Patent Application
20220276249
The present invention can be included in the medical field. Particularly, the present invention refers to an in vitro method for the diagnosis of colorectal cancer and/or pre-cancerous stage thereof.
Colorectal cancer (CRC) (also known as colon cancer, rectal cancer, or bowel cancer) is the development of cancer in the colon or rectum (parts of the large intestine). The vast majority of colorectal cancers are adenocarcinomas. This is because the colon has numerous glands within the tissue. When these glands undergo a number of changes at the genetic level, they proceed in a predictable manner as they move from benign to an invasive, malignant colon cancer. The adenomas of the colon, particularly advanced colorectal adenoma (AA), are a benign version of the malignant adenocarcinomas but still with malignant potential if not removed (they are usually removed because of their tendency to become malignant and to lead to colon cancer).
Screening is an effective way for preventing and decreasing deaths from colorectal cancer and is recommended starting from the age of 50 to 75. The best known and most frequently used screening test for colorectal cancer is called Fecal Immunochemical Test (FIT). FIT detects blood in the stool samples which can be a sign of pre-cancer or cancer. If abnormal results are obtained, usually a colonoscopy is recommended which allows the physician to look at the inside of the colon and rectum to make a diagnosis. During colonoscopy, small polyps may be removed if found. If a large polyp or tumor is found, a biopsy may be performed to check if it is cancerous. The gastroenterologist uses a colonoscopy to find and remove these adenomas and polyps to prevent them from continuing to acquire genetic changes that will lead to an invasive adenocarcinoma.
Although, as explained above, FIT is nowadays used for screening colorectal cancer, it is important to note that FIT offers a low sensitivity for AA (around 20-30% depending on literature) which means that most of said kind of patients can be wrongly classified as not having the disease. Consequently, FIT is not able to identify adenomas due to its low sensitivity. Moreover, since FIT uses stool samples, it offers a low compliance. On the other hand, the colonoscopy is an invasive technique wherein the most severe complication generally is the gastrointestinal perforation. On the other hand, colonoscopy is nowadays a procedure involving anesthesia, and the laxatives which are usually administered during the bowel preparation for colonoscopy are associated with several digestive problems.
It is important to note that the methods used today for screening general population at risk of suffering for CRC or AA are associated with a high rate of false positives. Consequently, a high amount of unnecessary follow-up colonoscopies are nowadays performed.
The present invention offers a clear solution to the problems cited above because it is focused on an in vitro method for identifying or screening human subjects at risk of suffering from colorectal cancer or colorectal adenomas (particularly advanced colorectal adenomas), departing from the concentration level of protein biomarkers isolated from minimally-invasive samples such as blood, serum or plasma. Since the method of the invention is based on blood, serum or plasma samples, it is expected to improve compliance to colorectal cancer screening. Moreover, the method of the invention offers high sensitivity and specificity, which means that it is a strong and cost-effective method for the detection of both colorectal cancer and colorectal adenomas.
The present invention refers to an in vitro method for diagnosing, identifying or screening human subjects at risk of suffering from colorectal cancer and/or advanced colorectal adenomas, departing from the concentration level of protein biomarkers isolated from minimally-invasive samples such as blood, serum or plasma. The method of the invention offers high sensitivity and specificity, which means that it is a strong and cost-effective method for the detection of both colorectal cancer and colorectal adenomas.
Since the method of the invention has higher sensitivity and specificity as compared to the method used today (FIT) for screening general population at risk of suffering from CRC or AA, it is associated with a lower percentage of false positives. Consequently, the method described in the present invention clearly helps in reducing the number of follow-up colonoscopies, thus improving the way that the patients are nowadays screened or diagnosed. Once the method of the invention is performed, if it is determined that the patients might be suffering from colorectal cancer and/or precancerous stage, the result is confirmed by colonoscopy. However, if it is not determined that the patient might be suffering from colorectal cancer and/or precancerous stage, there is no need to perform a colonoscopy and routine testing with the method of the invention defined below is recommended.
Particularly, the first embodiment of the present invention refers to an in vitro method (hereinafter “method of the invention”) for the diagnosis of colorectal cancer and/or a pre-cancerous stage thereof which comprises: a) Measuring the concentration level of at least Flt3L, in a biological sample obtained from the subject and b) wherein if a deviation or variation of the concentration level of at least Flt3L is identified, as compared with the reference concentration level measured in healthy control subjects, this is indicative that the subject is suffering from colorectal cancer and/or a pre-cancerous stage.
In a particularly preferred embodiment, the method of the invention comprises measuring the concentration level of at least the combination [Flt3L and CYFRA21-1] in a biological sample obtained from the subject.
In this regard, it is important to consider that all the most reliable signatures claimed in the present invention comprise Flt3L, preferably [Flt3L and CYFRA21-1], thus obtaining various biomarker signatures comprising Flt3L, preferably [Flt3L and CYFRA21-1], such as [Flt3L and CYFRA21-1 and AREG], [Flt3L and CYFRA21-1 and AREG and ErbB4] or [Flt3L and CYFRA21-1 and AREG and CLEC2C] with an Area Under the Curve (AUC) around 0.9 for the detection of CRC a with a good performance also for the detection of AA (see Table 12). Although any of the signatures comprising Flt3L, preferably [Flt3L and CYFRA21-1], can be efficiently used according to the present invention, the following signatures comprising Flt3L, preferably [Flt3L and CYFRA21-1], are particularly preferred since they offer an AUC value above 0.9 for the detection of CRC and a good performance for the detection of AA: [Flt3L and CYFRA21-1 and AREG and ErbB4] (AUC for CRC=0.931) or [Flt3L and CYFRA21-1 and AREG and CLEC2C] (AUC for CRC=0.915) (see Table 12).
The second embodiment of the present invention refers to a kit of parts comprising reagents for the determining the concentration level of any of the above cited signatures. In a preferred embodiment, the present invention refers to the in vitro use of a kit comprising reagents for the determination of the concentration level of Flt3L, or the combination [Flt3L and CYFRA21-1], preferably [Flt3L and CYFRA21-1 and AREG], [Flt3L and CYFRA21-1 and AREG and ErbB4] or [Flt3L and CYFRA21-1 and AREG and CLEC2C] for the diagnosis of colorectal cancer and/or a pre-cancerous stage thereof.
According to the method of the invention, after measuring the concentration level of any of the above cited combinations of biomarkers, a score value is obtained for the signature and this score value is compared with a threshold value which defines the diagnostic rule. If this score value is higher than the threshold, then the corresponding sample is classified as a positive sample, which is an indication that the patient might be suffering from colorectal cancer and/or pre-cancerous stage thereof. The threshold value has been defined in order to optimize sensitivity and specificity values. Consequently, in a preferred embodiment, the method of the invention comprises: a) Measuring the concentration level of any of the above cited combinations of biomarkers, in a biological sample obtained from the subject, b) processing the concentration values in order to obtain a risk score and c) wherein if a deviation or variation of the risk score value obtained for any of the above cited combinations of biomarkers is identified, as compared with a reference value, this is indicative that the subject is suffering from colorectal cancer and/or a pre-cancerous stage.
The third embodiment of the present invention refers to the in vitro use of any of the above cited biomarkers or signatures for the diagnosis of colorectal cancer and/or a pre-cancerous stage thereof.
In a preferred embodiment, the pre-cancerous stage of colorectal cancer is advanced colorectal adenoma.
In a preferred embodiment, the diagnosis of the colorectal cancer and/or a pre-cancerous stage thereof is confirmed by an image technique, preferably colonoscopy.
In a preferred embodiment the present invention refers to an in vitro method for detecting colorectal cancer and/or a precancerous stage thereof, said method comprising: a) obtaining a plasma sample from a human patient; and b) detecting whether any of the above cited protein biomarkers or signatures are present in the plasma sample by contacting the plasma sample with an antibody directed against said protein biomarkers or signatures and detecting binding between the proteins and the antibody.
The fourth embodiment of the present invention refers to a method for diagnosing and treating colorectal cancer or a pre-cancerous stage thereof, which comprises: a) obtaining a plasma sample from a human patient; b) detecting whether any of the above cited protein biomarkers or signatures are present in the plasma sample; c) diagnosing the patient with colorectal cancer or a pre-cancerous stage thereof when the presence of said protein biomarkers or signatures in the plasma sample is detected; and performing a colonoscopy to the patient and removing the colorectal cancer or polyps afterwards.
Alternatively, the fifth embodiment of the present invention refers to an in vitro method (hereinafter “method of the invention”) for the diagnosis of colorectal cancer and/or a pre-cancerous stage thereof which comprises: a) Measuring the concentration level of at least AREG, in a biological sample obtained from the subject and b) wherein if a deviation or variation of the concentration level of at least AREG is identified, as compared with the reference concentration level measured in healthy control subjects, this is indicative that the subject is suffering from colorectal cancer and/or a pre-cancerous stage.
In a particularly preferred embodiment, the method of the invention comprises measuring the concentration level of at least the combination [AREG and CYFRA21-1] in a biological sample obtained from the subject.
In this regard, it is important to consider that all the most reliable signatures claimed in the present invention comprises AREG, preferably [AREG and CYFRA21-1], thus obtaining various biomarker signatures comprising AREG, preferably comprising [AREG and CYFRA21-1], with an Area Under the Curve (AUC) around 0.9 for the detection of CRC a with a good performance also for the detection of AA (see Table 12bis). Consequently, although any of the signatures comprising AREG, preferably comprising [AREG and CYFRA21-1], could be effectively used according to the present invention, the following signatures comprising AREG, preferably comprising [AREG and CYFRA21-1], are particularly preferred since they offer an AUC value above 0.9 for the detection of CRC and a good performance for the detection of AA: [AREG and CYFRA21-1 and Flt3L and ErbB4] (AUC for CRC=0.931) or [AREG and CYFRA21-1 and Flt3L and CLEC2C] (AUC for CRC=0.915) (see Table 12bis).
So, in a particularly preferred embodiment, the method of the invention comprises measuring the concentration level of at least the combination [AREG and CYFRA21-1 and Flt3L], or the combination of [AREG and CYFRA21-1 and CLEC2C], or the combination of [AREG and CYFRA21-1 and ErbB4], or the combination [AREG and CYFRA21-1 and FasL], or the combination [AREG and CYFRA21-1 and CD147], or the combination [AREG and CYFRA21-1 and HGFR], or the combination [AREG and CYFRA21-1 and Flt3L and ErbB4], or the combination of [AREG and CYFRA21-1 and Flt3L and CLEC2C], or the combination of [AREG and CYFRA21-1 and HGFR and CD147] in a biological sample obtained from the subject.
In a particularly preferred embodiment, the method of the invention comprises measuring the concentration level of at least the combination [AREG and CD147], or the combination of [AREG and CLEC2C], or the combination of [AREG and HGFR], or the combination [AREG and CD147 and HGFR] in a biological sample obtained from the subject.
The sixth embodiment of the present invention refers to the in vitro use of any of the above cited signatures for the diagnosis of colorectal cancer and/or a pre-cancerous stage thereof.
According to the method of the invention, after measuring the concentration level of any of the above cited combinations of biomarkers, a score value is obtained for the signature and this score value is compared with a threshold value which defines the diagnostic rule. If this score value is higher than the threshold, then the corresponding sample is classified as a positive sample, which is an indication that the patient might be suffering from colorectal cancer and/or pre-cancerous stage thereof. The threshold value has been defined in order to optimize sensitivity and specificity values. Consequently, in a preferred embodiment, the method of the invention comprises: a) Measuring the concentration level of any of the above cited combinations of biomarkers, in a biological sample obtained from the subject, b) processing the concentration values in order to obtain a risk score and c) wherein if a deviation or variation of the risk score value obtained for any of the above cited combinations of biomarkers is identified, as compared with a reference value, this is indicative that the subject is suffering from colorectal cancer and/or a pre-cancerous stage.
The seventh embodiment of the present invention refers to a kit of parts comprising reagents for the determining the concentration level of any of the above cited signatures. In a preferred embodiment, the present invention refers to the in vitro use of a kit comprising reagents for the determination of the concentration level of any of the above cited combinations of biomarkers for the diagnosis of colorectal cancer and/or a pre-cancerous stage thereof.
In a preferred embodiment, the pre-cancerous stage of colorectal cancer is advanced colorectal adenoma.
In a preferred embodiment, the diagnosis of the colorectal cancer and/or a pre-cancerous stage thereof is confirmed by an image technique, preferably colonoscopy.
In a preferred embodiment the present invention refers to an in vitro method for detecting colorectal cancer and/or a precancerous stage thereof, said method comprising: a) obtaining a plasma sample from a human patient; and b) detecting whether any of the above cited protein biomarkers or signatures are present in the plasma sample by contacting the plasma sample with an antibody directed against said protein biomarkers or signatures and detecting binding between the proteins and the antibody.
The last embodiment of the present invention refers to a method for diagnosing and treating colorectal cancer or a pre-cancerous stage thereof, which comprises: a) obtaining a plasma sample from a human patient; b) detecting whether any of the above cited protein biomarkers or signatures are present in the plasma sample; c) diagnosing the patient with colorectal cancer or a pre-cancerous stage thereof when the presence of said protein biomarkers or signatures in the plasma sample is detected; and performing a colonoscopy to the patient and removing the colorectal cancer or polyps afterwards.
For the purpose of the present invention the following terms are defined:
For the purpose of the present invention the following proteins are identified according to Uniprot data base:
A total of 96 subjects from eight Spanish hospitals (Hospital de Burgos, Hospital de Vigo, Hospital de Donosti, Hospital de Ourense, Hospital del Bierzo, Hospital de Beltvigte and Hospital de Zaragoza) were prospectively included in this study: 64 patients newly diagnosed with sporadic colorectal neoplasia (32 with CRC and 32 with AA) and 32 healthy individuals without personal history of any cancer and with a recent colonoscopy confirming the lack of colorectal neoplastic lesions. Patients with AA were those with adenomas having a size of at least 10 mm or histologically having high grade dysplasia or >20% villous component. The characteristics of participants are shown in Table 1. Blood samples were collected prior to endoscopy or surgery in all individuals.
The study was approved by the Institutional Ethics Committee of each Hospital, and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki.
Ten mL of whole blood from each participant were collected in EDTA K2 containing tubes. Blood samples were placed at 4° C. until plasma separation. Samples were centrifuged at 1,600×g for 10 min at 4° C. to spin down blood cells, and plasma was transferred into new tubes, followed by further centrifugation at 16,000×g for 10 minutes at 4° C. to completely remove cellular components.
The concentration of the biomarkers in plasma samples was established using commercial ELISA (Enzyme-linked immunosorbent assay) and CLIA (Chemiluminescence immunoassay) test and following their corresponding instruction manual. HGFR and ErbB4 was analyzed with ELISA kit from Cloud clone Corp. Level of CD147, CLEC2C, Flt3L, and FasL was measured using ELISA Kit form Elabscience. In the case of the IFNgamma, ELISA kit from Abcam was used. Related to CLIA test, CYFRA21-1 and AREG were analyzed with CLIA test from Cloud Clone Corp.
For the protein quantification step the samples were processed with the corresponding kit (ELISA/CLIA) and distributed in experimental plates. Each plate contained also control data used to construct a standard curve. Fluorescence data obtained from each run (expressed as integer numbers) have been background corrected for each sample and quantified using a standard curve generated using a 2-degree polynomial regression model.
Three groups of individuals were considered in the analysis. CRC (Individuals diagnosed with colorectal cancer), AA (Individuals diagnosed with advanced adenoma) and CTL (Individuals with no disease).
Raw quantification data have been transformed by applying square root function, and then centering and scaling so that, after the transformation, each protein measure have mean 0 and standard deviation 1. Quantification values are summarized in Table 2 and Table 3, where each protein is described as median and interquartile range in the different groups considered.
Non-normality of the data was confirmed by Shapiro-Wilk test and, consequently, Wilcoxon rank-sum test was used to compare either CRC cases or AA cases against CTL individuals.
Diagnostic performance for the individual proteins and some of their combinations has been assessed by their receiver operating characteristic (ROC) curves, and the area under the ROC curves (AUC). Moreover, sensitivities, specificities, positive predictive and negative predictive values (PPV and NPV) for the different tests were calculated at the optimal cutoff point defined by the best Youden's Index (or equivalently, the point of the ROC that maximizes the sum of sensitivity and specificity).
Scores used for deriving the ROC-AUCs and the rest of performance values were obtained using univariate logistic regression model for the individual proteins and multivariate logistic regression models for the different combination of proteins considered. 95% CI for the AUCs was obtained with the DeLong methodology both in individual markers and combination of them.
Different metrics to evaluate the individual proteins were determined, also perming the following comparisons: CRC/AA vs CTL. It can be seen that AREG, CYFRA21-1 and Flt3L are significantly different between CRC and CTL groups and their AUC are significantly different from 0.5 (as their 95% confidence interval do not include 0.5). In the case of AA group, AREG also shows statistically differences compared to CTL group.
Table 2 and Table 3 shows metrics for individual proteins, including p-value from Wilcoxon test (p.Wilc), area under the ROC curve (AUC), and Sensitivity (Sens.), Specificity (Spec.), and positive (VPP) and negative (VPN) predictive values computed in the cut-off point of the ROC curve with the best Youden's index. The sign column indicates, for the biomarkers with p-value<0.25, whether high levels of the marker increase or decrease the risk of disease (+ and − respectively).
With the aim of improving individual diagnostic capability, combinations of proteins have been explored with the following procedure: Based on markers with p<0.25 either in CRC. vs. CTL (Table 3) or AA. vs. CTL (Table 4) comparisons, we used multivariate logistic regression to explore all possible combinations of two, three and four of these proteins taken at the same time.
Table 4, Table 5, Table 5 bis, Table 6 and Table 6 bis show the AUC achieved for the combinations of two, three and four biomarkers respectively, discriminating CRC vs CTL.
Table 7, Table 8 and Table 9 show the AUC achieved for the combinations of two, three and four biomarkers respectively, discriminating AA vs CTL.
Based on their respective AUCs, the best models have been selected. Table 10 and Table 10bis show the best results for CRC. Table 11 and Table 11bis show the best results for AA. The metrics for the best combinations of proteins are included, comprising area under the ROC curve (AUC), Sensitivity (Sens.), Specificity (Spec.), and positive (PPV) and negative (NPV) predictive values computed in the cut-off point of the ROC curve with the best Youden's index.
Finally, Table 12 and Table 12bis have been designed to show the overlapping of the most important signatures claimed in the present invention. It is clearly shown that all the best signature signatures comprise [Flt3L and CYFRA21-1] and [AREG and CYFRA21-1].
X
X
0.931
X
X
0.727
X
X
X
X
X
X
0.931
X
X
X
X
X
X
X
X
X
X
X
X
0.769
X
X
X
X
X
X
| Number | Date | Country | Kind |
|---|---|---|---|
| 19382156.8 | Mar 2019 | EP | regional |
| 19382157.6 | Mar 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/055277 | 2/28/2020 | WO |
