Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-01
2001-09-11
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091200, C536S024300
Reexamination Certificate
active
06287775
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of molecular biology and cancer diagnostics. More specifically, it is in the field of early detection of carcinomas of the ovarian epithelium.
BACKGROUND OF THE INVENTION
Ovarian cancer is the forth most common cause of death from cancer in women. Further, ovarian cancer is the leading cause of death among women with cancer of the female reproductive tract. As is the case with other common human carcinomas, a series of multiple genetic alterations are believed to be involved in the development of ovarian cancer. Some genetic abnormalities can alter the normal function of tumor suppressor gene products quantitatively and/or qualitatively and contribute to carcinogenesis. However, the genetic alterations involved in ovarian carcinoma remain largely unknown.
Despite the advances in imaging techniques and the availability of serum tumor markers (such as CA125), the majority of ovarian cancer patients are still diagnosed at an advanced stage of the disease—Stage III or IV. Although surgery and intensive chemotherapy have improved to a limited extent the response of ovarian cancer patients to treatment, recurrence and mortality among these patients remains a major problem. Clearly, the development of an early indicator of risk of ovarian cancer will be useful as a tool for early diagnosis and improving prognosis.
The p16 gene (or MSTI gene) has also been identified as a putative tumor suppressor gene. By binding to and inhibiting cyclin-dependent kinase (CDK4), which is activated by cyclin-D in the G1 phase of the cell cycle. It plays a critical role in regulation of normal cell growth. p16 could suppress cell division in a similar fashion to p21 by inhibiting the activity of cyclin-CDK complex. In addition, the p16 gene has already been shown on a high frequency of mutation in tumor cell lines however a much lower mutation frequency was detected in primary tumors.
SUMMARY OF THE INVENTION
An object of the present invention is a method for detecting change in the ovarian epithelium of a human subject, and especially detection of ovarian tumors and carinomas. This may be accomplished by taking a biological sample from the subject containing a p16 gene product, such as p16 mRNA, derived from said subject's ovarian epithelium; measuring the p16 geneproduct, such as by isolating the p16 mRNA from the sample; preparing complementary cDNA to the mRNA; combining the prepared cDNA with primers complementary to p16 DNA target sequences and to a control DNA target sequences; amplifying the DNA in the sample to produce amplification products; quantitating the amplification products; and comparing the quantity of p16 target sequence amplification product in the subject's sample against the quantity of pl
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target sequence amplification product from a similarly treated different sample to detect a change in the subject's ovarian epithelium relative to the different sample. The preferred p16 target sequences are about. 274 bp to 546 bp long.
The different sample may be taken from the same subject that is being tested, from a subject known to have a normal ovarian epithelium, or from a subject known to have an ovarian carcinoma. The biological sample may be peritoneal fluid or any other sample that contains p16 gene product. “p16 gene product” is defined as the p16 gene itself, including p16 cDNA, p16 mRNA and p16 proteins. Both internal controls (in the same reaction mixture as the sample) and external controls (in a separate reaction mixture from the sample) may be practiced in this method. Preferred controls are non p16 gene products that appear at substantially the same level in both normal and tumor samples. &bgr;-tubulin represents one such control.
Measurement and quantitation may be accomplished by any of a number of means known to one skilled in the art, including gel electrophoresis, isotopic labeling and immunoassay with radioactive or other detection method.
Another object of the present invention is to provide a kit for detecting change in the ovarian epithelium of a human subject. The kit comprises a container; reagents for measuring the relative level of a p16 gene product in a biological sample. Preferably the kit will include instructions. Reagents for measuring include those for quantitating the level of a p16 gene product in a biological sample. Additionally, the kit may include a calibrated chart in which the response of the reagents to known levels of a p16 gene product is plotted, and incorporates use of a control, such as a &bgr;-tubulin gene product, as a calibration standard.
A further object of the present invention is a method for early detecting an ovarian carcinoma in a human subject. This is accomplished by taking a biological sample from said subject, the sample containing a p16 gene product derived from said subject's ovarian epithelium; isolating the p16 gene product from the sample; and measuring the pl
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expression. For example isolating p16 mRNA from the sample, preparing complementary cDNA to the mRNA; combining the prepared cDNA with primers complementary to p16 DNA target sequences and to one or more control DNA target sequences; amplifying the DNA in the sample to produce amplification products; quantitating the amplification products; and comparing the quantity of p16 target sequence amplification product in the subject's sample against the quantity of p16 target sequence amplification product from a similarly treated reference sample to detect a change in the subject's ovarian epithelium relative to the reference sample. The reference sample may be taken from the group consisting of a sample from the same subject, a normal sample from a subject known to be ovarian tumor free, and a sample from a subject known to have an ovarian tumor.
Another object of the present invention is a method for producing a calibrated chart by collecting a biological sample from each member of a first group of subjects known to bear an ovarian tumor, and from each member of a second group of subjects known to be free of an ovarian tumor; quantitating the presence of a p16 gene product in each sample, the gene product selected from the group consisting of p16 cDNA, p16 mRNA and p16 protein; and then making a chart that shows the quantity of the p16 gene product for the tumor-bearing versus the tumor-free groups of subjects The quantity of p16 target sequence amplification product in the subject's sample is compared against the calibrated chart of p16 target sequence amplification product from similarly treated normal samples to detect a change in the subject's ovarian epithelium relative to normal subjects. Alternatively, early detection of an ovarian tumor can be accomplished in a subject by comparing the quantity of p16 target sequence amplification product in the subject's sample is compared against the quantity of p16 target sequence amplification product from similarly treated samples from subjects known to have ovarian tumors. Such early detection of ovarian tumors can be accomplished by determining p16 protein expression in the sample to early detect the presence of an ovarian tumor and comparing it with the calibrated chart.
The data base for this chart will allow comparison of p16 gene product expression levels and mutation status with other possible cancer related genes in individual ovarian tumors. The relationship of gene product expression and mutation and the effect this relationship has on malignant potential can provide important insight into candidate genes which have diagnostic potential and which may be candidates for gene therapy.
While a number of objects and advantages are disclosed above, it is well within the ability of one skilled in the art, in view of the present teachings, to conceive of additional objects and advantages that are still within the scope and spirit of the invention as a whole as disclosed herein.
REFERENCES:
Stratagene Catalogue p. 39, Jan. 1988.
O'Brien Timothy J.
Shiqemasa Kazushi
Adler Benjamin Aaron
Board of Trustees of the University of Arkansas
McKelvey Terry
Sandals William
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