WO2013181430A2

WO2013181430A2 - Use of protein epitope of satb2 as an ihc marker for diagnosis and differential diagnosis of human gastrointestinal malignancies

Info

Publication number: WO2013181430A2
Application number: PCT/US2013/043422
Authority: WO
Inventors: Paul ARDI; George Yang; Gary CIMENT
Original assignee: Cell Marque Corporation
Priority date: 2012-05-30
Filing date: 2013-05-30
Publication date: 2013-12-05
Also published as: WO2013181430A3

Abstract

The present invention relates to the use of a protein epitope of SATB2 as a marker for diagnosis and differential diagnosis and treatment of human gastrointestinal malignancies, as well as towards SATB2 antibodies useful in the diagnosis and differential diagnosis and treatment of human gastrointestinal malignancies.

Description

USE OF PROTEIN EPITOPE OF SATB2 AS AN IHC MARKER FOR DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS OF HUMAN GASTROINTESTINAL MALIGNANCIES

[001] This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 61 /653,361, filed on May 30, 2012, which is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION

[002] The present invention relates to the use of a protein epitope of SATB2 as an immunohistochemistry (IHC) marker for diagnosis and differential diagnosis and treatment of human gastrointestinal malignancies, as well as to the use of SATB2 antibodies in the diagnosis and differential diagnosis and treatment of human gastrointestinal malignancies.

BACKGROUND OF THE INVENTION

[003] Cancer is one of the most common causes of disease and death in the world and predominantly arises from epithelial origin. Gastrointestinal (GI) carcinomas are common and include upper GI (esophageal carcinoma and gastric carcinoma), lower GI (colorectal carcinoma), hepatocellular carcinoma, and pancreatic ductal carcinoma. Colorectal carcinoma is one of the most common cancers and accounts for

approximately 1 million new cases per year in the world (O'Connell, JB, et al., J Natl Cancer Inst 2004;96: 1420-5). It is the third most common cancer in men and women. An estimated 145,000 cases of colorectal carcinoma occur each year, and more than 51 ,000 deaths result from colorectal carcinoma in the USA each year (American Cancer Society 2012). Therefore, early detection by morphologic examination on tissue sections and differential diagnosis by immunohistochemistry from the carcinomas of other tissue origin, such as upper GI, liver, pancreas, lung, breast, female reproductive tract, and prostate are very important in the establishment of an early treatment regimen.

[004] The accurate diagnosis of colorectal adenocarcinoma and its differential diagnosis from other GI malignancies, such as esophageal adenocarcinoma, gastric carcinoma, pancreatic carcinoma, and hepatocellular carcinoma and metastatic carcinoma from other organs are of paramount importance for patients to get appropriate treatment. Traditionally, cancer diagnosis has typically been performed by microscopic examination of a tissue sample. This technique generally involves the biopsy of a tissue sample from a tumor that is suspected to be malignant, preparing it via fixation and processing procedures, and then sectioning and staining the sample for observation under the microscope. A person trained in pathology then analyzes and evaluates the sample based on visual observation to determine whether the sample is malignant, and if so, what the grade of the tumor is. The grade of the tumor general ly refers to the likelihood that the tumor will grow and spread to other parts of the body. Grades range from well- differentiated, low-grade tumors, which grow and spread more slowly, to

undifferentiated, high-grade tumors, which grow and spread more rapidly and aggressivly. Accurate determination of tumor grade is very important to obtaining a proper prognosis and course of treatment for the patient from which the sample was obtained. However, because this process so heavily relies on subjective visual examination of the sample, the information obtained from this method can be limited and can potentially lead to problems in obtaining an accurate prognosis.

[005] Accordingly, it is now common to use im m noh i toc h em i s try (IHC) methods as a supplement to or in place of microscopic evaluation of tissue samples that are suspected to be malignant. IHC involves detecting certain antigens, typically proteins, in a given tissue sample through the use of antibodies that are known to specifically bind to those antigens. Because of the high level of specificity with which antibodies bind to a given antigen, IHC can provide very high accuracy with regard to characterizing the expression of the specified antigen in a tested tissue sample. Further, the antibodies are typically labeled (e.g.. with enzymes or fluorescent dyes) to aid in the visualization of the antigen within the tissue sample. IHC is thus able to determine the presence, quantity, localization, and other characteristics of a specified antigen in a tested sample. Because the protein expression of a given tissue sample will vary not only between healthy and malignant tissues, but also between different types of malignant tissues, obtaining a sample's protein expression via IHC is therefore an extremely important diagnostic tool. However, because of the nature of antibody-antigen binding, obtaining the greatest diagnostic and prognostic value from II IC will necessarily rely on identifying and testing for the proper antigen or antigens and using the proper antibodies that can provide the specificity required to do so.

[006] Currently, there exists a notable lack of specificity in the technologies described above and in similar technologies known in the art, especially when differentiating tumors within the intestinal system. Thus, there is a need in the art for methods with greater specificity for diagnosing and treating intestinal system tumors.

SUMMARY OF INVENTION [007] The present invention provides protein epitopes of human special- AT-rich sequence-binding protein 2 (SATB2). The epitopes comprise amino acid residues of the SATB2 protein, as identified below in SEQ ID NOs 1-5 :

SEQ ID NO: l : CL RDS PDR RSG SPD VK (residues #9-24).

SEQ ID NO:2: CV VEQ LDG SLE YDN REE HAH (residues #64-83).

SEQ ID NO:3: CQ EFG RWY KY KKI KVE RVE RE (residues #219-240).

SEQ ID N():4: CZ ERD VI Y EEE SRH HHS ERM QH (CZ plus residues #551-

570).

SEQ ID NO:5 : CZ EAE EEN ADK SKA APA EID QR (CZ plus residues #714- 733).

It has been discovered that these sequences can be used as markers to detect, diagnose, and differentially diagnose gastrointestinal malignancies, including colorectal carcinoma, and typically colorectal adenocarcinoma, with increased accuracy and specificity over technologies existing in the prior art. Furthermore, it has been discovered that detection and measurement of the expression of SATB2 in colonic mucosa and other tissues by immunohistochemistry (IHC) methods can provide improved diagnosis, differential diagnosis, prognosis and treatment of gastrointestinal malignancies, including colorectal carcinoma. Accordingly, the SATB2 sequences of the present invention may be used to generate antibodies or antibody fragments that selectively bind to one or more of these protein epitopes, or portions thereof, for use in IHC and other methods. Thus, the present invention also provides antibodies and antibody fragments for such use. The antibodies may be monoclonal or polyclonal and may also be labeled (e.g., with an enzyme, fluorophore, or other label known in the art). Furthermore, the present invention also provides methods that use one or more of the protein epitope sequences and/or antibodies or antibody fragments generated from those sequences to detect, diagnose, differentially diagnose, prognose, and treat gastrointestinal malignancies, including colorectal carcinoma.

[008] In one embodiment of the invention, a SATB2 peptide or polypeptide comprising one or more of SEQ ID NOs 1-5, or portions thereof, is provided. The peptide or polypeptide may be used as an immunogen or antigen.

[009] In another embodiment of the invention, a SATB2 peptide or polypeptide comprising one or more of residues #9-24, residues #64-83, residues #219-240, residues #551-570, and residues #714-733 of SATB2, or portions thereof, is provided. The peptide or polypeptide may be used as an immunogen or antigen. [0010] In another embodiment of the invention, antibodies may be provided that selectively bind to peptide sequences or epitopes comprising one or more of SEQ ID NOs 1-5, or portions thereof. Furthermore, antibody fragments that also exhibit such specific binding may also be provided. The antibodies and antibody fragments may be monoclonal or polyclonal, and may be labeled.

[0011] In another embodiment of the invention, antibodies or antibody fragments that selectively bind to peptide sequences or epitopes comprising one or more of SEQ ID NOs 1 -5, or portions thereof, but that do not bind to SATB l , may be provided. The antibodies may be monoclonal or polyclonal, and may be labeled.

[0012] In another embodiment of the invention, antibodies or antibody fragments may be provided that selectively bind to certain portions of human SATB2, including portions of SATB2 comprising residues #9-24, residues #64-83, residues #219-240, residues #551-570, and residues #714-733. The antibodies may be monoclonal or polyclonal, and may be labeled.

[0013] In another embodiment of the invention, antibodies or antibody fragments that selectively bind to peptide sequences or epitopes comprising one or more of residues #9-24, residues #64-83, residues #219-240, residues #551-570, and residues #714-733 of SATB2, or portions thereof, but that do not bind to SATB l, may be provided. The antibodies may be monoclonal or polyclonal, and may be labeled.

[0014] In another embodiment of the invention, a method for preparing antibodies is provided. The method may comprise an immunization step wherein an antigen or immunogen comprising one or more of SEQ ID NOs 1-5, residues #9-24, residues #64- 83, residues #219-240, residues #551-570, and residues #714-733 of SATB2 is used. The antibody may be a monoclonal or polyclonal antibody. Also, the antibody may also not be capable of binding to SATB 1.

[0015] In another embodiment of the invention, a method for diagnosing, differentially diagnosing, obtaining a prognosis for, or treating a gastrointestinal malignancy is provided. The method may comprise detecting a SATB2 marker, wherein the marker comprises an epitope sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. The marker may also comprise an epitope sequence selected from the group consisting of residues #9-24, residues #64-83, residues #219-240, residues #551-570, and residues #714-733 of SATB2. The marker may be detecting using immunohistochemistry methods. The marker may also be detecting using an antibody or antibody fragment. The antibody may be prepared using an antigen that consists essentially of an amino acid sequence selected from the group consisting of SEQ ID N(): l . SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID O: 5. The antibody may also be prepared using an antigen that consists essentially of an amino acid sequence selected from the group consisting of residues #9-24, residues #64-83, residues #219-240, residues #551-570, and residues #714-733 of SATB2. The antibody used to detect the marker may also not be capable of binding to SATB l . The gastrointestinal malignancy may be colorectal carcinoma or colorectal adenocarcinoma.

DETAILED DESCRIPTION

[0016] The inventors have discovered novel methods for diagnosing and treating tumors of the gastrointestinal tract. The inventors have also discovered certain protein epitopes of human SATB2 and the antibodies that bind to them, which may be used in the diagnostic and treatment methods. In several preferred embodiments, protein epitopes of human SATB2 that exhibit little or no homology with human protein SATBl are used in conjunction with IHC methods to obtain a diagnosis, and/or preferably a differential diagnosis, of colorectal carcinoma with greater accuracy over existing diagnostic methods.

[0017] Special-AT-rich sequence-binding protein 2 (SATB2) (SEQ ID NO:6) is a nuclear matrix protein. As is shown by SEQ ID NO:6, Human SATB2 is a 733 amino acid protein. It is encoded by the SATB2 gene, and is involved in nuclear gene expression and chromatin-loop remodeling (www.uniprot.org). It is thought to play an important role in palate formation, and defects in the SATB2 gene have been associated with cleft palates and mental retardation. SATB2 is expressed at varying levels in tissues in the brain, liver, kidney, and spinal cord. Expression of the protein is also observed in the colonic mucosa, and early studies indicate an association between SATB2 expression and the prognosis of colorectal carcinoma. In fact, using

immunohistochemistry (IHC) analysis, with anti-SATB2 antibody, of 146 colorectal tumor samples that displayed underexpression of SATB2, there was strong correlation with poor prognosis, tumor invasion, lymph node metastasis, distant metastasis, and dukes' classification for colorectal carcinoma (Wang, S., et al., J Pathology

2009;219: 1 14-122).

[0018] Furthermore, studies with anti-SATB2 antibody and immunohistochemical (IHC) methods (Yang, GG, et al., unpublished observation), as well as others

(Magnusson, K, et al., Am J Surg Patho! 201 1 ;35:937-948) indicate that by using anti- SATB2 antibody and immunohistochemical detection methodologies, SATB2 can be detected in colorectal carcinoma with high sensitivity (>85%) and specificity (95%) in detection. However, in gastrointestinal (Gl ) carcinomas, only colorectal carcinoma expressed this protein and none of the other GI carcinomas showed positive expression. A few cases of epithelial malignant neoplasms, such as upper Gl carcinomas, pancreatic carcinomas, and hepatocellular carcinomas are negative for SATB2 expression.

[0019] Special-AT-rich sequence-binding protein 1 (SATBl) (SEQ ID NO: 7) is also a nuclear matrix protein. As is shown by SEQ ID NO: 7, human SATB 1 is a 763 amino acid protein. Like SATB2, SATB l is thought to play a role in gene regulation and chromatin remodeling. Expression of SATB 1 has been observed in thymocytes, and has also been associated with breast cancer growth and metastasis. Importantly, SATB 1 is a close homologue of SATB2 (Patani et ah, The mRNA Expression of SATB l and SATB2 in Human Breast Cancer, Cancer Cell International 2009, 9: 18).

[0020] The present invention is based in part on the discovery that the novel transcription factor SATB2 is expressed in colonic mucosa and brain tissue. Moreover, SATB2 II IC detection, combined with other GI malignancy biomarkers. such as CDX2 and cytokeratin 20, is able to identify and separate colorectal carcinomas from upper GI carcinomas, and carcinomas of other tissue origin. The present invention is also based in part on the discovery that antibodies directed against SATB2 could also be used as diagnostic biomarkers and/or therapeutic biomarkers for early detection of epithelial cancers of the gastrointestinal tract, especially colorectal carcinoma.

[0021] As shown below, five amino acids sequences were selected and isolated based on the least homology between SATB2 and SATB l . The SATB2 residues included in each sequence are listed at the right. In certain embodiments, the sequences may also include additional amino acid residues that are in addition to the residues indicated. For example, SEQ ID NOs 4 and 5 both include a "CZ" portion (representing Cys-Glx) in addition to the residues indicated. Equivalent examples may include amino acids other than C and Z and amino acids that are located at the C-tcrminus and/or the N- terminus. These additional amino acids may be desirable, for example, to aid in the synthesis and/or binding of the peptide sequence.

SEQ ID NO: I : CL RDS PDR RSG SPD VK (residues #9-24).

SEQ ID NO:2: CV VEQ LDG SLE YDN REE HAE (residues #64-83).

SEQ ID NO:3 : CQ EFG RWY KKY KKI KVE RVE RE (residues #219-240). SEQ ID NO:4: CZ ERD VIY EEE SRH HHS ERM QH (CZ plus residues #551- 570).

SEQ ID N0:5: CZ EAE EE ADK SKA APA EID QR (CZ plus residues #714- 733).

[0022] SEQ ID NO:5 was employed as the immunogen to produce a monoclonal antibody for SATB2. It comprises a polypeptide located at the C-terminus of human SATB2, and specifically amino acids 714 to 733. Using immunohistochemistry methods, antibodies directed against the amino acid epitope comprising amino acids 714 to 733 of SATB2 demonstrated that most tumors in other organ systems are not expressing the SATB2 antigen.

[0023] In some embodiments the present invention provides antibodies that bind to SATB2 but do not bind to SATB 1. In some embodiments, the antibodies bind to the C- terminal of SATB2 but do not bind to SATB ! . This differentiation is important because of the homology exhibited by SATB 1 and SATB2, and the inventors have made the surprising discovery that by utilizing protein epitopes of SATB2 that exhibit little or no homology with SATB 1 , more accurate diagnoses, differential diagnoses, and prognoses of colorectal cancer are possible.

[0024] In some embodiments, the present invention provides antibodies that bind to one or more epitopes from SEQ ID NOs 1-5, or portions of these sequences.

Monoclonal or polyclonal antibodies may be used, and may be produced using antigens based on one or more of SEQ ID NOs 1 -5 or on the SATB2 residue corresponding to those sequences. The antibodies can be created using routine methods known in the art. Generally, these methods involve immunization of an organism, wherein the target antigen is introduced into an animal to evoke high expression of antibodies that bind specifically to the antigen. Polyclonal antibodies may be collected directly from serum. Monoclonal antibodies that bind to the epitopes may be produced, for example, by known methods involving hybridomas created by fusing antibody-producing cells with immortal myeloma cells. Monoclonal antibodies obtained in this manner may be produced indefinitely in a laboratory. These methods also may typically require known purification and characterization steps in order to obtain the most usable antibodies.

[0025] In some embodiments, fragments of antibodies, such as Fab and F(ab')2 fragments, may be used in addition to, or in place, of whole antibodies. The antibody fragments may bind to one or more protein epitopes of SEQ ID NOs 1 -5 or the SATB2 residues corresponding to those sequences, or portions of any of these sequences. Antibody fragments may be obtained from methods that are routine and well-known in the art. Generally, these methods typically may involve cleaving whole antibodies and removing portions of the antibody that are not necessary for antigen binding.

[0026] In some embodiments, the antibodies or antibody fragments may be labeled. When antibodies are used as probes, labeling the antibodies provides a means for detecting the antibodies. This, in turn, allows for detection of the antigen that is bound to the antibody. Antibodies may be labeled according to methods that are routine and well- known in the art. Typical labels may include enzymes, lluorophores, fluorescent dyes, radioactive labels, and other labels known in the art that aid in visualizing the antibody- antigen interaction.

[0027] In some embodiments, the present invention provides antibodies or antibody fragments that can compete for at least 50%, 60%, 70%, 80%, 90% or 100% of the binding between antibodies or antibody fragments against one or more epitopes from SEQ ID NOs 1-5, and any peptide comprising one or more sequences of SEQ ID NOs 1- 5.

[0028] In some embodiments, the present invention provides antibodies or antibody fragments that bind to one or more epitopes from the following SATB2 regions or bind to part or whole of the following regions: residues #9-24, residues #64-83, residues #219-240, residues #551-570, or residues #714-733. In some embodiments, the antibody or antibody fragment binds to one or more epitopes from residues #714-733 or part or whole region of #714-733. In some embodiments, the antibodies or antibody fragments bind to one or more epitopes from residues #714-733 and bind to part or whole of SATB2 but do not bind to SATB 1.

[0029] In some embodiments, the present invention provides peptides for use as immunogens for the preparation of SATB2 antibodies, including peptides comprising one or more of SEQ ID NOs 1-5, as well as residues #9-24, residues #64-83, residues #219-240, residues #55 1 -570, or residues #714-733 of SATB2 (SEQ ID NO:6). In some embodiments, the present invention provides for immunogenic fragments of these sequences for use in the preparation of SATB2 antibodies.

[0030] In some embodiments, the present invention provides antibodies useful in diagnosis and differential diagnosis and treatment of human gastrointestinal

malignancies, such as for example colon cancer and colorectal malignancies. [0031] In some embodiments, the antibodies of the present invention ca be employed to differentiate colorectal carcinomas from upper GI carcinomas as well as from carcinomas of other tissue origins.

[0032] In some embodiments the present invention provides a method of using one or more protein epitopes from certain SATB2 regions, e.g., a SATB2 region comprising amino acid residues 9-24, 64-83, 219-240. 551-570, and/or 714-733 as a marker for diagnosis and differential diagnosis of human gastrointestinal malignancies. In some embodiments the present invention provides a method of using a protein epitope from SATB2 comprising amino acids 714-733 as a marker for diagnosis and differential diagnosis of human gastrointestinal malignanc ies. The marker can be detected by any means known in the art. In some embodiments, the marker is detected by

immunohistochemistry.

[0033] In some embodiments the present invention provides a method of using one or more protein epitopes from certain SATB2 regions, e.g., a SATB2 region comprising amino acids 9-24, 64-83, 219-240, 551-570, and/or 714-733 for the preparation of antibodies for use in the treatment of human gastrointestinal malignancies. In some embodiments the present invention provides a method of using a protein epitope from a SATB2 region comprising amino acids 714-733 for the preparation of antibodies for use in the treatment of human gastrointestinal malignancies.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1:

[0034] The DNA sequences and protein sequences of SATB2 were compared between human beings and other species for homologic sequences in human SATB2. SATB2 and SATB 1 were also compared since the latter is expressed in breast epithelial cells and possibly could be detected in breast cancer if there were a homologic sequence in SATB2 as an immunogen for the generation of antibody.

[0035] As described above, five amino acids sequences were selected based on the least homology between SATB2 and SATB l . SEQ ID NO:5 was employed as the immunogen to produce monoclonal antibody for SATB2.

SEQ ID NO:5 : CZ EAE EEN ADK SKA APA EID QR (CZ plus residues #714- 733). [0036] The polypeptide of SEQ ID NO:5 includes amino acids 714-733

( A EF. EN ADKSK A A PAEIDQR) of the C -terminus of SATB2 protein, in addition to amino acid residues C (Cys) and Z (Glx). This polypeptide was selected as an immunogen for immunizing mouse to generate monoclonal antibody against SATB2. The sequence is a polypeptide located at the C -term inns, including amino acids 714 to 733. The resultant monoclonal antibody directed against SATB2 C-terminus was produced. The antibody was tested using immunohistoehemislry methods on whole section and tissue microarray spec imens of colorectal adenocarcinoma and normal colon tissue.

[0037] Cases of colorectal adenocarcinoma were tested using mouse monoclonal anti-SA ΓΒ2 and immunohistochemical methods performed using a Ventana Ultra IHC automated IHC stainer.

[0038] SATB2 was expressed in normal colonic mucosal epithelial cells in the nuclei. S ATB2 was positive in eighteen of twenty-one cases of colorectal carcinoma with a sensitivity of 85%. Two cases of gastric adenocarcinoma and three cases of esophageal adenocarcinoma did not express SATB2 antigen in tumor cells. Normal mucosal epithelial cells in the upper gastrointestinal tract did not show expression of SATB2. A case of pancreatic ductal carcinoma did not display nuclear staining in tumor cells as well as adjacent normal ducts and acini. Three hepatocellular carcinoma cases demonstrated no staining by anti-SATB2 and normal liver tissue is completely negative for the antigen.

[0039] Using i mmunohistoch em i stry methods, antibody directed against the epitope of amino acids 714 to 733 of SATB2 can recognize tumor cells of colorectal adenocarcinoma, but does not stain tumor cells of the upper GI tract, pancreas and liver. Antibodies directed against the epitope of SATB2 amino acid 714 to 733 by using immunohistochemistry methods demonstrated that most of tumors in other organ systems are not expressing the SATB2 antigen. This antibody, combined with accompanied immunohistochemistry qualitative methods, is a powerful tool to make an accurate diagnosis for human malignancies.

[0040] These data have demonstrated that this antibody is of high sensitivity in identification of colorectal adenocarcinoma and of high specificity in differentiating primary and secondary colorectal carcinoma from carcinomas of other origin, especially in separating upper Gl carcinoma from lower GI carcinoma. Example 2:

[0041] Monoclonal antibodies generated by using the peptide sequence of SEQ ID NO:5 was validated with an immunohistochemistry method. Tissue microarray and whole tissue sections were made for the antibody evaluation. A total of fifty cases of colorectal adenocarcinoma (CRC) were tested. Forty six of fifty cases have shown expression of SATB2 protein in neoplastic cells and four carcinomas were negative for SATB2 protein expression. The sensitivity for this monoclonal antibody was 92%. Of the forty six carcinomas, eleven neoplasms (22%) showed strong staining; twenty neoplasms (40%) displayed moderate expression; and 15 neoplasms (30%) presented weak labeling. Table 1 demonstrates the results of sensitivity and specificity testing of the monoclonal antibody. Tabic 2 reveals the staining intensity of the antibody in tumor cells. All SATB2-positive CRC cases showed SATB2 protein expression in more than 10% of tumor cells.

Table 1. Sensitivity and Specificity of SATB2 Monoclonal Antibody in Detection of Colorectal Adenocarcinoma and Carcinomas of Other Tissue Origins

Table 2. Immunohistochemistry Staining Intensity of SATB2 Monoclonal of Colorectal Adenocarcinoma.

4/50 (8%) 15/50 (30%) 20/50 (40%) 1 /50 (22%)

[0042] Upper gastrointestinal tract malignancies were also tested with this antibody by immunohistochemistry. Seven gastric adenocarcinomas were tested and carcinoma cells in one of the seven (1/7) cases were weakly highlighted by the antibody with approximately 25% of cancer cells being positive, in three cases of esophageal adenocarcinoma, tumor cells in one case were weakly highlighted in approximately 25% of tumor cells. Eight cases of pancreatic ductal carcinoma were studied and two of eight malignant tumors displayed SATB2 protein expression in approximately 10% of tumor cells. In nine cases of hepatocellular carcinoma studied, one of them demonstrated the protein expression in the nuclei of the carcinoma cells weakly. Table 1 shows the results.

[0043] We have also studied SATB2 protein expression in twenty cases of invasive ductal carcinoma of the breast. None of the breast carcinomas revealed SATB2 protein expression by the antibody (Table 1, Table 3).

Table 3. Sensitivity and Specificity of SATB2 Monoclonal Antibody in Detection of Lower Gastrointestinal Tract, Upper Gastrointestinal Tract and Non-Gastrointestinal tract Carcinomas

[0044] All publications discussed and cited herein are incorporated herein by reference in their entireties. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. [0045] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1 . An isolated SATB2 peptide or polypeptide consisting essentially of a sequence of amino acids selected from the group consisting of SF.Q ID NO: 1 , SEQ ID NO: 2, SEQ ID NO:3. SEQ I D NO:4, SEQ ID NO:5, residues #551 -570 of SEQ ID NO:6. and residues #714-733 of SEQ ID NO:6.

2. An antibody or fragment of said antibody that binds to an epitope sequence

consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: l , SEQ ID NO:2, SEQ ID N():3. SEQ ID NO:4. SEQ ID NO:5. residues #551 -570 of SEQ ID NO:6, and residues #714-733 of SEQ ID NO:6.

3. The antibody or fragment of said antibody of claim 2. wherein the antibody is a monoclonal antibody.

4. The antibody or fragment of said antibody of claim 2, wherein the antibody is an isolated polyclonal antibody.

5. The antibody or fragment of said antibody of claim 2, wherein the antibody or

fragment of said antibody is labeled with an enzyme.

6. The antibody or fragment of said antibody of claim 2, wherein the antibody or

fragment of said antibody is fluorescently labeled.

7. The antibody or fragment of said antibody of claim 2, wherein the antibody or

fragment of said antibody does not bind to SATB 1 .

8. A method of preparing an antibody, or fragment thereof, comprising an

immunization step wherein the peptide or polypeptide of claim 1 is used as an antigen.

9. The method of claim 8, wherein the antibody is a monoclonal antibody.

10. The method of claim 8, wherein the antibody is an isolated polyclonal antibody.

1 1. The method of claim 8, wherein the antibody does not bind to S ATB 1.

12. A method for diagnosing or differentially diagnosing a gastrointestinal malignancy comprising the detection of a SATB2 marker, wherein the marker consists essentially of an epitope sequence selected from the group consisting of SEQ ID NO:l , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5. residues #551-570 of SEQ ID NO:6, and residues #714-733 of SEQ ID NO:6.

13. The method of claim 12. wherein the marker is detected by immunohistochemistry.

14. The method of claim 12, wherein the gastrointestinal malignancy is colorectal

carcinoma.

15. The method of claim 12, wherein the marker is detected by an antibody, or fragment thereof.

16. The method of claim 15, wherein the antibody is prepared using an antigen that consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, residues

#551-570 of SEQ ID NO:6, and residues #714-733 of SEQ ID NO:6.

1 7. The method of claim 15, wherein the antibody does not bind to SATB1.