AU707348B2 - Method for detection and treatment of breast cancer - Google Patents

Description

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WO 95/19369 PCT/US95/00608

DESCRIPTION

"METHOD FOR DETECTION AND TREATMENT OF BREAST CANCER" TECHNICAL FIELD The present invention relates generally to methods of detection and diagnosis of breast cancer and more particularly to a diagnostic method which relies on the identification of marker genes expressed in pre-invasive cancers by microscopicallydirected cloning. Furthermore, this invention concerns the prevention, detection, and diagnosis of breast cancer by addressing the molecular events which occur during the earliest alterations in breast tissue.

The present invention also relates generally to methods of treatment of breast cancer, and more particularly to gene therapy methods and methods for screening compounds that induce expression of the BRCA1 gene product.

BACKGROUND ART It will be appreciated by those skilled in the art that there exists a need for a more sensitive and less invasive method of early detection and diagnosis of breast cancer than those methods currently in use. Breast cancer presents inherent difficulties in regard to the ease with which it is detected and diagnosed. This is in contrast to detection of some other common cancers, including skin and cervical cancers, the latter of which is based on cytomorphologic screening techniques.

There have been several attempts to develop improved methods of breast cancer detection and diagnosis. In the attempts to improve methods of detection and diagnosis of breast cancer, numerous studies have searched for oncogene mutations, gene amplification, and loss of heterozygosity in invasive breast cancer (Callahan, et al., 1992; Cheickh, et al., 1992; Chen,et al, 1992; and, Lippman, et al, 1990). However, few studies of breast cancer have analyzed gene mutations and/or altered gene expression in ductal carcinoma in situ (DCIS). Investigators have demonstrated high levels of p53 protein in 13-40% of DCIS lesions employing a monoclonal antibody to p53, and subsequent sequencing demonstrated mutations in several cases (Poller et al, 1992). The neu/erbB2 gene appears to be amplified in a subset of DCIS lesions (Allred et al, 1992; Maguire et al, 1992). Histologic analysis of DCIS cases suggests that mutations and altered gene expression events, as well as changes in chromatin and WO 95/19369 PCT/US95/00608 2 DNA content, occur predominantly in comedo DCIS (B6cker et al, 1992; Killeen et al, 1991; and, Komitowski et al, 1990), which has a rapid rate of local invasion and progression to metastasis. Thus, there are presently no reliable marker genes for noncomedo DCIS (NCDCIS, hereafter).

Cancer in humans appears to be a multi-step process which involves progression from pre-malignant to malignant to metastatic disease which ultimately kills the patient.

Epidemiologic studies in humans have established that certain pathologic conditions are "pre-malignant" because they are associated with increased risk of malignancy. There is precedent for detecting and eliminating pre-invasive lesions as a cancer prevention strategy: dysplasia and carcinoma in-situ of the uterine cervix are examples of premalignancies which have been successfully employed in the prevention of cervical cancer by cytologic screening methods. Unfortunately, because the breast cannot be sampled as readily as cervix, the development of screening methods for breast premalignancy involves more complex approaches than cytomorphologic screening now currently employed to detect cervical cancer.

Pre-malignant breast disease is also characterized by an apparent morphological progression from atypical hyperplasias, to carcinoma in-situ (pre-invasive cancer) to invasive cancer which ultimately spreads and metastasizes resulting in the death of the patient. Careful histologic examination of breast biopsies has demonstrated intermediate stages which have acquired some of these characteristics but not others.

Detailed epidemiological studies have established that different morphologic lesions progress at different rates, varying from atypical hyperplasia (with a low risk) to comedo ductal carcinoma-in-situ which progresses to invasive cancer in a high percentage of patients (London et al, 1991; Page et al, 1982; Page et al, 1985; Page et al, 1991; and Page et al, 1978). Family history is also an important risk factor in the development of breast cancer and increases the relative risk of these pre-malignant lesions (Dupont et al, 1985; Dupont et al, 1993; and, London et al, 1991). Of particular interest is non-comedo carcinoma-in-situ which is associated with a greater than ten-fold increased relative risk of breast cancer compared to control groups (Ottesen et al, 1992; Page et al, 1982). Two other reasons besides an increased relative risk support the concept that DCIS is pre-malignant: 1) When breast cancer occurs in

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WO 95/19369 PCT/US95/00608 3 these patients it regularly occurs in the same region of the same breast where the DCIS was found; and 2) DCIS is frequently present in tissue adjacent to invasive breast cancer (Ottesen et al, 1992; Schwartz et al, 1992). For these reasons DCIS very likely represents a rate-limiting step in the development of invasive breast cancer in women.

DCIS (sometimes called intraductal carcinoma) is a group of lesions in which the cells have grown to completely fill the duct with patterns similar to invasive cancer, but do not invade outside the duct or show metastases at presentation. DCIS occurs in two forms: comedo DCIS and non-comedo DCIS. Comedo DCIS is often a grossly palpable lesion which was probably considered "cancer" in the 19th and early 20th century and progresses to cancer (without definitive therapy) in at least 50% of patients within three years (Ottesen et al, 1992; Page et al, 1982). Most of the molecular alterations which have been reported in pre-malignant breast disease have been observed in cases of comedo DCIS (Poller et al, 1993; Radford et al, 1993; and, Tsuda et al, 1993). Non-comedo DCIS is detected by microscopic analysis of breast aspirates or biopsies and is associated with a 10 fold increased risk of breast cancer, which corresponds to a 25-30% absolute risk of breast cancer within 15 years (Ottesen et al, 1992; Page et al, 1982; and, Ward et al, 1992).

Widespread application of mammography has changed the relative incidence of comedo and non-comedo DCIS such that NCDCIS now represents the predominant form of DCIS diagnosed in the United States (Ottesen et al, 1992; Page et al, 1982; and Pierce et al, 1992). Both forms of DCIS generally recur as invasive cancer at the same site as the pre-malignant lesion (without definitive therapy). The precursor lesions to DCIS are probably atypical ductal hyperplasia and proliferative disease without atypia which are associated with lower rates of breast cancer development, but show further increased risk when associated with a family history of breast cancer (Dupont et al, 1985; Dupont et al, 1989; Dupont et al, 1993; Lawrence, 1990; London et al, 1991; Page et al, 1982; Page et al, 1985; Page et al, 1991; Page et al, 1978; Simpson et al, 1992; Solin et al, 1991; Swain, 1992; Weed et al, 1990).

What is needed, then, is a sensitive method of detection and diagnosis of breast cancer when the cancerous cells are still in the pre-invasive stage. To illustrate the usefulness in early breast cancer detection of a marker gene and its encoded protein, 0 WO 95/19369 PCT/US95/00608 4 consider the dramatic impact that prostate specific antigen has had on early stage prostate cancer. This method of early detection and diagnosis of breast cancer is presently lacking in the prior art.

Breast cancer occurs in hereditary and sporadic forms. Recently the BRCA 1 gene has been cloned and shown to be mutated in kindreds with hereditary breast and ovarian cancer (Hall et al. 1990, Miki, Y. et al. 1994, Friedman et al. 1994, Castilla et al. 1994, Simard et al. 1994). Although 92% of families with two or more cases of early-onset breast cancer and two cases of ovarian cancer have germ-line mutations in BRCA 1 (Narod et al. in press), the gene has not been shown to be mutated in any truly sporadic case to date (Futreal et al. 1994). Despite the surprising paucity of somatically acquired mutations in sporadic breast cancer, it is still a likely tumor suppressor gene with a key role in breast epithelial cell biology. The BRCA 1 gene encodes a protein of 1863 amino acids with a predicted zinc finger domain observed in proteins which regulate gene transcription. Until the discovery of the function of the BRCA1 gene in conjucntion with the delopment of the present invention, the function was unknown.

DISCLOSURE OF THE INVENTION Epidemiologic studies have established that NCDCIS of the breast is associated with a ten-fold increased risk of breast cancer (absolute risk of 25-30%). It seems likely that this pre-invasive lesion is a determinate precursor of breast cancer because the subsequent development of breast cancer is regularly in the same region of the same breast in which the NCDCIS lesion was found. Important aspects of the present invention concern isolated DNA segments and those isolated DNA segments inserted into recombinant vectors encoding differentially expressed marker genes in abnormal tissue, specifically in NCDCIS, as compared with those expressed in normal tissue, and the creation and use of recombinant host cells through the application of DNA technology, which express these differentially expressed marker genes (Sambrook et al, 1989).

Because there are no cell lines or animal models which clearly display known characteristics of pre-invasive breast disease, human breast tissue samples are essential WO 95/19369 PCT/US95/00608 for studying pre-invasive breast disease. Using human tissue samples, we subsequently have developed a method for cDNA cloning from histologically identified lesions in human breast biopsies. We have used this method to clone genes which are differentially expressed in pre-invasive breast lesions such as NCDCIS lesions as compared to genes expressed in normal tissue. The differentially expressed genes detected in pre-invasive breast cancer are called marker genes. Identification of marker genes for pre-invasive breast disease provides improved methods for detection and diagnosis of pre-invasive breast cancer tissue, and further provides marker genes for studies of the molecular events involved in progression from pre-invasive to malignant breast disease.

Analysis of marker gene expression in NCDCIS presents the advantage that cancerous breast tissue at that stage is non-invasive. Detection and diagnosis of NCDCIS by means of differentially expressed marker genes compared to the same marker genes in normal breast tissue, would allow a greater ability to detect, prevent and treat the disease before it becomes invasive and metastasizes. The stage or intermediate condition of NCDCIS is a particularly good candidate for early intervention because it is 1) prior to any invasion and thus prior to any threat to life; 2) it is followed by invasive carcinoma in over 30% of cases if only treated by biopsy; and, 3) there is a long "window" of opportunity (4-8 years) approximately before invasive neoplasia occurs. Thus, NCDCIS is an ideal target for early diagnosis. While these morphologically defined intermediate endpoints have been widely accepted, progress in defining the molecular correlates of these lesions has been hampered by an inability to identify and sample them in a manner which would allow the application of molecular techniques.

Frozen tissue blocks from breast biopsies were used to construct and screen cDNA libraries prepared from NCDCIS tissue, normal breast tissue, breast cancer tissue, and normal human breast epithelial cells. Several cDNAs which were differentially expressed in human DCIS epithelial cells compared to normal breast epithelial cells were cloned and sequenced. One gene which is differentially expressed is the M2 subunit of RibRed which is expressed at low levels in human breast epithelial cells but at higher levels in 4 out of 5 DCIS tissue samples. It is presumed that the WO 95/19369 PCT/US95/00608 6 altered morphologic appearance and determinant biologic behavior of DCIS results from altered expression of genes (such as RibRed) which is important in the induction of breast cancer in humans.

This invention, therefore, provides a method of detecting and diagnosing preinvasive breast cancer by analyzing marker genes which are differentially expressed in non-comedo DCIS cells. Histopathologic studies have demonstrated that these morphologic patterns in breast tissue lead to invasive breast cancer in at least 20-30% of patients. The present method analyzes gene expression in normal, pre-malignant and malignant breast biopsies; and, it allows simultaneous comparison and cloning of marker genes which are differentially expressed in pre-invasive breast cancer. These marker genes can then be used as probes to develop other diagnostic tests for the early detection of pre-invasive breast cancer.

The present invention concerns DNA segments, isolatable from both normal and abnormal human breast tissue, which are free from total genomic DNA. The isolated DCIS-1 protein product is the regulatory element of the RibRed enzyme. This and all other isolatable DNA segments which are differentially expressed in preinvasive breast cancer can be used in the detection, diagnosis and treatment of breast cancer in its earliest and most easily treatable stages. As used herein, the term "abnormal tissue" refers to pre-invasive and invasive breast cancer tissue, as exemplified by collected samples of non-comedo or comedo DCIS tissues.

As used herein, the term "DNA segment" refers to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a differentially expressed protein (as measured by the expression of mRNA) in abnormal tissue refers to a DNA segment which contains differentially expressed-coding sequences in abnormal tissue as compared to those expressed in normal tissue, yet is isolated away from, or purified free from, total genomic DNA of Homo sapiens sapiens. Furthermore, a DNA segment encoding a BRCA1 protein refers to a DNA segment which contains BRCA1 coding sequences, yet is isolated away from, or purified free from, total genomic DNA of Homo sapiens sapiens. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, WO 95/19369 PCT/US95/00608 7 phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified differentially expressed gene or comprising an isolated or purified BRCA1 gene refers to a DNA segment including differentially expressed coding sequences or BRCA1 coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. "Isolated substantially away from other coding sequences" means that the gene of interest, in this case, any differentially expressed marker gene or the BRCA1 gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions.

Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode differentially expressed genes in pre-invasive breast cancer, each which includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, all seq id no:s 1-7 are derived from non-comedo DCIS samples from Homo sapiens sapiens. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode the M2 subunit of human RibRed that includes within its amino acid sequence the similar amino acid sequence of hamster RibRed corresponding to the M2 subunit of hamster RibRed.

In certain embodiments, the invention concerns isolated DNA segments and recombinant vectors which partially or wholly encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as partially or wholly encoded, respectively, by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. Naturally, where the DNA segment or vector encodes a full length differentially expressed protein, or is intended I WO 95/19369 PCT/US95/00608 8 for use in expressing the differentially expressed protein, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 and which encode a protein that exhibits differential expression, as may be determined by the differential display or differential sequencing assay, as disclosed herein.

The term "a sequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7" means that the sequence substantially corresponds to a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, respectively, and has relatively few nucleotides which are not identical to, or a biologically functional equivalent of, the nucleotides of the respective SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein, for example see pages 24 through Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 will be sequences which are "essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7", respectively.

In particular embodiments, the invention concerns a drug screening method and a gene therapy method that use isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a protein that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:49, SEQ ID NO:49 derived from breast tissue from Homo sapiens. In other particular embodiments, the invention concerns isolated DNA sequences and recombinant DNA vectors incorporating DNA sequences wich encode a protein taht includes with its amino acid sequence the amino acid sequence of the BRCA1 gene product from human breast tissue.

WO 95/19369 PCT/US95/00608 9 In certain embodiments, the invention concerns methods using isolated DNA segments and recombinant vectors which partially or wholly encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:49. Naturally, where the DNA segment or vector encodes a full length BRCA1 protein, or is intended for use in expressing the BRCA1 protein, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:47 and which encode a protein that retains activity as a negative growth regulator in human breast cells, as may be determined by antisense assay, as disclosed herein.

The term "a sequence essentially as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7" means that the sequence substantially corresponds to a portion of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, respectively, and has relatively few nucleotides which are not identical to, or a biologically functional equivalent of, the nucleotides of the respective SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein, for example see pages 24 through Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 will be sequences which are "essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7", respectively.

The term "a sequence essentially as set forth in SEQ ID NO:49" means that the sequence substantially corresponds to a portion of SEQ ID NO:49 and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the nucleotides of SEQ ID NO:49. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein, for example see pages 24 through 25. Accordingly, sequences which have between about 70% and about or more preferably, between about 81% and about 90%; or even more WO 95/19369 PCT/US95/00608 preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:49 will be sequences which are "essentially as set forth in SEQ ID NO:49".

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. The term "essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

Again, DNA segments which encode proteins exhibiting differential expression will be most preferred. The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see Figure 8).

In certain other embodiments, the invention concerns a method for screening drugs and a gene therapy method which involve the use of isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:47 and SEQ ID NO:48. The term "essentially as set forth in SEQ ID NO:47 and SEQ ID NO:48" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:47 and SEQ ID NO:48 respectively, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:47 and SEQ ID NO:48, respectively. Again, DNA segments which encode proteins exhibiting the negative regulatory activity of the BRCA1 will be most preferred. The term "functionally equivalent codon" is used herein to refer to codons WO 95/19369 PCT/US95/00608 11 that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see Figure 8).

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, introns, which are known to occur within genes.

Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences which have between about 20% and about 50%; or more preferably, between about 50% and about 70%; or even more preferably, between about 70 and about 99 of nucleotides which are identical to the nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 will be sequences which are "essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7", respectively. Sequences which are essentially the same as those set forth in SEQ ID NO: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID SEQ ID NO:6, and SEQ ID NO:7, respectively, under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art (Sambrook et al, 1989).

Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences which have between about 20% and about 50%; or more preferably, between about 50% and about 70%; or even more preferably, between about 70% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:47 and SEQ ID NO:48 will be sequences which are "essentially as set forth in WO 95/19369 PCT/US95/00608 12 SEQ ID NO:47 and SEQ ID NO:48", respectively. Sequences which are essentially the same as those set forth in SEQ ID NO:47 and SEQ ID NO:48 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:47 and SEQ ID NO:48, respectively, under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art (Sambrook et al, 1989).

It is also important to understand the molecular events which lead to progression from pre-invasive to invasive breast cancer. Breast cancer is a disease that is presumed to involve a series of genetic alterations that confer increasing growth independence and metastatic capability on somatic cells. Identifying the molecular events that lead to the initial development of a neoplasm is therefore critical to understanding the fundamental mechanisms by which tumors arise and to the selection of optimal targets for gene therapy and chemopreventive agents. As intermediate endpoints in neoplastic development, some pre-malignant breast lesions represent important, and possibly rate-limiting steps in the progression of human breast cancer, and careful epidemiological studies have established the relative risk for breast cancer development for specific histologic lesions. In particular, invasive breast cancer develops in the region of the previous biopsy site in at least 25-30% of patients following diagnosis of non-comedo DCIS providing strong evidence that this pre-malignant lesion is a determinant event in breast cancer progression. While these morphologically defined intermediate endpoints have been widely accepted, progress in defining the molecular correlates of these lesions has been hampered by an inability to identify and sample them in a manner which would allow the application of molecular techniques.

The present invention includes a comparison of gene expression between multiple breast tissue biopsy samples as a means to identify differentially expressed genes in pre-malignant breast disease compared with normal breast tissue. These genetic markers should be extremely useful reagents for early diagnosis of breast cancer, and for the delineation of molecular events in progression of breast cancer.

Identification of gene markers which are expressed in the majority of preinvasive breast cancer tissue samples involves cDNA library preparation from both WO 95119369 PCT/US95/00608 13 normal and abnormal tissue. This is followed by either a modified differential display method or a differential screening method to identify differential expression of genes which is subsequently confirmed by RT-PCR, nuclease protection assays and in situ hybridization of DCIS tissue RNA and control tissue RNAs (Sambrook et al, 1989).

Use of genetic engineering methods can bias the screening to specifically identify genes whose encoded proteins are secreted or are present at the cell surface, in order to find proteins which will be useful markers for diagnostic blood tests (secreted proteins) or for diagnostic imaging studies (cell surface proteins).

Thus, the method of the present invention begins with the collection of at least one tissue sample by a microscopically-directed collection step in which a punch biopsy is obtained exclusively from abnormal tissue which exhibits histological or cytological characteristics of pre-invasive breast cancer. Preferably, the sample site will be an isolatable tissue structure, such as ductal epithelial cells from pre-invasive breast cancer tissue. The mRNA is purified from the sample. Then, a cDNA library is prepared from the mRNA purified from the abnormal tissue sample (Sambrook et al, 1989).

A normal tissue sample is then obtained from the patient, using a sample site from an area of tissue which does not exhibit histological or cytological characteristics of pre-invasive cancer. A cDNA library is also prepared from this normal tissue sample.

The abnormal tissue cDNA library can then be compared with the normal tissue cDNA library by differential display or differential screening to determine whether the expression of at least one marker gene in the abnormal tissue sample is different from the expression of the same marker gene in the normal tissue sample.

Further diagnostic steps can be added to the method by cloning the marker gene using sequence-based amplification to create a cloned marker gene which can then be DNA-sequenced in order to derive the protein sequence. The protein sequence is then used to generate antibodies which will recognize these proteins by antibody recognition of the antigen. The presence of the antibody-recognized antigen can then be detected by means of conventional medical diagnostic tests.

WO 95/19369 PCT/US95/00608 14 This invention also includes methods of screening for compounds and gene therapy methods using the BRCA1 gene. BRCA1 mRNA is expressed at 5-10 fold higher levels in normal mammary tissue than in invasive breast cancer samples.

Having demonstrated that mRNA expression levels of BRCA1 are higher in normal mammary cells than in cancer cells, antisense methods were used to test the hypothesis that BRCA1 expression inhibits cell growth. These tests showed that diminished expression of BRCA1 increased the proliferative rate of breast cells.

An object of the present invention, then, is to provide a method of early detection of pre-invasive breast cancer in human tissue.

It is a further object of this invention to identify early marker genes for preinvasive breast disease which can be used in screening methods for early pre-invasive breast cancer.

It is also an object of this invention to produce a cDNA library from preinvasive breast cancer tissue resulting in a permanent genetic sample of that preinvasive breast cancer tissue.

It is also an object of this invention to provide a drug or biological screening method using the BRCA 1 promoter region and gene therapy method using the BRCA 1 gene.

List of Abbreviations TPA Phorbol 12-myristate 13-acetate MCF-7 An immortalized cell line derived from a metastasis of human breast cancer HMEC A primary (non-immortalized) cell line derived from breast epithelial cells obtained during reduction mammoplasty DCIS Ductal Carcinoma-in-situ NCDC Non-Comedo Ductal Carcinoma in situ cDNA Complementary DNA obtained from an RNA template DNA Deoxyribonucleic Acid RT-PCR Reverse Transcriptase-Polymerase Chain Reaction RibRed Ribonucleotide Reductase WO 95/19369 PCT/US95/00608 Fig. 1 shows Table I which describes anatomic lesion types in the human breast with pre-malignant implication.

Fig. 2 shows a model for pre-malignant conditions, highlighting magnitude of risk for progression to clinical malignancy.

Fig. 3 contains color photos of DCIS tissue, before (upper left panel) and after microscopically-directed excisional punch biopsy (upper right panel). The lower panels show tissue samples of normal breast tissue (lower left panel), and invasive breast cancer (lower right panel).

Fig. 4 shows expression of collagen III mRNA in tissue mRNA samples, analyzed by RNase protection assay methods.

Fig. 5 shows differential display of cDNAs obtained from patient tissue samples and controls.

Fig. 6 shows a comparison of the sequence between DCIS-1 and the human and hamster genes.

Fig. 7 shows expression of DCIS-1 mRNA in tissue mRNA samples analyzed by RNase protection assay as described in the legend to Figure 4.

Fig. 8 is Table II which displays the genetic code.

Fig. 9 is a Table which lists differentially expressed marker genes.

Figs. 10A and 10B shows expression of BRCA1 mRNA during breast cancer progression by PCR detection and nuclease protection assay, respectively.

Figs. 11A and 11B is a comparison of BRCA1 expression in normal breast and invasive breast cancer using nuclease protection assay of RNA, respectively.

Figs. 12A, 12B, and 12C show that antisense inhibition of BRCA1 accelerates mammary cell proliferation.

Figs. 13A and 13B includes a Northern blot of mRNA and nuclear runon studies that show that ribonucleotide reductase M2 mRNA is cell cycle regulated in MCF-7 cells.

Fig. 14 includes a nuclease protection assay that shows that antisense inhibition of BRCA1 in human mammary cells decreases BRCA1 mRNA and increases ribonucleotide reductase mRNA.

WO 95/19369 PCTUS95/00608 16 UTILITY STATEMENT The detection of differentially expressed genes in pre-invasive breast tissue, specifically in non-comedo ductal carcinoma in situ as compared to genes expressed in normal tissue, is useful in the diagnosis, prognosis and treatment of human breast cancer. Such differentially expressed genes are effective marker genes indicating the significantly increased risk of breast cancer in a patient expressing these differentially expressed marker genes. These marker genes are useful in the detection, early diagnosis, and treatment of breast cancer in humans.

The discovery of the function of the BRCA 1 gene has broad utility including, in the present invention, development of methods to treat familial and sporadic breast cancers as well as screen for therapeutic drugs through production of important indicator compounds.

ACTIVITY STATEMENT Of the differentially expressed genes described in this invention, DCIS-1 encodes a gene similar to the M2 subunit of hamster ribonucleotide reductase. The M2 subunit of ribonucleotide reductase (RibRed, hereafter) is responsible for regulation of RibRed. The differential levels of expression of the marker genes described in this invention (Seq ID No.s indicate genetic changes which have been linked to the presence of pre-invasive breast cancer.

The BRCA1 gene (Seq. ID No. 47) is differentially expressed in invasive breast cancer cells. The BRCA1 gene product is a negative regulator of mammary cell proliferation which is expressed at diminished levels in sporadic breast cancer.

BEST MODE FOR CARRYING OUT THE INVENTION For the purposes of the subsequent description, the following definitions will be used: Nucleic acid sequences which are "complementary" are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, that the larger purines will always base pair with the smaller pyrimidines to form only combinations of Guanine paired with Cytosine and Adenine paired with WO 95/19369 PCT/US95/00608 17 either Thymine in the case of DNA or Adenine paired with Uracil in the case of RNA.

"Hybridization techniques" refer to molecular biological techniques which involve the binding or hybridization of a probe to complementary sequences in a polynucleotide. Included among these techniques are northern blot analysis, southern blot analysis, nuclease protection assay, etc.

"Hybridization" and "binding" in the context of probes and denatured DNA are used interchangeably. Probes which are hybridized or bound to denatured DNA are aggregated to complementary sequences in the polynucleotide. Whether or not a particular probe remains aggregated with the polynucleotide depends on the degree of complementarity, the length of the probe, and the stringency of the binding conditions.

The higher the stringency, the higher must be the degree of complementarity and/or the longer the probe.

"Probe" refers to an oligonucleotide or short fragment of DNA designed to be sufficiently complementary to a sequence in a denatured nucleic acid to be probed and to be bound under selected stringency conditions.

"Label" refers to a modification to the probe nucleic acid that enables the experimenter to identify the labeled nucleic acid in the presence of unlabeled nucleic acid. Most commonly, this is the replacement of one or more atoms with radioactive isotopes. However, other labels include covalently attached chromophores, fluorescent moeities, enzymes, antigens, groups with specific reactivity, chemiluminescent moeities, and electrochemically detectable moeities, etc.

"Marker gene" refers to any gene selected for detection which displays differential expression in abnormal tissue as opposed to normal tissue. It is also referred to as a differentially expressed gene.

"Marker protein" refers to any protein encoded by a "marker gene" which protein displays differential expression in abnormal tissue as opposed to normal tissue.

"Tissuemizer" describes a tissue homogenization probe.

"Abnormal tissue" refers to pathologic tissue which displays cytologic, histologic and other defining and derivative features which differ from that of normal WO 95/19369 PCT/US95/00608 18 tissue. This includes in the case of abnormal breast tissue, among others, pre-invasive and invasive neoplasms.

"Normal tissue" refers to tissue which does not display any pathologic traits.

"PCR technique" describes a method of gene amplification which involves sequenced-based hybridization of primers to specific genes within a DNA sample (or library) and subsequent amplification involving multiple rounds of annealing, elongation and denaturation using a heat-stable DNA polymerase.

"RT-PCR" is an abbreviation for reverse transcriptase-polymerase chain reaction. Subjecting mRNA to the reverse transcriptase enzyme results in the production of cDNA which is complementary to the base sequences of the mRNA.

Large amounts of selected cDNA can then be produced by means of the polymerase chain reaction which relies on the action of heat-stable DNA polymerase produced by Thermus aquaticus for its amplification action.

"Microscopically-directed" refers to the method of tissue sampling by which the tissue sampled is viewed under a microscope during the sampling of that tissue such that the sampling is precisely limited to a given tissue type, as the investigator requires.

Specifically, it is a collection step which involves the use of a punch biopsy instrument.

This surgical instrument is stereotactically manually-directed to harvest exclusively from abnormal tissue which exhibits histologic or cytologic characteristics of pre-invasive cancer. The harvest is correlated with a companion slide, stained to recognize the target tissue.

"Differential display" describes a method in which expressed genes are compared between samples using low stringency PCR with random oligonucleotide primers.

"Differential screening" describes a method in which genes within cDNA libraries are compared between two samples by differential hybridization of cDNAs to probes prepared from each library.

"Nuclease protection assay" refers to a method of RNA quantitation which employs strand specific nucleases to identify specific RNAs by detection of duplexes.

"Differential expression" describes the phenomenon of differential genetic expression seen in abnormal tissue in comparison to that seen in normal tissue.

i rl i- 1 ;I -_l~llli^~l~=l-i=-Li ii. i WO 95/19369 PCTIUS95/00608 19 "Isolatable tissue structure" refers to a tissue structure which when visualized microscopically or otherwise is able to be isolated from other different surrounding tissue types.

"In situ hybridization of RNA" refers to the use of labeled DNA probes employed in conjunction with histological sections on which RNA is present and with which the labeled probe can hybridize allowing an investigator to visualize the location of the specific RNA within the cell.

"Comedo DCIS cells" refers to cells comprising an in situ lesion with the combined features of highest grade DCIS.

"Non-comedo DCIS cells" refers to cells of DCIS lesions without comedo features.

"Cloning" describes separation and isolation of single genes.

"Sequencing" describes the determination of the specific order of nucleic acids in a gene or polynucleotide.

The present invention provides a method for detecting and diagnosing cancer by analyzing marker genes which are differentially expressed in early, pre-invasive breast cancer, specifically in non-comedo DCIS cells. Our histopathologic studies have demonstrated that certain morphologic patterns in breast tissue are pre-malignant, leading to invasive breast cancer in at least 20-30% of patients. We have developed a new method for analyzing gene expression in normal, pre-malignant and malignant breast biopsies which allows simultaneous comparison and cloning of marker genes which are differentially expressed in pre-invasive breast cancer. These marker genes (which appear as differentially expressed genes in pre-invasive breast cancer) can be used as probes to develop diagnostic tests for the early detection of pre-invasive breast cancer (Sambrook, 1989).

The present invention thus comprises a method of identification of marker genes which are expressed in the majority of pre-invasive breast cancer tissue samples. It involves cDNA library preparation followed by a modified differential display method.

Use of genetic engineering methods (Sambrook, 1989) can bias the screening to specifically identify genes whose encoded proteins are secreted or are present at the cell WO 95/19369 PCT/US95/00608 surface, in order to find proteins which will be useful markers for diagnostic blood tests (secreted proteins) or for diagnostic imaging studies (cell surface proteins).

Naturally, the present invention also encompasses DNA segments which are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:47 and SEQ ID NO:48. Nucleic acid sequences which are "complementary" are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:47 and SEQ ID NO:48 under relatively stringent conditions such as those described herein.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared which include a short stretch complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:47 and SEQ ID NO:48, such as about nucleotides, and which are up to 10,000 or 5,000 base pairs in length, with segments of 500 being preferred in most cases. DNA segments with total lengths of about 1,000, 500, 200, 100 and about 50 base pairs in length are also contemplated to be useful.

It will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49. Recombinant vectors and isolated DNA WO 95/19369 PCT/US95/00608 21 segments may therefore variously include the differentially expressed coding regions or the BRCAI coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides which nevertheless include differentially expressed-coding regions and the BRCA1 coding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acids sequences.

The DNA segments of the present invention encompass biologically functional equivalent differentially expressed proteins and peptides biologically functional equivalent proteins of BRCA 1. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, to introduce improvements to the antigenicity of the protein or to test site-directed mutants or others in order to examine carcinogenic activity of the differentially expressed marker genes at the molecular level.

If desired, one may also prepare fusion proteins and peptides, where the differentially expressed marker gene coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes proteins which may be purified by affinity chromatography and enzyme label coding regions, respectively).

Recombinant vectors form important further aspects of the present invention.

Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment is positioned under the control of a promoter. The promoter may be in the form of the promoter which is naturally associated with a RIBRED gene, in human cells, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein.

WO 95/19369 PCT/US95/00608 22 In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a differentially expressed marker gene or the BRCA1 gene in its natural environment. Such promoters may include MMTV promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.

Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to appropriate bacterial promoters.

As mentioned above, in connection with expression embodiments to prepare recombinant differentially expressed marker gene encoded proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire differentially expressed protein or subunit being most preferred.

However, it will be appreciated that the use of shorter DNA segments to direct the expression of differentially expressed peptides or epitopic core regions, such as may be used to generate anti-marker protein antibodies, also falls within the scope of the invention (Harlow et al, 1988).

DNA segments which encode peptide antigens from about 15 to about amino acids in length, or more preferably, from about 15 to about 30 amino acids in length are contemplated to be particularly useful. The C terminus of proteins provide an excellent region for peptide antigen recogition (Harlow et al, 1988). DNA segments encoding peptides will generally have a minimum coding length in the order of about 45 to about 147, or to about 90 nucleotides. DNA segments encoding partial length peptides may have a minimum coding length in the order of about 50 nucleotides for WO 95/19369 PCT/US95/00608 23 a polypeptide in accordance with seq id no:3, or about 264 nucleotides for a polypeptide in accordance with SEQ ID NO: 1.

In addition to their use in directing the expression of the differentially expressed marker proteins, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that oligonucleotide fragments corresponding to the sequences of SEQ ID NO: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 for stretches of between about 10 to 15 nucleotides and about 20 to 30 nucleotides will find particular utility.

Longer complementary sequences, those of about 40, 50, 100, 200, 500, 1000, and even up to full length sequences of about 2,000 nucleotides in length, will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to differentially expressed marker gene sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

Nucleic acid molecules having stretches of 20, 30, 50, or even of 500 nucleotides or so, complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 are particularly contemplated as hybridization probes for use in, Southern and Northern blotting.

This would allow differentially expressed structural or regulatory genes to be analyzed, both in patients and sample tissue from pre-invasive and invasive breast tissue. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between about and about 100 nucleotides, but larger complementary stretches of up to about 300 nucleotides may be used, according to the length complementary sequences one wishes to detect.

i ii WO 95/19369 PCT/US95/00608 24 Nucleic Acid Hybridization The use of a hybridization probe of about 10 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired.

Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequences set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 and to select any continuous portion of one of the sequences, from about 10 nucleotides in length up to and including the full length sequence, that one wishes to utilise as a probe or primer. The choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the termini of the total sequence, or from the ends of the functional domain-encoding sequences, in order to amplify further DNA; one may employ probes corresponding to the entire DNA, or to the 5' region, to clone markertype genes from other species or to clone further marker-like or homologous genes from any species including human; and one may employ randomly selected, wild-type and mutant probes or primers with sequences centered around the RibRed M2 subunit encoding sequence to screen DNA samples for differentially expressed levels of RibRed, such as to identify human subjects which may be expressing differential levels of RibRed and thus may be susceptible to breast cancer.

The process of selecting and preparing a nucleic acid segment which includes a sequence from within SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 may alternatively be described as "preparing a nucleic acid fragment". Of course, fragments may also be obtained by other techniques such as, by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly lil; WO 95/19369 PCT/US95/00608 practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of differentially expressed marker genes or cDNAs. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, one will select relatively low salt and\or high temperature conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C to 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating specific differentially expressed marker genes.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate marker gene sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as 0.15M-0.9M salt, at temperatures ranging from to 55C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

it WO 95/19369 PCT/US95/00608 26 In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label. (Sambrook et al, 1989).

In a preferred embodiment of the method, certain preliminary procedures are necessary to prepare the sample tissue and the probes before the detection of differential expression of marker genes in abnormal tissue as compared to that in normal tissue can be accomplished.

SAMPLE PREPARATION RNA purification RNA was isolated from frozen tissue samples by mincing of microdisected frozen tissue fragments with a razor blade and then adding 800 microliter of 5.6M WO 95/19369 PCTfUS95/00608 27 guanidinium to increase mixing, followed by a 30 second microcentrifuge centrifugation at 14,000 rpm to remove particulate matter. The supernatant was then removed and the viscosity was reduced by multiple aspirations through a 22 gauge needle and then 200 ul of chloroform was added and the sample was incubated on ice for 15 minutes (during this time the sample was vortexed multiple times). Following incubation with chloroform, the sample was centrifuged for 15 minutes at 14,000 rpm and the aqueous layer was removed and ethanol precipitated. This extraction method produces RNA which is primarily derived from cells of epithelial origin. In order to obtain RNA samples which presumably includes RNA derived from these stromal cells; the particulate material (remaining in the pellet from the 30 second centrifugation) was homogenized with a tissuemizer, washed with PBS, treated with collagenase at 37°C for 30 minutes, sonicated, extracted with phenol/chloroform and ethanol precipitated.

cDNA libraries were constructed in lambda phage using polyA-selected mRNA from the following samples; cultured human breast epithelial cells, tissue from three reduction mammoplasty patients, tissue from three DCIS patients, and tissue from one DCIS patient (patient #10) that showed a focus of microinvasion adjacent to an area of DCIS. Multiple punches were needed to obtain sufficient RNA for polyA selection and library construction. 200 ug of total RNA was obtained by pooling 20 punches from normal breast tissue (reduction mammoplasty samples) and 5-8 punches from DCIS lesions, presumably reflecting the greater cellularity of the DCIS samples. cDNA libraries were constructed by first and second strand cDNA synthesis followed by the addition of directional synthetic linkers (ZAP-cDNA Synthesis Kit, Stratagene, La Jolla, California). The Xho I-Eco RI linkered cDNA was then ligated into lambda arms, packaged with packaging extracts, and then used to infect XLl-blue bacteria resulting in cDNA libraries.

PROBE PREPARATION The collagen III probe employed for nuclease protection assays was constructed by subcloning the 208 bp Hinc II-Pst I fragment from the 3' untranslated region of the human type In procollagen gene into pGem4Z. This region of the human procollagen III gene was obtained by PCR amplification of published sequence (Ala-Kokko et al, 1989) followed by restriction with Hinc II and Pst I. For a control probe to assure WO 95/19369 PCT/US95/00608 28 equal loading and recovery of RNA, we used a T7 polymerase-generated probe for human glyceraldehyde phosphate dehydrogenase (GADP) which protects a 140 bp Sac I-Xba I fragment; (a generous gift from Janice Nigro, Vanderbilt University). Probe DCIS-1 was generated by linearizing the rescued plasmid with Pvu II, which should generate a 200 bp protected fragment. RNase protection assays were performed with 1 ug of unselected RNA and the above-cited probes using the methods we have reported previously (Holt, 1993).

Differential Display-based cloning of cDNAs: Rescued cDNA library samples were used as templates for low stringency PCR with the either a pair of 25 bp primers or an anchored 14 bp primer paired with a random 25 bp primer. Random 25 bp primers were generated by a computer-based algorithm (Jotte and Holt, unpublished). Samples were denatured for two minutes at followed by 40 cycles, each cycle consisting of denaturation for 1 minute at 94*C., annealing for 2 minutes at 25°C., and extension for 1 minute at 72*C. The samples were then run on an 6% non-denaturing polyacrylamide gel, which was dried and autoradiographed. Specific bands were excised then reamplified with the same primers used for their generation. Specificity was confirmed on 6% polyacrylamide gel, and samples were purified by ethanol precipitation of the remainder of the PCR reaction. Fragments were then individually cloned into Srfl cut vectors by standard methods using PCR-ScriptTSK(+) Cloning Kit (Stratagene, LaJolla, California) and then sequenced.

EXAMPLE 1 Studies showing Increased Risk of Breast Cancer in Patients with DCIS Since the 1970's, studies of pre-invasive lesions associated with the development of breast cancer have been undertaken in an attempt to refine histologic and cytologic criteria for the hyperplastic lesions analogous to those of the uterine cervix and colon.

Because of the availability of tissue from breast biopsies done many years previously, cohorts of women who underwent breast biopsies 15 to 20 years ago, can be studied to determine the risk for development of breast cancer attributable to specific lesions.

WO 95/19369 PCT/US95/00608 29 Many concurrent studies evaluating lesions associated with cancer at time of cancer diagnosis led the way in pointing out lesions of potential interest (Wellings et al, 1975).

Hopefully, these intermediate stages in cancer development will serve to provide indicators of breast cancer development sufficiently precise to guide prevention and intervention strategies (Weed et al, 1990; Lippman et al, 1990). Such intermediate elements prior to the development of metastatic capable cancers also provide the opportunity to define the molecular biology of these elements. Studies of the development of pre-invasive breast disease have provided insight into different types of lesions with different implications for breast cancer risk and the process of carcinogenesis (See Figure Pre-invasive breast disease is herewith defined to be any reproducibly defined condition which confers an elevated risk of breast cancer approaching double that of the general population (Komitowski et al, 1990). The specifically-defined atypical hyperplasias and lobular carcinoma in situ confer relative risks of four to ten times that of the general population. This risk is for carcinoma to develop anywhere in either breast (Page et al, 1985; Page et al, 1991). The statistical significance of these observations have regularly been <.0001. Thus, absolute risk figures of 10-20% likelihood of developing into invasive carcinoma in 10 to 15 years arise. DCIS is a very special element in this story because the magnitude of risk is as high as any other condition noted .00005), but remarkably, the developing invasive cancer is in the same site in the same breast. This local recurrence and evolution to invasiveness marks these lesions as determinate precursors of invasive breast cancer (Betsill et al, 1978; Page et al, 1982). These figures are for the type of DCIS which has become detected very commonly since the advent of mammography, the small and NCDCIS variety. It is likely that the comedo DCIS variety indicates a much greater risk, often presenting as larger lesions, and treated regularly by mastectomy in the past 50 years making follow-up studies impossible (Figure 1).

The precision of histopathologic diagnosis in this area as noted in Table I (shown in Figure 1) was most convincingly confirmed in a large, prospective study (London et al, 1991). There has also been a recent review of the reproducibility of the assignment of diagnosis by a panel of pathologists (Schnitt et al, 1992). The precision has been fostered by combining histologic pattern criteria with cytologic and extent of WO 95/19369 PCT/US95/00608 lesion criteria. Classic surgical pathology criteria were predominantly derived from histologic pattern only. A further point of relevance to the importance of these histopathologically defined lesions of pre-malignancy in the breast is the relationship to familiality. A family history of breast cancer in a first degree relatives confers about a doubling of breast cancer risk. However, women with the atypical hyperplasias at biopsy and a family history of breast cancer are at 9-10 times the risk of developing invasive breast cancer as the general population (Dupont et al, 1985; Dupont et al, 1989).

Careful consideration of all of the above-mentioned epidemiologic data has led to the following model for progression from generalized pre-malignant lesions to determinant lesions to invasive cancer. Figure 2 shows this model for the induction and progression of pre-invasive breast disease based on study of the Vanderbilt cohort (Dupont et al, 1985) of more than 10,000 breast biopsies (follow-up rate 85%; median time of 17 years; 135 women developed breast cancer).

EXAMPLE 2 Identification of genes which are differentially expressed in DCIS Construction of cDNA libraries from DCIS lesions In order to study differential gene expression in DCIS, we collected cases of NCDCIS. The diagnosis of DCIS is made on histomorphologic grounds based on architectural, cytologic, and occasionally extent criteria. NCDCIS lacks comedo features and consists of microscopic intraductal lesions which fill and extend the duct, contain rigid internal architecture, and often have hyperchromatic and monomorphic nuclei.

Study of non-comedo DCIS for differential marker gene expression indicates the diagnostic utility of comparison of marker gene expression in these tissues. Although the morbidity and mortality of breast cancer clearly results from invasion and metastasis, the development of breast cancer is clearly significant in its early stages for two basic reasons: 1) The molecular changes will presumably be simpler in early lesions than in later lesions which may have acquired numerous mutations or "hits"; WO 95/19369 PCT/US95/00608 31 and 2) Successful prevention strategies may require attacking cancer before it develops the capacity to invade or metastasize.

Non-comedo DCIS is the earliest determinant lesion which recurs locally as invasive cancer. Although comedo DCIS may be technically easier to study because the tumors are larger, its aggressiveness and the presence of numerous genetic alterations (such as p53 and erbB2) suggest that it may have advanced beyond the earliest stages of carcinogenesis.

The commercial utility of a method for prevention of cancer is clear. In order to study differential gene expression in DCIS, breast tissue with extensive microscopic non-comedo DCIS was identified and banked in a frozen state. cDNA libraries were constructed from mRNA isolated from frozen sections of DCIS lesions. Tissue samples from patients with mammographic results consistent with DCIS were cryostat frozen and a definitive diagnosis was made by the histopathologic criteria which we have described (Jensen et al, Submitted for publication; Holt et al, In press).

Control mRNA was obtained from frozen tissue samples obtained from reduction mammoplasties and from cultured human breast epithelial cells. Because non-comedo DCIS is a microscopic lesion, we had to microlocalize regions of DCIS in biopsy samples. To accomplish this we prepared frozen sections in which we located regions of DCIS and then employed a 2 mm punch to obtain an abnormal tissue sample only from those regions that contained DCIS. This selective harvesting was accomplished by carefully aligning the frozen section slide with the frozen tissue block and identifying areas of interest. The harvest of the appropriate area was then confirmed with a repeat frozen section. A similar approach was used to isolate mRNA from lobules of normal breast in samples collected from a reduction mammoplasty. Prior studies have shown that breast lobules are approximately 2.5 mm in diameter, thus the 2 mm punch provided a well-tailored excision. This microlocation and collection step, in which abnormal tissue samples are collected from an isolatable tissue structure, was performed with extreme care and was absolutely crucial to the success of these studies.

Contamination by normal breast epithelial cells or by breast stromal cells would clearly negatively skew the differential screening approach. If the punch biopsy did not cleanly WO 95/19369 PCT/US95/00608 32 excise DCIS without contamination by other cell types or tissues then the sample was not used for mRNA isolation (Jensen et al, Submitted for publication). Figure 3 contains color photos of DCIS (abnormal) tissue, before (upper left panel) and after excisional punch biopsy (upper right panel). The lower panels show tissue samples of normal breast tissue (lower left panel), and invasive breast cancer (lower right panel).

Following microlocation punch harvesting of the frozen tissue, RNA was isolated, purified, and employed to construct cDNA libraries. RNA was isolated following mincing of tissue in 5.6M guanidinium isothiocyanate and 40% phenol, centrifugation to remove particulate matter, viscosity reduction by repeated aspiration through a 22 gauge needle, chloroform extraction and ethanol precipitation. In most samples there was particulate matter resistant to guanidinium-phenol extraction that was white in color and fibrous in appearance and was presumed to represent breast stroma. This stromal material was sparse in DCIS samples but abundant in samples obtained from normal breast tissue derived from reduction mammoplasties. The stromal material was minced with a tissuemizer, washed with PBS, treated with collagenase at 37°C for 30 minutes, sonicated, extracted with phenol/chloroform and ethanol precipitated. 200 ug of total RNA was obtained by pooling 20 punches from normal breast tissue (reduction mammoplasty samples) and 5-8 punches from DCIS lesions, presumably reflecting the greater cellularity of the DCIS samples. All libraries had greater than 50% inserts and contained between 2 X 106 and 7 X 107 phage recombinants with an average insert size varying between 500 and 1000 base pairs.

EXAMPLE 3 Development of an extraction method which produces breast epithelial RNA It was necessary that tissue samples not be contaminated by non-epithelial stromal cells. Such contamination would complicate efforts to compare gene expression between samples. In order to test the extent of stromal contamination of the mRNA samples, we determined the level of expression of collagen III mRNA by an RNase protection assay. RNase protection assays were employed in these and subsequent studies because it is a quantitative method and can be performed on small amounts of unselected RNA. Collagen II mRNA was identified in the presumed stromal fraction WO 95/19369 PCT/US95/00608 33 of the normal breast tissue and to a lesser extent in the microinvasive breast cancer sample, but no expression of collagen II was detected in the DCIS samples which were subsequently employed for cDNA library construction. Figure 4 compares expression in NL 2 and #10CA with other patient samples and NL1 to determine collagen III expression.

Expression of Collagen Il mRNA in tissue mRNA samples was analyzed by RNase protection assay by methods we have reported previously (Holt, 1993). One /g of mRNA was hybridized with two labeled RNA probes: a T7 polymerase-generated probe for human glyceraldehyde phosphate dehydrogenase (GADP) which protects a 140 bp Sac I-Xba I fragment; and a T7 polymerase-generated probe which protects a 208 bp Hinc II-Pst I fragment from the 3' untranslated region of the human type I procollagen gene (Coll III) obtained by PCR subcloning of the published sequence (Ala- Kokko et al, 1991). RNA samples were labeled as follows: NL1 is RNA from cultured human breast epithelial cells (Hammond et al, 1984), NL2 is RNA from normal breast tissue, NL3 is RNA derived from the fibrous stromal fraction of breast tissue as described (Jensen et al, Submitted for publication), NL4 is another sample from normal breast tissue. This is described in greater detail on page 30 of this patent application.

and #10 are from patient samples with DCIS. Sample #10CA is RNA obtained from the small focus of microinvasion shown in Figure 3. Con is a control sample using tRNA.

EXAMPLE 4 Screening of cDNA libraries Following successful testing which demonstrated that stromal contamination was not a problem, cDNA libraries were constructed in lambda phage using polyA-selected mRNA from the following samples: cultured human breast epithelial cells, tissue from three reduction mammoplasty patients, tissue from three DCIS patients, and tissue from one DCIS patient (patient #10) that showed a small focus of invasion adjacent to an area of DCIS. Multiple punches were needed to obtain sufficient RNA for polyA selection and library construction. Selective handling of tissue was accomplished.

WO 95/19369 PCT/US95/00608 34 Comparison of gene expression between samples was performed by either differential screening or a modification of differential display (Liang et al, 1992a; Liang et al, 1992b; Saiki et al, 1988; Melton et al, 1984). Plasmid DNA was prepared from the cDNA libraries following helper phage rescue and screened by two independent methods. Figure 5 below shows the results of differential display comparing cDNAs of several patient DCIS samples with cDNA obtained from normal breast epithelial cells and an early invasive cancer. Although few genes shown in this Figure are differentially expressed in the majority of samples with DCIS, the heterogeneity of gene expression in patient samples is seen.

The differential display method (Liang et al, 1992a and 1992b) allows simultaneous comparison of multiple tissue samples. Initial studies using this method (reverse transcriptase followed by PCR) were unsatisfactory because of unwanted amplification of contaminating DNA in tissue samples and the small size of many of the fragments identified by display. To circumvent some of these problems, we have attempted to combine the advantages of cDNA library screening with the advantages of differential display by: 1) Constructing cDNA libraries from the tissue mRNA samples; 2) Performing differential display on the plasmid DNA prepared from the cDNA libraries; 3) Subcloning the fragments identified by differential display; 4) Using the subcloned fragment as a probe to clone the cDNA from the appropriate library.

Example Identification of a gene (RibRed) which is differentially expressed in multiple NCDCIS cases Employing these methods, 10 differentially expressed clones were identified and the seven that showed the greatest difference in expression between multiple samples were further characterized by DNA sequencing. Comparison of the sequenced clones with GenBank demonstrated that six of the clones are apparently unique sequences (although further DNA sequencing is necessary); but that one of the clones (here termed DCIS-1 and described in Sequence Listing No. 1) showed 90% homology to the i WO 95/19369 PCTIUS95/00608 previously cloned hamster gene encoding the M2 subunit of ribonucleotide reductase (Pavloff et al, 1992; Hurta et al, 1991; Hurta et al, 1991). Although human M2 ribonucleotide reductase has been cloned previously, comparison of the hamster cDNA sequence with our clone and with the prior human clone indicates that DCIS-1 is homologous to an alternatively poly-adenylated form of the human ribonucleotide reductase which has not been cloned previously. Figure 6 shows a comparison of the sequence between DCIS-1 and the human and hamster genes.

Because of our concern that different patients may have differential gene expression which is idiosyncratic (or related to morphological differences in biopsy appearance) and not necessarily related to the induction or progression of DCIS, we simultaneously analyzed gene expression in multiple DCIS samples compared to multiple control samples. We constructed cDNA libraries from the following samples: 1) Cultured HMEC epithelial cells; 2) Reduction mammoplasty: 11 year old with virginal hyperplasia; 3) Reduction mammoplasty: 28 year old patient; 4) Reduction mammoplasty: 35 year old patient; DCIS patient #12; 6) DCIS patient #8; 7) DCIS patient 8) DCIS patient #10 from an area of invasive cancer adjacent to DCIS; In addition to the samples we employed to construct cDNA libraries shown above, we also obtained frozen tissue samples from 7 more DCIS patients, 2 cellular fibroadenoma samples, and samples of "usual hyperplasia" and atypical hyperplasia.

Because the DCIS clones were identified by cloning methods which include selection and amplification, it was important to confirm by nuclease protection assays that the genes were differentially expressed in the original unselected, unamplified RNA samples (Figure 7).

This approach allowed identification of a human gene similar to the hamster RibRed gene (coding for the M2 subunit) and 7 other human genes as genes which are differentially expressed in a majority of cases of DCIS in human breast tissue. The table of differentially expressed genes lists the genes which have been identified as WO 95/19369 PCT/US95/00608 36 differentially expressed genes in DCIS tissue samples as compared to that in normal tissue (Figure 9).

EXAMPLE 6 Methods for studying potential use of differentially expressed genes for diagnostic screening One advantage of the differential display method is that it allows comparison of multiple tissue samples of pre-invasive or invasive breast cancer. For example, use of this method has successfully demonstrated that the M2 subunit ribonucleotide reductase gene is differentially expressed in 4 out of 5 pre-invasive breast cancer tissue samples.

It is significant that the M2 subunit is involved in the regulation of the ribonucleotide reductase gene and is found to be over-expressed in abnormal tissue samples.

Identification of differentially expressed genes may lead to discovery of genes which are potentially useful for breast cancer screening. Of particular interest are genes whose expression is restricted to breast epithelial cells and whose gene products are secreted. Screening for secreted proteins is possible by using the known hydrophobic sequences which encode leader sequences as one primer for differential display. The identification of secreted proteins which are specific for early breast premalignancy (or even early invasive cancer) would provide an important tool for early breast cancer screening programs. If a differentially expressed gene has not been cloned previously (or if details of its expression are unknown or uncertain) then nuclease protection assays or Northern blots can be performed on RNA prepared from tissue samples from a variety of tissues to determine if expression of this gene is restricted to breast. If necessary cDNA libraries prepared from other tissues can be added to the differential display screen as a way to identify only those genes which are expressed in early breast cancer and, in addition, are only expressed in breast tissue.

Once differentially expressed genes have been initially characterized for expression in pre-malignant and malignant breast disease, antibodies to the protein products of potentially useful genes can be developed and employed for immunohistochemistry (Harlow et al, 1988). This will provide an additional test to determine whether the expression of this gene is restricted to the breast. Subsequently, these antibodies will s~l WO 95/19369 PCT/US95/00608 37 be used to detect the presence of this protein present in the blood of patients with preinvasive and/or invasive cancer. By assaying for serum protein levels in the same patients who exhibited elevated expression of the gene in their tissue samples it will be possible to determine whether a gene product is being secreted into the blood.

EXAMPLE 7 Decreased expression of BRCA1 accelerates growth and is observed during breast cancer progression Breast cancer occurs in hereditary and sporadic forms. Recently the BRCA 1 gene has been cloned and shown to be mutated in kindreds with hereditary breast and ovarian cancer (Hall et al. 1990, Miki, Y. et al. 1994, Friedman et al. 1994, Castilla et al. 1994, Simard et al. 1994). Although 92% of families with two or more cases of early-onset breast cancer and two cases of ovarian cancer have germ-line mutations in BRCA 1 (Narod et al. in press), the gene has not been shown to be mutated in any truly sporadic case to date (Futreal et al. 1994). Despite the surprising paucity of somatically acquired mutations in sporadic breast cancer, it is still a likely tumor suppressor gene with a key role in breast epithelial cell biology. The BRCA 1 gene encodes a protein of 1863 amino acids with a predicted zinc finger domain observed in proteins which regulate gene transcription.

As an initial characterization of the regulation and function of the BRCA 1 gene, we analyzed and manipulated expression of BRCA 1 mRNA levels. The results taken together indicate that the BRCA 1 gene product is a negative regulator of mammary cell proliferation which is expressed at diminished levels in sporadic breast cancer.

Expression of BRCA1 mRNA during breast cancer progression As described above, microscopy-directed cloning has been employed to compare gene expression in normal mammary epithelium, carcinoma in-situ, and invasive breast cancer. This method produces predominantly epithelial mRNA with minimal contamination from stromal elements and we used this approach to obtain mRNA from normal neoplastic tissues from patients without a family history of breast cancer.

Expression of BRCA1 exon 24 in human breast tissue samples is shown in Fig. 1. The legend of Fig. 1 is as follows.

WO 95/19369 PCT/US95/00608 38 The following tissue samples were used for mRNA isolation: Normal tissue samples: NLl-cultured human breast epithelial cells, NL2- Histologically normal breast tissue from an 11 year old undergoing a reduction mammoplasty, NL4- histologically normal breast tissue from an 14 year old undergoing a reduction mammoplasty.

Carcinoma-in-situ samples are #10, #12, #23 (comedo type), #41, #55; and invasive cancer samples #10CA (invasive cancer from the same patient with carcinomain-situ), 36CA, 1CA. All of these tissue samples were obtained from patients who had no family history of hereditary breast cancer and RNA preparation was performed as described above.

PCR detection of BRCA1 exon 24 in cDNA libraries from the following tissue samples is described in Figure 10A. Lane 1: human genomic DNA, lane 2: NL1, lane 3: NL4, lane 4: lane 5: #12, lane 6: #10, lane 7: #10CA, lane 8: #41, lane 9: #23, lane 10: 36CA, lane 11: lambda DNA. The arrow points to the expected 113 bp band.

Nuclease protection assays of microdissected mRNA from tissue samples are described in Fig. 10B. One ug of mRNA from each tissue sample was hybridized with 32P-labelled, T7 polymerase-generated RNA probes for BRCA1 and human glyceraldehyde-3-phosphate dehydrogenase (GAPD) which produce expected protected fragments of 113 and 140 respectively as indicated by the lines on the right. Data were quantitated by phosphorimaging. The hybridizing intensity of each BRCA1 band was normalized to its respective GAPD band. The normalized values of NL1, NL2, and NL4 were intensity in each sample relative to 1. Sample 1 employs human leukocyte mRNA; Samples 2-4 are NL1, NL2, and NL4; Samples 5-9 are 8(3.7), 10(2.8), 12 and 55 and 10-12 are #10CA 36CA and 1CA Fig. 10 shows that BRCA1 exon 24 mRNA is expressed at 5-10 fold higher levels in normal mammary tissue than in invasive breast cancer samples. Initial studies showed detectable levels of BRCA1 cDNA in a cDNA library prepared from a tissue sample with preinvasive carcinoma-in-situ but not in normal breast cancer invasive breast cancer cDNA libraries (Figure 10A). Because this method is relatively insensitive we directly quantitated BRCA1 mRNA by nuclease protection assays in RNA samples obtained by our microdissection method described above. These assays li-. _ilE WO 95/19369 PCT/US95/00608 39 indicate that expression of BRCA1 mRNA in micro-dissected normal mammary epithelial tissue (lanes 2-4, Figure 10B) is 5-15 fold higher than that in breast cancer (lanes 10-12, Figure 10B). The highest levels of BRCA1 are observed in samples from non-comedo ductal carcinoma-in-situ (lanes 5-9, Figure 10B), a premalignant breast lesion with a finite, but relatively low rate of progression to invasion (Betsill et at., 1978, Page, D.L. et al., 1982, Page and Dupont, 1990).

Because these studies suggested that invasive breast cancer exhibited lower mRNA levels than normal breast epithelial cells, we compared expression of paired samples of normal breast and invasive cancer from the same patient (Figure 11A; compare lanes 2 and 3, 4 and 5, 6 and The legend of Fig. 11 is as follows.

Nuclease protection assays of RNA obtained from paired samples of invasive breast cancer and histologically normal breast tissue are shown in Fig. 11A. Samples in lanes 2 and 3 (first patient), 4 and 5 (second patient), 6 and 7 (third patient) are from invasive cancer and normal breast tissue respectively. Lane 1 is NL1 mRNA as described in legend to Fig. 10 and lane 8 is human leukocyte mRNA. Ratios of BRCA1/GAPD for each sample: lane 1: 25.9, lane 2: 1.8, lane 3: 7.6, lane 4: lane 5: 12.4, lane 6: 0.7, lane 7: 6.0. The probes and methods are as described in Fig.

except the GAPD probe was of lower specific activity to improve quantitation.

Nuclease protection assays of RNA from a series of invasive breast cancer tissue samples (lanes 2-9 compared with NL1 (lane 1) and leukocyte mRNA (lane 10) are shown in Fig. 11B. Ratios of BRCA1/GAPD for each sample: lane 1: 19.1, lane 2: 0.3, lane 3: 1.8, lane 4: 1.6, lane 5: 0.2, lane 6: 0.3, lane 7: 1.9, lane 8: 0, lane 9: 0.6.

Although the samples were paired in Fig. 11A, they were not microdissected so this approach overestimates the relative expression level of invasive samples because they have a greater percentage of epithelial cells. RNA levels were four to eight fold higher in samples derived from normal breast than in samples derived from invasive breast cancer. We next analyzed expression levels in 8 non-hereditary invasive cancer samples (Figure 11B: lanes Although these samples showed some variability in expression level, all had lower levels of BRCA1 mRNA (determined by ratio of WO 95/19369 PCT/US95/00608 BRCA1 to GAPD) than the primary breast epithelial cell line or the normal breast samples shown in Figure 11A.

Effects of BRCA1 gene inhibition on proliferative rate and gene expression Having demonstrated that mRNA expression levels of BRCA1 are higher in normal mammary cells than in cancer cells, we used antisense methods to test the hypothesis that BRCA1 expression inhibits cell growth. Unmodified 18 base deoxyribonucleotide complementary to the BRCA1 translation initiation site were synthesized and added to cultures of primary mammary epithelial cells (Stampfer et al.

1980) or MCF-7 breast cancer cells (Soule and McGrath, 1980). Figure 12 is graph showing growth rate of human primary mammary epithelial cells MCF-7 cells retinal pigmented epithelial cells cultured as described below. Points and bars represent the mean and the 95% confidence interval of triplicate counts of cells incubated with a single bolus of the indicated concentration of antisense or control sense deoxyribonucleotide.

The morphologic appearance of the cell lines was not noticeably changed by addition of antisense oligonucleotide, but the proliferative rate was faster. Incubation of cells with 40 uM anti-BRCAl oligonucleotide produced accelerated growth of both normal (Figure 12A) and malignant mammary cells (Figure 12B), but did not affect the growth of human retinal pigmented epithelial cells (Figure 12C). An intermediate dose of anti-BRCA1 oligonucleotide produced a less pronounced but significant increase in cell growth rate. This was not a toxic effect of the oligonucleotide since a control "sense" oligomer with the same GC content did not increase the proliferation rate, and because an addition of a 10 fold excess of sense oligomer to the anti-BRCAl oligomer reversed the growth activation.

In order to critically evaluate the function of BRCA1 gene inhibition on growth stimulation and cell cycle progression it was necessary to identify a gene whose expression is cell cycle regulated in human mammary cells. The gene encoding the M2 subunit of ribonucleotide reductase is amplified in conditions of nucleotide starvation (Hurta and Wright 1992) and as shown above, exhibits elevated levels of expression in premalignant breast disease. Because ribonucleotide reductase constitutes the rate limiting step in DNA synthesis, we reasoned that it might be cell cycle regulated in a WO 95/19369 PCT/US95/00608 41 synchronous growth model such as MCF-7 cells which can be growth arrested by tamoxifen and then restimulated by estrogen (Aitken et al. 1985, Arteaga et al. 1989).

MCF-7 cells were growth arrested by tamoxifen for 48 hours and then stimulated at time zero with luM estradiol or control vehicle Inhibition of DNA synthesis by tamoxifen and induction of synthesis by estrogen were confirmed by nuclear labelling studies with tritiated thymidine.

Fig. 13 panels A and B show that transcription of the ribonucleotide reductase M2 gene is cell cycle regulated, inhibited by tamoxifen, and induced by estrogen. Fig.

13A is a Northern blot of mRNA from synchronized MCF-7 cells. At the indicated time in hours, total cellular RNA was isolated and Northern blotting performed using the 1.6 Kb Eco RI fragment from our cloned human ribonucleotide reductase cDNA described above. Two mRNA species of 1.6 and 3.4 Kb are observed in these studies.

Fig. 13B shows nuclear runon studies of synchronized MCF-7 cells were performed by our published methods (Holt et al 1988) employing the 1.6 Kb fragment of ribonucleotide reductase described above the 1.8 Kb fragment of Topoisomerase II (Topo) described in the Olsen et al. 1993); the 1.0 Kb cyclophilin gene (Thompson et al. 1994) used as a constitutive control; and 18S ribosomal RNA (Thompson et al. 1994). Con represents cells which were grown for 48 hours but not treated with tamoxifen.

Antisense inhibition is a useful strategy for studying gene expression which is dependent on expression of the antisense target gene (Robinson-Benion and Holt, in press, 1995), e.g. genes whose expression is directly or indirectly dependent on BRCA1 levels. Fig. 14 demonstrates that antisense inhibition of BRCA1 results in a corresponding increased expression of M2 ribonucleotide reductase mRNA. A nuclease protection assay of mRNA derived from primary mammary epithelial cells (lanes 1-4, 9-10) or MCF-7 cells (lanes 5-8, 11-12) cultured for 4 days with antisense or control oligonucleotide was performed under the following conditions: no oligonucleotide (lanes 1 and 40uM antiBRCAl (lanes 2,6,10,12); 4uM antiBRCA1 (lanes 3 and sense control (lanes 4,8,9,11). Probes for BRCA1 and GAPD are as described for WO 95/19369 PCT/US95/00608 42 Figure 10, and the ribonucleotide reductase M2 probe (RR) detects the 200 bp probe is described above.

Ribonucleotide reductase mRNA levels are highest in samples treated with uM anti-BRCA1 oligonucleotide for both primary mammary epithelial cells and for MCF-7 cells (Fig. 14). Antisense inhibition of BRCA1 results in a 70-90% inhibition of mRNA levels in anti-BRCAl treated cells compared with cells treated with the "sense" control oligonucleotide (compare lanes 9 and 10, Fig. 14). Note that MCF-7 cells have lower levels of BRCA1 than the normal mammary epithelial cells (compare lanes 1 and 5, Fig. 14) anti-BRCA 1 since the antisense inhibition may drop BRCA1 levels below a critical threshold which normally functions to inhibit growth.

Methodology Tissue samples. Freshly obtained breast biopsy or reduction mammoplasty specimens were frozen and then RNA was obtained following the microdissection method described above. Lesions were selected which were microlocalized and homogenous so that pure lesions could be obtained by 2 mm punches. Samples which had admixed normal epithelial, carcinoma-in-situ, or invasive cancer were not used for this study. Family history was obtained by chart review and/or interview to exclude familial breast cancer cases.

Nuclease Protection Assays. PCR primers were derived from BRCA1 sequence in GenBank (Accession number U14680); forward CAATTGGGCAGATGTGT 3' and reverse 5' CTGGGGGATCTGGGGTATCA 3' which amplify a 113 bp region from exon 24, corresponding to bases 5587 to 5699 of the human BRCA1. This region was selected because this exon has not been reported to be differentially spliced unlike more 5' exons. The BRCA1 probe was cloned by subcloning this 113 bp band from normal human genomic DNA into PCRscriptSK and screening for correct orientation. One ug of mRNA from each tissue sample was hybridized with 32P-labelled, T7 polymerase-generated RNA probes for BRCA1 and human glyceraldehyde-3-phosphate dehydrogenase (GADP) which would produce expected protected fragments of 113 and 140 respectively. The construction and use of the GADP probe for RNA standardization has been described above. The probe for WO 95/19369 PCT/US95/00608 43 ribonucleotide reductase M2 mRNA is the same as above and detects a 200 bp protected fragment.

Antisense oligonucleotide studies. Unmodified deoxyribonucleotide were analyzed by gel electrophoresis and UV shadowing and shown to be homogenous and of appropriate size. These oligonucleotide were purified by multiple lyophilization and solubilized in buffered media as described (Holt et al. 1988). Sequence of the unmodified antiBRCAl oligonucleotide 5' AAGAGCAGATAAATCCAT 3' and the complementary sense oligonucleotide 5' ATGGATITATCTGCTCTT 3' correspond to the presumed translation initiation site at bases 12-137 of the GenBank sequence. The antisense oligonucleotide sequence was searched against Genbank and no significant homologies were identified to genes except BRCA1. Oligonucleotides were used according to our published methods (Holt et al. 1988). Primary mammary epithelial cells were cultured in serum-free medium supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, phosphorylethanolamine, and bovine pituitary extract. MCF-7 cells were cultured in Minimum Essential Medium Eagle (Modified) with Earle's salts and 2g/L sodium bicarbonate m supplemented with 2mM Lglutamine, GMS-A (Gibco Cat. #680-1300AD), nonessential amino acids, and fetal calf serum. Retinal pigmented perithelial cells were cultured in DMEM and calf serum.

Our results indicate that the BRCA1 gene is expressed at higher levels in normal mammary cells than in breast cancer cells and that diminished expression of BRCA1 increased the proliferative rate of breast cells. This correlates well with the recent finding that patients with BRCA1 gene-linked hereditary breast cancer have tumors that grow more rapidly than comparable sporadic tumors (Marcus, J. et al. 1994). The decreased mRNA levels which were observed in sporadic breast cancers are not a consequence of differential splicing of the gene since the RNAs were quantitated with probes from the 3' end of the mRNA which is not a region where differential splicing is reported to occur (Miki, Y. et al 1994). Invasive sporadic cancers have BRCA1 mRNA levels which vary from 0 (in one case) to 20% of the levels observed in normal human mammary epithelium.

WO 95/19369 PCT/US95/00608 44 Examples 8 and 9 describe applications of the discovery of the function of the BRCA1 gene. Example 8 describes a gene therapy method and example 9 describes a drug screening method. The discovery of the diminished expression of the BRCA1 mRNA in breast cancer using the microdissection techniques of this invention provides an important scientific basis for these examples.

Example 8 Gene Therapy method based on determination of the function of the BRCA1 Gene Viral vectors containing a DNA sequence that codes for a protein having an amino acid sequence as essentially set forth in SEQ ID NO:49 can be constructed using techniques that are well known in the art. This sequence includes the BRCA1 gene product. Viral vectors containing a DNA sequence essentially as set forth in SEQ ID NO:47 (the BRCA1 gene) can be also constructed using techniques that are well known in the art. Retroviral vectors, adenoviral vectors, or adeno-associated viral vectors are all useful methods for delivering genes into breast cancer cells. An excellent candidate for use in breast cancer gene therapy is a Moloney-based retroviral vector with a breast selective MMTV promoter which we have reported previously (Wong et al). The viral vector is constructed by cloning the DNA sequence essentially as set forth in SEQ ID:47 into a retroviral vector such as a breast selective vector. Most preferably, the full-length (coding region) cDNA for BRCA1 is cloned into the retroviral vector. The retroviral vector would then be transfected into virus producing cells in the following manner: Viruses are prepared by transfecting PA317 cells with retroviral vector DNAs which were purified as described in Wong et al. Following transfection, the PA317 cells are split and then treated with G418 until individual clones can be identified and expanded. Each clone is then screened for its titer by analyzing its ability to transfer G418 resistance (since the retroviral vector contains a Neomycin resistance gene). The clones which have the highest titer are then frozen in numerous aliquots and tested for sterility, presence of replication-competent retrovirus, and presence of mycoplasm. The methods generally employed for construction and production of retroviral vectors have been described in Muller, 1990.

Once high titer viral vector producing clones have been identified, then patients with breast cancer can be treated by the following protocol: Viral vector expressing WO 95/19369 PCT/US95/00608 BRCA1 is infused into either solid tumors or infused into malignant effusions as a means for altering the growth of the tumor (since it is shown above that the BRCA1 gene product decreases the growth rate of breast cancer cells). Because viral vectors can efficiently transduce a high percentage of cancer cells, the tumors would be growth inhibited.

Example 9 Method of Screening Compounds Capable of Activating Promoter Region of the BRCA1 Gene The discovery of the function of the BRCA1 gene provides a clear utility in that induction of expression of the gene and the resulting increase in level of protein encoded by the gene in the breast cancer cell should slow the proliferation of the breast cancer cells. Induction of expression of the gene can be caused by administering a compound to a patient that stimulates the regulatory regions of this gene, such as the promoter.

A method for screening compounds that activate the promoter of the BRCA1 gene is designed in the following way. A promoter sequence is a DNA segment that upregulates the expression of a gene. A sequence essentially as set forth in SEQ ID NO:48 can be ligated into a suitable vector, such as a plasmid, that contains a reporter gene using standard recombinant DNA techniques of restriction enzyme digests, ligation of fragment into vector, and transformation of bacteria. SEQ ID NO:48 includes the promoter sequence of the BRCA1 gene. A reporter gene is a gene that produces a readily detectable product. Examples of appropriate reporter genes which could be employed for this purpose include Beta-galactosidase or the chloramphenicol acetyltransferase gene.

The BRCA1 promoter/reporter gene combination can then be cloned into an expression vector or viral vector by standard recombinant DNA methods. Breast cancer cells can then be transfected with the expression vector containing the BRCA1 promoter/reporter gene using standard transfection methods which we have reported previously (Holt et al. PNAS 1986). A stable transformant with appropriate low level expression (breast cancer cells have low level BRCA1 expression as shown above) will be identified and then characterized to demonstrate proper DNA integration and II- ~iri-- WO 95/19369 PCT/US95/00608 46 expression. Methods of establishing and characterizing stable transformants have been described (Holt. MCB, 1994). Once an appropriate stable transformant cell line is identified, then we can plate the cell line in a manner than permits screening of hundreds or thousands of drugs or biological agents (for example in multiple 96 well microtiter plates). Level of expression of the reporter gene can be quantitated and agents which activate expression are thus identified. A positive result induction of the promoter region) results in increased levels of the reporter gene resulting in either an increase in color (Beta-galactosidase assay) or specific radioactivity (Chloramphenicol acetyltransferase activity) through a reaction between the protein encoded by the reporter gene and a compound in the reaction medium. The compound produced by the reaction between the reporter gene protein and the compound in the reaction medium is the cause of the increase in color or specific radioactivity. These compounds can be called indicator compounds in that their presence indicates that the drug or biologial agent activitated the BRCA1 promoter. Methods for standardizing and performing Beta-galactosidase or chloramphenicol acetyltransferase assays have been reported (Holt et. al. MCB 1994). This method would be useful for initial screening of agents which increase BRCA1 expression. These agents could then be tested in more rigorous assays of breast cancer growth such as nude mouse tumor assays (Arteaga et al). This approach allows mass screening of large numbers of agents, sparing more rigorous animal tests for only promising compounds which score in the reporter gene assay described herein.

Thus, although there have been described particular embodiments of the present invention of a new and useful "Method for Detection and Treatment of Breast Cancer", it is not intended that such embodiments be construed as limitations upon the scope of this invention except as set forth in the following claims. It will be apparent to those skilled in the art that many changes and modifications may be made without departing from the invention in its broader aspects. For example, the above described techniques may be used in the diagnosis of other diseases and detection of differential genetic expression from microscopically-directed tissue samples of pathologic tissue. The production of a cDNA library produced as a result of the differential expression of genes in pathologic tissue in comparison to normal tissue provides the opportunity for WO 95/19369 PCT/US95/00608 47 further adiagnostic capabilities. Further, although there have been described certain experimental conditions used in the preferred embodiment, it is not intended that such conditions be construed as limitations upon the scope of this invention except as set forth in the claims.

The following references are included to provide details of scientific technology herein incorporated by reference to the extent that they provide additional information for the purposes of indicating the background of the invention or illustrating the state of the art.

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ADDITIONAL DESCRIPTION OF THE FIGURES Figure 2: Model for premalignant conditions, highlighting magnitude of risk for progression to clinical malignancy. Terms from human breast neoplasia are used: no proliferative disease (No Pro), proliferative disease without alypia (PDWA), typical hyperplasia carcinoma in situ (CIS). As is proposal of tumor progression each stage is more likely to proceed to the next (dotted lines), but could also remain stable (horizontal lines, probably fairly frequent), or directly proceed to develop a clone of cells with malignant behavior (vertical lines, becoming more likely further to right.) Figure 5: Differential display of cDNAs obtained from patient tissue samples and controls. Rescued cDNA library samples were used as templates for low stringency PCR with the primers 5'GATGAGTTCGTGTCCGTACAACTGG3' and GGTTATCGAAATCAGCCACAGCGCC3'; 40 cycles were performed at conditions described above. Samples (See legend to Figure Lane 1 #12; Lanes 2 and 3: separate phage rescues of NL1 to show reproducibility of the assay; Lane 4 Lane #10; Lane 6 #10CA; Lane 7 control from the rescued phage vector without cDNA inserts. Arrows mark cDNAs which are overexpressed in DCIS versus normal.

Arrowheads mark cDNAs which are differentially expressed in the invasive cancer (note this may reflect contamination from stromal cells). The bar marks a cDNA which is expressed in normal breast cells at higher levels than in DCIS or invasive cancer.

Figure 7: Expression of DCIS-1 mRNA in tissue mRNA samples analyzed by RNase protection assay. Probes: GADH probe and DCIS-1 clone probe which was generated by linearizing the rescued plasmid with Pvu I and should generate a 200 bp protected fragment. RNA samples were labeled as in the legend to Figure 4.

SUBSTITUTE SHEET (RULE 26) _Es_17777-7777=__ WO 95/19369 PCT/US95/00608 54 SEQUENCE LISTINGS GENERAL INFORMATION: APPLICANT: HOLT, JEFFREY T.

JENSEN, ROY A.

PAGE, DAVID L.

OBERMILLER, PATRICE S.

ROBINSON-BENION, CHERYL L.

THOMPSON, MARILYN E.

(ii) TITLE OF INVENTION: METHOD FOR DETECTION AND TREATMENTS OF BREAST CANCER (iii) NUMBER OF SEQUENCES: 49 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: I.C. WADDEY, JR.

STREET: 27TH FLOOR, L C TOWER, 401 CHURCH CITY: NASHVILLE STATE: TENNESSEE COUNTRY: USA ZIP: 37219 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette, 3.50 inch, 800 kB storage COMPUTER: IBM PC/XT/AT compatible OPERATING SYSTEM: MS-DOS (version SOFTWARE: WordPerfect 5.1/WordPerfect Editor (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: U.S. 08/182,961 FILING DATE: 14 JAN 1994 WO 95/19369 PCTIUS95/00608 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: I.C. WADDEY, JR.

REGISTRATION NUMBER: 25,180 REFERENCE/DOCKET NUMBER: 0216-9409 (ix) TELECOMMUNICATION INFORMATION TELEPHONE: (615) 242-2400 TELEFAX: (615) 242-2221

TELEX:

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 264 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens INDIVIDUAL/ISOLATE: sample of non-comedo DCIS DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: ductal carcinoma in situ CELL LINE: not derived from a cell line ORGANELLE: no (vii) IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained from identification of differential gene expression WO 95/19369 PCTIUS95/00608 56 (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown OiX) FEATURE: NAME/KEY: DCIS-1 LOCATION: GenBank accession no. L2736 IDENTIFICATION METHOD: microscopically-directed sampling and differential display OTHER INFORMATION: gene encoding M2 subunit of humanribonucleotide reductase (x PUBLICATION INFORMATION: unpublished RELEVANT RESIDUES IN SEQ ID NO: 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l1: TTGGGAATTG GGTACGCGGG CCCCCCACTG TGCCGAATTC CTGCATGCGG GGGATCCACT AGTTCAGAGC AGGCCGCCAC CCGTAGGACT CCAGCTTTTG TTCGTTCCCT TTAGTGAGGG 120 TTAATTTTCG AGCTTGGCGT AATCATGGTC ATAGCTGTTT CCTGTGTGAA ATTGTTATCC 180 GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAA GTGTAAAGCC TGGGGTGCCT 240 AATGAGTGAG CTAACTCACA TTAA 264 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 73 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens INDIVIDUAL/ISOLATE: sample of non-comedo DCIS DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast WO 95/19369 PCT/US95/00608 57 CELL TYPE: ductal carcinoma in situ CELL LINE: not derived from a cell line ORGANELLE: no (vii) IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained from identification of differential gene expression (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown (ix) FEATURE: NAME/KEY: DCIS-2 LOCATION: GenBank accession no. L27637 IDENTIFICATION METHOD: microscopically-directed sampling and differential display PUBLICATION INFORMATION: unpublished RELEVANT RESIDUES IN SEQ ID NO: 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TAGCCCGGTT ATCGAAATAG CCACAGCGCC TCTTCACTAT CAGCAGTACG CCGCCCAGTT GTACGGACAC GGA 73 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 46 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens WO 95/19369 PCTIUS95/00608 INDIVIDUAL/ISOLATE: sample of non-comedo DCIS DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: ductal carcinoma in situ CELL LINE: not derived from a cell line ORGANELLE: no IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained from identification of differential gene (vii) expression (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown (ix) FEATURE: NAME/KEY: DCIS-3 LOCATION: L27638 IDENTIFICATION METHOD: microscopically-directed sampling and differential display PUBLICATION INFORMATION: unpublished RELEVANT RESIDUES IN SEQ ID NO: 3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGCCCGATGT GTGTCGTACA ACTGGCGCTG TGGCTGATTT CGATAA 46 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 72 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no ~-~rrxn~ 9i~l- T~ WO 95/19369 PCTUS95/00608 (v) (vii) ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens INDIVIDUAL/ISOLATE: sample of non-comedo DCIS DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: ductal carcinoma in situ CELL LINE: not derived from a cell line ORGANELLE: no IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained from identification of differential gene expression (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown (ix) FEATURE: NAME/KEY: DCIS-4 LOCATION: L27640 IDENTIFICATION METHOD: micros sampling and differential display PUBLICATION INFORMATION: unpublished RELEVANT RESIDUES IN SEQ ID NO: 4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TAGCCCATGA GTTCGTGTCC GTACAACTGG GGCGCTGTGG CTGATTTCGA TANNNNNAGC ATCAGCCCGA CG 72 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 84 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear copically-directed WO 95/19369 PCTIUS95/00608 (ii) (iii) (iv) (v) (vii) MOLECULE TYPE: cDNA to mRNA HYPOTHETICAL: no ANTI-SENSE: no ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens INDIVIDUAL/ISOLATE: sample of non-comedo DCIS DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: ductal carcinoma in situ CELL LINE: not derived from a cell line ORGANELLE: no IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained from identification of differential gene expression (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown (ix) FEATURE: NAME/KEY: LOCATION: L27641 IDENTIFICATION METHOD: micros sampling and differential display PUBLICATION INFORMATION: unpublished RELEVANT RESIDUES IN SEQ ID NO: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TAGCCCGGTT ATCGAAATCA GCCACAGCGC CTAACTTCTG CAGAAGCCTT TGACCATCAC CAGTTGTACG GACACGAACT CATC 84 INFORMATION FOR SEQ ID NO:6: rcopically-directed WO 95/19369 PCT/US95/00608 (i) (ii) (iii) (iv) (v) (vii) expression (viii) (ix) SEQUENCE CHARACTERISTICS: LENGTH: 99 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA HYPOTHETICAL: no ANTI-SENSE: no ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens INDIVIDUAL/ISOLATE: sample of non-comedo DCIS DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: ductal carcinoma in situ CELL LINE: not derived from a cell line ORGANELLE: no IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained from identification of differential gene POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown

FEATURE:

NAME/KEY: DCIS-6 LOCATION: L27642 IDENTIFICATION METHOD: microscopically-directed sampling and differential display PUBLICATION INFORMATION: unpublished RELEVANT RESIDUES IN SEQ ID NO: 6 WO 95/19369 PCT/US95/00608 62 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTGGTTTCCG AAATTCCTGG GAAGGGGGGT GCTGGCGTGT GGAATTGTCG CGGCCCCTGG TCTGCCGCGG CGTTTTTTGT CTACATTCGT CGTAGCTCG 99 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 88 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens INDIVIDUAL/ISOLATE: sample of non-comedo DCIS DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: ductal carcinoma in situ CELL LINE: not derived from a cell line ORGANELLE: no (vii) IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained rom identification of differential gene expression (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown (ix) FEATURE: NAME/KEY: DCIS-7 LOCATION: L27643 i WO 95/19369 PCT/US95/00608 63 IDENTIFICATION METHOD: microscopically-directed sampling and differential display PUBLICATION INFORMATION: unpublished RELEVANT RESIDUES IN SEQ ID NO: 7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATCAGCGCGC GACATTCGGG TACCCGCGCC CCCCCCTCCG TCGGAATTCC TCGAGCCGGG ATCCATAGGA TGTGGAGTTA GTTTTGTT 88 INFORMATION FOR SEQ ID NO:8 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CGCGACGGCC GCGCGTCTGC CAGGG INFORMATION FOR SEQ ID NO:9 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no WO 95/19369 PCTUS95/00608 64 FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGCCCCTGCG TTACCCTCCC CGCCG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGATGGCGTC CTGTAACCCG ACGCT INFORMATION FOR SEQ ID NO: 11 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ACTGGGCTGT CCTGCGGTGG CGGGG INFORMATION FOR SEQ ID NO:12 ;I WO 95/19369 PCTIUS95/00608 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CTGAGAGGTA GCCGCGCGGA GGCTG INFORMATION FOR SEQ ID NO: 13 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GCCTGGCCGC GACACGGATT ACCGC INFORMATION FOR SEQ ID NO:14 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single WO 95/19369 PCT/US95/00608 66 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TTAGCGCATG GTGGACCTGG AGACG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGTGGTTACG TCAGCGAAGG TAATA INFORMATION FOR SEQ ID NO: 16 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no WO 95/19369 PCT/US95/00608 67 FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AGTCGCACGC ATGTCACGCT CCGCC INFORMATION FOR SEQ ID NO:17 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TATCCAAGCG GCAGGCTACG AGGCC INFORMATION FOR SEQ ID NO: 18 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GGCGCGCCCG ACGGTCTGGT ATCTA INFORMATION FOR SEQ ID NO: 19 i

F

WO 95/19369 PCT/US95/00608 68 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CTCCCTCCCC GGACTCGGGG TTAGT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATGCGGGCGG CTCGGGCCTG GTCGC INFORMATION FOR SEQ ID NO:21 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 95/19369 PCTIUS95/00608 69 (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CGTGAAGCCT ATGCCCTCCC TCAAC INFORMATION FOR SEQ ID NO:22 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GTGCCGTCGT AGCCCTTCAG CGATC INFORMATION FOR SEQ ID NO:23 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide

I

WO 95/19369 PCTUS95/00608 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: GCGACACTAG GCTCCCGGAG GAGGG INFORMATION FOR SEQ ID NO:24 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TGGGCCAGGC CTCCGGGCCC GGTAT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCGGAACTGC GATAGCGTCC GTCCC INFORMATION FOR SEQ ID NO:26 WO 95/19369 PCT/US95/00608 71 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: AGCGGACACC TGTTTCCCGA GAGCC INFORMATION FOR SEQ ID NO:27 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: AACGGGTGGA CATCCGCCTG CCGCC INFORMATION FOR SEQ ID NO:28 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear i. -II. 1_ WO 95/19369 PCT/US95/00608 72 (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TGAACCACGA TGTCAATCGT CCCGA INFORMATION FOR SEQ ID NO:29 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TCATCCCCGC CGAAAGACGC TCGCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide WO 95/19369 PCT/US95/00608 73 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATAGGCTGCG GCACGCGCTG GGACT INFORMATION FOR SEQ ID NO:31 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GACCAGGTGC GCACGAGCAT GTACA INFORMATION FOR SEQ ID NO:32 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: AGCGTAGTCA TCGGCCTTCG CGCCC INFORMATION FOR SEQ ID NO:33 WO 95/19369 PCT/US95/00608 74 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: GGCCCCTAGC CCAGGGTGAA GCCCA INFORMATION FOR SEQ ID NO:34 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CCCAGTGCTA CGGGCCGCCC CAAGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single WO 95/19369 PCT/US95/00608 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCTTCCTGGG TTACCTGCCC TCGGG INFORMATION FOR SEQ ID NO:36 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: TCCGGACAGC AGCCACGCCA AGGGC INFORMATION FOR SEQ ID NO:37 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no WO 95/19369 PCT/US95/00608 76 FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: ACGCGCTGGT CCACCGAGGC CTGAT INFORMATION FOR SEQ ID NO:38 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: CGATGCAAGG CCAGCAGCAC TCGAC INFORMATION FOR SEQ ID NO:39 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: CCCCCGGAGC GGACCACCGG ACGTG INFORMATION FOR SEQ ID WO 95/19369 PCT/US95/00608 77 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGCGGGGAGG GATCGGGGGC CAAGC INFORMATION FOR SEQ ID NO:41 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: GCCTGGTGTA GGCAGGCAGC TCTTA INFORMATION FOR SEQ ID NO:42 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 95/19369 PCT/US95/00608 78 (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: CCACCCCTGT AGTGCGGGCT GCGAG INFORMATION FOR SEQ ID NO:43 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GGAACCCGAC GCCCGTCCAG GGTTC INFORMATION FOR SEQ ID NO:44 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no WO 95/19369 PCT/US95/00608 79 FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: TCGGGCAGCA AGGCCGGGAC GCTCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GACGGGGGAC GGGCTAGGTG GCTTA INFORMATION FOR SEQ ID NO:46 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA DESCRIPTION: PCR primer (iii) HYPOTHETICAL: yes (iv) ANTI-SENSE: no FRAGMENT TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: CTTGTTGCCG GCGGAGAGGG CTGCC INFORMATION FOR SEQ ID NO:47: r WO 95/19369 WO 95/19369 PCT/US95/00608 SEQUENCE CHARACTERISTICS: LENGTH: 5712 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens

INDIVIDUAL/ISOLATE:

DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: ductal carcinoma in situ, invasive breast cancer and normal breast tissue CELL LINE: not derived from a cell line ORGANELLE: no (vii) IMMEDIATE

SOURCE:

LIBRARY: cDNA library derived from human CLONE: obtained using published sequence (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown (ix) FEATURE: NAME/KEY: BRCA1 LOCATION: GenBank accession no. U14680 IDENTIFICATION METHOD: microscopically-directed sampling and nuclease protection assay OTHER INFORMATION: gene encoding BRCA1 protein WO 95/19369 WO 9519369PCT/US95/00608 81 Wx PUBLICATION INFORMATION: AUTHORS: Mild, et. al.

TITLE: A strong candidate gene for the breast and ovarian cancer susceptibility gene BRCAL1 JOURNAL: Science VOLUME: 266 PAGES: 66-71 DATE: 1994 RELEVANT RESIDUES IN SEQ ID NO: 47 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: agctcgctga gacttcctgg accccgcacc aggctgtggg gtttctcaga taactgggce cctgcgctca ggaggccttc accctctgct ctgggtssag ttcattggaa cagaaagaa 119 atg gat tta tct get ctt cgc gtt gaa gs gta esa sat gtc att sat 167 Met Asp Leu Ser AMa Leu Arg Vat GLu GLu Vat Gin Asn Vat lie Asn 1 5 10 get atg eag aaa ate tta gag tgt ccc ate tgt ctg gag ttg ate sag 215 Ala Met Gin Lys lie Leu Giu Cys Pro lie Cys Leu GLu Leu lie Lys 25 gas cct gtc tce sea sag tgt gac ese ats ttt tgc sa ttt tgc atg 263 GLu Pro Val Ser Thr Lys Cys Asp His lie Ph. Cys Lys Ph. Cys Met 40 ctg sa ett etc sac cag sag sa ggg ect tea eag tgt ect tta tgt 311 Leu Lys Leu Leu Asn Gin Lys Lys GLy Pro Ser Gin Cys Pro Leu Cys 55 sag sat gat ateascc sa agg sgc eta csa gas agt seg age ttt sgt 359 Lys Asn Asp Iie Thr Lys Arg Ser Leu Gin GLu Ser Thr Arg Ph. Ser 70 75 cas ctt gtt gas gag eta ttg sa atc att tgt get ttt cag ctt gac 407 Gin Lau Vai GLu Giu Leu Leu Lys lie lie Cys Ala Ph. G~n Lau Asp 90 sea ggt ttg gag tat gcs sac age tat sat ttt ges sa sag gas sat 455 Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Ph. Ala Lys Lys Glu Asn 100 105 110 sac tet cet gs eat eta sa gat gas gtt tet ate ate cas agt atg 503 Asn 5cr Pro Giu His Leu Lys Asp Giu Vat Ser lie lie Gin Ser Met 115 120 125 ggc tac aga sac cgt gee sa aga ett cta cag agt gas ccc gs sat 551 GLy Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gin Ser Giu Pro Giu Asn 130 135 140 cct tcc ttg eag gas acc agt etc agt gtc cas cte tet sac ctt gga 599 Pro Ser Leu Gin Giu Thr 5cr Lau Ser Vat Gin Leu 5cr Asn Leu Gly 145 150 155 160 WO 95/19369 WO 9519369PCTIUS95/00608 cact gtg aga act ctg agg aca aag cag cgg ata caa cct caa aag acg Thr Vat Arg Thr Lau Arg Thr Lys Gin Arg lie Gin Pro Gin Lys Thr 165 170 175 tct gtc tac att gias ttg gga tct gat tct tct gaa gat acc gtt aat Set VaL Tyr lie Gtu Leu Gly Ser Asp Ser Ser Gtu Asp Thr VaL Asn 180 ag gca act tat tgc agt Lys Ata Thr Tyr Cys Set 195 cct caa gga acc agg gat Pro Gin GLy Thr Arg Asp 210 gct tgt gaa ttt tct gag Ala Cys GLu Phe Set GLu gat caa gan ttg Asp Gin GLu Leu agt ttg gat tct Ser Leu Asp Set 220 gta aca aat act Vai Thr Asn Thr 190 tta can atc acc Leu Gin lie Thr 205 gca aaa aag gct Ala Lys Lys Ala gaa cat cat caa Glu His His Gin acg gat Thr Asp 230 agt aat aat gat ttg aac acc act 235 240 gag aag cgt gca gct gag agg Pro Set Asn Asn Asp Leu Asn Thr Thr GLu Lys Arg Ala Ala Giu Arg 245 250 255 cat cca gaa aag tat cag ggt agt tct gtt tca aac ttg cat gtg gag His Pro Glu Lys Tyr Gin Gly Set Set Val Set Asn Leu His Vai GLu 260 265 270 cca tgt ggc aca aat act cat gcc agc tca tta cag cat gag aac agc Pro Cys Gly Thr Asn Thr His Ala Ser Set Leu Gin His Glu Asn Ser 275 agt tta tta Set Leu Leu 290 tgt at aaa Cys Asn Lys 280 285 ctc act aaa gac aga atg aat gta gaa aag gct gaa ttc Leu Thr Lys Asp Arg Met Asn Vat Giu Lys Ala Giu Phe 295 300 agc aaa cag cct ggc tta gca agg agc caa cat aac aga Set Lys Gin Pro GLy Leu Ala Atg Set Gin His Asn Arg 305 310 tqg gct gga agt aag gaa aca tgt Trp Ala Gly Ser Lys Gtu Thr Cys 325 gaa aaa aag gta gat ctg aat gct Glu Lys Lys Val Asp Leu Asn Ala 340 aat gat Asn Asp 330 gat ccc Asp Pro agg Arg cgg act ccc agc Arg Thr Pro Set 335 tgg aat aag cag aaa ctg cca tgc tca gag Trp Asn Lys Gin Lys Leu Pro Cys Set Glu 355 360 gat gtt cct tgg ata aca cta aat agc agc Asp Vat Pro Trp lie Thr Leu Asn Set Set 370 375 tgg ttt tcc aga agt gat gaa ctg tta ggt Trp Phe Set Arg Set Asp Glu Leu Leu Gly 385 390 ggg gag tct gaa tca aat gcc aaa gta gct ctg tgt gag aga aaa gaa Leu Cys GLu Atg Lys Giu 350 aat cct aga gat act gaa Asn Pro Arg Asp Thr GLu 365 att cag aaa gtt aat gag lie Gin Lys Val Asn GLu 1031 1079 1127 1175 1223 1271 1319 1367 380 tct gat gac tca cat gat Set Asp Asp Set His Asp 395 400 gat gta ttg gac gtt cta GLy GLu Set GLu Set Asn Ala Lys Vai Ala Asp Val Leu Asp Val Leu 405 410 415 WO 95/19369 PTU9/00 PCT/US95/00608 eat gag gte get gee tat tct ggt Asn Giu Val Asp GLu Tyr Ser Gty 420 9CC agt gat ect cat gag gct tta Ala Ser Asp Pro His Giu Ata Lau 435 440 tcc aaa tca gta gag agt mat mtt Ser Lys Ser VaL Giu Ser Asp iLe 450 455 tat cgg aag meg gca agc ctc ccc tca gag saae gac tta ctg Ser GLu Lys lie Asp Lou Leu 430 tgt ma agt gee age gtt cec Cys Lys Ser Asp Arg VaL His gee sma ate ttt ggg saa cc Asp Lys lie Phe GLy Lys Thr gee Giu.

460 eec tte agc cat Tyr Arg Lys Lys Ala Ser Lou Pro Asn Lou Ser His 465 470 475 eta att ae gge gce ttt gtt act gag cee ceg ate Lau ie Ilie GLy Alm Phe VaL 5cr GLu Pro Gin lie 485 490 ccc ctc ace eat a tte seg cgt sma agg age cct Pro Lou Thr Asn Lys Leu Lys Aog Lys Arg Arg Pro 500 505 cat cct gag get ttt etc sag ama gee get ttg gee His Pro GLu Asp Phe lIe Lys Lys Ata Asp Leu Ala 515 520 ect gee etg ae eat ceg gga act eec caa meg gag Pro Giu Met lie Asn Gin Giy Thr Asn Gin Thr GLu 530 535 gtg etg eat att act eat agt ggt eat gag eat sa Vet Met Asn ILe Thr Asn Ser Gty His Giu Asn Lys 545 550 555 tct att eag eat gag ma met ect ae cc&ae gee Ser ILe Gin Asn Giu Lys Asn Pro Asn Pro Ile Giu gta act gem mat VaL Thr Giu Asn 480 ate cam gag lie Gin Gtu 495 ace tee ggc Thr Ser Gly 510 egt Arg gtt cee meg act Vai Gin Lys Thr 525 eag eat ggt cee Gin Asn Giy Gin 540 saema ggt get Thr Lys GLy Asp tee etc gee sa Ser Leu Giu Lys 575 age agt ate age Ser 5cr lie Ser 590 gem ect saa g 1415 1463 1511 1559 1607 1655 1703 1751 1799 1847 1895 1943 1991 2039 2087 2135 560 gee tet get Giu Ser Aie eat atg gae Asn Giu Lou mat egg ctg Asn Arg Leu 610 eta gte gte Leu Vai Vai tte Phe etc 565 570 ecg sa get gee cet ae age Thr Lys Ate Giu Pro lie Ser 585 tta eat etc cec eat tca saa Giu Lou Asn Ilie Met His Asn Ser Lys Aim Pro Lys Lys 595 600 605 egg egg meg tet tet ace egg cat mtt eat geg ett gee Arg Arg Lys Sor Ser Thr Arg His lie His Ais Lou GLu 615 620 agt ae mat eta age eca ect eat tgt act gee ttg eme 5cr Arg Asn Leu Ser Pro Pro Asn Cys Thr Giu Lau Gin 630 635 agt tgt tet age agt gee gag ate ag ee a eag Ser Cys Ser Ser Ser GLu Giu lie Lys Lys Lys Lys 645 650 eca gte egg eec agc age eec eta eme etc atg gem Pro Vet Arg His Ser Arg Asn Leu Gin Lau Met Gtu 660 665 tee mac Tyr Asn 655 ggt eae Giy Lys 670 cam etg Gin Met WO 95/19369 PCTUS95IOO608 gaa ect gca act gga gcc aag aag Giu Pro ALa Thr Gty Ala Lys Lys 675 agt aaa aga cat gac agc gat act Ser Lys Arg His Asp Ser Asp Thr aac aag cca aat Asn Lys Pro Asn CCa gag ctg aag Pro GLu Lou Lys gaa cag sa Gtu Gin Thr 685 tta aca aat Leu Thr Asn 690 ggt tct Gty Ser 695 700 ttt act aag tgt tea aat ace agt gaa ctt aaa gaa Phe Thr Lys Cys Ser Asn Thr Ser GLu Lou Lys Gtu 710 715 ttt gte Phe Vat 720 aca gtt Thr Vat aat cet age ctt cca aga gaa gaa aaa gaa gag aaa eta gaa Asn Pro Ser Lau Pro Arg GLu GLu Lys GLU Gtu Lys Lou GLu 725 aaa gtg tct aat Lys Vat Ser Asn 740 aat get gaa gac Asn Ala Glu Asp 745 Caa act gaa aga Gin Thr Gtu Arg 760 agt gga gaa agg gtt ttg Ser Gty GLu Arg VaL Lau 755 ccc aaa gat etc atg Pro Lys Asp Lau Met 750 tet gta gag agt agc Ser VaL Giu Ser Ser 765 att tea ttg gta eet ggt act gat tat ggc act cag gaa agt ate teg lie Ser Leu Vat Pro Gty Thr Asp Tyr Giy Thr Gin Gtu Ser lie Ser 770 775 780 tta etg gaa gtt age act eta ggg aag gca aaa aca gas eea aat aaa Leu GLu Vai 785 5cr Thr Lau GLy Lys Ala Lys Thr Glu Pro Asn Lys 2183 2231 2279 2327 2375 2423 2471 2519 2567 2615 2663 2711 2759 2807 2855 2903 tgt gtg agt cag tgt gea gea Cys VaL Ser Gin Cys Ala Ala 800 805 ggt tgt tee aaa gat aat aga Gly Cys Ser Lys Asp Asn Arg 820 ttg gga eat gaa gtt aae eac Lau Gly His GLu Vat Asn His 835 gaa agt gaa ett gat get cag ttt gaa aac Phe Giu Asn aat gac aea Asn Asp Thr 825 agt egg gaa Ser Arg Giu 840 ccc aag gga eta att Pro Lys Gly Lau ie 810 gaa ggc ttt aag tat Gtu Gly Phe Lys Tyr 830 aea age ata gaa atg Thr Ser lie Gtu Met 845 tat ttg cag aat aea tte aag gtt tea Gku Ser Giu Leu Asp ALa Gin Tyr Lou Gin Asn Thr Phe Lys Vat Ser cge eag Arg Gin 865 855 860 tea ttt get ceg ttt tea aat eea gga aat gca gaa gag Ser Phe Aia Pro Phe Ser Asn Pro Gly Asn Ala Giu Giu 870 875 gaa tgt gea aca ttc Giu Cys Ala Thr Phe 880 eea aaa gte act ttt Pro Lys Vat Thr Phe 900 tct Ser 885 gaa Giu gee cac tet ggg tee tta aag aaa Ala His Ser Gty Ser Leu Lys Lys 890 caa agt Gin Ser 895 tgt gaa eaa aag gaa gaa aat eaa gga aag Cys Giu Gin Lys GLu Giu Asn Gin GLy Lys 905 910 ect gta cag aca gtt aat ate act gea ggc aat gag tet aat ate aag Asn Giu Ser Asn lie Lys Pro VaL Gin Thr Vat Asn lie Thr Ala Gly 915 920 925 WO 95/19369 PCTIUS95/00608 ttt cct gtg gtt ggt cag aae Phe Pro vat VaL GLy Gin Lys 930 sgt atc aaa gge ggc tct agg Ser Ile Lys Gty Giy Ser Arg 945 950 eec gae act gga ctc att act Asn Giu Thr Gty Lau Ile Thr 960 965 cca tat Cgt ate cca cca ctt Pro Tyr Arg Ile Pro Pro Leu ag cca gtt get aet gcc aaa tgt Lys Pro VaL Asp Asn ALa Lys Cys 940 tgt cte tca tct cag ttc age ggc Cys Leu Ser Ser Gin Phe Arg Giy 955 et sa cat gge ctt tta cee eec Asn Lys His Gly Leu Lau Gin Asn cce Pro 970 ttt ccc etc ag Phe Pro Ile Lys tca ttt gtt see act Ser Phe VaL Lys Thr 990 980 as tgt eag sa Lys Cys Lys Lys 995 tca cct gee age Ser Pro Giu Arg 1010 ace att egc cgt Thr lie Ser Arg eat ctg cta gag gee eec ttt gag Asn Lau Lou GLu Gtu Asn Phe Giu 1000 gee etg gge eat gag eec att cca Giu Met Gty Asn Giu Asn Ile Pro 1015 gee cat tce etg Giu His 5cr Met 1005 sgt ace gtg agc 5cr Thr Vat Ser 1020 2951 2999 3047 3095 3143 3191 3239 3287 3335 3383 3431 3479 aet eec att aga gee eat gtt ttt ea gee gcc egc Asn Asn Ilie Arg GLu Asn VaL Phe Lys GLu Ala Ser 1030 1035 tce agc et ett et gee gte ggt tcc sgt act et gee gtg ggc tcc Ser Ser Asn Ilie Asn Giu Vat Gty Ser Ser Thr Asn GLu Vat GLy Ser 1040 1045 1050 1055 sgt att aet gaa ate ggt tcc sgt get gasaaec ett cee gca gee cte Ser lie Asn Giu lie Giy 5cr 5cr Asp Giu Asn Iie Gin Aia Giu Leu 1060 1065 1070 ggt age eec age ggg cca ass ttg eat gct etg ctt age tta ggg gtt Giy Arg Asn Arg Gty Pro Lys Lau Asn Aia Met Lou Arg Lau GLy Vat 1075 1080 1085 ttg cee cct gag gtc tat aas caa sot ctt cct gga sot aat tgt aag Lau Gin Pro Giu Vat Tyr Lys Gin Ser Lau Pro Giy Ser Asn Cys Lys 1090 1095 1100 cat cct gee eta ea ag cee gae tat gee gee gte gtt cag act gtt His Pro Giu Ilie Lys Lys Gin Giu Tyr Giu Giu Vat Vat Gin Thr Vai 1105 1110 1115 et ace get ttc Asn Thr Asp Phe 1120 atg gga sot sot Met Giy Ser Ser tct cca tat Ser Pro Tyr 1125 cat gce tct His Aie Ser 1140 ctg att tce get eec tte ga ceg cct 3527 Lau ies Ser Asp Asn Leu Giu Gin Pro 1130 1135 cag gtt tgt tct gag ace cct get gec 3575 Gin Vat Cys 5cr Giu Thr Pro Asp Asp 1145 1150 ctg tte get get ggt gee ate eag gee get act sgt ttt gct gee et 3623 Lau Leu Asp Asp Giy Giu Ile Lys Giu Asp Thr Ser Phe Ale Giu Asn 1155 1160 1165 gac ett ag gee egt tct gct gtt ttt agc ass agc gtc cag aea gge 3671 Asp lie Lys Giu 5cr Ser Aia Vat Phe Ser Lys Ser VaL Gin Lys Giy 1170 1175 1180 WO 95/19369 PCT/US95/00608 gag ctt agc egg agt cct agc cct Gtu Lou Ser Arg Ser Pro Ser Pro 1185 1190 ggt tac cge aga ggg gcc aag aaa GLy Tyr Arg Arg Gty Ala Lys Lys 1200 1205 tct egt gag gat gee gag ctt ccc Ser Ser Gtu Asp GLu Gtu Lau Pro 1220 aaa gta aac aat ea cct tct cag Lys VaL Asn Asn ite Pro Ser Gin 1235 acc gag tgt ctg tct eag aac ace Thr Glu Cys Leu Ser Lys Asn Thr ttc ccc cat ace cat ttg gct ceg Phe Thr His Thr His Leu Aia Gin 1195 tte geg tcc tca gee gag eec tte Lau Gtu Ser Ser Gtu Gtu Asn Lou 1210 1215 tgc ttc caa cac ttg tta ttt ggt Cys Phe Gin His Lau Lou Phe Gty 1225 1230 tct act egg cet cgc acc gtt gct Ser Thr Arg His Ser Thr Vet ALa 1240 1245 gag geg et tte tta tce ttg ag GLu Gtu Asn Leu Leu Ser Lau Lys 1260 ceg gtaea ttg gcc ag qca tct 1250 cat agc tte aat gec tgc egt eec Asn Ser Leu Asn Asp Cys Ser Asn Gin Vat lie Lou ALa Lys Ats Ser 1265 1270 1275 cag gee cet ccc ctt agt gag gee ac e a tgt tct gct Gin Giu His His Leu Ser Giu Giu Thr Lys Cys Ser Ale 1280 1285 1290 tct tca ccg tgc agt gee ttg gee gac ttg act gca et Ser Ser Gtn Cys Ser Gtu Lau Giu Asp Leu Thr Ale Asn 1300 1305 cag get cct ttc ttg ett ggt tct tcc ea cee etg egg Gin Asp Pro Phe Lou ite Gty Ser Ser Lys Gin Met Arg 1315 1320 gee egc cag gge gtt ggt ctg egt gec sag gee ttg gtt Gtu Ser Gin Gty Vai Giy Lau Ser Asp Lys Giu Leu Val egc ttg ttt Ser Leu Phe 1295 ace eec cc Thr Asn Thr 1310 cat ceg tct His Gin Ser 1325 tca get get 1330 1335 1340 gee gac age gge ccg ggc ttg gee gee act cat ccc gee gag cee egc Giu Giu Arg Giy Thr Gty Lou Giu Gtu Asn Asn Gin Giu Giu Gin Ser 1345 1350 1355 etg get tce eec tta ggt gee gca gca tct ggg tgt gag agt gee ac Met Asp Ser Asn Lau Giy GLu Ale Aia 5cr GiY Cys Giu Ser Gtu Thr 3719 3767 3815 3863 3911 3959 4007 4055 4103 4151 4199 4247 4295 4343 4391 4439 1360 1365 cgc gtc tct gee gec tgc tce Ser Vat Ser Giu Asp Cys Ser 1380 acc act ceg cag agg get cc Thr Thr Gin Gin Arg Asp Thr 1395 1370 ggg cte tcc tct cag cgt gac Giy Lau 5cr Ser Gin Ser Asp 1385 atg ccc cat eec ctg ea ag Met Gin His Asn Lou Ilie Lys 1400 1405 1375 att tte ILie Lou 1390 ctc cag Lou Gin cag gee etg gct gee cte gee gct gtg tte gee cag cat ggg Gin Gtu Met Aia Gtu Lau Giu Ale Vet Lou Giu Gin His Giy Ser Gin 1410 1415 1420 cct tct eec egc tec cct tcc etc a agt gec tct tct gcc ctt gag Pro Ser Asn Ser Tyr Pro Ser ite ite 5cr Asp Ser Ser Aia Lou Gtu 1425 1430 1435 WO 95/19369 PCTIUS95/00608 gac ctg cga aat eca gas caa age aca tca gaa ga gca gta tta act Asp Leu Arg Asn Pro GLu Gin Ser Thr Ser GLu Lys VaL Leu GLn Thr 1440 1445 1450 1455 tea cag ga agt agt gaa tac cct ata age eag aat cca gag gge ctt Ser GLn Lys Ser Ser GLu Tyr Pro ite Ser GLn Asn Pro Giu GLy Xaa 1460 1465 1470 tct get gac aag ttt gag gtg tct gca gat agt tct ace agt saa ant Ser Ata Asp Lys Phe Giu Vat Ser ALa Asp Ser Ser Thr Scr Lys Asn 1475 1480 1485 saa gag cca ggs gtg gas agg tea tee cet tet a tge eca tea tta Lys GLu Pro GLy VaL Giu Arg Ser Ser Pro Ser Lys Cys Pro Ser Leu 1490 1495 1500 gat gat agg tgg tae atg cac agt tge tet ggg agt ctt eag aat aga Asp Asp Arg Trp Tyr Met His Ser Cys 5cr GLy Ser Leu GLn Asn Arg 1505 -1510 1515 1520 sac tac eca tet eaa gag gag etc att aag gtt gtt gat gtg gag gag Asn Tyr Pro Pro Gin Giu Gtu Lau Rie Lys VaL VaL Asp Vat Giu Giu 1525 1530 1535 eaa egg etg gas gag tct ggg ces eac gat ttg aeg gag aca tet tac Gin Gin Lau GLu Giu Ser Giy Pro His Asp Leu Thr Giu Thr Ser Tyr 1540 1545 1550 ttg eca agg egg gat eta gag gga ace cet tae ctg gas tct gga ate Lau Pro Arg Gin Asp Leu Giu Giy Thr Pro Tyr Leu Giu Ser Giy lie 1555 1560 1565 age etc ttc tet gat gac cet gas tet gat cet tet gas gac aga gee Ser Lau Phe Scr Asp Asp Pro Giu Ser Asp Pro Ser Giu Asp Arg Ala 1570 1575 1580 ces gag tea get egt gtt ggc ace ate ce tct tea ace tct ges ttg Pro Giu Ser Aia Arg Vat Giy Asn lie Pro Ser Ser Thr Ser Ata Leu 1585 1590 1595 1600 aga gtt ccc egg ttg sa gtt geg gaa tet gee egg agt eca get get Lys Vat Pro Gin Leu Lys Vat Ata Gtu Ser Ala Gin Ser Pro Ala Ate 1605 1610 1615 get eat act act gat act get ggg tat nat geg atg gas gas agt gtg Ala His Thr Thr Asp Thr Ata Gty Tyr Asn Ate Met Giu Giu Ser Vat 1620 1625 1630 age agg gag gag cca gs ttg aca get tea aca gag agg gte sac sa Ser Arg Giu Lys Pro Giu Leu Thr Ata Scr Thr Giu Arg Vat Asn Lys 1635 1640 1645 aga atg tee atg gtg gtg tct ggc etg ace cca gas gag ttt atg etc Arg Met Ser Met Vat Val Scr GLy Leu Thr Pro Gtu Gtu Phe Met Leu 1650 1655 1660 gtg tac gag ttt gee ag ga cac eae ate act tta act aat eta att Vat Tyr Lys Phe Ala Arg Lys His His ies Thr Leu Thr Asn Leu Lie 1665 1670 1675 1680 act gaa gag act act eat gtt gtt atg aga aca gat get gag ttt gtg Thr Giu Giu Thr Thr His Vat Vat Met Lys Thr Asp Ala Gtu Phe Vat 1685 1690 1695 4487 4535 4583 4631 4679 4727 4775 4823 4871 4919 4967 5015 5063 5111 5159 5207 WO 95/19369 PCTIUS95/00608 tgt gaa cgg aca ctg aaa tat Cys GLu Arg Thr Leu Lys Tyr 1700 gta gtt agc tat ttc tgg gtg VaL VaL Ser Tyr Phe Trp VaL 1715 ctg aat gag cat gat ttt gee Leu Asn Gtu His Asp Phe Gtu 1730 1735 eec ccc caa ggt cca eag cga ttt cta gga ett Phe Leu Gty lie 1705 ecc cag tct att Thr Gin Ser lie 1720 gtc age gga get Vat Arg GLy Asp gce age gee tcc gcg gga gge eec tgg Ate GLy GLy Lys Trp 1710 aaa goo age ace etg Lys GLu Arg Lys Met 1725 gtg gtc at gga age VaL Vat Asn GLy Arg 1740 ceg gec age eag etc Asn His Gin GLy Pro Lys Arg Ate Arg GLu Ser Gin Asp Arg Lys lie 1745 1750 1755 1760 ttc egg ggg cte goe etc tgt tgc tat ggg ccc ttc acc eec ctg ccc Phe Arg Gly Leu GLu lie Cys Cys Tyr GLy Pro Phe Thr Asn Met Pro 1765 1770 1775 ae get ccc ctg gee tgg atg gte cag ctg tgt ggt gct tct gtg gtg Thr Asp Gin Leu GLu Trp Met Vet Gin Leu Cys GLy Ate Ser Vat Vet 1780 1785 1790 5255 5303 5351 5399 5447 5495 5543 5591 5639 5687 5712 aeg gag Lys Gtu gtt gtg Val Vat 1810 ctt tce toe ttc cc ctt ggc ace ggt gtc cac ccc att gtg Leu Ser Ser Phe Thr Leu Gty Thr Gty Vat His Pro lie Vel 1795 1800 1805 cag cce get gcc tgg ace gag gacoeat ggc ttc cat gee ctt Gtn Pro Asp Ate Trp Tht Gtu Asp Asn Gty Phe His Ata lie 1815 1820 cag etg tgt gag gce cct gtg gtg ccc o gag tgg gtg ttg gac Gin Met Cys Gtu Ate Pro Vat VaL Thr Arg Gtu Trp Vat Lau Asp 1830 1835 1840 gte gc ctc tao cag tgc cag gag ctg gec aco tac ctg ate ccc Vat Ata Leu Tyr Gin Cys Gin Glu Leu Asp Thr Tyr Leu lite Pro 1845 1850 1855 cog atc ccc Ccc Gin lie Pro His 1860 ae ccc tec tgat Ser His Tyr INFORMATION FOR SIEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 1237 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: DNA regulatory sequence HYPOTHETICAL: no (iv) ANTI-SENSE: no II .li WO 95/19369 PCTIUS95/00608 89 ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens

INDIVIDUAL/ISOLATE:

DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: normal breast CELL LINE: not derived from a cell line ORGANELLE: no (vii) IMMEDIATE SOURCE: LIBRARY: cDNA library derived from human CLONE: obtained using published sequence (viii) POSITION IN GENOME: CHROMOSOME/SEGMENT: unknown MAP POSITION: unknown UNITS: unknown (ix) FEATURE: NAME/KEY: BRCA1 promoter

LOCATION:

IDENTIFICATION METHOD: restriction enzyme digest OTHER INFORMATION: DNA sequence regulating gene encoding BRCA1 protein PUBLICATION INFORMATION: AUTHORS: Brown et al.

TITLE: Scientific Correspondence JOURNAL: Nature VOLUME: 372 PAGES: 733 DATE: 22/29 DECEMBER 1994 RELEVANT RESIDUES IN SEQ ID NO: 48 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: TTCCGGGACT CTACTACCTT TACCCAGACG AGAGGGTGAA GGCCTCCTGA TCGCAGGGGC CCAGTTATCT GAGAAACCCC ACAGCCTGGT GCGGGGTCCA GGAAGTCTCA GCGAGCTCAC 120 WO 95/19369 PCTIUS95/00608 GCCGCGCAGT CGCAGTTTTA ATTTATCTGT AATTCCCGCG CTTTTCCGTT GCCACGGAAA 180 CCAAGGGGCT ACCGCTAAGC AAGGGAGGGA CAGAAAGAGC CCCCGGATGA CGTAAAAGGA AAGCGCCCGC CCTCTCGCCT TGTACCAAGG TCAGAATCGC CAGTCTTAAG AAGAGGTCCC AGGTAAATAA AAGGATTGTT GCGGAACGAA AGGCCTTGGC ATCCAATACC AAAGCGGGCA GCGGGCTTAT ACATGTCAAC AGCAGCCTCT CAGAATACGA CAAGCGTCTC TCGGGGCTCT AAGAGACGGA AGAGGAAGAA CTACGCTTCC AGTTGCGGCT CACCTGAGGC CTGAATATCA ATTACCCCAC TCTTTCCGCC AATCAAGGTA CAATCAGAGG GGATTGGCCA CCCAGTCTGC TTCTACCTGA GTTCGCCGTA TATTACGTCA CAGTAATTGC GCGTAAGATA GTGTCCAAAG CTAATGGAGT CCTCCAGTTT GGGAGGTGGA GGGAAAGAAC TACTATTTCC AACATGCATT CACACTGTTC CTTGGAAACT GTAGTCTTAT GGAGAGGAAC CAATTCTCAC GGAAATCCAG TGGATAGATT GGAGACCTCC AGTAATATTG GGTTGTTATG TTCTCCTATC TTGAGAGCAG AGACTAGGCC AAAAAAAGAT ATAGGAAGAC TACGATTCCC ATCCAGCCCC ACGAGTCTCG 840 GGCAAGTAGT CCTCTAAGGT CAGTGGCCTG CGGGGACGCA GTGGGCGCCG AATTTGCCTG 900 GGGAAGGGGA AATCCCTCTC TGGTCACATC TGCGCACTCC TAGTTCCGCC CCTCAGCATC 960 AATGTTTGTT ATTGTTGTTC GGGTTCAGGT TGCTTCTGCC CCGCCCCATC GACGCAATCT 1020 CCACCAATCA ATGGCGTGGT CGTTTTGAGG GACAAGTGGT GAGAGCCAAT CATCTTGGCG 1080 AACACTCGGA GAAACAGGGG ACTAGTTACT GTCTTTATCC GCCATGTTAG ATTCACCCCA 1140 CAGGGATAGC GGCAGAGCCG GTAGCGGACG GTCCTTGCAT TGGCCTCCGG CAGGCGCCCC 1200 CCGGGGGCGG GAAGCTGGTA AGGAAGCAGC TGCGGTT 1237 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 1863 TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown MOLECULE TYPE: protein HYPOTHETICAL: no ANTI-SENSE: no (iii) (iv) ORIGINAL SOURCE ORGANISM: Homo sapiens sapiens

INDIVIDUAL/ISOLATE:

DEVELOPMENTAL STAGE: adult TISSUE TYPE: female breast CELL TYPE: normal breast tissue CELL LINE: not derived from a cell line ORGANELLE: no FEATURE: (iX) NAME/KEY: BRCA1 protein WO 95119369 PCTUS9s5rjo608j 91 2 LOCATION: 1 to 1863 IDENTIFICATION METHOD: observation of mRNA and antisense inhibition of BRCA1 gene OTHER INFORMATION: BRCA1 protein has a negative regulatory effect on growth of human mammary cells.

(x PUBLICATION

INFORMATION:

AUTHORS: Mild, et. al.

TITLE: A strong candidate gene for the breast and ovarian cancer susceptibility gene BRCA1.

JOURNAL: Science VOLUME: 266 PAGES: 66-71 DATE: 1994 RELEVANT RESIDUES IN SEQ ID NO: 49 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: Met Asp Leu Ser Ate Lou Arg Val Giu GLu VaL Gin Asn Vat lie Asn 1 5 10 Ate Met Gin Lys lie Leu Giu Cys Pro lie Cys Leu GLu Leu ILe Lys 25 Giu Pro Vat Ser Thr Lys Cys Asp His lie Phe Cys Lys Phe Cys Met 40 Leu Lys Leu Lau Asn Gin Lys Lys GLy Pro Ser Gin Cys Pro Lou Cys 55 Lys Asn Asp lie Thr Lys Arg Ser Leu Gin Giu Ser Thr Arg Phe Ser 70 75 Gin Lou Val Giu Giu Leu Lou Lys lie lie Cys Ala Phe Gin Lou Asp 90 Thr GLy Lou Giu Tyr Ata Asn Ser Tyr Asn Phe Ala Lys Lys Giu Asn 100 105 110 Asn Ser Pro GLu His Lau Lys Asp GLu Vat Scr lie lIe Gin 5cr Met 115 120 125 Giy Tyr Arg Asn Arg Aia Lys Arg Leu Lau Gin 5cr Giu Pro Giu Asn 130 135 140 Pro Ser Leu Gin Giu Thr Ser Leu Ser Vat Gin Lou Ser Asn Lou Gly 145 150 155 160 Thr Vai Arg Thr Leu Arg Thr Lys Gin Arg lie Gin Pro Gin Lys Thr 165 170 175 Scr Vol Tyr lie Giu Lou Gly Ser Asp Ser Ser Giu Asp Thr Val Asn 180 185 190 WO 95/19369 PCT/US95/00608 92 Lys Ala Thr Tyr Cys Ser Vat Gly Asp Gin GLu Leu Leu Gin lie Thr 195 200 205 Pro Gin Gly Thr Ar; Asp Gtu lie Ser Leu Asp Ser ALa Lys Lys Ala 210 215 220 Ala Cys GLu Phe Ser Glu Thr Asp Vat Thr Asn Thr Giu His His Gin 225 230 235 240 Pro Ser Asn Asn Asp Leu Asn Thr Thr GLu Lys Ar; Ala Ala GLu Arg 245 250 255 His Pro GLu Lys Tyr Gin Gly 9cr Ser Vol Ser Asn Leu His Val Glu 260 265 270 Pro Cys Gly Thr Asn Thr His Ala Ser Ser Leu Gin His Glu Asn Ser 275 280 285 9cr Lau Leu Lau Thr Lys Asp Ar; Met Asn Vol Giu Lys Ala GLu Phe 290 295 300 Cys Asn Lys Ser Lys Gin Pro GLy Leu Ala Ar; Ser Gin His Asn Ar; 305 310 315 320 Trp Ala Giy Ser Lys Glu Thr Cys Asn Asp Ar; Ar; Thr Pro 9cr Thr 325 330 335 GLu Lys Lys Val Asp Leu Asn Ala Asp Pro Leu Cys Giu Ar; Lys Giu 340 345 350 Trp Asn Lys GIn Lys Lau Pro Cys Ser Glu Asn Pro Ar; Asp Thr GLu 355 360 365 Asp Vol Pro Trp lIe Thr Lau Asn 9cr Ser lIe GIn Lys Vol Asn GLu 370 375 380 Trp Phe 9cr Ar; Ser Asp Giu Leu Leu GLy Ser Asp Asp Ser His Asp 385 390 395 400 GLy Giu Ser GLu Ser Asn Ala Lys Vol Ala Asp Vol Lau Asp Vol Leu 405 410 415 Asn GLu Vol Asp GLu Tyr Ser GLy Ser 9cr GLu Lys lie Asp Leu Lau 420 425 430 Ala Ser Asp Pro His Glu Ala Leu lie Cys Lys 9cr Asp Ar; Val His 435 440 445 Ser Lys Ser Val Giu Scr Asp lie Giu Asp Lys lie Phe GLy Lys Thr 450 455 460 Tyr Ar; Lys Lys Ala Ser Leu Pro Asn Leu Ser His Vol Thr GLu Asn 465 470 475 480 Leu lie lie GLy Ala Phe Vol Ser Giu Pro Gin lie lie GIn GLu Ar; 485 490 495 Pro Leu Thr Asn Lys Lau Lys Ac; Lys Ar; Ar; Pro Thr Scr GLy Lau 500 505 510 His Pro GLu Asp Ph. lIe Lys Lys Ala Asp Lau Ala Vol Gin Lys Thr 515 520 525 Pro GLu Met lie Asn GIn Giy Thr Asn GIn Thr Glu Gin Asn Gly Gin 530 535 540 Val Met Asn lie Thr Asn 9cr Giy His Gtu Asn Lys Thr Lys GLy Asp 545 550 555 9cr lic GIn Asn GLu Lys Asn Pro Asn Pro lIe Glu 9cr Lau Glu Lys 560 565 570 575 WO 95/19369 PCTIUS95/00608 93 GLu Ser AMa Phe Lys Thr Lys Ala Giu Pro lie Ser Ser Ser lie Ser 580 585 590 Asn GLu Leu Giu Leu Asn lie Met His Asn Ser Lys ALa Pro Lys Lys 595 600 605 Asn Arg Lou Arg Arg Lys Ser Ser Thr Arg His lie His AMs Leu Giu 610 615 620 Leu Val VaL Ser Arg Asn Leu Ser Pro Pro Asn Cys Thr Giu Leu Gin 625 630 635 lie Asp Ser Cys Ser Ser Ser Giu Giu liLe Lys Lys Lys Lys Tyr Asn 640 645 650 655 Gin Met Pro Vat Arg His Ser Arg Asn Leu Gin Leu Met Gtu Gly Lys 660 665 670 Giu Pro ALa Thr Gly ALa Lys Lys Ser Asn Lys Pro Asn GLu Gin Thr 675 680 685 Ser Lys Arg His Asp Ser Asp Thr Ph. Pro GLu Leu Lys Leu Thr Asn 690 695 700 Ala Pro Gly Ser Ph. Thr Lys Cys Ser Asn Thr 5cr GLu Leu Lys GLu 705 710 715 Ph. Vai Asn Pro Ser Leu Pro Arg Giu GLu Lys Giu Giu Lys Leu Giu 720 725 730 735 Thr Vai Lys VaL Ser Asn Asn Ala Glu Asp Pro Lys Asp Leu Met Lou 740 745 750 Ser Gly Gtu Arg VaL Lou Gin Thr Giu Arg Ser Vat GLu Ser Ser 755 760 765 lie 5cr Lou VaL Pro Giy Thr Asp Tyr GLy Thr Gin Glu Ser lie 770 775 780 Leu Leu Giu VaL Ser Thr Leu Giy Lys Ala Lys Thr Giu Pro Asn Lys 785 790 795 Cys VaL 5cr Gin Cys Ala Ala Phe GLu Asn Pro Lys Giy Lou lie His 800 805 810 815 GLy Cys Ser Lys Asp Asn Arg Asn Asp Thr GLu Giy Phe Lys Tyr Pro 820 825 830 Leu Gly His GLu VaL Asn His Ser Arg Giu Thr 5cr lie Giu Met Giu 835 840 845 GLu Ser GLu Leu Asp ALa Gin Tyr Lou Gin Asn Thr Phe Lys VaL Sor 850 855 860 Lys Arg Gin Ser Ph. Aia Pro Ph. Ser Asn Pro Giy Asn Ala Giu Gtu 865 870 875 Giu Cys Ala Thr Phe Ser Ala His Ser Gly 5cr Lau Lys Lys Gin Ser 880 885 890 895 Pro Lys Vai Thr Phe GLu Cys Giu Gin Lys GLu GLu Asn Gin Gly Lys 900 905 910 Asn Glu Ser Asn lie Lys Pro Vat Gin Thr Vai Asn lie Thr Ala Gly 915 920 925 Ph. Pro Vat Vat Gly Gin Lys Asp Lys Pro Val Asp Asn Ala Lys Cys 930 935 940 Ser lie Lys GLY Gly Ser Arg Ph. Cys Lou Ser Ser Gin Ph. Arg Gly 945 950 955 WO 95/19369 608 94 Asn GLu Thr Gty Leu lie Thr Pro Asn Lys His GLy Lau Lau Gin Asn 960 965 970 975 Pro Tyr Arg lie Pro Pro Lau Ph. Pro lie Lys Ser Ph. Vat Lys Thr 980 985 990 Lys Cys Lys Lys Asn Lou Lau GLu GLu Asn Phe Giu GLu His Ser Met 995 1000 1005 Ser Pro Gtu Arg Giu Met GLy Asn Giu Asn lie Pro Ser Thr VaL Ser 1010 1015 1020 Thr lie Ser Arg Asn Asn lie Arg Gtu Asn VaL Ph. Lys Giu ALa Ser 1025 1030 1035 Ser Ser Asn lie Asn Gtu Vat GLy Ser Ser Thr Asn GLu Vat Giy Ser 1040 1045 1050 1055 Ser lie Asn Gtu lie Giy Sor Ser Asp Giu Asn lie Gin Ala Giu Lau 1060 1065 1070 Gty Arg Asn Arg Gly Pro Lys Lou Asn Ata Met Lou Arg Lou Gly Vat 1075 1080 1085 Lou Gin Pro Gtu Vat Tyr Lys Gin Ser Lau Pro Gty Ser Asn Cys Lys 1090 1095 1100 His Pro Giu lie Lys Lys Gin GLu Tyr Gtu Giu Vat Vat Gin Thr Vat 1105 1110 1115 Asn Thr Asp Ph. Ser Pro Tyr Lou Ile 5cr Asp Asn Lou Gtu Gin Pro 1120 1125 1130 1135 Net Giy Ser 5cr His Ala Ser Gin Vat Cys 5cr Gtu Thr Pro Asp Asp 1140 1145 1150 Lou Lou Asp Asp GLY Gtu lie Lys GLu Asp Thr Sor Ph. Aim Giu Asn 1155 1160 1165 Asp lie Lys Giu Ser Sor Ata Vat Ph. Ser Lys 5cr Vat Gin Lys GLy 1170 1175 1180 Giu Lou Sor Arg Ser Pro Ser Pro Ph. Thr His Thr His Lou Ala Gin 1185 1190 1195 Giy Tyr Arg Arg GLy Ala Lys Lys Lou Giu Sor 5cr Giu GLu Asn Lou 1200 1205 1210 1215 Ser Sor Giu Asp Giu Giu Lou Pro Cys Pho Gin His Lou Lou Ph. Giy 1220 1225 1230 Lys Vat Asn Asn Ilo Pro 5cr Gin Sor Thr Arg His 5cr Thr Vat Ala 1235 1240 1245 Thr Giu Cys Lou Sor Lys Asn Thr Giu GLu Asn Lau Lou Sor Lou Lys 1250 1255 1260 Asn Ser Lou Asn Asp Cys 5cr Asn Gin Vat Ilo Lou Ala Lys ALs Ser 1265 1270 1275 Gin Giu His His Leu Ser Giu Giu Thr Lys Cys Ser ALa Ser Lou Ph.

1280 1285 1290 1295 Ser Gin Cys Ser GLu Lou Giu Asp Lou Thr ALa Asn Thr Asn Thr 1300 1305 1310 Gin Asp Pro Ph. Lou Iie GLy 5cr 5cr Lys Gin Met Arg His Gtn Sor 1315 1320 1325 Giu Ser Gin Gly Vat Giy Lou Ser Asp Lys Giu Lou Val 5cr Asp Asp 1330 1335 134.0 WO 95/19369 PCTIUS95/00608 Glu GLu Arg GLy Thr Sly Leu Glu Glu Asn Asn Gin Glu GLu Gin Ser 1345 1350 1355 Met Asp Ser Asn Leu Sly SLu Ala Ala Ser Gly Cys GLu Ser SLu Thr 1360 1365 1370 1375 Ser VaL Ser Gtu Asp Cys Ser GLy Leu Ser Ser Gin Ser Asp lie Leu 1380 1385 1390 Thr Thr Sin SIn Arg Asp Thr Met Sin His Asn Leu Ilie Lys Lau Gin 1395 1400 1405 GIn Slu Met Ala Glu Lau GLu Ala Vat Leu Glu GIn His Sly Ser Gin 1410 1415 1420 Pro Ser Asn Ser Tyr Pro Ser Ile lie Ser Asp Ser Ser Ala Leu GLu 1425 1430 1435 Asp Lau Arg Asn Pro Gtu Sin Ser Thr Ser Giu Lys Val Leu SIn Thr 1440 1445 1450 1455 Ser GIn Lys Ser Ser GLu Tyr Pro lie Ser Gin Asn Pro Glu SLy Xaa 1460 1465 1470 Ala Asp Lys Phe Glu Val Ser Ala Asp Ser Ser Thr Ser Lys Asn 1475 1480 1485 Lys Glu Pro Sly Val Glu Arg Ser 5cr Pro Ser Lys Cys Pro Ser Leu 1490 1495 1500 Asp Asp Arg Trp Tyr Met His Ser Cys 5cr Sly Ser Leu GIn Asn Arg 1505 1510 1515 1520 Asn Tyr Pro Pro GIn G~u Glu Lau lie Lys Vat Val Asp Vat Glu Giu 1525 1530 1535 GIn SIn Leu Slu Slu 9cr Sly Pro His Asp Leu Thr SLu Thr Ser Tyr 1540 1545 1550 Lau Pro Arg SIn Asp Leu Glu Sly Thr Pro Tyr Leu S~u Ser Sly Ilie 1555 1560 1565 9cr Leu Phe Ser Asp Asp Pro Slu 5cr Asp Pro 9cr Stu Asp Arg Ala 1570 1575 1580 Pro S~u Ser Ala Arg Val Sly Asn Ile Pro Ser 9cr Thr 9cr Ala Leu 1585 1590 1595 1600 Lys Val Pro SIn Leu Lys Val Ala Stu Ser Ala SIn Ser Pro Ala Ala 1605 1610 1615 ALa His Thr Thr Asp Thr Ala Gly Tyr Asn Ala Met Slu S~u Ser Vat 1620 1625 1630 Arg S~u Lys Pro SLu Lau Thr Ala 9cr Thr Slu Arg Vat Asn Lys 1635 1640 1645 Arg Met 5cr Met Val Vat 5cr Sly Lau Thr Pro Stu Siu Phe Met Leu 1650 1655 1660 Val Tyr Lys Phe Ala Arg Lys His His lie Thr Lau Thr Asn Leu lie 1665 1670 1675 1680 Thr Glu SLu Thr Thr His Vat Val Met Lys Thr Asp Ala G~u Phe VaL 1685 1690 1695 Cys Slu Arg Thr Leu Lys Tyr Phe Leu Sly lie Ala Sly Sly Lys Trp 1700 1705 1710 Val Vat 9cr Tyr Phe Trp Vat Thr Sin Ser lie Lys Slu Arg Lys Met 1715 1720 1725 WO 95/19369 PCTUS95OO6O8 96 Leu Asn GLu His Asp Ph. Gtu Vat Arg GLy Asp VaL Vat Asn Gty Arg 1730 1735 1740 Asn His GLn Gly Pro Lys Arg ALa Arg Gtu Ser GLn Asp Arg Lys lie 1745 1750 1755 1760 Ph. Arg Gty Leu GLu Ile Cys Cys Tyr Gty Pro Ph. Thr Asn Met Pro 1765 17"70 1775 Thr Asp GLn Lau GLu Trp Met VaL GLn Lau Cys Gty ALa Ser Vat Vat 1780 1785 1790 Lys GLu Lau Ser Ser Ph. Thr Leu GLy Thr M~y VaL His Pro Ile Vat 1795 1800 1805 Vat Vat GLn Pro Asp ALa Trp, Tht GLu Asp Asn Gty Ph. His Ata ILe 1810 1815 1820 GLy Gin Met Cys Gtu Ala Pro VaL Vat Thr Arg Gtu Trp Vat Leu Asp 1825 1830 1835 1840 Ser Vat ALa Lau Tyr Gin Cys GLn Gtu Lau Asp Thr Tyr Lau ILe Pro 1845 1850 1855 Gin ILe Pro His Ser His Tyr 1860

Claims (7)

1. A method of treating breast cancer in a patient comprising the steps of ligating a gene that encodes a protein having an amino acid sequence as essentially set forth in SEQ ID NO:49 with a promoter capable of inducing expression of the gene in a breast cancer cell and introducing the ligated gene into a breast cancer cell.

2. The method of treating breast cancer described in claim 1 wherein the gene has a DNA sequence selected from among: a. the DNA sequence as essentially set forth in SEQ ID NO:47 or its complementary strands; b. a DNA sequence with hybridizes to SEQ ID NO:47 or fragments thereof; and c. DNA sequences which but for the degeneracy of the genetic code would hybridize to the DNA sequences defined in and 3

3. The method of treating breast cancer described in either claim 1 or claim 2 wherein the gene has a DNA sequence having 20-99% homology with SEQ ID NO:47. C C

4. The method according to any of the preceding claims wherein the ligated gene is introduced into the cell in a viral expression vector. C

5. The method according to any of the preceding claims wherein the breast cancer is gene-linked hereditary breast cancer.

6. The method described in any one of claims 1 to 4 wherein the breast cancer is sporadic breast cancer. -98-

7. A method of treating breast cancer as claimed in claim 1, substantially as hereinbefore described. DATED this 10 day of May, 1999 VANDERBILT UNIVERSITY By Its Patent Attorneys A TATLOCK ASSOCIATES a.. a a a 9 a a a a. a ass a a a a a a a S a a. a a a *SaSa. a AU~ m K:\V~5 \SPEC\18317-95 EDITORIAL NOTE NUMBER 18317/95 THIS SPECIFICATION DOES NOT CONTAIN PAGES NUMBERED 99 TO 113