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Biomarkers for breast cancer

Abstrict

Methods and compositions are provided for the detection of breast cancer based upon the identification of three biomarkers for non-normal breast cells. The biomarkers were identified based upon multiple sampling of reference breast tissue samples from independent cases of breast cancer. Two biomarkers display increased expression in non-normal cells while the third biomarker displays decreased expression in non-normal cells.

Claims

We claim:

1. A method to determine the presence of non-normal or abnormal breast cells in a sample from a human subject comprising assaying said sample for increased expression of one or more human CRIP1 or HN1 sequences.

2. The method of claim 1 wherein said assaying is for increased expression of human CRIP1 sequences.

3. The method of claim 1 wherein said assaying is for increased expression of human HN1 sequences.

4. The method of claim 1 wherein said sample is from a subject afflicted with, or suspected of having, breast cancer.

5. The method of claim 1 wherein said sample is obtained by solid tissue biopsy or a non-invasive procedure.

6. The method of claim 5 wherein said non-invasive procedure is selected from ductal lavage, fine needle aspiration, or a needle biopsy.

7. The method of claim 5 wherein microdissection is used to isolate breast cells from said sample before assaying for nucleic acid expression.

8. The method of claim 1 wherein said assaying is by hybridization to a polynucleotide comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human CRIP1.

9. The method of claim 1 wherein said assaying is by hybridization to a polynucleotide comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human HN1.

10. The method of claim 1 wherein said assaying is by PCR amplification of said sequences.

11. The method of claim 10 wherein said PCR is quantitative PCR.

12. The method of claim 1 wherein said non-normal cells are ADH, DCIS, or IDC cells.

13. A method to determine the presence of non-normal or abnormal breast cells in a sample from a human subject comprising assaying said sample for decreased expression of human ESE-2/ELF5 sequences.

14. The method of claim 13 wherein said sample is from a subject afflicted with, or suspected of having, breast cancer.

15. The method of claim 13 wherein said sample is obtained by solid tissue biopsy or a non-invasive procedure.

16. The method of claim 15 wherein said non-invasive procedure is selected from ductal lavage, fine needle aspiration, or a needle biopsy.

17. The method of claim 16 wherein microdissection is used to isolate breast cells from said sample before assaying for nucleic acid expression.

18. The method of claim 17 wherein said assaying is by hybridization to a polynucleotide comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human ESE-2/ELF5.

19. The method of claim 13 wherein said assaying is by PCR amplification of said ESE-2/ELF5 sequence.

20. The method of claim 19 wherein said assaying is by quantitative PCR.

21. The method of claim 13 wherein said assaying is for inactivation or methylation of ESE-2/ELF5 sequences.

22. The method of claim 13 wherein said assaying comprises detection of increased mRNA degradation.

23. The method of claim 13 wherein said non-normal cells are ADH, DCIS, or IDC cells.

24. A polynucleotide comprising a segment consisting of a fragment of an HN I sequence selected from SEQ ID NOS: 29-83 of between 24 and 500 nucleotides and one or more non-HN1 nucleic acid molecules.

25. A polynucleotide consisting of a fragment of an HN1 sequence selected from SEQ ID NOS: 29-83 of between 24 and 500 nucleotides.

26. A population of singled stranded nucleic acid molecules comprising one or both strands of a human CRIP1 or HN1 sequence wherein at least a portion of said population is hybridized to one or both strands of a nucleic acid molecule quantitatively amplified from RNA of a non-normal or abnormal breast cell.

27. The population of claim 26 wherein the population is immobilized on a solid support.

28. The population of claim 27 wherein said solid support is a microarray.

29. The population of claim 26 wherein said nucleic acid molecules amplified from a non-normal or abnormal breast cell are amplified by quantitative real time PCR (RT-PCR).

30. The population of claim 29 wherein said quantitative RT-PCR is of amplified RNA of said breast cancer cell.

31. The population of claim 26 wherein said population of single stranded molecules is equal to or in excess of all of one or both strands of the nucleic acid molecules amplified from a non-normal or abnormal breast cell such that the population is sufficient to hybridize to all of one or both strands.

32. The population of claim 26 wherein said population of single stranded molecules comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human CRIP1.

33. The population of claim 26 wherein said population of single stranded molecules comprising sequences of at least 24 nucleotides from the 3' untranslated region, the coding region, or the 5' untranslated region, of human HN1.

34. The population of claim 26 wherein said non-normal cells are ADH, DCIS, or IDC cells.

Description

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent applications Ser. Nos. 10/028,018, filed Dec. 21, 2001, and 10/211,015, filed Aug. 1, 2002, which are hereby incorporated in their entireties as if fully set forth.

FIELD OF THE INVENTION

[0002] The invention relates to the identification and use of gene sequences which are differentially expressed in breast cancer. In particular, the invention provides the identities of three sets of sequences that may be used to identify the presence of breast cancer in tissue and cell samples. The expression of these sequences, whether embodied in nucleic acid expression, protein expression, nucleic acid amplification and/or activation, or other formats, are used in the diagnosis and/or treatment of breast cancer as well as for the study and/or determination of prognosis of a patient. When used for diagnosis, the expression levels of these sequences are used to identify the presence of breast cancer and provide guidance as to the treatment thereof.

BACKGROUND OF THE INVENTION

[0003] Breast cancer is by far the most common cancer among women. Each year, more than 180,000 and 1 million women in the U.S. and worldwide, respectively, are diagnosed with breast cancer. Breast cancer is the leading cause of death for women between ages 50-55, and is the most common non-preventable malignancy in women in the Western Hemisphere. An estimated 2,167,000 women in the United States are currently living with the disease (National Cancer Institute, Surveillance Epidemiology and End Results (NCI SEER) program, Cancer Statistics Review (CSR), www-seer.ims.nci.nih.gov/Publications/CSR1973 (1998)). Based on cancer rates from 1995 through 1997, a report from the National Cancer Institute (NCI) estimates that about 1 in 8 women in the United States (approximately 12.8 percent) will develop breast cancer during her lifetime (NCI's Surveillance, Epidemiology, and End Results Program (SEER) publication SEER Cancer Statistics Review 1973-1997). Breast cancer is the second most common form of cancer, after skin cancer, among women in the United States. An estimated 250,100 new cases of breast cancer are expected to be diagnosed in the United States in 2001. Of these, 192,200 new cases of more advanced (invasive) breast cancer are expected to occur among women (an increase of 5% over last year), 46,400 new cases of early stage (in situ) breast cancer are expected to occur among women (up 9% from last year), and about 1,500 new cases of breast cancer are expected to be diagnosed in men (Cancer Facts & Figures 2001 American Cancer Society). An estimated 40,600 deaths (40,300 women, 400 men) from breast cancer are expected in 2001. Breast cancer ranks second only to lung cancer among causes of cancer deaths in women. Nearly 86% of women who are diagnosed with breast cancer are likely to still be alive five years later, though 24% of them will die of breast cancer after 10 years, and nearly half (47%) will die of breast cancer after 20 years.

[0004] Every woman is at risk for breast cancer. Over 70 percent of breast cancers occur in women who have no identifiable risk factors other than age (U.S. General Accounting Office. Breast Cancer, 1971-1991: Prevention, Treatment and Research. GAO/PEMD-92-12; 1991). Only 5 to 10% of breast cancers are linked to a family history of breast cancer (Henderson IC, Breast Cancer. In: Murphy G P, Lawrence W L, Lenhard R E (eds). Clinical Oncology. Atlanta, Ga.: American Cancer Society; 1995:198-219).

[0005] The relationship between the expression of cellular factors and breast cancer has been an area of interest. STK15, a centrosomal protein kinase, has been observed as frequently amplified in breast cancer, and its quantitative expression levels positively correlate with tumor grade (Zhou, H. et al. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet 20, 189-93, (1998)). RAD51 has recently been shown to interact with the tumor suppressor BRCA1 (Chen, J. J., et al. BRCA1, BRCA2, and Rad51 operate in a common DNA damage response pathway. Cancer Res 59, 1752s-1756s (1999)), and its expression also positively correlates with tumor grade in breast cancer (Maacke, H. et al. Over-expression of wild-type Rad51 correlates with histological grading of invasive ductal breast cancer. Int J Cancer 88, 907-13 (2000)).

[0006] Citation of documents herein is not intended as an admission that any is pertinent prior art. All statements as to the date or representation as to the contents of documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of the documents.

SUMMARY OF THE INVENTION

[0007] The present invention relates to the identification and use of gene sequences identified as differentially expressed in breast cancer. The sequences of two of the genes display increased expression in non-normal (or abnormal) breast cells, such as those that would be identified as atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) by standard pathology techniques based upon cytological criteria. The sequences of the third gene display decreased expression the same non-normal cells.

[0008] The first set of sequences found to be more highly expressed in non-normal breast cells are those of human cysteine-rich intestinal protein 1 (CRIP1 or hCRIP1, also known as human cysteine-rich heart protein or HCRHP). CRIP1 has been mapped to human chromosomal segment 7q11.23 (see Garcia-Barcelo et al. Genomics, 47(3):419-422, 1998).

[0009] The second set of sequences found to be more highly expressed in non-normal breast cells are those of the "hematological and neurological expressed sequence 1" (HN1 or Hn1). Murine HN1 has been identified as being expressed in mouse hemopoietic and brain tissues (see Tang et al. Mamm. Genome, 8:695-696, 1997).

[0010] The set of sequences found to be expressed at lower levels in non-normal breast cells are those expressed with a "second epithelium restricted Ets transcription factor" termed ESE-2 (see Oettgen et al., J. Biol. Chem., 274(41):29439-52, 1999). The coding region sequence of ESE-2b is identical to that of the E74 like factor 5, termed ELF5 (see Zhou et al., Oncogene, 17(21):2719-32, 1998).

[0011] The identified sequences may thus be used in methods of detecting the presence of non-normal breast cells in a tissue or cell containing sample from a subject. The presence of non-normal breast cells may also be used in methods of diagnosing the presence of breast cancer in a tissue or cell containing sample from a subject. A subject, from which a sample is taken, may be one afflicted with, or suspected of having, breast cancer.

[0012] The present invention provides a non-subjective means for detecting the presence of non-normal breast cells. This provides advantages over the use of histomorphological or cytological criteria in standard pathology techniques, which requires some level of interpretation by a pathologist trained in assessing the presence and/or progression of breast cancer. The expression levels of these sequences may also be used as a means to assay small, node negative tumors that are not readily assessed by conventional means.

[0013] The expression levels of the identified sequences may be used alone or in combination with other sequences capable of identifying the presence of non-normal cells or of various stages and/or grades of breast cancer. Preferably, the sequences of the invention are used alone or in combination with each other.

[0014] The present invention provides means for correlating a molecular expression phenotype with a physiological (cellular) stage or state of a non-normal or abnormal breast cell. This correlation provides a way to molecularly diagnose and/or monitor a cell's status in comparison to normal breast cell phenotypes as disclosed herein. Additional uses of the sequences are in the classification of cells and tissues; determination of diagnosis and/or prognosis. Use of the sequences to identify cells of a sample as non-normal or abnormal may also be used to determine the choice, or alteration, of therapy used to treat such cells in the subject from which the sample originated.

[0015] The ability to identify non-normal and abnormal breast cells is provided by the recognition of the relevancy of the level of expression of the identified sequences and not by the form of the assay used to determine the actual level of expression. An assay may utilize a means related to the expression level of the sequences disclosed herein as long as the assay reflects, quantitatively or qualitatively, expression of the sequence. Preferably, however, a quantitative assay means is preferred. Identifying features of the sequences include, but are not limited to, unique nucleic acid sequences used to encode (DNA), or express (RNA), the disclosed sequences or epitopes specific to, or activities of, proteins encoded by the sequences. Alternative means include detection of nucleic acid amplification as indicative of increased expression levels (CRIP1 and HN1 sequences) and nucleic acid inactivation, deletion, or methylation, as indicative of decreased expression levels (ESE-2 and ELF5 sequences). Stated differently, the invention may be practiced by assaying one or more aspect of the DNA template(s) underlying the expression of the disclosed sequence(s), of the RNA used as an intermediate to express the sequence(s), or of the proteinaceous product expressed by the sequence(s). As such, the detection of the amount of, stability of, or degradation (including rate) of, such DNA, RNA and proteinaceous molecules may be used in the practice of the invention.

[0016] The practice of the present invention is unaffected by the presence of minor mismatches between the disclosed sequences and those expressed by cells of a subject's sample. A non-limiting example of the existence of such mismatches are seen in cases of sequence polymorphisms between individuals of a species, such as individual human patients within Homo sapiens. Knowledge that expression of the disclosed sequences (and sequences that vary due to minor mismatches) is correlated with the presence of non-normal or abnormal breast cells and breast cancer is sufficient for the practice of the invention with an appropriate cell containing sample via an assay for expression.

[0017] In one aspect, the invention provides for the identification of the expression levels of the disclosed sequences by analysis of their expression in a sample containing breast cells. In one preferred embodiment, the sample contains single cells or homogenous cell populations which have been dissected away from, or otherwise isolated or purified from, contaminating cells beyond that possible by a simple biopsy. Multiple means for such analysis are available, including detection of expression within an assay for global, or near global, gene expression in a sample (e.g. as part of a gene expression profiling analysis such as on a microarray) or by specific detection, such as quantitative PCR or real time quantitative PCR.

[0018] Preferably, the sample is isolated via non-invasive means. The expression of the disclosed sequence(s) in the sample may be determined and compared to the expression of said sequence(s) in reference data of non-normal breast cells. Alternatively, the expression level may be compared to expression levels in normal cells, preferably from the same sample or subject.

[0019] When individual breast cells are isolated in the practice of the invention, one benefit is that contaminating, non-breast cells (such as infiltrating lymphocytes or other immune system cells) are not present to possibly affect detection of expression of the disclosed sequence(s). Such contamination is present where a biopsy is used to generate gene expression profiles.

[0020] While the present invention has been described mainly in the context of human breast cancer, it may be practiced in the context of breast cancer of an animal known to be potentially afflicted by breast cancer by use of the corresponding sequences of the animal. Preferred animals for the application of the present invention are mammals, particularly those important to agricultural applications (such as, but not limited to, cattle, sheep, horses, and other "farm animals") and for human companionship (such as, but not limited to, dogs and cats).

BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 shows log.sub.2 plots of the ratio of expression in ADH, DCIS, and IDC cells to normal cells for CRIP1 and ELF5 sequences. The horizontal line is at "0" such that the ratio is "1" and all points above the line represent increases in expression relative to normal breast cells while all points below the line represent decreases in expression relative to normal breast cells.

[0022] FIG. 2 shows the results of in situ hybridization with sense and anti-sense CRIP1 sequences to locate its expression at the cellular level in normal versus IDC cells of the same sample. CRIP1 signal localized to the epithelial cells, and its intensity was markedly increased in the IDC compartment of the same biopsy.

[0023] FIG. 3 shows the results of in situ hybridization with sense and anti-sense CRIP1 sequences to locate its expression at the cellular level in normal versus DCIS and IDC cells. CRIP1 signal again localized to the epithelial cells, and its intensity was markedly increased in non-normal cells.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0024] Definitions of terms as used herein:

[0025] A "sequence" or "gene sequence" as used herein is a nucleic acid molecule or polynucleotide composed of a discrete order of nucleotide bases. The term includes the ordering of bases that encodes a discrete product (i.e. "coding region"), whether RNA or proteinaceous in nature, as well as the ordered bases that precede or follow a "coding region". Non-limiting examples of the latter include 5' and 3' untranslated regions of a gene. It is appreciated that more than one polynucleotide may be capable of encoding a discrete product. It is also appreciated that alleles and polymorphisms of the disclosed sequences may exist and may be used in the practice of the invention to identify the expression level(s) of the disclosed sequences or the allele or polymorphism. Identification of an allele or polymorphism depends in part upon chromosomal location and ability to recombine during mitosis.

[0026] The terms "correlate" or "correlation" or equivalents thereof refer to an association between expression of one or more sequences and a physiologic state of a breast cell to the exclusion of one or more other states by use of the methods as described herein. The invention provides for the correlation between increases in CRIP1 and HN1 sequences and non-normal or abnormal breast cells. Similarly, the invention provides for the correlation between decreases in ESE-2/ELF5 sequences and non-normal or abnormal breast cells. Increases and decreases may be readily expressed in the form of a ratio between expression in a non-normal cell and a normal cell such that a ratio of one (1) indicates no difference while ratios of two (2) and one-half indicate twice as much, and half as much, expression in the non-normal cell versus the normal cell, respectively. Expression levels can be readily determined by quantitative methods as described below.

[0027] For example, increases in CRIP1 expression can be indicated by ratios of or about 1.1, of or about 1.2, of or about 1.3, of or about 1.4, of or about 1.5, of or about 1.6, of or about 1.7, of or about 1.8, of or about 1.9, of or about 2, of or about 2.5, of or about 3, of or about 3.5, of or about 4, of or about 4.5, of or about 5, of or about 5.5, of or about 6, of or about 6.5, of or about 7, of or about 7.5, of or about 8, of or about 8.5, of or about 9, of or about 9.5, of or about 10, of or about 15, of or about 20, of or about 30, of or about 40, of or about 50, of or about 60, of or about 70, of or about 80, of or about 90, of or about 100, of or about 150, of or about 200, of or about 300, of or about 400, of or about 500, of or about 600, of or about 700, of or about 800, of or about 900, or of or about 1000. A ratio of 2 is a 100% (or a two-fold) increase in expression. Similar ratios can be used with respect to increases in HN1 expression. Decreases in ESE-2/ELF5 expression can be indicaed by ratios of or about 0.9, of or about 0.8, of or about 0.7, of or about 0.6, of or about 0.5, of or about 0.4, of or about 0.3, of or about 0.2, of or about 0.1, of or about 0.05, of or about 0.01, of or about 0.005, of or about 0.001, of or about 0.0005, of or about 0.0001, of or about 0.00005, of or about 0.00001, of or about 0.000005, or of or about 0.000001. Non-limiting examples of such ratios are shown in FIG. 1.

[0028] A "polynucleotide" is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides linked by phosphodiester bonds and encompasses the strand of a given sequence as disclosed herein as well as the complementary strand of a given sequence. The term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA as well as analogs thereof comprising a non-phosphodiester backbone. It also includes known types of modifications including labels known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), as well as unmodified forms of the polynucleotide.

[0029] The term "amplify" is used in the broad sense to mean creating an amplification product can be made enzymatically with DNA or RNA polymerases. "Amplification," as used herein, generally refers to the process of producing multiple copies of a desired sequence, particularly those of a sample. "Amplification" may also be used in the context of DNA amplification wherein copies of coding sequences within the cellular genome are increased. "Multiple copies" mean at least 2 copies. A "copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence. Methods for amplifying mRNA are generally known in the art, and include reverse transcription PCR (RT-PCR) and those described in U.S. patent application Ser. No. 10/062,857 entitled "Nucleic Acid Amplification" filed on Oct. 25, 2001 as well as U.S. Provisional Patent Application Nos. 60/298,847 (filed Jun. 15, 2001) and 60/257,801 (filed Dec. 22, 2000), all of which are hereby incorporated by reference in their entireties as if fully set forth.

[0030] By corresponding is meant that a nucleic acid molecule shares a substantial amount of sequence identity with another nucleic acid molecule. Substantial amount means at least 95%, usually at least 98% and more usually at least 99%, and sequence identity is determined using the BLAST algorithm, as described in Altschul et al. (1990), J. Mol. Biol. 215:403-410 (using the published default setting, i.e. parameters w=4, t=17). Alternatively, RNA may be directly labeled as the corresponding cDNA by methods known in the art.

[0031] A "microarray" is a linear or two-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support such as, but not limited to, glass, plastic, or synthetic membrane. The density of the discrete regions on a microarray is determined by the total numbers of immobilized polynucleotides to be detected on the surface of a single solid phase support, preferably at least about 50/cm.sup.2, more preferably at least about 100/cm.sup.2, even more preferably at least about 500/cm.sup.2, but preferably below about 1,000/cm.sup.2. Preferably, the arrays contain less than about 500, about 1000, about 1500, about 2000, about 2500, or about 3000 immobilized polynucleotides in total. As used herein, a DNA microarray is an array of oligonucleotides or polynucleotides placed on a chip or other surfaces used to hybridize to amplified or cloned polynucleotides from a sample. Since the position of each particular group of primers in the array is known, the identities of a sample polynucleotides can be determined based on their binding to a particular position in the microarray.

[0032] Because the invention relies upon the identification of sequences that are over- or under-expressed, one embodiment of the invention involves determining expression by hybridization of mRNA, or an amplified or cloned version thereof, of a sample cell to a polynucleotide of a disclosed sequence. Preferred polynucleotides of this type contain at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, at least about 34, at least about 36, at least about 38, at least about 40, at least about 42, at least about 44, or at least about 46 consecutive bases of a sequence that is not found in other human sequences. The term "about" as used in the previous sentence refers to an increase or decrease of 1 from the stated numerical value. Longer polynucleotides may of course contain minor mismatches (e.g. via the presence of mutations) which do not affect hybridization to the nucleic acids of a sample. Such polynucleotides may be label to assist in their detection; alternatively, the nucleic acids to which such polynucleotides will hybridize may be labeled. Such polynucleotides may also be immobilized, such as by attachment to a solid support.

[0033] Even more preferred are polynucleotides of at least or about 50, at least or about 100, at least about or 150, at least or about 200, at least or about 250, at least or about 300, at least or about 350, at least or about 400, at least or about 450, or at least or about 500 consecutive bases of a sequence that is not found in other sequences in the human genome. The term "about" as used in the preceding sentence refers to an increase or decrease of 10% from the stated numerical value. The polynucleotides may of course contain minor mismatches which do not affect hybridization to the nucleic acids of a sample.

[0034] In another embodiment of the invention, all or part of a disclosed sequence may be amplified and detected by methods such as the polymerase chain reaction (PCR) and variations thereof, such as, but not limited to, quantitative PCR (QPCR), reverse transcription PCR (RT-PCR), and real-time PCR, optionally real-time RT-PCR. Such methods would utilize one or two primers that are complementary to portions of a disclosed sequence, where the primers are used to prime nucleic acid synthesis. The newly synthesized nucleic acids are optionally labeled and may be detected directly or by hybridization to a polynucleotide of the invention. The newly synthesized nucleic acids may be contacted with polynucleotides (containing sequences) of the invention under conditions which allow for their hybridization.

[0035] Alternatively, and in another embodiment of the invention, expression of a sequence may be determined by analysis of expressed protein encoded by said sequence in a cell sample of interest by use of one or more antibodies specific for one or more epitopes of the individual products (proteins) in said cell sample. Such antibodies are preferably labeled to permit their easy detection after binding to the gene product. In the case of a protein that may be found in the blood, serum or other bodily fluid, the assay may modified to use such materials in place of a breast cell containing sample.

[0036] The term "label" refers to a composition capable of producing a detectable signal indicative of the presence of the labeled molecule. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

[0037] The term "support" refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes and silane or silicate supports such as glass slides.

[0038] As used herein, a "breast tissue sample" or "breast cell sample" refers to a sample of breast tissue or fluid isolated from an individual, preferably suspected of being afflicted with, or at risk of developing, breast cancer. Such samples are primary isolates (in contrast to cultured cells) and may be collected by a non-invasive means, including, but not limited to, ductal lavage, fine needle aspiration, needle biopsy, the devices and methods described in U.S. Pat. No. 6,328,709, or another suitable means recognized in the art. Alternatively, the "sample" may be collected by an invasive method, including, but not limited to, surgical biopsy.

[0039] "Expression" and "gene expression" include transcription and/or translation of nucleic acid material, such as the sequences of the invention.

[0040] As used herein, the term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates.

[0041] Conditions that "allow" an event to occur or conditions that are "suitable" for an event to occur, such as hybridization, strand extension, and the like, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Such conditions, known in the art and described herein, depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions. These conditions also depend on what event is desired, such as hybridization, cleavage, strand extension or transcription.

[0042] Sequence "mutation," as used herein, refers to any sequence alteration in the sequence of a gene disclosed herein interest in comparison to a reference sequence. A sequence mutation includes single nucleotide changes, or alterations of more than one nucleotide in a sequence, due to mechanisms such as substitution, deletion or insertion. Single nucleotide polymorphism (SNP) is also a sequence mutation as used herein. Because the present invention is based on the relative level of sequence expression, mutations in non-coding regions of genes as disclosed herein may also be assayed in the practice of the invention. "Detection" includes any means of detecting, including direct and indirect detection of gene expression and changes therein. For example, "detectably less" products may be observed directly or indirectly, and the term indicates any reduction (including the absence of detectable signal). Similarly, "detectably more" product means any increase, whether observed directly or indirectly.

[0043] Increases and decreases in expression of the disclosed sequences are defined in the following terms based upon percent or fold changes over expression in normal cells. Increases may be of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200% relative to expression levels in normal cells. Alternatively, fold increases may be of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 fold over expression levels in normal cells. Decreases may be of 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% relative to expression levels in normal cells.

[0044] Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[0045] Specific Embodiments

[0046] The present invention relates to the identification and use of three sets of sequences for the detection of non-normal and cancerous breast cells. The differential expression of these sequences in non-normal or abnormal breast cells relative to normal breast cells is used to identify a breast cancer cell as being non-normal or otherwise abnormal. The invention is advantageously used to identify breast cells as being those of ADH, DCIS, and IDC as otherwise determinable standard pathological techniques. The invention may also be applied to the identification of breast cells as being those of atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), and invasive lobular carcinoma (ILC) as otherwise determinable by standard pathological techniques.

[0047] Other non-limiting examples of non-normal or abnormal cells include malignant cells, atypical cells (including reactive and pre-neoplastic), neoplastic cells, tumor cells, and cancer or cancerous cells.

[0048] The sequences(s) identified by the present invention are expressed in correlation with non-normal breast cells, and thus negatively correlated with normal breast cells. For example, CRIP1, identified by I.M.A.G.E. Consortium CloneID 1323448 and cluster NM.sub.--001311 ("The I.M.A.G.E. Consortium: An Integrated Molecular Analysis of Genomes and their Expression," Lennon et al., 1996, Genomics 33:151-152; see also image.llnl.gov) has been found to be useful in discriminations between normal and ADH or DCIS or IDC breast cells.

[0049] In preferred embodiments of the invention, any sequence, or unique portion thereof, of the CRIP1 sequences identified by the cluster, as well as UniGene Homo sapiens cluster Hs.17409, maybe used. The consensus sequence of the I.M.A.G.E. Consortium cluster is as follows, with the assigned coding region (ending with a termination codon) underlined and preceded by the 5' untranslated and/or non-coding region and followed by the 3' untranslated and/or non-coding region:

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