HK1079795B - Plasma or serum marker and process for detection of cancer - Google Patents

Abstract

This disclosures provide, in one aspect, a method for detecting non-clinically diagnosed cancer in patient. In one embodiment, the method includes extracting blood serum or plasma from the patient, and then detecting beta-catenin RNA in the blood serum or plasma. In addition, in this embodiment, the method includes determining the presence of the cancer based on the detected beta-catenin RNA. In another aspect, this disclosure provides another embodiment of a method for detecting non-clinically diagnosed cancer in a patient. In this embodiment, the method includes extracting blood serum or plasma from the patient, and then detecting beta-catenin DNA in the blood serum or plasma. In addition, in this embodiment, the method includes determining the presence of the cancer based on the detected beta-catenin DNA. Related methods for detecting non-clinically diagnosed cancer in a patient comprising detecting beta-catenin-associated gene RNA, and beta-catenin-associated gene DNA, in the blood serum or plasma are also disclosed.

Description

Plasma or serum markers and methods for cancer detection

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional international patent application claims priority from U.S. provisional application serial No.60/392,191, filed on 28.6.2002, entitled "plasma or serum markers and methods for cancer detection", which is the same as the owner of the present application and is hereby incorporated by reference in any sense.

Technical Field

The present invention relates to a PCR-based method for detecting plasma or serum markers for cancer diagnosis, early detection, monitoring and mass screening, more specifically for the detection of beta-catenin RNA and DNA in plasma or serum of colorectal cancer.

Background

Colorectal cancer (CRC) is one of the most common malignancies in the world. The number of new cases of CRC has increased since 1975. More than 70% of CRC cases develop from sporadic adenomas or adenomatous polyps. Early detection and surgical removal of polyps is believed to be the most effective method to prevent benign polyps from developing into malignant tumors, thereby reducing mortality due to CRC.

Traditional screening methods for colorectal cancer include sigmoidoscopy, fecal occult blood test, colonoscopy and double-control barium enema. However, these conventional methods are subject to limitations and are invasive, costly, have low predictive value or result in low detection rates. For example, WO0142504 (the teachings of which are hereby incorporated by reference) discloses a multi-step reaction method for detecting extracellular tumor-associated nucleic acids in plasma or serum. Further improvements are needed.

Beta-catenin was originally identified by its interaction with cadherin. Recent evidence suggests that it functions as a transcription factor and plays a key role in the Wnt-signaling pathway (Willert & Nusse, 1998). The accumulation of cytoplasmic and nuclear β -catenin signaling has been shown to be closely associated with the generation of a wide variety of tumors (Morin, 1999).

It has been found that staining of nuclear β -catenin levels using immunohistochemistry is highly correlated with the presumed subsequent stages in colorectal tumorigenesis, with positive staining of 0% in normal tissues, polyps of 8%, adenomas of 92%, and carcinomas of 100%. It has also been found that the nuclear β -catenin signal appears to clearly distinguish polyps (non-adenomatous polyps) from adenomas (adenomatous polyps). This would be a useful marker for clinical diagnosis or early detection of CRC, where adenomas are considered as endpoints of risk factors. However, this diagnostic method based on the evaluation of nuclear β -catenin requires a colonoscopy procedure followed by surgical removal of the suspect tissue.

Thus, there is an urgent need for an effective, less invasive, more accurate test for early detection of cancer. The present invention satisfies this need.

Summary of The Invention

The present invention provides PCR (polymerase chain reaction) -based methods or procedures for detecting serum or plasma marker RNA and DNA associated with beta-catenin to provide an efficient, less invasive and more accurate test for diagnosis, early detection, monitoring and group screening of colorectal and other cancer types. It will be appreciated that this method of detecting beta-catenin RNA and DNA in serum is applicable to other plasma and serum RNA and DNA encoding beta-catenin-related proteins. In one embodiment, the RNA or DNA is from the gene-encoded beta-catenin, alpha-catenin, E-catherin, and other beta-catenin-related proteins.

The methods of the invention include the detection of serum or plasma RNA or/and DNA from humans or animals as a tool for the diagnosis, early detection, monitoring, treatment and mass screening of neoplastic diseases in different stages of progression and clinical stage. One advantage of the present invention resides in the non-invasive nature of the method, while a second advantage resides in the improved convenience and sensitivity of sample collection.

Details of various embodiments of the invention are set forth below. These embodiments are for illustrative purposes only and the principles of the invention may be practiced in other embodiments. Other features and advantages of the present invention will become apparent from the following description and examples.

Drawings

For a more complete understanding of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying drawings. It is emphasized that certain elements may not be illustrated for clarity of discussion. Reference will now be made in detail to the following description, taken in conjunction with the accompanying drawings, in which:

FIG. 1a, FIG. 1b and FIG. 1c illustrate the detection of beta-catenin RNA from plasma of colorectal cancer patients using RT-PCR.

FIGS. 2a and 2b illustrate the detection of colorectal adenomas using RT-PCR on blood beta-catenin RNA from patients.

FIG. 2c illustrates the detection of colorectal adenoma using RT-PCR on blood β -actin RNA from patients.

FIGS. 3a, 3b, 3c, 3d and 3e illustrate the detection of serum beta-catenin DNA from adenoma or carcinoma patients and normal controls.

Detailed Description

The present invention discloses the study of sensitive and specific biomarkers for early detection of colorectal cancer. Advanced understanding of the molecular mechanisms that explain colorectal cancer tumorigenesis has helped identify few oncogenes and tumor suppressors as potential clinical biomarkers for colorectal cancer formation and early detection. These include the k-ras, APC, p53, MCC, DCC genes. However, none of the candidate markers alone provides a satisfactory detection rate. The recent PCR-based detection of k-ras, APC and p53 mutations in blood samples from cancer patients does greatly improve the convenience of sample collection. However, the detection rate is generally lower than that observed for the primary tumor. For example, in 14 colorectal cancer patients in the study, 7 confirmed k-ras mutations, the same mutation is found in 6 patients serum. The seropositivity rate was 86% (Anker 1997). Another study showed that the seropositivity rates for loss of heterozygosity (LOH), microsatellite instability, k-ras and the p53 mutation were 0, 19 and 70%, respectively (Hibi 1998). Similar results have been obtained in other types of cancer, where the genetic changes seen in serum DNA (deoxyribonucleic acid) tend to be lower than those seen in primary tumors (Kopreski 2001; Sozzi 1999; von Knobuch 2001).

Compared with other related studies, the early detection of colorectal cancer by using serum beta-catenin DNA according to the invention can meet the criteria as an early detection marker: 1. the markers are differentially present in the blood of normal and pre-cancerous or tumor-bearing patients; 2. the method has the ability to detect adenomatous polyps as small as 4mm in diameter; 3. the method is simple and has high accuracy; 4. the required blood sample volume is small (2-5ml) and sample collection is by non-invasive normal blood drawing procedures. Thus, the present invention suggests that the levels of β -catenin DNA, together with β -catenin RNA, in serum or plasma can provide a solution to the quest for effective and accurate testing of colorectal cancer using pre-existing instrumentation and reagents, and thus be suitable for broad-scale mass screening, early detection, and disease monitoring of this growing common cancer.

Examples

The following examples are intended to illustrate, but not limit, embodiments of the invention described herein. In particular, in the following discussion, numerous specific details are set forth to provide a thorough understanding of the present disclosure. However, it will be understood by those skilled in the art that some of the techniques herein may be practiced without such specific details. In other instances, well-known elements or specific details have been reduced or omitted entirely because a detailed discussion of such features is not considered necessary to obtain a complete understanding of the present disclosure, and is considered to be within the understanding of persons of ordinary skill in the relevant art.

Example 1

^*Detection of beta-catenin RNA in all plasma samples from patients with colorectal cancer

To detect the presence of plasma β -catenin, two blood samples from cancer patients were subjected to RT-PCR (reverse transcription polymerase chain reaction) using primer #1 which would produce the exon 3 region of the 224bp gene. RNA samples extracted from carcinomas expressing high levels of β -catenin were included as positive controls. The results show that in both plasma and positive control RNA samples a 224bp band was produced when Reverse Transcriptase (RT) was present in the reaction, but no band was present in the absence of reverse transcriptase (FIG. 1 a). Other 10 plasma RNA samples were analyzed by RT-PCR using intron-bridged primers (primer #2, Table 1).

The data show that the 250bp fragment was clearly detected in all 10 patient plasma samples (FIG. 1a, lanes 1-10), suggesting the presence of beta-catenin RNA in the circulating blood of cancer patients. The data also demonstrate that the reaction is RT-dependent (FIG. 1b, lane 12). The genomic DNA sample was included as a positive control for the PCR reaction and a 450bp band appeared as expected (FIG. 1b, lane 11).

To demonstrate that the 250bp band is from RNA in plasma rather than DNA template, three remaining plasma RNA samples without DNase I pre-treatment were tested.

Two PCR products appeared on the gel: a 250bp band amplified from RNA, and a 450bp band amplified from DNA-contaminated plasma RNA extract. All triplicate samples gave bands of 250 and 450bp in the presence of RT (FIG. 1b, lanes 13-15), whereas RNase treated DNA samples gave a single 450bp band in the absence of RT (FIG. 1b, lane 1).

Cancer tests were performed on 15 patients using the three slightly different experimental settings described above, and the data showed that 15 of the 15 patients were clearly positive for plasma β -catenin.

Example 2

^*Plasma RNA is present at high rates in cancer patients, but not in healthy individuals

17 plasma samples from individuals with suspected adenomas were screened for beta-catenin RNA. Of these 17 plasma samples from individuals with suspected adenomas, 11 of them were plasma positive as indicated by the presence of 250bp RT-PCR products; 6 of the samples were found to be negative (FIG. 2a, lanes 1-11; FIG. 2b, lanes 1-6). The 6 negative samples were subjected to RT-PCR assays using primers specific for the β -actin sequence (Table 1, primer # 3). Beta-actin RNA was detected in all 6 plasma samples (FIG. 2c, lanes 1-6), indicating that these 6 plasma RNA extracts have amplifiable grade quality. Of the 6 patients with negative β -catenin signals (table 2, patients #10, 14 and 16), later biopsies confirmed that three were diagnosed with adenomas, two had granulation tissue, and the other had an expanded lymphatic space (table 2, patients # 1-3). The percentage of detection in adenoma patients was 79% (11 out of 14). 10 healthy subjects were analyzed by RT-PCR in parallel. Of the 10 healthy controls, 9 showed a negative plasma β -catenin signal, but all showed a positive β -actin RNA signal (fig. 2d and 2e, lanes 1-10). Only 1 of them had a rather weak positive signal (FIG. 2d, lane 10).

To summarize, the plasma of 32 patients confirmed to be carcinoma or adenoma was examined for the presence of β -catenin using RT-PCR analysis. The results showed that 100% (15 out of 15) of the cancer patients, 79% (11 out of 14) of the adenomas patients, and 10% (1 out of 10) of the healthy volunteers carried β -catenin RNA in their circulating blood. It is worth mentioning that apparently healthy subjects with trace amounts of plasma β -catenin RNA are chronically afflicted with colorectal discomfort, occasionally with hematochezia and diarrhea, although no abnormal or ulcerative colitis was detected in the endoscopy. Three patients with suspicious adenomas were also consented to plasma β -catenin testing. All three patients who subsequently were biopsied to confirm not to have adenomas were negative for plasma signals.

Free DNA has been shown to be present in circulating blood of patients with physiological disorders and cancer, and this DNA can be detected using PCR assays.

In addition, it has been reported that genetic changes in a specific gene sequence can be detected in the serum of cancer patients (Anker P1997; Hibi K1998; Kopreski MS 2001; Sozzi G1999; von Knobuch R2001). In addition to plasma DNA, sequence-specific RNA has been detected in cancerous, but not healthy, individuals using RT-PCR analysis (Kopreski MS 1999; Lo KW 1999; Chen XQ 2000). Whether PCR methods that detect plasma and serum DNA or RNA can be implemented for the diagnosis and prognosis of cancer will depend primarily on how well the data can validate the tumor status or even pre-cancerous lesions. For example, carcinoembryonic antigen (CEA) is widely expressed in a variety of cancers as well as in certain normal tissues including colon tissue. Together with the carbohydrate antigen 19-9(CA 19-9), these are the two most common tumor markers in the treatment of CRC patients. Generally, CEA labeling yields positive detection rates ranging from 40 to 60% by routine immunochemical determination of protein content. Serum CEA RNA detection using RT-PCR has improved detection rates from 35% to about 70% (Guadagni, 2001), another recent study demonstrated that tyrosine mRNA is present in the serum of 60% (4 out of 6) malignant melanoma patients, but not in any normal control serum (Kopreski 1999). In our current study, the positive rate of CRC detection was 100% for patients with carcinoma and 79% for patients with adenoma. Thus, plasma β -catenin RNA appears to be a potent serum marker for CRC detection.

In the eye, the only non-invasive screening method for CRC is the Fecal Occult Blood Test (FOBT). Several studies found that FOBT screening reduced mortality by 16% in both common and high risk patients. However, the limitation of this test is the low prediction rate (less than 20%). Another method used for CRC screening, especially for early detection of adenomas, is flexible sigmoidoscopy, which is said to reduce mortality by 70% in a few case-control studies (for review see Scotiniotis I, 1999). This test is sensitive and specific; however, it is invasive in nature. In this regard, RT-PCR based methods for serum β -catenin detection may indeed provide an ideal tool for CRC screening in both general and high risk individuals. The method can be used for monitoring postoperative and chemotherapy patients. Since β -catenin is also known to be associated with other types of cancer, our current invention for detecting serum or plasma β -catenin can be extended to the use for detecting, monitoring, screening for cancers with different tissue origins. This is the first report and suggests that the presence of plasma beta-catenin RNA is of diagnostic value.

Example 3

^*Immunochemical staining of nuclear beta-catenin signals in adenoma and carcinoma tissues

In more than 200 cases examined, 92% of adenomas and 100% of carcinomas, but none of the normal tissues showed elevated nuclear β -catenin. To determine the nuclear β -catenin signal of adenomas and carcinomas obtained from the patients from examples 1 and 2, paraffin-embedded tissue blocks of adenomas and carcinomas from 32 patients were sectioned and examined for nuclear β -catenin. Immunohistochemical staining was scored according to intensity and percentage of positive cells. Table 2 shows that nuclear translocation of β -catenin was observed in all tissue samples.

Example 4

^*Quantification of beta-catenin RNA in blood of healthy individuals and patients with adenomas or carcinomas using real-time RT-PCR

Quantitative differences in plasma β -catenin between adenomas and carcinoma patients were studied using real-time reverse transcription polymerase chain reaction (RT-PCR). The results indicate the average copy of the beta-catenin mRNACompared to normal individuals (n-14; mean 36; range from 0 to 169), adenoma patients (n-12; 3 negatives; 8 positives; mean 1.1 × 10)³(ii) a Ranging from 0.69 x 10³To 1.80X 10³) 30 times higher, and cancer patients (n ═ 18; mean 2.2X 10⁴(ii) a Ranging from 0.67 x 10⁴To 4.4X 10⁴) The height is 598 times higher. The beta-catenin mRNA copy number in cancer patients was 19 times higher than in adenoma patients. These quantitative analyses provide clear evidence: plasma beta-catenin mRNA is differentially present and can be used as a diagnostic tool to distinguish between healthy subjects, adenomas, and carcinoma patients.

Example 5

^*Detection of beta-catenin DNA in serum of patients with colorectal adenoma and carcinoma

PCR analysis was first performed using serum DNA samples extracted from patients with colorectal carcinoma. The results showed that a 359bp band was observed in all 15 serum DNA samples (FIG. 3a, lanes 1-16). 10 patients identified as having adenomas with mild to severe dysplasia were tested. Positive bands were detected in 9 out of 10 patients (FIG. 3b, lanes 1-11). The detection rate is 90%. The only negative case (FIG. 3b, lane 8) was amplifiable, since it gave a positive band of 156bp after amplification with the RET specific primer (FIG. 3d, lower part of the graph, lane 13). Control 10 healthy volunteers were also PCR amplified for beta-catenin. None of the serum samples showed a positive signal for beta-catenin, whereas positive signals were clearly detected using RET specific primers (FIG. 3c, lanes 1-10 and 1D, lanes 1-11). In addition, a known positive carcinoma serum sample was run in parallel and showed a typical 359bp band on agarose gel (FIG. 3c, lane 11). Lanes 12 of FIGS. 3c and 3d are negative controls for PCR reactions.

The data show for the first time that serum β -catenin DNA was detectable in all patients with colorectal carcinoma as well as in 9 out of 10 patients with colorectal adenoma, whereas all 10 healthy individuals did not contain serum β -catenin DNA. The result indicates bloodThe presence of β -catenin DNA was significantly correlated with the presence of cancer in the pre-neoplastic and malignant stages, which also suggested that circulating β -catenin originated from the adenoma or cancerous tissues of the patient. Of the 10 adenoma patients of this example, the serum beta-catenin negative individual (patient #9, Table 4) had the smallest adenoma (diameter 3.5mm, 48 mm)³). With penultimate small adenoma (63 mm)³) Shows amplifiable beta-catenin DNA in the blood of patients, suggesting that the sensitivity of the method will allow us to detect at least as little as 63mm³Before malignant adenomatous polyps. It is proposed to quantify the copy number of beta-catenin DNA in a sample using real-time PCR analysis. The findings indicate that measuring the level of β -catenin DNA in blood provides a highly sensitive and non-invasive method for the early detection of colorectal cancer. This approach can be extended to cancers of different tissue origin.

Referring now to the drawings, FIG. 1 shows generally the detection of beta-catenin RNA in the plasma of colorectal cancer patients using RT-PCR. More specifically, FIG. 1a shows RT-PCR amplification of beta-catenin using primers for the beta-catenin exon. Lanes 1-4, RT-PCR reactions of blood RNA samples isolated from two cancer patients in the presence (lanes 1 and 3) and absence (lanes 2 and 4) of RT enzyme; lane 5, mRNA extracted from a β -catenin expressing cancer sample as a positive control; lane 6, buffer control. M: and (3) RNA labeling. FIG. 1b shows RT-PCR amplification of beta-catenin using beta-catenin intron-bridged primer pairs. Lanes 1-10, dnasa-treated plasma RNA isolated from ten cancer patients; lane 11, genomic DNA as a positive control for PCR reaction; lane 12, buffer control. Lanes 13-17, samples from lanes 8-12, respectively, without prior treatment with dnase. FIG. 1c shows lanes 1-3, beta-catenin RNA (250bp) isolated from three patients without DNAse treatment by RT-PCR using intron-bridged primers; lane 4, positive DNA control; lane 5, negative buffer control. M: and (3) DNA marking.

FIG. 2 shows the detection of RNA from β -catenin (FIGS. 2a and 2b) and β -actin (FIG. 2c) in blood from a patient suspected of having a colorectal adenoma (FIGS. 2a-2c) by RT-PCR. A. Lanes 1-17, plasma RNA isolated from 17 patients; lane 18, positive DNA control; lane 19, negative control. Detection of blood β -catenin (FIG. 2d) and β -actin (FIG. 2e) RNA from plasma of 10 healthy subjects (lanes 1-10). Lane 11, positive DNA control, lane 12, negative buffer control.

FIG. 3 shows the detection of β -catenin DNA in patients with adenoma or carcinoma and normal control sera. FIG. 3a, FIG. 3b and FIG. 3c show PCR analysis of serum samples isolated from colorectal cancer patients using beta-catenin specific primers: FIG. 3a, lanes 1-15; analysis of patients with colorectal adenomas: FIG. 3b, lanes 1-10; analysis of healthy individuals: FIG. 3c, lanes 1-10. FIG. 3 d: the RET specific primers were used to perform PCR reactions on serum samples with negative beta-catenin signals. Lanes 1-10, serum samples from the same healthy individuals as shown in FIG. 3 c; figure 3d, lane 13: as shown in the graph of FIG. 3b, the same serum sample is shown in lane 8. Positive control genomic DNA isolated from carcinoma: FIG. 3a, lane 16; FIG. 3b, lane 11; FIG. 3c, lane 11; FIG. 3d, lane 11. Negative no-cell control: FIG. 3a, lane 17; FIG. 3b, lane 12; FIG. 3c, lane 12; FIG. 3d, lane 12. M: hae III lambda DNA labeling.

The applied technique is:

blood sample and RNA extraction

6-ml blood samples were collected from each patient via a percutaneous needle into 8-ml vacuum vessels (Vacutaners) containing lithium heparin ethylenediaminetetraacetate. Blood samples were centrifuged at 4800rpm for 8 minutes. Plasma was aliquoted into polypropylene tubes and stored at-80 ℃ for later use in RNA extraction. RNA was extracted from plasma samples using the TRIZOL kit (Life Technologies, USA) and then purified using RNeasy columns (Qiagen, Germany) according to the manufacturer's manual. Briefly, 2ml of each plasma sample was mixed with 1.6ml of TRIZOL and 0.4ml of chloroform, and centrifuged at 12,000rpm at 4 ℃ for 15 minutes. The aqueous phase was collected and RNA was extracted using RNeasy column. The isolated RNA was dissolved in 15. mu.l of DEPC-treated water. The RNA samples were further treated with PCR-grade dnase i (dnase i) ((life technologies)). In the reaction, 1. mu.l each of 10 XDase I reaction buffer and DNase I was added to 15. mu.l of RNA sample and incubated at room temperature for 15 minutes, followed by inactivation of DNase I by addition of 1. mu.l of 15mM EDTA and heating at 65 ℃ for 5 minutes, and then cooling on ice for the RT-PCR reaction.

Primers and RT-PCR reactions for blood RNA samples

The detection of plasma β -catenin was performed by RT-PCR assay from a primer set (table 1) including intron sequences spanning between exons 3 and 4 of the β -catenin gene. For comparison, an additional primer sequence set located within exon 3 of the β -catenin gene was also incorporated in some PCR reactions. The reverse transcription reaction was performed according to the manufacturer's instructions (Qiagen, Germany). PCR was performed using AmpliTaq Gold as polymerase (Perkin-Elmer Corp., Foster City, Calif.) using reagents provided in the GeneAmp DNA amplification kit. The parameters used for PCR were 40 cycles with an initial denaturation at 95 ℃ for 10 min, followed by 1 min 15 sec at 94 ℃,1 min 30 sec at 59 ℃ (β -catenin), 1 min 30 sec at 72 ℃, and a final extension step of 10 min at 72 ℃. The PCR products were analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining. Negative (water) controls were included in each RT-PCR assay. All samples with negative results were subjected to RT-PCR assay for beta-catenin using intron-bridged primers (Table 3) as a control for RNA amplifiable by plasma extraction.

DNA extraction

Serum was removed from the supernatant of the coagulated blood sample and centrifuged at 4800rpm for 8 minutes, whereupon the serum was carefully aliquoted into polypropylene tubes and stored at-20 ℃ for later use in DNA extraction. DNA was isolated from 200. mu.l serum using the QIAamp DNA Mini kit (Qiagen, Hilden Germany) according to the manufacturer's protocol. The DNA sample was treated with 50. mu.l dd H₂And (4) eluting with O.

Primers and PCR reactions for blood DNA samples

Detection of β -catenin was performed using a PCR assay with a primer set (table 3) flanked by the second and third introns of the β -catenin gene. PCR was performed using AmpliTaq Gold as polymerase (Perkin-Elmer Corp., Foster City, CA) using reagents provided in the GeneAmpDNA amplification kit. The parameters used for PCR were 40 cycles with an initial denaturation at 95 ℃ for 10 min, followed by 1 min 15 sec at 94 ℃, 57 ℃ (β -catenin) and 69 ℃ (RET) for 1 min 30 sec, 1 min 30 sec at 72 ℃, and a final extension step at 72 ℃ for 10 min. The PCR products were analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining. The PCR product was confirmed by direct DNA sequencing. A negative (water) control was included in each PCR assay. All samples with negative results were subjected to PCR assay of the RET gene as a control for the amplifiable grade quality of the serum DNA samples. The RET gene sequence encoding the receptor tyrosine kinase is normally present in the circulating blood of healthy individuals (Matisa-Guiu 1998).

Immunohistochemical staining and evaluation

Monoclonal antibody to β -catenin (C19220) was purchased from transduction laboratory (u.s.a.). The antibody is prepared against the C-terminus of mouse beta-catenin (a.a.571-581) and is reactive with human, rat and mouse species of beta-catenin. Tissue sections 4 μm thick were placed on silane-coated (Sigma Chemicals, st. louis, MO) glass slides, air dried overnight, and rehydrated with xylene and gradient ethanol. Antigen search and immunochemical staining were performed as described in a Ventana-ES automated immunostaining machine (Ventana, Tucson, Az). Sections were counterstained with Harris hematoxylin and rehydrated in gradient ethanol followed by mounting with cover gel (permount). Negative controls were performed by replacing the beta-catenin antibody with TBS. Positive signals were evaluated under an optical microscope at a magnification of 10 × 40 in 4 fields without knowledge of clinical results. Results were manually evaluated by two independent observers, and data were expressed as IHC scores obtained by multiplying "staining intensity" by "percentage of positive cells" according to Remmele and schicketan z with minor modifications (Remmele & schicketan z, 1993; Wong et al, 2001). In this study, the IHC score is expressed as follows: "-", 1+ ═ weak, 2+ ═ moderate, 3+ ═ strong, and 4+ ═ strong.

Quantitative analysis of plasma beta-catenin RNA by real-time RT-PCR

The copy number of plasma beta-catenin RNA was measured by real-time RT-PCR using the TagMan detection system (Heid et al, 1996). The amount of fluorescent product in any given cycle during the PCR exponential phase is proportional to the initial number of copies of the template. The reactions were recorded and analyzed using an ABI Prism 7700 sequencer (Perkin-Elmer applied Biosystems, UK) equipped with a 96-well thermal cycler. Briefly, RNA samples (50-100ng) were incubated with 0.01 units of uracil N-glycosylase (2 min at 50 ℃) and reverse transcribed at 60 ℃ for 30 min in a 25- μ l oligo (dT) -primer reaction (system). The cDNA template was then initially denatured at 92 ℃ for 5 minutes and then labeled with a fluorescence quencher 6-carboxyfluorescein at the 5 'end and a fluorescence quencher molecule at the 3' end before 40 PCR cycles (20 seconds at 92 ℃ and 1 minute at 62 ℃ each) in the presence of the forward and reverse primers.

Table 1: sequences of primers used in PCR reactions

Table 2: association of plasma beta-catenin RNA with nuclear beta-catenin expression (IHC score) at their respective lesions in patients with colorectal adenoma and carcinoma

dys: abnormal hyperplasia; N.A.: not applicable; N.D.: and (5) not testing.

Table 3: primers used in PCR reactions

TABLE 4 patient records

dys: abnormal hyperplasia; N.A.: not applicable; N.D.: and (5) not testing.

Although various embodiments are disclosed herein, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents. Moreover, the above-described advantages and features operate in the described embodiments, but should not limit the application of the claims to operations and configurations that achieve any or all of the above-described advantages. Further, the teachings from the following references are hereby incorporated by reference in any sense:

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In addition, section headings herein are provided for consistency with the hints of the French title 37 CFR 1.77, or for providing organizational cues. These headings should not be used to limit or describe the features of the invention which may be produced by the disclosure of the invention, as set forth in any of the claims. In particular, and by way of example, although the headings refer to "technical field of the invention," the claims should not be limited by the language chosen under this heading to describe the so-called field of the invention. Furthermore, the description of technology in the "background of the invention" should not be construed as an admission that the technology is prior art to any of the inventions in the present disclosure. Neither should the "summary of the invention" be read as a description of the invention set forth in the claims set forth herein. Furthermore, the singular form of "the invention" referred to in these headings or elsewhere in this disclosure should not be taken to imply that there is only one inventive point claimed in this disclosure. Since many inventions may be set forth with limitations in the various claims appended hereto, the claims and their equivalents define the inventions protected thereby. In all cases, the scope of the claims should be considered in their own right with reference to the specification and should not be limited to the headings set forth herein.

Claims (1)

1. A primer pair for use in a method of detecting the presence and copy number of RNA or DNA encoding β -catenin in ex vivo serum or plasma, said primer pair consisting of TGATTTGATGGAGTTGGACAT and CATTGCATACTGTCCATCAAT specific for β -catenin.