# Patent

## Title
Tumor markers in ovarian cancer

## Abstract

The present invention features methods of diagnosing and prognosticating ovarian tumors by detecting increased expression of an ovarian tumor marker gene in a subject or in a sample from a subject. Also featured are kits for the aforementioned diagnostic and prognostic methods. In addition, the invention features methods of treating and preventing ovarian tumors, and methods of inhibiting the growth or metastasis of ovarian tumors, by modulating the production or activity of an ovarian tumor marker polypeptide. Further featured are methods of inhibiting the growth or metastasis of an ovarian tumor by contacting an ovarian tumor cell with an antibody that specifically binds an ovarian tumor marker polypeptide.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a 35 U.S.C. § 371 national phase application filed from, and claiming priority to, international application PCT/US01/10947, filed Apr. 3, 2001 (published under PCT Article 21(2) in English), which claims priority to U.S. provisional patent application Ser. No. 60/194,336, filed Apr. 3, 2000, which applications are herein incorporated by reference in their entireties.


This invention was made with intramural support from the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
This invention relates generally to the identification of ovarian tumor markers and diagnostic, prognostic, and therapeutic methods for their use, as well as kits for use in the aforementioned methods.
BACKGROUND OF THE INVENTION
Ovarian cancer is one of the most common forms of neoplasia in women. Early diagnosis and treatment of any cancer ordinarily improves the likelihood of survival. However, ovarian cancer is difficult to detect in its early stages, and remains the leading cause of death among women with cancer of the female reproductive tract.
The low survival rate of ovarian cancer patients is in part due to the lack of good diagnostic markers for the detection of early stage neoplasms, and in part due to a deficit in the general understanding of ovarian cancer biology, which would facilitate the development of effective anti-tumor therapies. The present invention overcomes these shortcomings by providing much-needed improvements for the diagnosis, treatment, and prevention ovarian tumors, based on the identification of a series of ovarian tumor marker genes that are highly expressed in ovarian epithelial tumor cells and are minimally expressed in normal ovarian epithelial cells. Over 75% of all ovarian tumors, and about 95% of all malignant ovarian tumors, arise from the ovarian surface epithelium (OSE). Because the tumor marker genes are broadly expressed in various types of ovarian epithelial tumors, the present invention should greatly improve the diagnosis and treatment of most ovarian cancers.
SUMMARY OF THE INVENTION
In a first aspect, the invention features a method of detecting an ovarian tumor in a subject. The method includes the step of measuring the expression level of an ovarian tumor marker gene in the subject, wherein an increase in the expression level of the ovarian tumor marker gene in the subject, relative to the expression level of the ovarian tumor marker gene in a reference subject not having an ovarian tumor, detects an ovarian tumor in the subject.
In a second aspect, the invention features a method of identifying a subject at increased risk for developing ovarian cancer. The method includes the step of measuring the expression level of an ovarian tumor marker gene in the subject, wherein an increase in the expression level of the ovarian tumor marker gene in the subject, relative to the expression level of the ovarian tumor marker gene in a reference subject not at increased risk for developing ovarian cancer, identifies an individual at increased risk for developing ovarian cancer.
In a preferred embodiment of the second aspect of the invention, the expression level of the ovarian tumor marker gene in the subject is compared to the expression level of the tumor marker gene in a reference subject that is identified as having an increased risk for developing ovarian cancer.
In a third aspect, the invention features a method of determining the effectiveness of an ovarian cancer treatment in a subject. The method includes the step of measuring the expression level of an ovarian tumor marker gene in the subject after treatment of the subject, wherein a modulation in the expression level of the ovarian tumor marker gene in the subject, relative to the expression level of the ovarian tumor marker gene in the subject prior to treatment, indicates an effective ovarian cancer treatment in the subject.
In a preferred embodiment of the first three aspects of the invention, the expression level of the ovarian tumor marker gene is determined in the subject by measuring the expression level of the tumor marker gene in a sample from the subject. The sample may be, for example, a tissue biopsy, ovarian epithelial cell scrapings, peritoneal fluid, blood, urine, or serum. In another preferred embodiment of the first three aspects of the invention, the expression level of the tumor marker gene is measured in vivo in the subject.
In yet another preferred embodiment of the first three aspects of the invention, the expression level of more than one ovarian tumor marker gene is measured. For example, the expression level of two, three, four, five, or more tumor marker genes may be measured.
In various other embodiments of the first three aspects of the invention, the expression level of the tumor marker gene may be determined by measuring the level of ovarian tumor marker mRNA. For example, the level of ovarian tumor marker mRNA may be measured using RT-PCR, Northern hybridization, dot-blotting, or in situ hybridization. In addition, or alternatively, the expression level of the ovarian tumor marker gene may be determined by measuring the level of ovarian tumor marker polypeptide encoded by the ovarian tumor marker gene. For example, the level of ovarian tumor marker polypeptide may be measured by ELISA, immunoblotting, or immunohistochemistry. The level of ovarian tumor marker polypeptide may also be measured in vivo in the subject using an antibody that specifically binds an ovarian tumor marker polypeptide, coupled to a paramagnetic label or other label used for in vivo imaging, and visualizing the distribution of the labeled antibody within the subject using an appropriate in vivo imaging method, such as magnetic resonance imaging.
In still another embodiment of the first three aspects of the invention, the expression level of the tumor marker gene may be compared to the expression level of the tumor marker gene in a reference subject diagnosed with ovarian cancer.
In a fourth aspect, the invention features a method of identifying a tumor as an ovarian tumor. The method includes the step of measuring the expression level of an ovarian tumor marker gene in a tumor cell from the tumor, wherein an increase in the expression level of the ovarian tumor marker gene in the tumor cell, relative to the expression level of the ovarian tumor marker gene in a noncancerous ovarian cell, identifies the tumor as an ovarian tumor.
In a fifth aspect, the invention features a method of treating or preventing an ovarian tumor in a subject. The method includes the step of modulating production or activity of a polypeptide encoded by an ovarian tumor marker gene in an ovarian epithelial cell in the subject.
In a sixth aspect, the invention features a method of inhibiting the growth or metastasis of an ovarian tumor cell in a subject. The method includes the step of modulating production or activity of a polypeptide encoded by an ovarian tumor marker gene in the ovarian tumor cell in the subject.
In a seventh aspect, the invention features a method of inhibiting the growth or metastasis of an ovarian tumor in a subject. The method includes the step of contacting an ovarian tumor cell with an antibody that specifically binds an ovarian tumor marker polypeptide encoded by an ovarian tumor marker gene, wherein the binding of the antibody to the ovarian tumor marker polypeptide inhibits the growth or metastasis of the ovarian tumor in the subject.
In various preferred embodiments of the seventh aspect of the invention, the ovarian tumor marker polypeptide may be on the surface of the ovarian tumor cell, and the antibody may be coupled to a radioisotope or to a toxic compound.
In an eighth aspect, the invention features a kit including an antibody for measuring the expression level of an ovarian tumor marker gene in a subject.
In a ninth aspect, the invention features a kit including a nucleic acid for measuring the expression level of an ovarian tumor marker gene in a subject.
In a tenth aspect, the invention features a method of diagnosing ovarian cancer in a subject. The method includes the step of measuring the amount of an ovarian tumor marker polypeptide in the subject, wherein an amount of ovarian tumor marker polypeptide that is greater than the amount of ovarian tumor marker polypeptide measured in a subject not having ovarian cancer diagnoses an ovarian cancer in the subject.
In various embodiments of the tenth aspect of the invention, the ovarian tumor marker polypeptide can be present at the surface of a cell (e.g., a cell-surface-localized polypeptide such as a cell adhesion molecule), or the ovarian tumor marker polypeptide may be in soluble form (e.g., secreted from a cell, released from a lysed cell, or otherwise detectable in a fluid-based assay).
In a preferred embodiment of all of the above aspects of the invention, the ovarian tumor may be an epithelial ovarian tumor. The epithelial ovarian tumor may be, for example, a serous cystadenoma, a borderline serous tumor, a serous cystadenocarcinoma, a mucinous cystadenoma, a borderline mucinous tumor, a mucinous cystadenocarcinoma, an endometrioid carcinoma, an undifferentiated carcinoma, a cystadenofibroma, an adenofibroma, or a Brenner tumor. The epithelial ovarian tumor may also be a clear cell adenocarcinoma.
In preferred embodiments of all of the above aspects of the invention, the ovarian tumor marker gene can be, but is not limited to, alpha prothymosin; beta polypeptide 2-like G protein subunit 1; tumor rejection antigen-1 (gp96)1; HSP90; Hepatoma-Derived Growth Factor (HGDF); DKFZp5860031; CD63 antigen (melanoma 1 antigen); protein kinase C substrate 80K-H; Polymerase II cofactor 4 (PC4); mitochondrial Tu translation elongation factor; hNRP H1; Solute carrier family 2; KIAA0591 protein; X-ray repair protein; DKFZP564M2423 protein; growth factor-regulated tyrosine kinase substrate; and eIF-2-associated p67. The ovarian tumor marker gene may also be HSP60 or Lutheran blood group (3-CAM). In other preferred embodiments of all aspects of the invention, the ovarian tumor marker gene may also be HLA-DR alpha chain; cysteine-rich protein 1; claudin 4; claudin 3; ceruloplasmin (ferroxidase), glutathione perroxidase 3; secretory leukocyte protease inhibitor; HOST-1 (FLI4303 fis); interferon-induced transmembrane protein 1; apolipoprotein J/clusterin; serine protease inhibitor, Kunitz type 2; apoplipoprotein E; complement component 1, r subcomponent; G1P3/IFI-6-16; Lutheran blood group (BCAM); collagen type III, alpha-1; Mal (T cell differentiation protein); collagen type I, alpha-2; HLA-DPB1; bone marrow stroma antigen 2 (BST-2); or HLA-Cw.
The ovarian tumor marker gene may also be HOST-3 (Claudin-16) (e.g., Genbank® Accession No. XM—003150; SEQ ID NOs: 141 and 142); HOST-4 (e.g., a gene that comprises SEQ ID NO: 144); or HOST-5 (sodium dependent transporter isoform NaPi-Iib) (e.g., Genbank® Accession No. AF146796; SEQ ID NOs: 146 and 147).
In other preferred embodiments of all aspects of the invention, the ovarian tumor marker gene comprises a nucleotide sequence set forth in one of SEQ ID NOs: 84-102.
In still other preferred embodiments of all aspects of the invention, the ovarian tumor marker gene comprises a nucleotide sequence set forth in one of SEQ ID NOs: 103-129.
In yet other preferred embodiments of all aspects of the invention, the ovarian tumor marker gene comprises a nucleotide sequence set forth in one of SEQ ID NOs: 141, 143, or 145.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.


DETAILED DESCRIPTION OF THE INVENTION
The low survival rate of ovarian cancer patients is in part due to the lack of good diagnostic markers allowing early detection of the disease. Further compounding this difficulty in early diagnosis is the lack of effective treatments for ovarian cancer, development of which has been impeded by a deficit in the general understanding of ovarian cancer biology. The present invention overcomes these deficits in the art by providing ovarian tumor markers that are expressed at elevated levels in ovarian epithelial tumor cells, relative to their expression in normal ovarian epithelial cells.
To identify marker genes that are up-regulated in ovarian tumor cells, SAGE (Serial Analysis of Gene Expression; Velculescu et al., Science 270:484-487, 1995) was employed to obtain global gene expression profiles of three ovarian tumors, five ovarian tumor cell lines of various histological types, a pool of ten ovarian tumor cell lines of various histological types, and normal human ovarian surface epithelium (HOSE). The expression patterns were generated by acquiring thousands of short sequence tags that contain sufficient information to uniquely identify transcripts due to the unique position of each tag within the transcript. Comparing the SAGE-generated expression profiles between ovarian cancer and HOSE revealed an abundance of genes that are expressed at elevated levels in ovarian tumor cells, relative to their expression in normal HOSE.
Selected SAGE results were further validated through immunohistochemical analysis of archival ovarian serous carcinoma samples. Ovarian tumor marker genes implicated in immune response pathways, regulation of cell proliferation, and protein folding were identified, many of which are membrane-localized or secreted. The ovarian tumor marker genes identified from these SAGE profiles are useful both as diagnostic and prognostic markers to detect and monitor a broad variety of ovarian cancers, and as therapeutic targets for the treatment of such ovarian cancers.
Definitions
In this specification and in the claims that follow, reference is made to a number of terms that shall be defined to have the following meanings.
As used in the specification and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a cell” can mean a single cell or more than one cell.
By “ovarian cell” is meant a cell that is of ovarian origin or that is a descendent of a cell of ovarian origin (e.g., a metastatic tumor cell in the liver that is derived from a tumor originating in the ovary), irrespective of whether the cell is physically within the ovary at the time at which it is subjected to a diagnostic test or an anti-tumor treatment. For example, the ovarian cell may be a normal ovarian cell or an ovarian tumor cell, either within the ovary or at another location within the body. The ovarian cell may also be outside the body (for example, in a tissue biopsy). A preferred ovarian cell is an ovarian cell of epithelial origin.
By “ovarian tumor marker gene” is meant a gene of the invention, for which expression is increased (as described below) in ovarian tumor cells relative to normal ovarian cells. Preferably, an ovarian tumor marker gene has been observed to display increased expression in at least two ovarian tumor SAGE libraries (relative to a HOSE library), more preferably in at least three SAGE libraries, and most preferably in at least four SAGE libraries (relative to a HOSE library). Examples of ovarian tumor marker genes are provided in Tables 2 and 4 hereinbelow.
By “ovarian tumor marker polypeptide” is meant a polypeptide that is encoded by an ovarian tumor marker gene and is produced at an increased level in an ovarian tumor cell due to the increased expression of the ovarian tumor marker gene that encodes the polypeptide.
By “sample” is meant any body fluid (e.g., but not limited to, blood, serum, urine, cerebrospinal fluid, semen, sputum, saliva, tears, joint fluids, body cavity fluids (e.g., peritoneal fluid), or washings), tissue, or organ obtained from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a lysate (or lysate fraction) or extract derived from a cell; or a molecule derived from a cell or cellular material.
By “modulate” is meant to alter, by increase or decrease.
By “increase in gene expression level,” “expressed at an increased level,” “increased expression,” and similar phrases is meant a rise in the relative amount of mRNA or protein, e.g., on account of an increase in transcription, translation, mRNA stability, or protein stability, such that the overall amount of a product of the gene, i.e., an mRNA or polypeptide, is augmented. Preferably the increase is by at least about 3-fold, more preferably, by at least about: 4-fold, 5-fold, 7-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 70-fold, or more. For example, as described herein, the expression level of the ovarian tumor marker genes of the invention is generally increased by at least 3-fold in ovarian tumor cells, relative to normal ovarian surface epithelial cells.
By “decrease in gene expression level” is meant a reduction in the relative amount of mRNA or protein transcription, translation, mRNA stability, or protein stability, such that the overall amount of a product of the gene, i.e., an mRNA or polypeptide, is reduced. Preferably the decrease is by at least about 20%-25%, more preferably by at least about 26%-50%, still more preferably by at least about 51%-75%, even more preferably by at least about 76%-95%, and most preferably, by about 96%-100%.
By “about” is meant ±10% of a recited value.
By “modulating production or activity of a polypeptide encoded by an ovarian tumor marker gene” is meant to increase or decrease gene expression level, as described above, or to stimulate or inhibit the ability of an ovarian tumor marker polypeptide to perform its intrinsic biological function (examples of such functions include, but are not limited to, enzymatic activity, e.g., kinase activity or GTPase activity; cell-signaling activity, e.g., activation of a growth factor receptor; or cell adhesion activity. The modulation may be an increase in the amount of the polypeptide produced or an increase in the activity of the polypeptide, of at least about: 2-fold, 4-fold, 6-fold, or 10-fold, or the modulation may be a decrease in the amount of the polypeptide produced or a decrease in the activity of the polypeptide, of at least about: 20%-25%, 26%-50%, 51%-75%, 76%-95%, or 96%-100%. These increases and/or decreases are compared with the amount of production and/or activity in a normal cell, sample, or subject.
By “effective amount” of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired effect, e.g., modulation of ovarian tumor marker gene expression or modulation of ovarian tumor marker polypeptide activity. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity and type of disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with a molecule or compound of the invention (e.g., an antibody or nucleic acid molecule) without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
By “having an increased risk” is meant a subject that is identified as having a higher than normal chance of developing an ovarian tumor, compared to the general population. Such subjects include, for example, women that have a hereditary disposition to develop ovarian cancer, for example, those identified as harboring one or more genetic mutations (e.g., a mutation in the BRCA-1 gene) that are known indicators of a greater than normal chance of developing ovarian cancer, or who have a familial history of ovarian cancer. In addition, a subject who has had, or who currently has, an ovarian tumor is a subject who has an increased risk for developing an ovarian tumor, as such a subject may continue to develop new tumors. Subjects who currently have, or who have had, an ovarian tumor also have an increased risk for ovarian tumor metastases.
By “treat” is meant to administer a compound or molecule of the invention to a subject in order to: eliminate an ovarian tumor or reduce the size of an ovarian tumor or the number of ovarian tumors in a subject; arrest or slow the growth of an ovarian tumor in a subject; inhibit or slow the development of a new ovarian tumor or an ovarian tumor metastasis in a subject; or decrease the frequency or severity of symptoms and/or recurrences in a subject who currently has or who previously has had an ovarian tumor.
By “prevent” is meant to minimize the chance that a subject will develop an ovarian tumor or to delay the development of an ovarian tumor. For example, a woman at increased risk for an ovarian tumor, as described above, would be a candidate for therapy to prevent an ovarian tumor.
By “specifically binds” is meant that an antibody recognizes and physically interacts with its cognate antigen and does not significantly recognize and interact with other antigens.
By “probe,” “primer,” or “oligonucleotide” is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the “target”). The stability of the resulting hybrid depends upon the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules, and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for ovarian tumor marker nucleic acids (e.g., genes and/or mRNAs) preferably have at least 50%-55% sequence complementarity, more preferably at least 60%-75% sequence complementarity, even more preferably at least 80%-90% sequence complementarity, yet more preferably at least 91%-99% sequence complementarity, and most preferably 100% sequence complementarity to the ovarian tumor marker nucleic acid to be detected. Probes, primers, and oligonucleotides may be detectably-labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art. Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).
By “specifically hybridizes” is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base-pairs) with a substantially complementary nucleic acid (e.g., an ovarian tumor marker mRNA of the invention) under high stringency conditions, and does not substantially base pair with other nucleic acids.
By “high stringency conditions” is meant conditions that allow hybridization comparable with the hybridization that occurs using a DNA probe of at least 500 nucleotides in length, in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V), at a temperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC, 0.2 M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42° C. (these are typical conditions for high stringency Northern or Southern hybridizations). High stringency hybridization is relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization. In contrast to Northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually 16 nucleotides or longer for PCR or sequencing, and 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and may be found, for example, in F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997, herein incorporated by reference.
Examples of Ovarian Tumor Marker Genes
Examples of ovarian tumor marker genes of the invention include alpha prothymosin (e.g., Genbank® Accession No. M14483; SEQ ID NOs: 1 and 2); beta polypeptide 2-like G protein subunit 1 (e.g., Genbank® Accession No. M24194; SEQ ID NOs: 3 and 4); tumor rejection antigen-1 (gp96)1 (e.g., Genbank® Accession No. NM—003299; SEQ ID NOs: 7 and 8); HSP90 (e.g., Genbank® Accession No. AA071048; SEQ ID NOs: 9 and 10); Hepatoma-Derived Growth Factor (HGDF) (e.g., Genbank® Accession No. D16431; SEQ ID NOs: 13 and 14); DKFZp5860031 (e.g., Genbank® Accession No. AL117237; SEQ ID NOs: 15 and 16); CD63 antigen (melanoma 1 antigen) (e.g., Genbank® Accession No. AA041408; SEQ ID NOs: 17 and 18); protein kinase C substrate 80K-H (e.g., Genbank® Accession No. J03075; SEQ ID NOs: 19 and 20); Polymerase II cofactor 4 (PC4) (e.g., Genbank® Accession No. X79805; SEQ ID NOs: 21 and 22); mitochondrial Tu translation elongation factor (e.g., Genbank® Accession No. L38995; SEQ ID NOs: 23 and 24); hHRP H1 (e.g., Genbank® Accession No. L22009; SEQ ID NOs: 25 and 26); Solute carrier family 2 (e.g., Genbank® Accession No. AF070544; SEQ ID NOs: 27 and 28); KIAA0591 protein (e.g., Genbank® Accession No. AB011163; SEQ ID NOs: 29 and 30); X-ray repair protein (e.g., Genbank® Accession No. AF035587; SEQ ID Nos: 31 and 32); DKFZP564M2423 protein (e.g., Genbank® Accession No. BC003049; SEQ ID NOs: 35 and 139); growth factor-regulated tyrosine kinase substrate (e.g., Genbank® Accession No. D84064; SEQ ID NOs: 36 and 37); and/or eIF-2-associated p67 (e.g., Genbank® Accession No. U29607; SEQ ID NOs: 38 and 39). The ovarian tumor marker gene may also be HSP60 (e.g., Genbank® Accession No. M22382; SEQ ID NOs: 11 and 12) and Lutheran blood group protein (B-CAM) (e.g., Genbank® Accession No. NM—005581; SEQ ID NOs: 5 and 6).
Other examples of ovarian tumor marker genes of the invention include HLA-DR alpha chain (e.g., Genbank® Accession No. K01171; SEQ ID NOs: 40 and 41); cysteine-rich protein 1 (e.g., Genbank® Accession No. NM—001311; SEQ ID NOs: 42 and 43); claudin 4 (e.g., Genbank® Accession No. NM—001305; SEQ ID NOs: 44 and 45); HOST-2 (e.g., SEQ ID NO: 46); claudin 3 (e.g., Genbank® Accession No. NM—001306; SEQ ID NOs: 47 and 48); ceruloplasmin (ferroxidase) (e.g., Genbank® Accession No. M13699; SEQ ID NOs: 49 and 50); glutathione perroxidase 3 (e.g., Genbank® Accession No. D00632; SEQ ID NOs: 51 and 52); secretory leukocyte protease inhibitor (e.g., Genbank® Accession No. M114471; SEQ ID NOs: 53 and 54); HOST-1 (FLJ14303 fis) (e.g., Genbank® Accession No. AK024365; SEQ ID NOs: 55 and 56); interferon-induced transmembrane protein 1 (e.g., Genbank® Accession No. J04164; SEQ ID NOs: 57 and 58); apolipoprotein J/clusterin (e.g., Genbank® Accession No. J02908; SEQ ID NOs: 59 and 60); serine protease inhibitor, Kunitz type 2 (e.g., Genbank® Accession No. AF027205; SEQ ID NOs: 61 and 62); apoplipoprotein E (e.g., Genbank® Accession No. BC003557; SEQ ID NOs: 63 and 64); complement component 1, r subcomponent (e.g., Genbank® Accession No. M14058; SEQ ID NOs: 65 and 66); G1P3/IIFI-6-16 (e.g., Genbank® Accession No. X02492; SEQ ID NOs: 67 and 68); Lutheran blood group (BCAM) (e.g., Genbank® Accession No. X83425; SEQ ID NOs: 69 and 70); collagen type III, alpha-1 (e.g., Genbank® Accession No. X14420; SEQ ID NOs: 71 and 72); Mal (T cell differentiation protein) (e.g., Genbank® Accession No. M15800; SEQ ID NOs: 73 and 74); collagen type I, alpha-2 (e.g., Genbank® Accession No. J03464; SEQ ID NOs: 75 and 76); HLA-DPB1 (e.g., Genbank® Accession No. J03041: SEQ ID NOs: 77 and 78): bone marrow stroma antigen 2 (BST-2) (e.g., Genbank® Accession No. D28137; SEQ ID NOs: 79 and 80); and HLA-Cw (e.g., Genbank® Accession No. X17093; SEQ ID NOs: 81 and 82).
Still other examples of ovarian tumor marker genes of the invention include HOST-3 (Claudin-16) (e.g., Genbank® Accession No. XM—003150; SEQ If) NOs: 141 and 142); HOST-4 (e.g., a gene that comprises SEQ ID NO: 144); or HOST-5 (sodium dependent transporter isoform NaPi-Iib) (e.g., Genbank® Accession No. AF146796; SEQ ID NOs: 146 and 147).
Ovarian tumor marker genes of the invention may also be described by SAGE tags, as disclosed herein. For example, an ovarian tumor marker genes of the invention can include a nucleotide sequence set forth in one of SEQ ID NOs: 84-102; 103-129; or 141, 143, or 145.
Diagnostic Uses of Ovarian Tumor Marker Genes and Polypeptides
The ovarian tumor marker genes of the invention are overexpressed in a broad variety of ovarian epithelial tumor cells, relative to normal ovarian epithelial cells. This differential expression can be exploited in diagnostic tests for ovarian cancer, in prognostic tests for assessing the relative severity of ovarian cancer, in tests for monitoring a subject in remission from ovarian cancer, and in tests for monitoring disease status in a subject being treated for ovarian cancer. Increased expression of an ovarian tumor marker gene, i.e., detection of elevated levels of ovarian tumor marker mRNA and/or protein in a subject or in a sample from a subject (i.e., levels at least three-fold higher than in a normal subject or in an equivalent sample, e.g., blood, cells, or tissue from a normal subject) is diagnostic of ovarian cancer.
One of ordinary skill in the art will understand that in some instances, higher expression of a given ovarian tumor marker gene will indicate a worse prognosis for a subject having ovarian cancer. For example, relatively higher levels of ovarian tumor marker gene expression may indicate a relative large primary tumor, a higher tumor burden (e.g., more metastases), or a relatively more malignant tumor phenotype.
The diagnostic and prognostic methods of the invention involve using known methods, e.g., antibody-based methods to detect ovarian tumor marker polypeptides and nucleic acid hybridization- and/or amplification-based methods to detect ovarian tumor marker mRNA. One of ordinary skill in the art will understand how to choose the most appropriate method for measuring ovarian tumor marker expression, based upon the combination of the particular ovarian tumor marker to be measured, the information desired, and the particular type of diagnostic test to be used. For example, immunological tests such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and Western blots may be used to measure the level of an ovarian tumor marker polypeptide in a body fluid sample (such as blood, serum, sputum, urine, or peritoneal fluid). Biopsies, tissue samples, and cell samples (such as ovaries, lymph nodes, ovarian surface epithelial cell scrapings, lung biopsies, liver biopsies, and any fluid sample containing cells (such as peritoneal fluid, sputum, and pleural effusions) may be tested by disaggregating and/or solubilizing the tissue or cell sample and subjecting it to an immunoassay for polypeptide detection, such as ELISA, RIA, or Western blotting. Such cell or tissue samples may also be analyzed by nucleic acid-based methods, e.g., reverse transcription-polymerase chain reaction (RT-PCR) amplification, Northern hybridization, or slot- or dot-blotting. To visualize the three-dimensional distribution of tumor cells within a tissue sample, diagnostic tests that preserve the tissue structure of a sample, e.g., immunohistological staining, in situ RNA hybridization, or in situ RT-PCR may be employed to detect ovarian tumor marker polypeptide or mRNA, respectively. For in vivo localization of tumor masses, imaging tests such as magnetic resonance imaging (MRI) may be employed by introducing into the subject an antibody that specifically binds an ovarian tumor marker polypeptide (particularly a cell surface-localized polypeptide), wherein the antibody is conjugated or otherwise coupled to a paramagnetic tracer (or other appropriate detectable moiety, depending upon the imaging method used); alternatively, localization of an unlabeled tumor marker-specific antibody may be detected using a secondary antibody coupled to a detectable moiety.
The skilled artisan will understand that selection of a particular ovarian tumor marker polypeptide as the target for detection in any diagnostic test and selection of the particular test to be employed will depend upon the type of sample to be tested. For example, measurement of ovarian tumor marker polypeptides that are secreted from a cell (e.g., HDGF) may be preferred for serological tests. Moreover, ovarian tumor marker polypeptides that are not normally actively secreted from cells (e.g., intracellular or membrane-associated polypeptides), but that are found in blood and other fluid samples (e.g., peritoneal fluid or washings) at detectable levels in subjects having tumors (e.g., due to tumor cell lysis) are considered to be soluble ovarian tumor marker polypeptides that may be used in serological and other diagnostic assays of body fluids.
A fluid sample (such as blood, peritoneal fluid, sputum, or pleural effusions) from a subject with ovarian cancer, particularly metastatic cancer, may contain one or more ovarian tumor cells or ovarian tumor cell fragments. The presence of such cells or fragments allows detection of a tumor mRNA using an RT-PCR assay, e.g., but not limited to, real-time quantitative RT-PCR using the Taqman method (Heid and Stevens, Genome Res. 6:986-94, 1996).
In addition, since rapid tumor cell destruction often results in autoantibody generation, the ovarian tumor markers of the invention may be used in serological assays (e.g., an ELISA test of a subject's serum) to detect autoantibodies against ovarian tumor markers in a subject. Ovarian tumor marker polypeptide-specific autoantibody levels that are at least about 3-fold higher (and preferably at least 5-fold or 7-fold higher, most preferably at least 10-fold or 20-fold higher) than in a control sample are indicative of ovarian cancer.
Cell-surface localized, intracellular, and secreted ovarian tumor marker polypeptides may all be employed for analysis of biopsies, e.g., tissue or cell samples (including cells obtained from liquid samples such as peritoneal cavity fluid) to identify a tissue or cell biopsy as containing ovarian tumor cells. A biopsy may be analyzed as an intact tissue or as a whole-cell sample, or the tissue or cell sample may be disaggregated and/or solubilized as necessary for the particular type of diagnostic test to be used. For example, biopsies or samples may be subjected to whole-tissue or whole-cell analysis of ovarian tumor marker polypeptide or mRNA levels in situ, e.g., using immunohistochemistry, in situ mRNA hybridization, or in situ RT-PCR. The skilled artisan will know how to process tissues or cells for analysis of polypeptide or mRNA levels using immunological methods such as ELISA, immunoblotting, or equivalent methods, or analysis of mRNA levels by nucleic acid-based analytical methods such as RT-PCR, Northern hybridization, or slot- or dot-blotting.
All of the above methods are well-known in the art. For example, generation of antibodies against a given protein, ELISA, immunoblotting, selection of nucleic acid primers for PCR, RT-PCR, Northern hybridization, in situ hybridization, in situ RT-PCR, and slot- or dot-blotting are all well-described in Current Protocols in Molecular Biology (Ausubel et al., eds.), John Wiley and Sons, Inc., 1996.
Kits for Measuring Expression Levels of Ovarian Tumor Marker Genes
The present invention provides kits for detecting an increased expression level of an ovarian tumor marker gene in a subject. A kit for detecting ovarian tumor marker polypeptide will contain an antibody that specifically binds a chosen ovarian tumor marker polypeptide. A kit for detecting ovarian tumor marker mRNA will contain one or more nucleic acids (e.g., one or more oligonucleotide primers or probes, DNA probes, RNA probes, or templates for generating RNA probes) that specifically hybridize with a chosen ovarian tumor marker mRNA.
Particularly, the antibody-based kit can be used to detect the presence of, and/or measure the level of, an ovarian tumor marker polypeptide that is specifically bound by the antibody or an immunoreactive fragment thereof. The kit can include an antibody reactive with the antigen and a reagent for detecting a reaction of the antibody with the antigen. Such a kit can be an ELISA kit and can contain a control (e.g., a specified amount of a particular ovarian tumor marker polypeptide), primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates and color reagents as described above. The diagnostic kit can, alternatively, be an immunoblot kit generally comprising the components and reagents described herein.
A nucleic acid-based kit can be used to detect and/or measure the expression level of an ovarian tumor marker gene by detecting and/or measuring the amount of ovarian tumor marker mRNA in a sample, such as a tissue or cell biopsy (e.g., an ovary, ovarian cell scrapings, a bone marrow biopsy, a lung biopsy or lung aspiration, etc.). For example, an RT-PCR kit for detection of elevated expression of an ovarian tumor marker gene will contain oligonucleotide primers sufficient to perform reverse transcription of ovarian tumor marker mRNA to cDNA and PCR amplification of ovarian tumor marker cDNA, and will preferably also contain control PCR template molecules and primers to perform appropriate negative and positive controls, and internal controls for quantitation. One of ordinary skill in the art will understand how to select the appropriate primers to perform the reverse transcription and PCR reactions, and the appropriate control reactions to be performed. Such guidance is found, for example, in F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997. Numerous variations of RT-PCR are known in the art. One example of a quantitative RT-PCR assay is the real-time quantitative RT-PCR assay described by Heid and Stevens (Genome Res. 6:986-94, 1996), in which the primers are labeled by a fluorescent tag, and the amount of amplification product may be measured in a Taqman® apparatus (Perkin-Elmer; Norwal, Conn.).
Targeted Delivery of Immunotoxins to Ovarian Tumor Cells
The tumor marker genes of the invention can be employed as therapeutic targets for the treatment or prevention of ovarian cancer. For example, an antibody molecule that specifically binds a cell surface-localized ovarian tumor marker polypeptide can be conjugated to a radioisotope or other toxic compound. Antibody conjugates are administered to the subject such that the binding of the antibody to its cognate ovarian tumor marker polypeptide results in the targeted delivery of the therapeutic compound to ovarian tumor cells, thereby treating an ovarian cancer.
The therapeutic moiety can be a toxin, radioisotope, drug, chemical, or a protein (see, e.g., Bera et al. “Pharmacokinetics and antitumor activity of a bivalent disultide-stabilized Fv immunotoxin with improved antigen binding to erbB2” Cancer Res. 59:4018-4022 (1999)). For example, the antibody can be linked or conjugated to a bacterial toxin (e.g., diptheria toxin, pseudomonas exotoxin A, cholera toxin) or plant toxin (e.g., ricin toxin) for targeted delivery of the toxin to a cell expressing the ovarian tumor marker. This immunotoxin can be delivered to a cell and upon binding the cell surface-localized ovarian tumor marker polypeptide, the toxin conjugated to the ovarian tumor marker-specific antibody will be delivered to the cell.
In addition, for any ovarian tumor polypeptide for which there is a specific ligand (e.g., a ligand that binds a cell surface-localized protein), the ligand can be used in place of an antibody to target a toxic compound to an ovarian tumor cell, as described above.
Antibodies That Specifically Bind Ovarian Tumor Marker Polypeptides
The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, so long as they exhibit any of the desired properties (e.g., specific binding of an ovarian tumor marker polypeptide, delivery of a toxin to an ovarian tumor cell expressing an ovarian tumor marker gene at an increased level, and/or inhibiting the activity of an ovarian tumor marker polypeptide) described herein.
Whenever possible, the antibodies of the invention may be purchased from commercial sources. The antibodies of the invention may also be generated using well-known methods. The skilled artisan will understand that either full length ovarian tumor marker polypeptides or fragments thereof may be used to generate the antibodies of the invention. A polypeptide to be used for generating an antibody of the invention may be partially or fully purified from a natural source, or may be produced using recombinant DNA techniques. For example, a cDNA encoding an ovarian tumor marker polypeptide, or a fragment thereof, can be expressed in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells), after which the recombinant protein can be purified and used to generate a monoclonal or polyclonal antibody preparation that specifically bind the ovarian tumor marker polypeptide used to generate the antibody.
In addition, one of skill in the art will know how to choose an antigenic peptide for the generation of monoclonal or polyclonal antibodies that specifically bind ovarian tumor antigen polypeptides. Antigenic peptides for use in generating the antibodies of the invention are chosen from non-helical regions of the protein that are hydrophilic. The PredictProtein Server or an analogous program may be used to select antigenic peptides to generate the antibodies of the invention. In one example, a peptide of about fifteen amino acids may be chosen and a peptide-antibody package may be obtained from a commercial source such as Anaspec (San Jose, Calif.). One of skill in the art will know that the generation of two or more different sets of monoclonal or polyclonal antibodies maximizes the likelihood of obtaining an antibody with the specificity and affinity required for its intended use (e.g., ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy). The antibodies are tested for their desired activity by known methods, in accordance with the purpose for which the antibodies are to be used (e.g., ELISA, immunohistochemistiy, immunotherapy, etc.; for further guidance on the generation and testing of antibodies, see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988). For example, the antibodies may be tested in ELISA assays, Western blots, immunohistochemical staining of formalin-fixed ovarian cancers or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e.; the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
Monoclonal antibodies of the invention may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fe fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
The antibody fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody fragment. (Zoller, M. J. Curr. Opin. Biotechnol. 3:348-354, 1992).
The antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993)). Human antibodies can also be produced in phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). The techniques of Cote et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].
Administration of Therapeutic and Diagnostic Antibodies
Antibodies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered.
The antibodies can be administered to the subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. The antibodies may also be administered by intratumoral or peritumoral routes, to exert local as well as systemic therapeutic effects. Local or intravenous injection is preferred.
Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of antibody used and other drugs being administered. Guidance in selecting appropriate doses for antibodies is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
Following administration of an antibody for treating ovarian cancer, the efficacy of the therapeutic antibody can be assessed in various ways well known to the skilled practitioner. For instance, the size, number, and/or distribution of ovarian tumors in a subject receiving treatment may be monitored using standard tumor imaging techniques. A therapeutically-administered antibody that arrests tumor growth, results in tumor shrinkage, and/or prevents the development of new tumors, compared to the disease course that would occurs in the absence of antibody administration, is an efficacious antibody for treatment of ovarian cancer.
Antisense and Gene Therapy Approaches for Inhibiting Ovarian Tumor Marker Gene Function
Because the ovarian tumor marker genes of the invention are highly expressed in ovarian tumor cells and are expressed at extremely low levels in normal ovarian cells, inhibition of ovarian tumor marker expression or polypeptide activity may be integrated into any therapeutic strategy for treating or preventing ovarian cancer.
The principle of antisense therapy is based on the hypothesis that sequence-specific suppression of gene expression (via transcription or translation) may be achieved by intracellular hybridization between genomic DNA or mRNA and a complementary antisense species. The formation of such a hybrid nucleic acid duplex interferes with transcription of the target tumor antigen-encoding genomic DNA, or processing/transport/translation and/or stability of the target tumor antigen mRNA.
Antisense nucleic acids can be delivered by a variety of approaches. For example, antisense oligonucleotides or antisense RNA can be directly administered (e.g., by intravenous injection) to a subject in a form that allows uptake into tumor cells. Alternatively, viral or plasmid vectors that encode antisense RNA (or RNA fragments) can be introduced into cells in vivo. Antisense effects can also be induced by sense sequences; however, the extent of phenotypic changes are highly variable. Phenotypic changes induced by effective antisense therapy are assessed according to changes in, e.g., target mRNA levels, target protein levels, and/or target protein activity levels.
In a specific example, inhibition of ovarian tumor marker function by antisense gene therapy may be accomplished by direct administration of antisense ovarian tumor marker RNA to a subject. The antisense tumor marker RNA may be produced and isolated by any standard technique, but is most readily produced by in vitro transcription using an antisense tumor marker cDNA under the control of a high efficiency promoter (e.g., the T7 promoter). Administration of antisense tumor marker RNA to cells can be carried out by any of the methods for direct nucleic acid administration described below.
An alternative strategy for inhibiting ovarian tumor marker polypeptide function using gene therapy involves intracellular expression of an anti-ovarian tumor marker antibody or a portion of an anti-ovarian tumor marker antibody. For example, the gene (or gene fragment) encoding a monoclonal antibody that specifically binds to an ovarian tumor marker polypeptide and inhibits its biological activity is placed under the transcriptional control of a specific (e.g., tissue- or tumor-specific) gene regulatory sequence, within a nucleic acid expression vector. The vector is then administered to the subject such that it is taken up by ovarian tumor cells or other cells, which then secrete the anti-ovarian tumor marker antibody and thereby block biological activity of the ovarian tumor marker polypeptide. Preferably, the ovarian tumor marker polypeptide is present at the extracellular surface of ovarian tumor cells.
Nucleic Acid Delivery
In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for inhibition of ovarian tumor marker protein expression. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCOBRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art. In addition, the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).
As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells antisense nucleic acid that inhibits expression of an ovarian tumor marker gene. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science 272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747, 1996). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478, 1996). This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
As one example, if the antisense nucleic acid of this invention is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 107 to 109 plaque forming units (pfu) per injection but can be as high as 1012 pfu per injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997). Ideally, a subject will receive a single injection. If additional injections are necessary, they can be repeated at six month intervals for an indefinite period and/or until the efficacy of the treatment has been established.
Parenteral administration of the nucleic acid or vector of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein. For additional discussion of suitable formulations and various routes of administration of therapeutic compounds, see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995.
EXAMPLE I
Identification of Ovarian Tumor Marker Genes Using SAGE
Serial Analysis of Gene Expression is a method that enables the global analysis of gene expression from a tissue of interest (Velculescu et al., Science 270:484487, 1995; Zhang et al., Science 276:1268-72, 1997). The advantages of SAGE over cDNA arrays, another method for the global analysis of gene expression, include: 1) the possibility of identifying novel genes, 2) determination of absolute levels of gene expression, which is difficult in hybridization-based techniques, and, 3) examination of gene expression as a whole instead of as a subset of genes.
Construction and Screening of SAGE Libraries
The SAGE technique has been described in detail (Velculescu et al., Science 270:484-487, 1995). The SAGE libraries disclosed herein were made as described by Velculescu, supra. First, total RNA was purified from the cells. Poly A+RNA was then isolated and reverse transcription was performed using a biotinylated poly dT primer for first strand synthesis. The cDNA mixture was cut with NlaIII and the biotinylated 3′fragments were collected using streptavidin beads. The beads were divided into two aliquots (A and B) and linkers containing PCR primer sites and a site for class II restriction enzyme BsmFI were ligated to the DNA fragments attached to the beads from samples A and B. The mixture was treated with the restriction enzyme BsmFI, which recognizes the site in the linker but cuts 14 bp downstream. The resulting fragments contained the linker and 10 bp of “cDNA sequence” that is referred to as “tag”. The tags from samples A and B were ligated together to form ditags, which were then amplified by PCR. Any repeated ditag (tags containing the same two individual tags) are an indication of PCR bias and were eliminated by the SAGE software (Velculescu et al., Science 270:484-487, 1995; Zhang et al., Science 276:1268-72, 1997). The tags were concatemerized and cloned into a sequencing vector. Sequencing revealed the identity and frequency of the different tags. As described above, the 10 bp tag is sufficient to identify cDNA and the frequency of a particular tag represents the frequency of a particular message in the population. The SAGE software developed in the laboratories of Bert Vogelstein and Kenneth Kinzler at Johns Hopkins extracts the tags from the raw sequencing data, matches the tags to the corresponding genes (present in Genbank®) and makes frequency comparisons between the tags from an individual library or other libraries.
Verification of Ovarian Tumor Marker Genes Identified by SAGE
The most promising candidates are selected and verified by any expression analysis method, e.g., Northern analysis or reverse transcription-polymerase chain reaction (RT-PCR). For Northern analysis, radioactive probes are generated from expressed sequence tags (ESTs) corresponding to the candidate genes and are used to hybridize to membranes containing total RNA from various ovarian cancers and controls. The candidates may also be verified by real-time PCR using the Taqman® method (Heid and Stevens, Genome Res. 6:986-94, 1996). Amplification primers and fluorescent probes are synthesized according to instructions from the manufacturer (Perkin-Elmer; Norwalk, Conn.). Quantitative PCR is performed using a PE 5700 apparatus or an analogous instrument.
Sources of RNA for SAGE Library Construction
Eleven SAGE libraries were constructed, as shown in Table 1. The human ovarian surface epithelial cell (HOSE) library was constructed using RNA from HOSE cells that were obtained by gently scraping the ovarian surface from a hysterectomy patient followed by short-term in vitro culture (three passages) of the cells. Three of the ovarian tumor libraries (designated OVT6, OVT7, and OVT8) were constructed using RNA from one of three primary high grade serous adenocarcinomas libraries from individual ovarian tumor cell lines were generated using RNA from OV1063 (derived from an ovarian papillary adenocarcinoma; obtained from the American Type Culture Collection (ATCC; Manassas, Va.; CRL-2183)); ES-2 (derived from a clear cell adenocarcinoma; from the ATCC; CRL-1978); A2780 (derived from an ovarian cancer; obtained from Dr. Vilhelm Bohr, Baltimore, Md.); OVCA432 (derived from an ovarian serous cystadenocarcinoma; Bast et al., J. Clin. Invest. 68:1331-1337, 1981); ML10 (derived from an ovarian cystadenoma; Luo et al. Gyn. Oncol., 67:277-284, 1997); or IOSE29 (simian virus 40-immortalized OSE cells; Auersperg et al., Proc. Natl. Acad. Sci. USA 96:6249-6254, 1999).
The pooled library was generated using RNA from a pool of 10 cell lines: A2780; BG-1 (poorly differentiated ovarian cancer; obtained from Dr. Carl Barrett, Durham, N.C.); ES-2; OVCA432; MDAH 2774 (endometrioid adenocarcinoma; obtained from the ATCC); and five cell lines obtained from Dr. Michael Birrer (Rockville, Md.): AD10 (an adriamycin-resistant derivative of A2780); A222 (ovarian carcinoma); UCI101 (papillary ovarian adenocarcinoma); UCI107 (papillary ovarian adenocarcinoma); and A224 (ovarian carcinoma).












TABLE 1





Library
Seq
Tags (raw)
Tags
Genes
At least 2




















HOSE
2,290
49,394
47,881
16,034
4,532


OVT6
2,104
43,891
41,620
18,476
4,799


OVT7
2,089
57,725
53,898
19,523
5,669


OVT8
2,076
36,813
32,494
16,363
3,815


OV1063
2,146
41,131
37,862
15,231
4,746


ES-2
1,775
36,430
35,352
14,739
3,952


A2780**
475
9,269
8,246
5,179
1,021


OVCA432
384
3,011
2,824
1,940
310


Pool
2,201
10,952
10,554
5,956
1,627


ML10
1,935
61,083
55,700
18,727
6,637


IOSE29
*
*
*
*
*


TOTAL
17,475
349,699
326,431
75,056
25,071





* To be sequenced


**Incomplete






Results of SAGE
Eleven ovarian SAGE libraries were constructed, ten of which have been sequenced to date. The overall data are summarized in Table 1 above. For each SAGE library, Table 1 shows the number of SAGE library clones sequenced, the number of raw tags sequenced, the number of tags obtained after correction for PCR bias, the total number of genes that are represented by the corrected pool of tags, and the number of genes that were represented at least twice in the corrected pool of tags. For most libraries, 35,000-61,000 tags were obtained, yielding anywhere from 14,000-20,000 genes. In total, 75,056 genes were identified.
In order to identify genes that are up-regulated in ovarian tumors and that may serve as diagnostic markers and therapeutic targets, we compared gene expression between the normal ovarian cells (HOSE) and the cancer cells (OVT6, OVT7, OVT8, OV1063, ES2, A2780, Pool). OVCA432 was not included in this analysis because of the poor number of tags obtained from this library. We looked for genes for which expression was absent or low (frequency smaller or equal to 2 tags per 100,000) in HOSE and at least 7- to 10-fold up-regulated in the majority of the tumor libraries, and detected a number of genes matching these criteria. Table 2 shows the libraries that were screened, the SAGE tags that were identified in the library screens, along with their corresponding genes and Genbank® accession numbers, and the relative expression of each gene in each library. Any one of these ovarian tumor marker genes may be used in the diagnostic and/or therapeutic methods of the invention.


















TABLE 2





SEQ. ID













NO. (Tag)
Tag
OVT8
OVT7
OVT6
A2780
OV1063
ES2
Pool
HOSE
Gene Product
Genbank


























83
TCAGACGCAG
52
149
91
97
49
214
82
2
Prothymosin, alpha
M14483


84
TTATGGGATC
57
80
57
140
83
126
274
2
G protein, beta polypeptide
M24194












2-like 1


85
CCCGCCCCCG
136
166
52
22
7
0
146
2
Lutheran blood group
NM_005581












(B-CAM)


86
GAGGAAGAAG
14
38
57
76
53
80
100
2
Tumor rejection antigen-1
NM_003299












(gp96) 1


87
GAAGCTTTGC
27
43
43
22
27
66
73
2
HSP90
AA071048


88
TACCAGTGTA
30
16
14
140
22
30
100
2
HSP60
M22382


89
TCTTCTCCCT
8
42
32
22
27
25
46
2
Hepatoma-Derived Growth
D16431












Factor (HDGF)


90
TTGGCTTTTC
14
12
71
32
10
22
18
0
DKFZp5860031
AL117237


91
GGAAGGGAGG
30
14
16
11
12
44
55
2
CD63 antigen (melanoma 1
AA041408












antigen)


92
AAGCCAGCCC
19
17
36
22
17
27
18
2
Protein kinase C substrate
J03075












80K-H


93
TTTCAGATTG
16
26
25
32
22
19
18
0
Polymerase II cofactor 4
X79805












(PC4)


94
GCATAGGCTG
11
24
25
22
12
27
9
2
Tu translation elong. factor
L38995












(mitochondrial)


95
TTTGTTAATT
30
16
16
43
17
19
18
2
hNRP H1
L22009


96
GAGACTCCTG
11
23
23
22
12
3
64
2
Solute carrier family 2
AF070544


97
CCTGTAATTC
19
10
27
32
15
8
27
2
KIAA0591 protein
AB011163


98
GTGGTGCGTG
16
10
21
11
15
19
27
2
X-ray repair protein
AF035587


99
TTGGACCTGG
11
19
9
11
27
16
18
2
ATP synthase
AA524164












(delta subunit)


100
CTTAAGGATT
11
12
18
11
15
27
9
0
DKFZP564M2423 protein
BC003049


101
GTCTGTGAGA
8
17
9
22
12
22
18
0
Growth factor-regul. tyr
D84064












kinase substrate


102
GAAACTGAAC
16
10
14
32
12
3
9
2
eIF-2-associated p67
U29607









EXAMPLE II
Identification of Additional Ovarian Tumor Marker Genes Using SAGE
Serial Analysis of Gene Expression (SAGE) was used to generate global gene expression profiles from various ovarian cell lines and tissues, including primary cancers, ovarian surface epithelial (OSE) cells and cystadenoma cells. The profiles were used to compare overall patterns of gene expression and identify differentially expressed genes. We have sequenced a total of 385,000 tags, yielding over 56,000 genes expressed in ten different libraries derived from ovarian tissues.
In general, ovarian cancer cell lines showed relatively high levels of similarity to libraries from other cancer cell lines, regardless of the tissue of origin (ovarian or colon), indicating that these lines had lost many of their tissue specific expression patterns. In contrast, immortalized OSE (IOSE) and ovarian cystadenoma cells showed much higher similarity to primary ovarian carcinomas as compared to primary colon carcinomas. Primary tissue specimens therefore appeared to be a better model for gene expression analyses. Using the expression profiles described above and stringent selection criteria, we have identified a number of genes highly differentially expressed between non-transformed ovarian epithelia and ovarian carcinomas. Some of the genes identified are already known to be overexpressed in ovarian cancer but several represent novel candidates. Many of the genes up-regulated in ovarian cancer represent surface or secreted proteins such as Claudin-3 and -4, HE4, Mucin-1, Ep-CAM and Mesothelin. The genes encoding apolipoprotein E (ApoE) and apolipoprotein J (ApoJ), two proteins involved in lipid homeostasis are among the genes highly up-regulated in ovarian cancer. Selected SAGE results were further validated through immunohistochemical analysis of ApoJ, Claudin-3, Claudin-4 and Ep-CAM in archival material. These experiments provided additional evidence of the relevance of our findings in vivo.
A) Methods
Cell Culture and Tissue Samples
Ovarian cancer cell lines OV1063, ES2, and MDAH 2774 were obtained from the American Type Culture Collection (Manassas, Va.). Cell lines A222, AD10, UCI101 and UCI107 were obtained from Dr. Michael Birrer (Rockville, Md.). Cell line A2780 was obtained from Dr. Vilhelm Bohr (Baltimore, Md.). The SV40-immortalized cell lines IOSE29 (Auersperg, N., et al. Proc. Natl Acad. Sci. USA, 96:6249-6254, 1999) and ML10 (Luo, M. P., et al. Gynecol. Oncol. 67:277-284, 1997) were kindly provided by Dr. Nelly Auersperg (British Columbia, Canada) and Dr. Louis Dubeau (Los Angeles, Calif.), respectively. Except for IOSE29, ML-10 and HOSE-4, all cell lines were cultured in McCoy's 5A growth medium (Life Technologies, Inc, Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml of Penicillin and 100 ug/ml Streptomycin). IOSE29 was cultivated in Medium 199 (Life Technologies, Inc, Gaithersburg, Md.) supplemented with 5% newborn calf serum (NCS). ML10 was cultivated in MEM (Life Technologies, Inc, Gaithersburg, Md.) supplemented with 10% FBS and antibiotics as above.
Three high-grade serous ovarian cancer specimens, OVT6, OVT7, and OVT8, composed of at least 80% tumor cells as determined by histopathology, were chosen for SAGE. The ovarian tumor samples were frozen immediately after surgical resection and were obtained form the Johns Hopkins gynecological tumor bank in accordance with institutional guidelines on the use of human tissue. Normal human ovarian surface epithelial (HOSE-4) cells were cultured from the right ovary of a patient undergoing hysterectomy and bilateral salpingo-oophorectomy for benign disease. The OSE cells were obtained by gently scraping the surface of the ovary with a cytobrush and grown for 2 passages in RPMI 1640 medium supplemented with 10% FBS and 10 ug/ml insulin-like growth factor (IGF).
Serial Analysis of Gene Expression (SAGE)
Total RNA was obtained from guanidinium isothiocyanate cell lysates by centrifugation on CsCl. Polyadenylated mRNA was purified from total RNA using the Messagemaker® kit (Life Technologies, Gaithersburg, Md.) and the cDNA generated using the eDNA Synthesis System (Life Technologies, Gaithersburg, Md.). For the “Pool” library, 100 ug of total RNA from each of 10 ovarian cancer cell lines (A222, A2780, AD10, BG-1, ES-2, MDAH 2774, OVCA432, OV1063, UCI101 and UCI107) were combined and mRNA purified. SAGE was performed essentially as described (Velculescu, V. E., et al, Science 270:484-487, 1995) for all the libraries except HOSE. To create the HOSE library, MicroSAGE, a modified SAGE technique developed for limited sample sizes (Datson, N. A., et al. Nucleic Acids Res. 27:1300-1307, 1999), was used. Approximately 1×106 OSE cells in short-term culture were lysed and the mRNA purified directly using Oligo (dT)25 Dynabeads® (Dynal, Norway). As part of the Cancer Genome Anatomy Project (CGAP) SAGE consortium, the SAGE libraries were arrayed at the Lawrence Livermore National Laboratories and sequenced at the Washington University Human Genome Center or NISC (Nil, Bethesda, MD). The data has been posted on the CGAP website as part of the SAGEmap database (Lal, A., et al. Cancer Res. 59:5403-5407, 1999.).
Sequence data from each library were analyzed by the SAGE software (Velculescu, V. E., et al. Science 270:484-487, 1995.) to quantify tags and identify their corresponding transcripts. The data for the colon libraries NC1, NC2, Tu98, Tu102, HCT116 and SW837 were obtained from the SAGEmap database and analyzed in the same way. Because the different libraries contained various numbers of total tags, normalization (to 100,000 tags) was performed to allow meaningful comparisons. The 10,000 most highly expressed genes in each of the 16 SAGE libraries of interest were formatted in a Microsoft Excel spreadsheet and Pearson correlation coefficients were calculated for each pair-wise comparison using normalized tag values for each library. The value for the Pearson correlation coefficient (r) represents the degree of similarity (the strength of the relationship) between two libraries and is calculated using the following equation:




  r
  =
  
    
      
        n
        ⁡
        
          (
          
            Σ
            ⁢
            
              
            
            ⁢
            x
            ⁢
            
              
            
            ⁢
            y
          
          )
        
      
      -
      
        
          (
          
            Σ
            ⁢
            
              
            
            ⁢
            x
          
          )
        
        ⁢
        
          (
          
            Σ
            ⁢
            
              
            
            ⁢
            y
          
          )
        
      
    
    
      
        
          [
          
            
              n
              ⁢
              
                
              
              ⁢
              Σ
              ⁢
              
                
              
              ⁢
              
                x
                2
              
            
            -
            
              
                (
                
                  Σ
                  ⁢
                  
                    
                  
                  ⁢
                  x
                
                )
              
              2
            
          
          ]
        
        [
        
          
            n
            ⁢
            
              
            
            ⁢
            Σ
            ⁢
            
              
            
            ⁢
            
              y
              2
            
          
          -
          
            
              (
              Σy
              )
            
            2
          
        
      
    
  




where, xi=number of tags per 100,000 for tag i in the first library and yi=number of tags per 100,000 for tag i in the second library. For our purposes n equals 10,000 since 10,000 tags are compared. A dendrogram representing the hierarchical relationships between samples was then generated using hierarchical cluster analysis as described risen, M. B., et al. Proc. Natl Acad. Sci. USA 95:14863-14868, 1998). In addition, the identification of differentially expressed genes was also done using this subset of the SAGE data.

Immunohistochemistry
Deparafinized 5-um sections of formalin-fixed ovarian cancer specimens were submitted to heat-induced antigen retrieval and processed using the LSAB2 system (DAKO, Carpinteria, Calif.) with 3,3′-diaminobenzidine as the chromatogen and a hematoxylin counterstain. Monoclonal antibody against ApoJ/Clusterin (Clone CLJ-9) was obtained from Alexis Corporation (San Diego, Calif.) and used at a 1:500 Dilution. Monoclonal antibody against Ep-CAM (Clone 323/A3) from NeoMarkers® (Fremont, Calif.) was used at a 1:500 dilution. Polyclonal antibodies against Claudin-3 and 4 were a generous gift from Drs. M. Furuse and S. Tsukita (Kyoto, Japan) and were used at a dilution of 1:1000.
B) Results
Ovarian SAGE Library Construction and Analysis
Gene expression alterations that arise during malignant transformation can be identified a number of ways. We chose the unbiased, comprehensive method SAGE to create global gene expression profiles from ten different ovarian sources. The expression patterns are generated by sequencing thousands of short sequence tags that contain sufficient information to uniquely identify the corresponding transcripts (Velculescu, V. E., et al. Science 270:484487, 1995). Ten different SAGE libraries were constructed and sequenced for this study (Table 3). Our libraries included two derived from OSE cells (IOSE29 and HOSE-4), one derived from immortalized cystadenoma cells (ML-10), three primary tumors (OVT-6, -7, -8) and four libraries derived from ovarian cancer cell lines (OV-1063, ES-2, A2780 and a pool of cell lines). Almost 20,000 sequencing reactions were performed yielding a total of 384,497 tags, of which, 82,533 were unique. Accounting for a SAGE tag error rate of 6.8% (due to sequencing errors; see Zhang, L., et al., Science 276:1268-1272, 1997), we estimate that we have identified a total of 56,387 genes expressed in ovarian tissues. Except for the A2780 cell line and the pooled lines (POOL) samples, a minimum of 12,000 genes were obtained from every library. Typically, for each library, 10% of the genes were expressed at levels of at least 0.01% and, collectively, these genes accounted for more than 50% of all the tags sequenced. Among the tags that appeared more than once, up to 95% matched to known sequences in the current Genbank nr database. For example, of the 6637 tags that appeared more than once in ML10, only 311 had no matches in the current database, excluding the EST databases.







TABLE 3







Summary of SAGE library analyses












Librarya
Sequence
Tagsb
Unique tagsc
Genesd
≧2 tagse















HOSE
2,290
47,881
16,034
12,778
4,532


IOSE
1,912
47,549
18,004
14,771
5,681


ML10
1,935
55,700
18,727
14,939
6,637


OVT6
2,104
41,620
18,476
15,646
4,799


OVT7
2,089
53,898
19,523
15,858
5,669


OVT8
2,076
32,494
16,363
14,153
3,815


OV1063
2,146
37,862
15,231
12,656
4,746


A2780
1,332
21,587
10,717
9,249
2,761


ES2
1,775
35,352
14,739
12,335
3,952


POOL
2,201
10,554
5,956
5,238
1,627


TOTAL
19,860
384,497
82,533
56,387
28,219





aThe libraries are: HOSE, human ovarian surface epithelium from short term culture; IOSE, SV40-immortalized ovarian surface epithelium; ML10, SV40-immortalized benign cystadenoma; OVT6, OVT7, and OVTB, primary ovarian serous adenocarcinomas; OV1063, A2780, and ES2, ovarian cancer cell lines; POOL, a pool of ten ovarian cancer cell lines.


bTag numbers after elimination of linker-based tags and duplicate ditags.


cThe number of unique tags identified in each library.


dThe number of genes identified after correction for sequencing errors.


eThe number of genes represented at least twice.






Comparison of Global Gene Expression Between Ovarian Tissue Samples
Although progression to malignancy requires a number of gene expression changes, the transcript levels from the vast majority of genes remain unaltered (Zhang, L., et al., Science 276:1268-1272, 1997; and Alon, U., et al., Proc. Natl Acad. Sci. USA 96:6745-6750, 1999). Similarities between the global expression profiles of two given samples can be readily visualized using scatterplots and quantitated through the calculation of Pearson correlation coefficients. Scatterplots of global gene expression analysis in lOSE (ovarian) vs. ML10 (ovarian), OVT6 (ovarian), or Tu98 (colon) cells were generated using the Spotfire® Pro 4.0 software (Cambridge, Mass.) and the Pearson correlation coefficients for each pair-wise comparison of the 16 ovarian and colon SAGE libraries were calculated.
As expected, the immortalized IOSE29 and ovarian cystadenoma strain ML10 are much more similar to ovarian tumors than to colon tumors (average correlation coefficients of 0.70 vs. 0.51, respectively). In addition, IOSE29 and ML10 are very similar to each other, with a correlation coefficient of 0.82. The primary culture of OSE cells (HOSE-4) exhibited higher similarities to the ovarian tumors than to the colon tumors, although the similarity levels were much lower than those observed for IOSE29. Interestingly, HOSE-4 and IOSE29 appear to be much more distantly related than expected considering the fact that they were both derived from “normal” OSE cells. The differences in gene expression between these cells may be due to a number of factors. The age of the patient, the pathological state of the ovaries, the presence of non-epithelial cells in the culture and the fact that IOSE29 is SV40-immortalized may all contribute to the gene expression differences observed. However, it is unlikely that the main differences are due to SV40-immortalization since IOSE29 is much more similar to normal colon (a non SV40-immortalized epithelium) than HOSE-4. It is, of course, possible that the lower degree of similarity between HOSE-4 and the ovarian tumors compared to IOSE29 and ML-10 reflects the fact that HOSE-4 represents a better approximation of the normal in vivo OSE cell.
Three dendrograms were created from hierarchical cluster analysis of all colon and ovarian SAGE libraries, ovarian samples only, and non-malignant ovarian and colon epithelia as well as ovarian and colon primary tumors, using Cluster software (Eisen, M. B., et al. Proc. Natl. Acad. Sci USA 95:14863-14868, 1998). When all the samples were included in the hierarchical clustering analysis, the primary colon tumors clustered with the normal colon epithelium, but colon cell lines clustered with the ovarian specimens. Clearly, the tissue clustering that was readily apparent when comparing primary tissues or immortalized lines was lost when including carcinoma cell lines. For example, A2780, a widely used ovarian cancer cell line was just as similar to colon cancer cell lines as it was to ovarian cancer cell lines. This observation supports the idea that in the process of establishment, cell lines may lose many of the gene expression characteristics of their tissue of origin, although tissue specific expression is clearly not completely lost in cancer cell lines (Ross, D. T., et al. Nat. Genet. 24:227-235,2000).
It is widely believed that epithelial ovarian cancer and benign ovarian cysts, while not necessarily part of a progression sequence toward malignancy, are both derived from the ovarian surface epithelium (Scully, R. E. J. Cell Biochem. 23, Suppl.: 208-218, 1995). OSE cells themselves are mesodermal in origin and are believed to undergo metaplasia before progressing to neoplasia (Scully, R. E. J. Cell Biochem. 23 Suppl.: 208-218, 1995; and Maines-Bandiera, S. L. and Auersperg, N. Int. J. Gynecol. Pathol. 16:250-255, 1997). On the other hand, it has also been argued that ovarian cancers are not derived from OSE but rather from the secondary Mullerian system, structures lined by Mullerian epithelium but located outside the uterus, cervix and fallopian tubes (Schink, J. C. Semin. Oncol. 26 Suppl. 1: 2-7, 1999). This hypothesis would explain some of the shortcomings of the OSE model, such as the requirement for metaplasia and the lack of well-defined precursors in the ovary. While not wishing to be bound by theory, our results are consistent with the widely accepted dogma of the OSE origin of ovarian cancer. Indeed, IOSE29 showed high degrees of similarity to the ovarian tumors and both IOSE29 and HOSE were much more closely related to ovarian than colon primary cancers.
E-cadherin expression has been proposed to be a major determinant in the formation of metaplastic OSE (Auersperg, N., et al. Proc. Natl Acad. Sci. USA, 96:6249-6254, 1999; and Maines-Bandiera, S. L. and Auersperg, N. Int. J. Gynecol. Pathol. 16:250-255, 1997). Consistent with this hypothesis, E-cadherin was absent in IOSE29, HOSE and ML10 but was expressed in all three ovarian tumors (Table 4). Other cadherins are also shown for comparison. Interestingly, VE-cadherin is absent in most libraries except in two of the pre-neoplastic ovarian samples, again suggesting metaplasia As expected, LI-Cadherin was expressed exclusively in the colon-derived libraries. Interestingly, vimentin, a mesenchymal marker, was present in essentially all the ovarian libraries but very low in the colon specimens. Although the specificity of vimentin as a mesenchymal marker has been questioned, this suggests that OSE may retain some of their mesenchymal characteristics, even after turning on the expression of E-cadherin.
The cytokeratins (CKs) and carcinoembryonic antigen (CEA) have been used to differentiate between colon cancer and ovarian cancer (Lagendijk, J. H., et al. Hum. Pathol. 29:491-497, 1998; and Berezowski, K., et al. Mod. Pathol. 9:426-429, 1996). Typically, colon cancer expresses CK20 and CEA while ovarian cancer expresses CK7. The expression patterns in our libraries were consistent with previously reported observations: CK20 and CEA were found in normal colon and colon tumors but absent from all of our ovarian samples (Table 4). Conversely, CK7 was expressed in all three primary ovarian tumors and, while not absent, was much lower in the colon samples. Examination of the differential expression patterns of a variety of established ovarian cancer markers thus provided validation of the SAGE database and cluster analysis.
Differential Gene Expression
The ultimate goal of comparing SAGE libraries is to identify differentially expressed genes. Criteria for differential expression can be determined for each comparison and transcripts within the determined range selected for study. We found a large number of genes that were up-regulated in only one or two of the three tumors on which SAGE was performed. For example, a total of 444 genes were up-regulated more than 10-fold in at least one of the three ovarian primary cancers compared to IOSE29. However, only 45 genes were overexpressed more than 10-fold in all three ovarian tumors analyzed compared to IOSE29.
Our analysis of three different primary ovarian cancers allowed us to reduce the number of candidates by looking for consistency between samples. In order to identify genes that are very likely to be frequently up-regulated during ovarian tumorigenesis we set the following conservative criteria for our analysis. First, the fold induction was calculated by adding the number of normalized tags from the three primary tumors and dividing this number by the total normalized tags in the three non-malignant specimens. Cell lines were not included here for reasons described above. In addition, although HOSE-4 appeared more distantly related to the other non-transformed specimens, we believe that the inclusion of HOSE-4, while possibly eliminating real candidates makes our analysis more conservative and more likely to identify truly overexpressed genes in ovarian cancer. Second, all three primary tumors were required to consistently show elevated levels (>12 tags/100,000) of the gene in question. This eliminated genes that may be very highly overexpressed in one tumor but not in others. Finally, the candidate genes were required to be expressed in at least one ovarian cell line at a level greater than 3 tags/100,000. This last criterion was used to reduce the possibility of identifying genes because of their high level of expression in inflammatory cells or in the stroma of the primary tumors. Using these criteria, the genes that exhibited more than 10-fold overexpression were identified and are shown in Table 4.
Two members of the Claudin family of tight junction proteins, Claudin-3 and 4 were found among the top six differentially expressed genes and likely represent transmembrane receptors. In addition, Apolipoprotein J (ApoJ) and Apolipoprotein E (ApoE) were both overexpressed in ovarian cancer.
Of the 27 overexpressed genes shown in Table 4, ten were relatively specific for the ovary (HLA-DR, two different ESTs, GA733-1, ceruloplasmin, glutathione peroxidase-3, the secretory leukocyte protease inhibitor, ApoJ, ApoE and mesothelin) while the others were also expressed in colon tissues. In any event, it is significant that MUC1, HE4, Ep-CAM and mesothelin, four genes already known to be up-regulated in epithelial ovarian cancer, were identified in this study. This fact validates our approach as well as our set of criteria used to determine the genes differentially expressed.
Similarly, stringent criteria were used to identify genes down-regulated in ovarian tumors compared to IOSE29, HOSE-4 and ML10. Again, the fold difference was calculated by adding tag frequency for all three “normal” specimens and dividing by the total number of tags in the three ovarian tumors. A candidate was required to be expressed at a level of 12 tags/100,000 or greater in all three normal samples. The genes found elevated more than ten-fold in normal tissue compared to tumors are shown in Table 4.







TABLE 4







A subset of genes differentially expressed in ovarian tumors compared to non-malignant ovarian samples










EXPRESSIONc
















SEQ ID



OSE
Ovarian
Colon
Colon



NO. (TAG)
TAG
GENE
Fold
ML10
Tumors
Epithelium
Tumors
FUNCTION




















up-regulateda








103
GGGCATCTCT
HLA-DR α chain
289
−
++
−
−
Major histocompatibility complex,










class II/antigen presentation


104
TTTGGGCCTA
Cysteine-rich protein 1
123
−
++
+
−
LIM/double zinc finger


105
ATCGTGGCGG
Claudin 4
109
−
+
++
+
Tight junction barrier function


106
TATTATGGTA
ESTs (HOST-2)
101
−
+
−
−
Unknown


107
GCCTACCCGA
Surface marker
93
−
+
−
−
Tumor Ag/Ca2+ signal transducer




1/GA733-1/TROP2


108
CTCGCGCTGG
Claudin 3
83
−
+
++
+
Tight junction barrier function


109
TTGCTTGCCA
Ceruloplasmin (ferroxidase)
79
−
+
−
−
Secreted metalloprotein/antioxidant


110
CCTGCTTGTC
HE4
72
−
++
+
−
Secreted protease inhibitor


111
AGGGAGGGGC
Glutathione peroxidase 3
69
−
+
−
−
Secreted selenoprotein/peroxidase




(plasma)


112
TGTGGGAAAT
Secretory leukocyte
60
−
++
−
−
Secreted serine protease inhibitor




protease inhibitor


113
CCTGATCTGC
ESTs (HOST-1)
56
−
+
−
−
Unknown


114
ACCATTGGAT
Interferon-induced
49
−
++
−
+
Receptor for interferon signaling




transmembrane protein 1


115
AGTTTGTTAG
Ep-CAM/EGP2/
48
−
+
++
+
Tumor Ag/Ca2+-independant










CAM/proliferation




TROP1/GA733-2


116
CCTGGGAAGT
Mucin 1
43
−
++
+
+
Tumor Ag/Type-I










membrane glycoprotein


117
CAACTAATTC
Apolipoprotein J/clusterin
39
−
++
−
−
Secreted chaperone/cytoprotection


118
GCCTGCAGTC
Serine protease inhibitor,
34
−
++
++
+
Transmembrane/protease inhibitor




Kunitz type, 2


119
CGACCCCACG
Apolipoprotein E
34
−
++
−
−
Lipoprotein particle binding,










internalization and catabolism


120
TTCTGTGCTG
Complement component 1,
24
−
+
−
−
Serine protease of complement










system/autoimmune diseases




r subcomponent


121
CGCCGACGAT
G1P3/IFI-6-16
24
−
++
+
+
Interferon primary response/










α IFN-inducible


122
CCCGCCCCCG
Lutheran blood group
17
−
++
−
−
Possible cell surface receptor/




protein/BCAM










immunoglobulin superfamily


123
GATCAGGCCA
Collagen Type III, alpha-1
16
−
++
−
+
Unknown


124
GTGGAAGACG
Mal (T cell differentiation
16
−
+
−
−
Trans-Golgi membrane protein










(epithelial cells)/T-cell










differentiation




protein)


124
GATGAGGAGA
ESTs (Collagen Type I,
13
+
++
−
+
Unknown




alpha-2


126
TTCCCTTCTT
HLA-DPB1
13
−
+
−
−
Major histocompatibility complex,










class II/antigen presentation


127
CCCCCTGCAG
Mesothelin
12
−
++
−
−
GPI-anchored/mesothelioma and










ovarian cancer antigen/










cell adhesion


128
TGCTGCCTGT
Bone marrow stroma
12
−
++
−
+
Type II transmembrane










protein/pre-B-cell growth




antigen 2/BST-2


129
TGCAGCACGA
HLA-Cw
10
−
++
++
+
Major histocompatibility complex,










class I/antigen presentation




down-regulatedb


130
GGTTATTTTG
Unknown
99
+
−
−
−
Unknown


131
TGTCATCACA
Lysyl oxidase-like 2
73
+
−
−
−
Secreted/collagen and elastin










crosslinker


132
AAAATAAACA
Chloride intracellular
29
+
−
−
−
Ion transport




channel 4 like


133
TAAAAATGTT
Plasminogen activator
26
++
−
−
−
Serine protease inhibitor




inhibitor, type 1





family/tPA inhibitor


134
GAGCTTTTGA
EST
14
+
−
−
−
Unknown


135
GGCTGATGTG
Glycine-t-RNA synthetase
13
+
−
−
−
Protein synthesis


136
CGACGAGGAG
Epithelial membrane protein-3
13
+
−
−
−
Proliferation, differentiation,










and apoptosis


137
GCCCCCAATA
Galectin-1
10
++
+
−
−
β-galactoside binding lectin/










ECM interaction and proliferation


138
GCAACTTGGA
Vinexin β
10
+
−
−
−
Cell-adhesion and cytoarchitecture





aCandidates up-regulated at least 30-fold in tumors


bCandidates down-regulated at least 10-fold in tumors


cExpression is defined as: −, 0–9 tags/100,000; +, 10–49 tags/100,000; ++, >49 tags/100,000






In order to validate the candidates identified by SAGE, we performed immunohistochemical analysis of thirteen cases of serous cancer of the ovary using antibodies against four of the genes identified as up-regulated in ovarian cancer (Table 5). This was particularly important since the SAGE analysis was initially performed from primary ovarian cancers, which contain a mixture of cell types. Ep-CAM exhibited diffuse, strong staining of tumor cell membranes in all thirteen tumors, without blood cell or stromal staining. Importantly, only one of six samples of the ovarian surface epithelium present in the cases showed weak focal staining, and the rest were negative. The strong immunoreactivity of all thirteen ovarian tumors confirms the validity of our approach to identify genes highly and consistently up-regulated in ovarian cancer. Similarly, ApoJ was found to be expressed in ovarian cancer cells and absent from the surface epithelium. While some expression was detected in non-tumor stroma and inflammatory cells, most of the immuno-reactivity was in tumor cells, and a majority (nine out of thirteen) of the cases showed staining. This observation represents the first report of ApoJ expression in ovarian cancer and provides a novel target for diagnosis or therapy. Claudin-3 and -4 also exhibited staining limited to the tumor component of the specimens. Most tumor cells showed strong membrane staining with weak cytoplasmic reactivity. Some tumors specimens showed decreased membrane staining with strong cytoplasmic reactivity. The normal surface epithelial component (or mesothelial cells) examined did not stain or only stained weakly with the Claudin-4 antibody, while the determination of Claudin-3 levels in normal epithelium was complicated by a low background reactivity with this antibody.
Incorporation by Reference
Throughout this application, various publications, patents, and/or patent applications are referenced in order to more fully describe the state of the art to which this invention pertains. The disclosures of these publications, patents, and/or patent applications are herein incorporated by reference in their entireties to the same extent as if each independent publication, patent, and/or patent application was specifically and individually indicated to be incorporated by reference.
OTHER EMBODIMENTS
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

## Claims

1. A method for detecting an ovarian carcinoma in a subject comprising, acquiring a first sample of ovarian tissue from the subject, measuring the amount of claudin-3 present in the first sample and comparing said measurement from said first sample with a value determined by measuring the amount of claudin-3 in a second sample of non-cancerous ovarian epithelium, wherein an increase in the amount of claudin-3 in the first sample, as compared to the amount of claudin-3 in the second sample indicates the presence of ovarian carcinoma cells in the first sample.

2. The method of claim 1, wherein said first sample is selected from the group consisting of an ovarian tissue biopsy and ovarian epithelial cell scrapings.

3. The method of claim 1, wherein the first sample comprises suspected ovarian carcinoma tissue.

4. The method of claim 1, wherein said second sample is selected from the group consisting of an ovarian tissue biopsy and ovarian epithelial cell scrapings.