Patent Application
20050014165
The field of art of this disclosure concerns biomarkers for colorectal cancer (CRC). These biomarkers are useful for risk assessment, early detection, establishing prognosis, evaluation of intervention, recurrence of CRC, and discovery of therapeutic intervention, and methods of use thereof.
In the field of medicine, clinical procedures providing for the risk assessment and early detection of CRC have been long sought. Currently, CRC is the second leading cause of cancer-related deaths in the Western world. One picture that has clearly emerged through decades of research into CRC is that early detection is critical to enhanced survival rates.
The currently accepted methods for CRC screening include the fecal occult blood test (FOBT), x-ray using double contrast between barium enema and air (DCBE), sigmoidoscopy, and colonoscopy. Sigmoidoscopy is an invasive procedure that visually examines the lower third of the colon using a lighted, flexible endoscope, while a related method, colonoscopy, is a procedure that examines the entire colon. In both cases, biopsy samples can be taken during the procedure.
Concerning the accepted methods for screening, none clearly possess what is desired in a screening examination for CRC. While FOBT is rapid, it is a very general, and therefore a very non-specific screening method for CRC. Though DCBE has proven useful in specifically imaging abnormalities in the colon, the drawbacks of the DCBE method include: 1.) Patient discomfort in preparation of and during the examination, creating reluctance for compliance of DCBE as a screening method. 2.) Exposure of a patient to x-ray radiation, limiting DCBE in terms of frequency of use as a screening method. 3.) Research indicating that DCBE is more effective in detecting larger growths, which contraindicates its use for early detection. 4.) Biopsy samples cannot be taken during the procedure. 5.) Due to the cost involved, not all insurance providers pay for DCBE screening exams. Though sigmoidoscopy has gained favor from many physicians, the drawbacks of this method include: 1.) Patient discomfort in preparation of and during the examination, creating reluctance for compliance of sigmoidoscopy as a screening method. 2.) Due to the cost involved, not all insurance providers pay for sigmoidoscopy screening exams. 3.) Since only the lower third of the colon is inspected, there is a suggestion by studies that many significant lesions are in the proximal end of the colon, rendering sigmoidoscopy inadequate. Though colonoscopy addresses the issue of complete inspection of the colon, the drawbacks of colonoscopy as a screening method include: 1.) Creating even more patient discomfort than sigmoidoscopy, therefore generally requiring sedation, and thereby exacerbating the issue with patient compliance. 2.) Due to the cost involved, not all insurance providers pay for colonoscopy screening exams. 3.) There are risks of colonoscopy that include bleeding, and puncture of the lining of the colon.
Emerging spectroscopic technologies, such as magnetic resonance imaging and tomographic imaging each have drawbacks that are drawn from the list of drawbacks for the currently accepted screening methodologies.
Accordingly, there is a need in the art for approaches that have value in early detection and treatment of CRC that are cost effective, rapid, and minimally or noninvasive. Additional utility would be realized from an approach that would also serve as the basis for establishing prognosis, monitoring patient treatment, and detecting relapse, as well as the discovery of therapeutic intervention of CRC.
Still another sought after approach apart from currently accepted methods for screening for CRC, has been the search for biomarkers that have value in detection and treatment of CRC. For more than four decades, since the discovery of alpha-fetoprotein (AFP) and carcinogenic embryonic antigen (CEA), the search for biomarkers for cancer detection and treatment in general has been in a state of evolution. Biomarkers for cancer have five potential uses in the management of patient care. Ideally, they would be used for risk assessment, for early diagnosis, for establishing prognosis, for monitoring treatment, and for detecting relapse. Additionally, such markers could play a valuable role in developing therapeutic interventions.
It is further advantageous for the sampling methods used in conjunction with biomarker analysis to be minimally invasive or non-invasive. Examples of such sampling methods include serum, stool, swabs, and the like. Non-invasive and minimally invasive methods increase patient compliance, and generally reduce cost.
Clinically, the two criteria that are important for assessing the effectiveness of biomarkers are selectivity and sensitivity. Selectivity of a biomarker defined clinically refers to percentage of patients correctly diagnosed. Sensitivity of a biomarker in a clinical context is defined as the probability that the disease is detected at a curable stage. Ideally, biomarkers would have 100% clinical selectivity and 100% clinical sensitivity. To date, no single biomarker has been identified that has an acceptably high degree of selectivity and sensitivity required to be effective in for the broad range of needs in patient care management. However, from the clinical perspective, single serum biomarkers, such as AFP and CEA have proven to provide value in some aspects of patient care management.
For example, elevated serum levels of CEA were first discovered in 1965 in patients with adenocarcinoma of the colon. Elevated levels can be found in a variety of benign and malignant conditions other than colon cancer. Additionally, the production of CEA by early localized tumors of the colon is in the normal range. Therefore CEA lacks both the sensitivity and selectivity required to be of value for risk assessment or early diagnosis. Further, elevated levels of CEA correlate poorly with colon tumor differentiation and stage, rendering CEA as a biomarker for prognosis of colon cancer of limited value. The two areas for which CEA has proven helpful clinically in managing patient care are in evaluating the effectiveness of treatment, and for detecting relapse. Illustrative of this, numerous studies have found that there is high correlation between elevated serum levels of CEA preceding clinical detection of recurrence of colon cancer. This has proven to be of value in managing the care of high-risk patents with second-look surgical procedures based on rising levels of CEA.
Currently, investigations across numerous areas of oncology research, including CRC, ovarian, breast, and head and neck, are finding increased sensitivity and selectivity in panels of markers. It is now generally held that many mutations must take place before normal cell processes are altered, resulting in a disease, such as cancer. Still, given the complexity of biological systems, discovery of panels useful in providing value in patient care management for CRC is in the nascent stage.
To date, a greater understanding of the biology of CRC has been gained through the research on adenomous polyposis coli (APC), p53, and Ki-ras genes, as well as the corresponding proteins, and related pathways involved regulation thereof. However, there is a distinct difference between research on a specific a gene, its expression, protein product, and regulation, and understanding what genes are critical to include in a panel used to for the analysis of CRC that is useful in the management of patient care for the disease. To date, panels that have been suggested for CRC are comprised of specific point mutations of the APC, p53, and Ki-ras, as well as BAT-26, which is a gene that is a microstatelite instability marker.
What is disclosed herein is based on studies conducted in mouse multiple intestinal neoplasia (MIN) model, in which expressions levels of genes were screened in adenomous polyps. In the mouse MIN subjects, a chemically induced mutation of the APC gene is effected. The normal control is defined by littermates for which there was no aberration of the APC gene, and are therefore designated wildtype. From studies based on the mouse MIN model, candidate genes were selected for studying human subjects. From these human subject studies, a panel of biomarkers is disclosed herein. Further, what is disclosed are methods for measuring gene and protein expression levels based on the panel. Additionally, another aspect of what is disclosed are kits which provide the reagents and instructions for measuring gene and protein expression levels based on the panel. The panel, methods and kits are useful in the management of patient care for CRC. Additionally, the panel, methods and kits are believed useful as the basis for discovery of therapeutic interventions for CRC.
One embodiment of what is disclosed is a panel of biomarkers with the selectivity and sensitivity required for managing patient care for CRC. In Table 1, entries 1-22 are the polynucleotide coding sequences for a panel of biomarkers, and include the name and abbreviation of the gene. Entries 23-44 in Table 1 are the protein, or polypeptide, amino acid sequences that correspond to the coding sequences for entries 1-22. A biomarker, as defined by the National Institutes of Health (NIH) is a molecular indicator of a specific biological property; a biochemical feature or facet that can be used to measure the progress of disease or the effects of treatment. A panel of biomarkers is a selection of biomarkers. Biomarkers may be from a variety of classes of molecules. As previously mentioned, there is still a need for biomarkers for CRC having the selectivity and sensitivity required to be effective for all aspects of patient care management. Therefore, the selection of an effective set of biomarkers is differentiating in providing the basis for effective determination of CRC.
In another embodiment of this disclosure, expression levels of polynucleotides for the biomarkers indicated in SEQ ID NOs 1-22, are used in the determination of CRC. Such analysis of polynucleotide expression levels is frequently referred to in the art as gene expression profiling. In gene expression profiling, levels of mRNA in a sample are measured as a leading indicator of a biological state, in this case, as an indicator of CRC. One of the most common methods for analyzing gene expression profiling is to create multiple copies from mRNA in a biological sample using a process known as reverse transcription. In the process of reverse transcription, the mRNA from the sample is used to create copies of the corresponding DNA sequence from which the mRNA was originally transcribed. In the reverse transcription amplification process, copies of DNA are created without the regulatory regions in the gene known as introns. These multiple copies made from mRNA are therefore referred to as copy DNA, or cDNA. Entries 45-88 are the sets of primers used in the reverse transcription process for each gene listed in entries 1-22.
Since the reverse transcription procedure amplifies copies of cDNA proportional to the original level of mRNA in a sample, it has become a standard method that allows the analysis of even low levels of mRNA present in a biological sample. Genes may either be up regulated or down regulated in any particular biological state, and hence mRNA levels shift accordingly.
In still another embodiment of this disclosure, expression levels of proteins listed in SEQ ID NOs 23-44, which correspond to the genes indicated in SEQ ID NOs 1-22, are disclosed. The term “polypeptide” or “polypeptides” is used interchangeably with the term “protein” or “proteins” herein. As discussed previously, proteins have been long investigated for their potential as biomarkers, with limited success. There is value in protein biomarkers as complementary to polynucleotide biomarkers. Reasons for having the information provided by both types of biomarkers include the current observations that mRNA expression levels are not good predictors of protein expression levels, and that mRNA expression levels tell nothing of the post-translational modifications of proteins that are key to their biological activity. Therefore, in order to understand the expression levels of proteins, and their complete structure, the direct analysis of proteins is required.
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Methods and kits for the polynucleotide and polypeptide expression profiling for the panel of molecular markers are also contemplated as part of the present disclosure.
In one embodiment, a method for gene expression profiling comprises measuring cDNA levels for biomarkers selected in the claimed panel. Such a method requires the use of primers, enzymes, and other reagents for the preparation, detection, and quantitation of cDNAs. The method of creating cDNA from mRNA in a sample is referred to as the reverse transcriptase polymer chain reaction (RT-PCR). The primers listed in SEQ ID NOs 45-88 are particularly suited for use in gene expression profiling using RT-PCR based on the claimed panel. A series of primers were designed using Primer Express Software (Applied Biosystems, Foster City, Calif.). Specific candidates were chosen, and then tested to verify that only cDNA was amplified, and not contaminated by genomic DNA. The primers listed in SEQ ID NOs 45-88 were specifically designed, selected, and tested accordingly. In addition to the primers, reagents such as one including a dinucleotide triphosphate mixture having all four dinucleotide triphosphates (e.g. dATP, dGTP, dCTP, and dTTP), one having the reverse transcriptase enzyme, and one having a thermostable DNA polymerase are required for RT-PCR. Additionally buffers, inhibitors and activators are also required for the RT-PCR process. Once the cDNA has been sufficiently amplified to a specified end point, the cDNA sample must be prepared for detection and quantitation. Though a number of detection schemes are contemplated, as will be discussed in more detail below, one method contemplated for detection of polynucleotides is fluorescence spectroscopy, and therefore chromophores that are suited to fluorescence spectroscopy are desirable for labeling polynucleotides. One example of such a fluorescent label is SYBR Green, though numerous related chromophores exist, and are known in the art.
In another embodiment, a method for protein expression profiling comprises using an antibody panel based on the claimed panel of biomarkers for measuring targeted polypeptide levels from a biological sample. In one embodiment contemplated for the method, the antibodies for the panel are bound to a solid support. The method for protein expression profiling may use a second antibody having specificity to some portion of the bound polypeptide. Such a second antibody may be labeled with molecules useful for detection and quantitation of the bound polypeptides, and therefore in binding to the polypeptide label it for detection and quantitation. Additionally, other reagents are contemplated for labeling the bound polypeptides for detection and quantitation. Such reagents may either directly label the bound polypeptide or, analogous to a second antibody, may be a moiety with specificity for the bound polypeptide having labels. Examples of such moieties include but are not limited to small molecules such as cofactors, substrates, complexing agents, and the like, or large molecules, such as lectins, peptides, olionucleotides, and the like. Such moieties may be either naturally occurring or synthetic.
Examples of detection modes contemplated for the disclosed methods include, but are not limited to spectroscopic techniques, such as fluorescence and UV-Vis spectroscopy, scintillation counting, and mass spectroscopy. Complementary to these modes of detection, examples of labels for the purpose of detection and quantitation used in these methods include, but are not limited to chromophoric labels, scintillation labels, and mass labels. The expression levels of polynucleotides and polypeptides measured using these methods may be normalized to a control established for the purpose of the targeted determination. These methods are believed useful in providing determinations as the basis of effective management of patient care for CRC. These methods may also be used in the discovery of therapeutic interventions for CRC. Additionally, not only biopsy samples from sigmoidoscopy, colonoscopy, or surgery may be analyzed by these methods, but biological samples from non-invasive or minimally evasive collection methods are indicated for these methods, as well.
It is further contemplated in what is disclosed to provide kits having the reagents and procedures that facilitate the ready implementation of the methods, and provide consistency and quality control thereby.
In one embodiment, a kit for gene expression profiling comprises the reagents and instructions necessary for the gene expression profiling of the claimed panel. Thus, for example, the reagents may include primers, enzymes, and other reagents for the preparation, detection, and quantitation of cDNAs for the claimed panel of biomarkers. As discussed above, the method of creating cDNA from mRNA in a sample is referred to as the reverse transcriptase polymer chain reaction (RT-PCR). The primers listed in SEQ ID NOs 45-88 are particularly suited for use in gene expression profiling using RT-PCR based on the claimed panel. The primers listed in SEQ ID NOs 45-88 were specifically designed, selected, and tested accordingly. In addition to the primers, reagents such as one including a dinucleotide triphosphate mixture having all four dinucleotide triphosphates (e.g. dATP, dGTP, dCTP, and dTTP), one having the reverse transcriptase enzyme, and one having a thermostable DNA polymerase are required for RT-PCR. Additionally buffers, inhibitors and activators used for the RT-PCR process are suitable reagents for inclusion in the kit embodiment. Once the cDNA has been sufficiently amplified to a specified end point, the cDNA sample must be prepared for detection and quantitation. One method contemplated for detection of polynucleotides is fluorescence spectroscopy, and therefore chromophores that are suited to fluorescence spectroscopy are desirable for labeling polynucleotides and may also be included in reagents of the kit embodiment. Instructions included with the kit embodiment for gene expression profiling preferably teach the user the following steps: to obtain a biological sample; to isolate cellular RNA from the sample; to amplify copies of cDNA from the sample for each biomarker in the panel, and the panel for which the reagents are provided; and to quantify levels of cDNA amplified from the sample. Though tissue samples from a variety of procedures may be used, the instructions for obtaining a biological sample are preferably whereby the user obtains a sample of colorectal cells in a minimally invasive manner, such as by use of a swab or collection of a stool sample. The instructions may also preferably include the step of comparing the cDNA levels quantified to a control.
In another embodiment, a kit for protein expression profiling comprises the reagents and instructions necessary for protein expression profiling of the claimed panel. Thus, in this embodiment, the kit for protein expression profiling includes supplying an antibody panel based on the claimed panel of biomarkers for measuring targeted polypeptide levels from a biological sample. One embodiment contemplated for such a panel includes the antibody panel bound to a solid support. Additionally, the reagents included with the kit for protein expression profiling may use a second antibody having specificity to some portion of the bound polypeptide. Such a second antibody may be labeled with molecules useful for detection and quantitation of the bound polypeptides, and therefore in binding to the polypeptide label it for detection and quantitation. Additionally, other reagents are contemplated for labeling the bound polypeptides for detection and quantitation. Such reagents may either directly label the bound polypeptide or, analogous to a second antibody, may be a moiety with specificity for the bound polypeptide having labels. Examples of such moieties include but are not limited to small molecules such as cofactors, substrates, complexing agents, and the like, or large molecules, such as lectins, peptides, olionucleotides, and the like. Such moieties may be either naturally occurring or synthetic. Instructions for the protein expression profiling kit preferably teach the user: to obtain a biological sample; to use the antibody panel supplied with the kit for each biomarker in the panel to bind the polypeptides from the sample; and to quantify levels of polypeptides bound from the sample to the antibody panel. Preferably, the kit instructions also include a step of comparing the polypeptide levels to a control. Preferably the biological sample is obtained by a minimally invasive procedure such as use of a swab to through a stool sample.
Additionally, consumable labware required for sample collection, preparation, and analysis may be provided with the kits.
What has been disclosed herein has been provided for the purposes of illustration and description. It is not intended to be exhaustive or to limit what is disclosed to the precise forms described. Many modifications and variations will be apparent to the practitioner skilled in the art. What is disclosed was chosen and described in order to best explain the principles and practical application of the disclosed embodiments of the art described, thereby enabling others skilled in the art to understand the various embodiments and various modifications that are suited to the particular use contemplated. It is intended that the scope of what is disclosed be defined by the following claims and their equivalence.
This application claims priority to provisional application Ser. No. 60/488660, entitled, “Molecular Marker Panel for Determination of Colorectal Cancer”, which was filed on Jul. 18, 2003, and incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60488660 | Jul 2003 | US |
