US20010039016A1 - Use of expression profiling for identifying molecular markers useful for diagnosis of metastatic cancer - Google Patents

Use of expression profiling for identifying molecular markers useful for diagnosis of metastatic cancer Download PDF

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US20010039016A1
US20010039016A1 US09/819,248 US81924801A US2001039016A1 US 20010039016 A1 US20010039016 A1 US 20010039016A1 US 81924801 A US81924801 A US 81924801A US 2001039016 A1 US2001039016 A1 US 2001039016A1
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Scott Waldman
Jason Park
Stephanie Schulz
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Thomas Jefferson University
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Definitions

  • the present invention relates to methods to isolate molecular markers that may be used to detect metastasized tumor cells.
  • Cancer represents a significant worldwide health problem. Cancer is an uncontrolled growth and spread of cells. For many cancers, metastasis to adjacent or distant tissues results in physiologic impairment and often death. Early diagnosis and the ability to diagnosis metastasis of primary tumors represent significant challenges in the effective treatment of neoplastic disease.
  • Stage at diagnosis is the single most important prognostic determinant for patients with cancer and dictates the role of adjuvant chemotherapy in this disease. Given the prognostic and therapeutic importance of staging, accurate histopathologic evaluation of lymph nodes to detect invasion by cancer cells is crucial. Specific diagnosis of cancer metastasis is currently preformed by histologic and cytologic resemblance to normal tissue. Cancer cells frequently maintain their phenotypic characteristics of their normal cell of origin.
  • the present invention relates to a method for the isolation of tissue-specific molecular markers that are useful in the diagnosis of metastatic cancer.
  • One aspect of the invention is a method to identify molecular markers useful for detecting tumor cells that have metastasized from an origin tissue to a destination tissue or fluid.
  • the method comprises the steps of down-regulating in a population of origin tissue cells the activity of a transcription factor associated with terminal differentiation in the origin tissue, comparing an expression profile of the population of down-regulated origin cells with an expression profile of a population of control origin cells, identifying candidate markers which are expressed in the population of control origin cells but not the population of down-regulated origin cells, and comparing expression of the candidate markers in populations of control origin cells, cancerous origin cells and destination cells, wherein a candidate marker which is expressed in population of control origin cells and cancerous origin cells, but not the population of destination cells is a useful marker for the detection of cancer metastasized from the origin tissue to the destination tissue.
  • the method may comprise the additional step of isolating the molecular marker.
  • the method may also comprise the additional steps of identifying the transcription factor that binds to regulatory regions of a gene associated with terminal differentiation of the origin tissue.
  • FIG. 1 Functional characterization of deletion mutants of the human GC-C gene promoter. Deletion mutants of the GC-C gene 5′-flanking region were linked to luciferase and co-transfected with the Renilla luciferase control plasmid pRL-TK into intestinal (T84, Caco2) and extra-intestinal (HepG2, HeLa, HS766T) cell lines. Data are expressed as luciferase activity relative to the pGL3 Basic promoterless construct (Relative Activity). Each bar represents the mean ⁇ the standard error of at least 3 independent transfections performed in duplicate.
  • FIG. 2 DNAse I protection of the proximal human GC-C promoter. Footprinting reactions included the indicated mg quantities (NE) of HepG2 or T84 nuclear extract and the ⁇ 46 to ⁇ 257 promoter fragment labeled at the 5′-end of the coding strand. A control digestion contained 60 mg of bovine serum albumin (BSA). Protected bases were identified by a Maxam-Gilbert sequencing reaction (G+A) of the labeled fragment. The sequence of FP1 is given. Arrowhead indicates DNAse I hypersensitivity site at base ⁇ 163.
  • BSA bovine serum albumin
  • FIG. 3 Regulation of reporter gene expression by intestine-specific protected elements. FP1 and FP3 were deleted from the ⁇ 835 luciferase construct by in vitro mutagenesis, and wild-type and deletion constructs were expressed in HepG2 and T84 cells. Results are expressed as luciferase activity relative to a promoterless construct and represent the mean ⁇ the standard error of 3 independent transfections performed in duplicate.
  • FIG. 4 Intestinal specificity of FP1 probe EMSA. Nuclear extracts from intestinal or extra-intestinal cells, or BSA (10 mg), were incubated with labeled FP1 for 30 min. at room temp prior to separation on a non-denaturing 6% polyacrylamide gel.
  • FIG. 5 Cdx2 binding element FP1 is required for GC-C reporter gene activation. Putative binding sites for Cdx2 and HNF-4a are indicated on the ⁇ 835 construct. T84 and HepG2 cells were transfected with the ⁇ 835 reporter construct from which FP1 was deleted, or that construct containing the ‘CCC’ mutation. Results are expressed as (luciferase activity of mutant construct, luciferase activity of wildtype construct) ⁇ 100, and represent the mean ⁇ the standard error of 3 independent transfections performed in duplicate.
  • the values expressed as relative luciferase activities are, respectively, (wildtype; FP1 deletion; ‘CCC’ mutation): T84 (16.2 ⁇ 2.7; 1.9 ⁇ 0.3; 2.3 ⁇ 0.1) and HepG2 (2.1 ⁇ 0.1; 2.9 ⁇ 0.3; 2.2 ⁇ 0.1).
  • the present invention relates to methods to identify and characterize molecular markers useful for detecting metastasized tumor cells.
  • molecule markers used to detect tumor cells are transcripts or proteins specifically expressed as a result of the hyperproliferative state of the cell.
  • the molecular markers that are identified and characterized by the method of the present invention are specifically expressed in terminally differentiated tissues and are not specific to tumor cells. Tumor cells continue to express the genes associated with terminal differentiation of their tissue of origin.
  • the transcripts and proteins of these genes are ideally suited to detect tumor cells that have metastasized to a destination tissue, such as a lymph node, because the origin tissue specific markers will be out of place in the destination tissue. Because these molecular markers are specific to the origin tissue and not a particular tumor, they will broadly recognize many tumors metastasized from the origin tissue.
  • the method for identifying molecular markers useful for detecting metastasized tumor cells identifies “candidate” tissue-specific molecule markers and determines which of these candidate markers are suitable for the detection of metastatic cancer.
  • Tissue-specific markers associated with the terminal differentiation of a desired origin tissue are characterized by down-regulating the activity of a transcription factor associated with terminal differentiation of origin tissue, comparing the expression profiles of the down-regulated origin tissue with unaltered control origin tissue, and identifying transcripts or proteins that are candidate tissue-specific markers by virtue of their expression being up- or down-regulated in conjunction with the down-regulation of the transcription factor.
  • the expression of the candidate tissue-specific markers are compared in the control origin tissue, tumors derived from the origin tissue, and destination tissues of interest for biopsy.
  • Candidate markers that are expressed in control origin tissue and tumors, but not destination tissue are useful markers for detecting metastatic tumor cells.
  • terminal differentiation refers to a differentiation state of a cell or tissue from which no further differentiation can occur.
  • metastasize refers to the process whereby cancer cells break loose from a tumor mass and form secondary tumors or metastases at other sites of the body.
  • colorectal tract refers to the tissues and organs than comprise the large intestine to and including the rectum. These tissues and organs comprise, but are not limited to, the terminal ileum and ileocecal valve, the cecum, the ascending, transverse, descending and sigmoid colon, the rectum and the anal sphincters.
  • the origin tissue of the invention is any terminally differentiated tissue of the body in which tumor cells first arise.
  • “arise” it is meant to confer to cells the hyperproliferative phenotype associated with tumor cells.
  • the origin tissue is preferably a tissue from which cancer cells are most likely to metastasize.
  • the tissue is mammalian, and in a most preferred embodiment, the tissue is human.
  • the origin tissue includes, but is not limited to, colorectal, intestine, stomach, liver, mouth, esophagus, throat, thyroid, skin, brain, kidney, pancreas, breast, cervix, ovary, uterus, testicle, prostate, bone, muscle, bladder and lung.
  • the cell lines of particular interest represent terminally differentiated cells of the origin tissue, including embryonic tissue cell lines and immortalized cell lines (Yeager and Reddel, 1999, Curr. Opin. Biotechnology 10:465-469).
  • Cell lines of particular interest include, but are not limited to, T84, Caco2, HT29, SW480, SW620, NCI H508, SW1116, SW1463, Hep G2, HS766T, and HeLa cells.
  • These and additional cell lines of origin tissue may be obtained from the American Type Culture Collection (Manassas, Va.), as well as from commercial sources.
  • Cancerous origin tissues are isolated from tumors that arise in the origin tissue. Cancerous cells may be obtained by removing tumors from patients. Established populations of tumor tissue, i.e. cell lines of tumor cells, can be used to advantage in the method of the invention. Cancer cell lines of interest include, but are not limited to, T84, Caco2, HT29, SW480, SW620, NCI H508, SW1116, SW1463, Hep G2, HS766T, and HeLa cells. These cell lines and other useful cell lines may be obtained from the American Type Culture Collection (Manassas Va.), as well as from commercial sources.
  • the destination tissue of the invention is any tissue or bodily fluid that may be biopsied to detect metastasized tumor cells.
  • tissue of the body are well known to those in the art for their propensity to accumulate metastasized tumor cells, and these tissues are preferred for the destination tissue.
  • the destination tissue may be any tissue of the body. Destination tissues of particular interest include, but are not limited to, lymph node, blood, cerebral spinal fluid, and bone marrow. Additional cell lines for origin tissue cells may be obtained from the American Type Culture Collection (Manassas, Va.), as well as from commercial sources. Preferably, biopsy or resected tissue is used as the destination tissue.
  • the transcription factors used in the method of the invention are transcription factors that are associated with terminal differentiation of the origin tissue. Many such transcription factors are already know to those skilled in the art.
  • the transcription factor is associated with the terminal differentiation of a preferred origin tissue.
  • the transcription factors include, but are not limited to, Cdx2 (intestine) (Mallo, G. V. et al., 1997 Int J Cancer 74:35-44; Genbank Accession No. BF591065), STAT5 (breast) (Hou, J. et al., 1995 Immunity 2:321-329; Genbank Accession No. L41142), NKX3.1 (prostate) (Genbank Accession No.
  • the method of the present invention may, in some embodiments, further comprise steps to identify a transcription factor gene associated with terminal differentiation. These additional steps comprise identifying the transcription factor that binds to the regulatory regions of a gene associated with terminal differentiation in the origin tissue.
  • electromobility shift assays and/ or supershift assays are used to characterize the transcription factor that binds to the regulatory region of a gene whose expression is associated with terminal differentiation.
  • Example 1 illustrates the characterization of transcription factor Cdx2 by its binding to the regulatory regions of the gene encoding the intestine-specific protein guanylyl cyclase C.
  • the activity of transcription factor associated with terminal differentiation is “down-regulated” in a population of origin tissue cells.
  • down-regulated it is meant that the activity of the transcription factor is reduced in the cell population as compared to a “normal” or control cell population.
  • a “cell population” refers to a cell culture, tissue culture, resected tissue or biopsy sample, or any group of cells from the desired tissue type.
  • a population of normal or control origin cells refers is a population of origin cells from the culture of origin tissue cells used for down-regulating the transcription factor, but without modification of the activity of the transcription factor.
  • the activity of the transcription factor may be down-regulated in cell populations by several means well known to those in the art.
  • the transcription factor gene is down regulated by site-directed mutagenesis of the coding or regulatory regions of the gene, or the transcription of an antisense gene constructed from the coding sequence of the transcription factor gene.
  • the activity of the transcription factor is blocked or inhibited by specific antibodies, DNA-binding molecules, or small molecules that interfere with the activity of the transcription factor by interfering with the assembly and/or initiation of the transcriptional complex.
  • Inhibitor polynucleotide molecules of interest include, but are not limited to, FP1, FP1B and SIFI (see Example 1).
  • the transcription factor may be down-regulated by activating a signaling event that inactivates the transcription factor, such as the addition of an extracellular ligand that initiates a cell-signaling event that phosphorylates and inactivates the transcription factor.
  • a signaling event such as the addition of an extracellular ligand that initiates a cell-signaling event that phosphorylates and inactivates the transcription factor.
  • the down-regulated origin cells are cdx2-null polyps.
  • Cdx2-null polyps can be resected from a mouse that is heterozygous for an inactive copy of the homeobox gene cdx2, which controls cell differentiation in the intestinal epithelium (Chawengsaksophak et al., 1997, Nature 386:84-87; Tamai et al., 1999, Cancer Res. 59:2965-2970; Beck et al., 1999, PNAS 96:7318-7323; incorporated by reference herein).
  • Cdx2 stimulates the markers of endocyte differentiation.
  • the method of the invention comprises the step of comparing the expression profile of the population of down-regulated origin cells with the expression profile of the population of control origin cells.
  • expression profile it is meant the array of nucleic acids or proteins that are expressed in a cell population. Most commonly, expression profiles are arrays of nucleic acid molecules, primarily mRNA molecules, that are found in the profiled cell population. Methods to compare RNA expression profiles are well known to those in the art. Some methods of particular interest include, but are not limited to, differential display (Welsh et al., 1992, Nucleic Acids Res. 20:4695-4970; Liang and Pardee, 1992, Science 257:967-970; Barnes, 1994, Proc. Natl. Acad. Sci.
  • kits for differential display may be purchased, such as the Delta® Differential Display Kit (Clontech, Palo Alto, Calif.), among others.
  • Commercial kits for subtractive hybridization may be purchased, such as Clontech PCR-Select® Subtraction (Clontech, Palo Alto, Calif.), among others.
  • Micro-arrays of popular cDNA populations may be purchased (Incyte Genomics, Inc, St. Louis. Mo.), or custom micro-arrays may be ordered from commercial sources (Radius Biosciences, Medfield Mass.; ProtoGene Laboratories, Inc., Menlo Park Calif.).
  • a preferred membrane-format microarray is LifeGridTM Sequence-Verified Gene Expression Array Kits (Incyte Pharmaceuticals, Inc., St. Louis, Mo.) and a preferred slide-format microarray is ®GEM® Gene Expression Microarray (Incyte Pharmaceuticals, Inc., St. Louis, Mo.).
  • Commercial kits for RAGE are available from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, Md.).
  • GeneTag® a proprietary technology developed by Celera Genomics (Rockville, Md.), may also be used to quantify gene expression in a profile of RNA transcripts.
  • Protein expression profiles may also be compared by methods that will be well known to those in the art. Methods of particular interest include, but are not limited to, 2-Dimensional Electrophoresis - Mass Spectroscopy (2DE-MS) (O'Farrell, 1975, J. Biol. Chem. 250: 4007-4021; Patterson and Aebersold, 1995, Electrophoresis 16: 1791-1814; Gygi et al., (2000) Curr. Opinion in Biotech. 11: 396-401; and refernces cited therein) and Isotope-Coded Affinity Tags (ICAT) (Gygi et al., 1999, Nature Biotech. 17: 994-999; Gygi et al., 2000, Curr. Opinion in Biotech. 11: 396-401; and references cited therein).
  • 2DE-MS 2-Dimensional Electrophoresis - Mass Spectroscopy
  • ICAT Isotope-Coded Affinity
  • Nucleic acid molecules or protein molecules of interest identified by the comparison of expression profiles may additionally be isolated using methods that will be well known to those skilled in the art. The isolation method chosen depends in many cases on the method used to compare the expression profiles, and the preferred method will often be described in the reference that describes the method of comparison (see aforementioned citations). For example, nucleic acid bands may be removed from a polyacrylamide gel, agarose gel or nitrocellulose, the nucleic acids eluted and cloned using techniques well known in the art (Ausubel et al. Current Protocols in Molecular Biology. New York: John Wiley & Sons, Inc., 2000).
  • the method of the invention comprises the step of comparing the expression of the candidate markers in several kinds of cells.
  • There are many methods to compare the expression of single genes which will be well know to those in the art (Ausubel et al. Current Protocols in Molecular Biology. New York: John Wiley & Sons, Inc., 2000), including but not limited to, northern analysis, Southern analysis with cDNA, RNase protection assays, quantitative PCR, competitive PCR, 5′ nuclease assays (Lie and Petropoulos, 1998, Curr. Opin. Biotech. 9:43-48 and the references cited therein), western analysis, dot blot western, ELISA and other immunoassays, and immunohistochemistry.
  • the molecular markers identified by the method of the invention may be used to diagnose and stage cancer in mammalian patients, including following the development of recurrence of cancer after surgery and screening normal patients for the development of cancer.
  • the molecular markers utilized would be identified ideally from the same tissue that the patients cancer arose.
  • a selection of molecular markers isolated from different origin tissues is preferred.
  • the metastases may be diagnosed by any technique that will detect the nucleic acid or protein molecular marker. The sensitively of the technique will determine in part the size of metastasis that can be detected. Preferred techniques utilize PCR, ELISA, and the like.
  • Example 2 illustrates a particularly preferred method to diagnose metastasized cancer with the molecular markers of the method.
  • Tissue specific molecular markers can also be utilized to localize therapeutics to specific tissue and organ systems. This use is particularly appropriate for tissue-specific molecular markers that are localized on the surface of the tissue cells.
  • These therapeutics include, but are not limited to, chemotherapeutics, analgesics, antibiotics, anti-inflamatories, hormones and stimulants.
  • Protein molecular markers may be used to generate antibodies that may be used in diagnosis method and to localize therapeutics.
  • Polyclonal antibodies and monoclonal antibodies, and fragments thereof, and various conjugates of them can be made by methods well known in the art.
  • Cdx2 is a Transcription Factor Associated with the Intestinal-specific Expression of Guanylyl Cyclase C
  • GC-C guanylyl cyclase C
  • Genomic Library Screening and Sequencing The GC-C gene 5′ regulatory region was cloned from a KFIXII human genomic library (Stratagene, La Jolla Calif.). The library was screened by hybridization with a probe specific for exon 1 of the guanylyl cyclase C (GC-C) cDNA. A 2.8 kb Xbal fragment that included 2 kb upstream of the start site of transcription was subcloned into Bluescript KS (Stratagene). All constructs were generated from this Bluescript/human GC-C gene construct. The nucleic acid sequence of each construct was confirmed by BigDye terminator® reaction chemistry for sequence analysis on the Applied Biosystems Model 377 DNA sequencing systems (Perkin-Elmer, Norwalk CN; Applied Biosystems, Foster City Calif.).
  • Assays of reporter gene activity were conducted with cells plated in 6-well seeded at either 5.0 ⁇ 10 5 (T84, Caco2, and HeLa) or 1.0 ⁇ 10 6 cells per well (HepG2 and HS766T). Cells were incubated overnight, washed one time with PBS, and supplemented with fresh media before transfection.
  • Plasmids purified with the Qiafilter Kit (Qiagen, Valencia Calif.) were transfected into cells with the non-liposomal lipid transfection reagent Effectene® (Qiagen). All cell lines were co-transfected with both 0.4 mg of firefly luciferase experimental reporter constructs, modified from pGL3-Basic, and 0.1 mg of the Renilla luciferase control reporter, pRL-TK, driven by a viral thymidine kinase promoter (Promega). Cells were incubated with transfection complexes for 24 h, rinsed with PBS, then supplemented with appropriate media and incubated for a further 24 h.
  • Nuclear Protein Extraction Nuclear extracts were prepared essentially as previously described (Ausubel et al. Current Protocols in Molecular Biology. New York: John Wiley & Sons, Inc., 2000). Nuclear protein concentration was determined using Coomassie Protein Assay Reagent (Pierce, Rockford Ill.).
  • Electromobility Shift Assay Protein-DNA binding reactions performed in the same buffer as the DNase I protection assay (4% glycerol, 10 mM Tris-HCl (pH 7.5) 50 mM NaCl, 2.5 mM MgCl 2 and 5 mM DTT) included 1 mg of Poly(dI.dC)-Poly(dI.dC) (Amersham Pharmacia Biotech, Piscataway, N.J.) and 30 kcpm of probe. Reactions were initiated by the addition of nuclear extract and incubated for 30 min at room temp to produce protein complexes which were separated on a 6% non-denaturing, polyacrylamide (37.5:1) gel in 0.5 ⁇ TBE running buffer.
  • Oligonucleotide probes for EMSA were synthesized. Complementary oligonucleotides in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA were annealed in a Hybaid Thermal Cycler by a programmed ramp in temp from 95° C. to 25° C. over the course of 1 h. The single stranded sequences of the probes were:
  • FP1 5′ CAGCTAATCTCTCTGTTTATAGCTCTGACCTTTC 3′ (SEQ ID NO:3)
  • FP1B 5′ ATCTCTCTGTTTATAGCTCTGACCTTTCTGGGTGC 3′ (SEQ ID NO:4)
  • FP1-CCC 5′ CAGCTAATCTCTCTGCCCATAGCTCTGACCTTTC 3′ (SEQ ID NO:5)
  • SIF1 5′ GATCCGGCTGGTGAGGGTGCAATAAAACTTTATGAGTA 3′ (SEQ ID NO:6)
  • the membrane was rinsed for 5 min in EMSA binding buffer and hybridized with 20 ml of EMSA binding buffer with 100 kcpm/ml of labeled FP1 probe for 1 h at room temp. The membrane was then washed for 5 min each in three changes of EMSA binding buffer, dried and visualized by autoradiography.
  • DNAse I protection by intestine-specific nuclear protein binding to the 5′ regulatory region of GC-C revealed two regions ( ⁇ 75 to ⁇ 83, FP1; ⁇ 164 to ⁇ 178, FP3) which were protected only by nuclear extracts from intestinal cells (T84; FIG. 2). Regions ⁇ 104 to ⁇ 137 (FP2) and ⁇ 180 to ⁇ 217 (FP4) were protected by nuclear extracts from either intestinal (T84) or extra-intestinal (HepG2) cells, although the proximal and distal ends of FP2 exhibited different patterns of protection. These data suggest that the protected regions designated FP1 and FP3 were specific binding sites for nuclear proteins from intestinal cells. In addition, an intestine-specific site of open chromatin structure in the proximal 5 ′-flanking region of the GC-C gene was identified by a DNAse I hypersensitive site at base ⁇ 163 (FIG. 2).
  • FP1 contains the consensus binding site for the homeodomain protein Cdx2 (Quandt et al., Nucleic Acids Res 1995; 23:4878-84). Since Cdx2 is a transcription factor that directs intestine-specific expression of several genes, FP1 was more closely examined (Traber and Silberg, 1996, Annu Rev Physiol 58:275-97).
  • Specific complexes are formed by intestinal nuclear extract and FP1 probe.
  • the ability of the protected site FP1 to form intestine-specific complexes was determined by incubating an oligonucleotide probe with nuclear extracts prepared from T84, Caco2, HepG2, or HeLa cells. Indeed, several complexes were obtained by EMSA when the FP1 probe was incubated with nuclear extracts from those cells (FIG. 4). However, only one complex satisfied criteria for intestinal specificity, including formation by nuclear extracts from T84 and Caco2 cells, but not from HepG2 or HeLa cells.
  • Extracts from T84 and Caco2 cells also formed complexes with SIF1 that were identical to those obtained previously with that probe, demonstrating the integrity of the extracts (Suh et al., 1994, Mol Cell Biol 14:7340-51). All of the EMSA complexes formed with T84 nuclear extracts were competed with increasing amounts of unlabelled FP1 probe in a concentration-dependent manner. In contrast, an unlabelled competitor in which the Cdx2 binding site was specifically mutated (FP1-CCC probe, see Materials and Methods) did not compete against the intestine-specific complex.
  • Cdx2 binds specifically to the FP1 probe.
  • labeled FP1 was incubated with in vitro transcribed and translated murine Cdx2. This resulted in a complex whose mobility was identical to the intestine-specific complex formed by T84 nuclear extract.
  • labeled FP1-CCC did not form the intestine-specific complex with either Cdx2 or T84 nuclear extract.
  • An antibody against Cdx2 decreased the mobility of the specific complex formed between labeled FP1 and either T84 nuclear extract or in vitro transcribed and translated Cdx2.
  • an antibody against a related homeodomain transcription factor, Cdx1 did not alter the mobility of the intestine-specific complex.
  • FP1B which is highly homologous to FP1 probe, specifically bound to an intestine-specific protein of ⁇ 40 kDa in T84 and Caco2, but not HepG2, nuclear extracts.
  • FP1B probe bound to a ⁇ 131 kDa protein present in all cell lines examined.
  • anti-Cdx2 antibody recognized a protein doublet of ⁇ 40 kDa expressed in T84, but not in HepG2 or HeLa, cell nuclear extracts, a pattern which is characteristic of Cdx2 (James et al., 1994, J Biol Chem 269:15229-37).
  • the FP1 protected region binds to an intestine-specific factor of the same molecular weight and antigenic recognition as Cdx2.
  • Southwestern blots revealed that FP1 probe binds directly to Cdx2.
  • This example illustrates the use of a tissue-specific molecule marker to diagnose metastases.
  • Detection of GCC mRNA by RT-PCR enhances the accuracy of colorectal cancer staging.
  • the expression in lymph nodes of GCC mRNA a molecular marker for colorectal cancer cells in extraintestinal tissues, is associated with disease recurrence in patients with histologically negative nodes (stage II).
  • Expression of GCC mRNA reflects the presence of colorectal cancer micrometastases below the limit of detection by standard histopathology.
  • GCC-specific RT-PCR can reliably and reproducibly detect a single human colorectal cancer cell (T84 cells, ATCCC, Rockville, Md.) in 10 7 nucleated blood cells (Carrithers et al., 1996, Proc Natl Acad Sci USA, 93:14827-32).
  • GCC guanylyl cyclase family of receptors
  • GCC expression persists in intestinal cells that undergo neoplastic transformation to colorectal cancer cells. Examination of >300 surgical specimens demonstrated that GCC was specifically expressed by all primary and metastatic colorectal cancer cells, but not by any other extraintestinal tissues or tumors. GCC is identified only in lymph nodes from stage II patients who suffered recurrence ⁇ 3 y, but not in lymph nodes from patients without recurrent disease 6 y, following diagnosis.
  • RNA PCR kit ver.2 (Takara Shuzo Co., Ltd., Kyoto, Japan; Carrithers et al., 1996, Proc Natl Acad Sci USA 93:14827-32; Waldman et al., 1996, Dis Colon Rectum 41:1-6). Only total RNA that yielded amplicons following ⁇ -actin-specific RT-PCR was employed in studies outlined below.
  • RT-PCR GCC-specific and nested carcinoembryonic antigen-specific RT-PCR was performed as described previously (Carrithers et al., 1996, Proc Natl Acad Sci USA 93:14827-32; Waldman et al., 1996, Dis Colon Rectum 41:1-6; Morriss et al., 1998, New Engl J Med 1998;339:223-8). RT-PCR reactions were separated by electrophoresis on 4% NuSieve 3:1 agarose® (FMC Bioproducts, Rockland, Me.) and amplification products visualized by ethidium bromide.
  • Positive controls consisting of RNA isolated from human colorectal cancer cells expressing GCC and carcinoembryonic antigen (Caco2 cells; American Type Culture Collection, Rockville, Md.) and negative controls, consisting of incubations in which no template was added and RNA from lymph nodes devoid of colorectal cancer, were included. Amplicon identity was confirmed by sequencing. Production of GCC-specific amplicons was confirmed by Southern analysis, employing a 32 P-labeled antisense probe complimentary to a sequence internal to primers used for amplification (Kroczek, 1993, J Chromatog 618:133-145).
  • RT-PCR analysis of RNA expression in lymph nodes For the 28 patients in the control and case groups, a total of 524 (18.4 ⁇ 12.5 lymph nodes/patient) lymph nodes collected at surgery were reported free of tumor by histologic review. The number of lymph nodes obtained from each patient at the time of initial operative staging was similar between control (19.9 ⁇ 13.2) and case (17.2 ⁇ 12.7) groups. Twenty-one patients (75%) yielded 159 paraffin-embedded lymph nodes (7.6 ⁇ 5.2 lymph nodes/patient) that could be adequately evaluated by RT-PCR.
  • Lymph nodes omitted from RT-PCR analysis were not available from pathology (326 lymph nodes from 28 patients; 62.2% of 524 lymph nodes obtained at surgery) or did not yield RNA (39 lymph nodes from 7 patients; 7.4% of 524 lymph nodes obtained at surgery; 19.7% of 198 lymph nodes available for RT-PCR analysis).
  • the number of lymph nodes available for RT-PCR analysis was balanced between control (6.4 ⁇ 3.0) and case (8.1 ⁇ 6.3) groups.
  • ⁇ -Actin-specific amplicons (an indicator of intact RNA) were not detected in total RNA from pooled sections of lymph nodes of 5 (41.7%) patients from the case group and 2 (16.7%) patients from the control group and these patients were excluded from further analysis.
  • GCC-specific amplicons were detected in all reactions using RNA from lymph nodes of patients in the case group (Table 1). The presence of GCC-specific amplicons in these reactions was confirmed by sequencing and/or Southern analyses and suggests the presence of colorectal cancer micrometastases in lymph nodes of patients with recurrent disease. Of note, GCC mRNA was not expressed in any of 39 lymph nodes from 21 other patients without colorectal cancer (negative controls) that have been analyzed by RT-PCR to date. TABLE 1 GCC mRNA expression in lymph nodes and patient outcome.
  • Carcinoembryonic antigen is a glycoprotein expressed by ⁇ 60% of colorectal cancers and by other tumors, normal cells, and in some non-malignant pathological conditions.
  • RT-PCR analysis of carcinoembryonic antigen expression has been suggested to be a marker of colorectal cancer micrometastases in lymph nodes.
  • total RNA extracted from pooled lymph node sections was analyzed by RT-PCR using carcinoembryonic antigen-specific primers (Liefers et al., 1998, New Engl J Med 339:223-8).
  • Nested RT-PCR failed to yield CEA-specific amplicons in reactions using total RNA from patients in the control group, but detected carcinoembryonic antigen-specific amplicons in 1 patient in the case group. The presence of carcinoembryonic antigen-specific amplicons was confirmed by sequence analysis.

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