Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1998-05-14
2001-01-30
Johnson, Nancy A. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S007230, C435S007210, C435S967000, C435S960000, C436S501000, C436S503000, C436S811000, C530S387100, C530S389100, C530S388100
Reexamination Certificate
active
06180333
ABSTRACT:
BACKGROUND OF THE INVENTION
The eukaryotic cell cycle is controlled by protein complexes composed of cyclins and cyclin-dependent kinases (cdks). The regulatory function of cdks is achieved by phosphorylation of key substrates, such as the members of the retinoblastoma gene family. Activity of cdks is regulated by post-translational modification and by the association or dissociation with inhibitory subunits designated cyclin-dependent-kinase inhibitors (CKIs). Two families of these inhibitors have been identified in mammalian cells. Members of each of the two families share a high percentage of sequence homology, in addition to their specificity of interaction with cdks. The first family, which includes p21 (also known as Cip1, Pic1, Sdi1, mda6 and Waf1), p27 (also known as Ick, Kip1 and Pic2) and p57 (also known as Kip2) preferentially inhibit cdk2. The second family, which includes p16 (also known as Ink4A, Mts1, Cdkn2 and Cdkn4i), p15 (also known as Ink4B and Mts2), p18 (also known as Ink4C and Ink6A) and p19/p20 (also known as Ink4D and Ink6B) preferentially bind to and inhibit cdk4 and cdk6. Results from several studies indicate CKIs to be the products of potential tumor-suppressor genes (MacLachlan et al. (1995)
Crit. Rev. Eukaryotic Gene Expression
5:127-156).
p27 was first identified in complexes with cdk2/cyclin E in Transforming Growth Factor-&bgr; (TGF-&bgr;) arrested cells (Koff et al. (1991)
Cell
66:1217-1228). p27 protein associates with cyclin E/cdk2 and Cyclin A/cdk2 complexes and inhibits their activities. It is a negative cell cycle regulator implicated in G1 phase arrest by TGF-&bgr; cell contact, inhibition agents that increase the level of cyclic AMP, staurosporin, lovastatin, tamoxifen and rapamycin. Overexpression of p27 protein in mammalian cells induces a G1 block of the cell cycle (Polyak et al. (1994)
Cell
79: 59-66; Toyoshima, T. and Hunter, T. (1994)
Cell
78: 67-74). In addition, high levels of p27 have been found in quiescent cells thus suggesting a role for p27 in maintaining cells in G0 (Nourse et al. (1994)
Nature
372:570-573). Levels of p27 decrease as cells reenter the cell cycle, mostly due to ubiquitin-proteosome dependent degradation (Pagano et al. (1995)
Science
269:682-685). No structural alteration of p27 gene has been reported in human neoplasms to date (Bullrich et al. (1995)
Cancer Research
55: 1199-1205; Cordon-Cardo, C. (1995)
Am. J. Pathol.
147:1-16; Kawamat et al. (1995)
Cancer Research
55:2266-2269; Pietenpol et al. (1995)
Cancer Research
55:1206-1210; Ponce-Casta{tilde over (n)}eda et al. (1995)
Cancer Research
55:1211-1214; Stegmaier et al. (1996)
Cancer Research
56:1413-1417). Thus, despite its potential role as a tumor suppressor, p27 gene does not appear to be mutated in human tumors.
However, recent evidence suggests an involvement of this CKI in neoplastic transformation. For example, p27 deficient mice have been shown to develop tumors of the pituitary gland with 100% of penetrance (Fero et al. (1996)
Cell
85:733-774; Kiyokawa et al. (1996)
Cell
85:721-732). In addition, p27 has been shown to be a target of the Adenovirus E1A (Mal et al. (1996)
Nature
380:262-265). Further, HPV E7 oncoproteins have been shown to dissociate p27 from the cyclin/cdk complexes (Zerfass-Thome et al. (1996)
Oncogene
2323-2330). p27 has also been implicated as a regulator of drug resistance in solid tumors and has been suggested as a target for development of antagonists which may be useful as chemosenstizers in conjunction with conventional anticancer therapy. St. Croix et al. (1996)
Nature Medicine
2(11):1204-1210.
It has now been found that levels of p27 expression in tumor cells correlate with the degree of malignancy of the tumor and can be used in prognosticating overall survival time in cancer patients.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of determining the degree of malignancy or grading of a tumor in a patient by determining levels of p27 expression in tumor cells isolated from the patient.
Another object of the present invention is to provide a method of prognosticating overall survival time in cancer patients by determining levels of p27 expression in tumor cells isolated from the patient.
DETAILED DESCRIPTION OF THE INVENTION
Lung cancer in the greatest single cause of cancer-related deaths in Western countries and despite continuing research efforts into new therapeutic strategies, survival in patients suffering from lung cancer (approximately 13%) has not improved over the past two decades. Only approximately 30% of patients suffering from lung cancer are eligible for radical lung resection, and only about one third of these patients are free from disease 5 years after surgery (Shottenfeld, D., “Epidemiology of lung cancer”, in
Lung Cancer: Principles and Practice,
ed. by Pass H. I., Mitchell J. B., Johnson, Turrisi A T. (1996). Lippincot-Raven Publishers, Philadelphia) . Thus, accurate determination of the cancer's malignant potential is important in order to select an appropriate course of therapy.
This determination, known as “grading”, is typically carried out by examination of the character and appearance of tumor sections by skilled pathologists and is based upon a number of histological criteria. However, a significant problem in the use of such histological criteria is that they are subjective, and variability exists not only between different pathologists but, oftentimes, within a single pathologist's reading of the same tissue specimen. In addition, there is heterogeneity within the tumor itself in both primary and metastatic sites. Accordingly, there is a need for more accurate determination of the malignant potential of a tumor.
It has now been found that expression levels of cell cycle regulators such as p27 in tumor cells correlate with the degree of malignancy (grading) (p=0.01). Further, expression levels of p27 in tumor cells were shown to correlate with survival time in cancer patients. Thus, a simple immunohistochemical assay measuring p27 expression on formalin fixed-paraffin embedded specimens can now be used to quickly and reproducibly to grade tumor and to evaluate the prognosis of cancer patients.
Accordingly, the present invention provides a method of grading tumors by determination of the levels of p27 expression in a tissue sample from a tumor. Tissue samples of a tumor can be obtained by various means including, but not limited, surgical resectioning or removal and biopsies. Once the tissue is obtained, levels of p27 expression in the sample are determined preferably via an immunohistochemical assay. As will be obvious to those of skill in the art upon this disclosure, however, other means of measuring p27 expression levels in tissues can be used. The levels are then compared to established control levels of p27 expression in normal noncancerous tissue, nonmetastic cancerous tissue and metastic cancerous tissue so that tissue sample can be graded.
The present invention also provides a method for prognosticating the survival rates of cancer patients by determination of p27 levels in tumor tissue obtained from these patients. For example, the determination of p27 levels in tissue samples from patients affected by non small cell lung cancer was demonstrated to have prognostic value in identifying those patients most likely to benefit from radical lung resection. By “non small cell lung cancer or NSCLC”, it is meant all forms of lung cancer except small cell lung cancer (SCLC) and includes, but is not limited to squamous cell carcinomas, adenocarcinomas, broncioaveolar carcinomas and large cell carcinomas. p27 protein expression levels were measured in a series of non small cell lung cancer specimens. Immunohistochemistry and western blot analysis were performed on each specimen. Tumors expressing low to undetectable levels of p27 were found to contain high p27 degradation activity. Expression levels were then correlated with the outcomes of these patients. It was found that p27 was a prognostic factor corr
Holleran Anne L.
Johnson Nancy A.
Seidel Gonda Lavorgna & Monaco, PC
Thomas Jefferson University
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