheritance of mutant PTEN alleles in Cowden syndrome causes multi-organ development of benign hamartomatous and malignant epithelial tumors (6-8), including an elevated incidence of endometrial adenocarcinoma.
A particular variant of endometrial cancer contributes the bulk of PTEN mutations. These are endometrioid endometrial adenocarcinomas (1;2), the most common type [80% (9;10)] of endometrial cancer in the U.S., readily discriminated by routine histopathology from “non-endometrioid” tumors such as papillary serous and clear cell adenocarcinomas, which also occur at this site. Risk for endometrioid endometrial adenocarcinomas increases in subjects with high estrogen levels unopposed by progestins (11), and appearance of a physically distinctive precancerous lesion (12). Interaction between genetic and hormonal events during the premalignant phases of endometrial tumorigenesis is expected, yet has never been precisely elucidated.
Inaccessibility of premalignant tissues, controversy in their diagnosis, and paucity of high-yield candidate genes are longstanding barriers to productive exploration of endometrial precancer biology. PCR methods have improved the analytical repertoire suited to physically small precancers, including detailed mutational (13), clonal (14), and even lineage reconstruction (15) analysis. However, accurate diagnosis of the precancers themselves, typically termed “hyperplasias” in the widely used World Health Organization nomenclature (16), has been difficult to standardize (17), and even when criteria are agreed upon, reproducibility (18) is suboptimal. Previous reports of PTEN mutation in putative endometrial precancers have used subjective diagnostic criteria (19-21).
Thus, there presently is a need for objective, reproducible, and sensitive methods for diagnosis of endometrial precancers.
SUMMARY OF THE INVENTION
Loss of PTEN function by mutational or other mechanisms is an early and progressive event in endometrial tumorigenesis that may occur in response to known endocrine risk factors, and offers an immunohistochemical biomarker for premalignant disease. Unexpectedly, it has been discovered that individual PTEN-negative glands in endometria (particularly in subjects exposed to unopposed estrogen) are distinguishable by immunohistochemistry using PTEN antibodies or antigen-binding fragments thereof, and that these glands are the earliest recognizable stage of endometrial carcinogenesis, followed by proliferation into dense clusters that form discrete premalignant lesions. Accordingly, the invention provides improved methods and compositions for diagnosing endometrial precancers. The methods of the invention also may be used for identifying individuals at risk for endometrial cancer, or individuals at risk for the recurrence of endometrial cancer after treatment. The methods of the invention may also be used for identifying pharmaceutical candidate compounds active in the onset, progression, or regression of endometrial cancer or precancer.
According to one aspect of the invention, methods for determining the likelihood of a group of endometrial cells or an endometrial gland to become cancerous are provided. The methods include performing PTEN antibody or antigen-binding fragment thereof immunohistochemistry on a group of endometrial cells or one or more endometrial glands and determining the binding of the PTEN antibody or antigen-binding fragment thereof to the group of endometrial cells or glands. A reduced amount of PTEN antibody or antigen-binding fragment thereof bound to the group of endometrial cells or glands relative to a control group of cells indicates that the group of endometrial cells or glands has an increased likelihood of becoming cancerous.
In preferred embodiments, the PTEN antibody or antigen-binding fragment thereof is 6H2.1 antibody or antigen-binding fragment thereof.
In certain embodiments, the group of endometrial cells or the one or more endometrial glands and the control group of cells are present in a tissue sample, such as a tissue biopsy. In other embodiments the control group of cells and the group of endometrial cells or glands are the same cell type. In certain preferred embodiments, the amount of binding of the PTEN antibody or antigen-binding fragment thereof to the group of endometrial cells or glands is 50% or less of the binding of the PTEN antibody or antigen-binding fragment thereof to the control group of cells.
In still other aspects of the invention, the methods also include determining the size of a group of endometrial cells or one or more endometrial glands which have reduced PTEN expression. The size of the cells or glands can be measured in control tissues or in a control biopsy of the same subject to establish a baseline size or average size for the cells or glands. Likewise, PTEN expression can be measured in control tissues or in control biopsies of the same subject. In these embodiments, an increased size of the group of endometrial cells or the glands relative to a control group of cells or glands indicates that the group of endometrial cells or glands has an increased likelihood of becoming cancerous.
In some embodiments of the foregoing methods, the group of endometrial cells or glands are obtained from a subject suspected of having endometrial cancer, or from a subject having or suspected of having elevated unopposed estrogen levels. In still other embodiments, the subject is receiving or has received unopposed estrogen treatment.
According to another aspect of the invention, methods for determining regression, progression or onset of a condition characterized by abnormal levels of PTEN protein are provided. The methods include obtaining a level of the amount of PTEN from a sample obtained from a subject and comparing the level to a control as a determination of regression, progression or onset of the condition. In preferred embodiments of these methods, the PTEN antibody is 6H2.1. In certain embodiments, the subject is undergoing drug therapy for a condition characterized by abnormal levels of PTEN protein.
According to still another aspect of the invention, methods for monitoring the progression of endometrial precancers are provided. The methods include determining the expression of PTEN in endometrial cells or glands by PTEN antibody or antigen-binding fragment thereof immunohistochemistry of an endometrial tissue sample obtained at a first time, determining the expression of PTEN in endometrial cells or glands by PTEN antibody or antigen-binding fragment thereof immunohistochemistry of an endometrial tissue sample obtained at a second time, and comparing the expression of PTEN in the endometrial cells or glands at the first time and the second time. Reduced expression of PTEN at the second time relative to the first time indicates progression of endometrial precancers to a cancerous stage. In preferred embodiments the PTEN antibody is 6H2.1.
In some embodiments, the methods also include determining the size of groups of endometrial cells or glands which have reduced PTEN expression in the endometrial tissue sample obtained at the first time and the endometri

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