CN116298295A

CN116298295A - Tumor autoantigen/antibody combination for early detection of colorectal cancer and application thereof

Info

Publication number: CN116298295A
Application number: CN202310558646.0A
Authority: CN
Inventors: 孙苏彭; 康美华; 阴亮; 孙立平
Original assignee: SHANGHAI HENGXIN BIOTECHNOLOGY CO Ltd
Current assignee: SHANGHAI HENGXIN BIOTECHNOLOGY CO Ltd
Priority date: 2023-05-18
Filing date: 2023-05-18
Publication date: 2023-06-23
Anticipated expiration: 2043-05-18
Also published as: CN116298295B

Abstract

The invention relates to a tumor autoantigen/antibody combination for early detection of colorectal cancer and application thereof. In particular, the invention provides the use of diagnostic reagents for autoantibodies. The research of the invention proves that the autoantibody combination can be used for early screening or differential diagnosis of colorectal cancer and efficacy evaluation of colorectal cancer patients after intervention treatment. The detection method is simple and quick, has high patient compliance and high sensitivity and high specificity, and is beneficial to more accurately and earlier diagnosis and prognosis evaluation of colorectal cancer.

Description

Tumor autoantigen/antibody combination for early detection of colorectal cancer and application thereof

Technical Field

The invention relates to the field of biotechnology and medical diagnosis, in particular to a tumor autoantigen/antibody combination for early detection of colorectal cancer and application thereof.

Background

Colorectal cancer (CRC) is the third most common cancer worldwide in men and the second most in women. It is estimated that 140 thousands of new cases of CRC account for 9.9% of the global cancer burden. At the same time, about 70 tens of thousands of deaths are caused annually, making them the fourth most common cause of cancer-related death. Men have higher CRC mortality than women, with significant regional differences.

In china, the incidence of colorectal cancer is fifth among all cancers in men, fourth among women, and fifth among both men and women. The incidence rate of CRC in China is 15-25/10 ten thousand people, and the death rate is 7-13/10 ten thousand people, and the trend is upward. The incidence rate of urban areas is higher than that of rural areas, the incidence rate of more developed areas is higher, and the incidence rate of the southeast coastal areas in recent times is faster.

Risk factors for CRC include age, sex, familial adenomatous polyposis, multiple adenomatous individuals or family history, inflammatory bowel disease, diabetes and insulin resistance, alcohol consumption, obesity, smoking, red meat and high fat intake, pelvic radiotherapy, and the like.

Colorectal cancer screening has greatly driven a downward trend in CRC morbidity and mortality over the last 20 years. The clinical value of screening is manifested in the aspects of preventing cancer occurrence, mortality and excessive treatment cost, and detection can timely discover the existence of tissue lesions before the lesions develop into cancers and early cancers spread to the intestinal wall, thereby playing a positive role.

Patients with early stage localized colorectal cancer (stage I and II) have a 5 year survival rate approaching 90%. Patients diagnosed with advanced CRC, often with distant organ metastasis, have a survival rate of only 13.1% and at this stage the treatment is often palliative, with the economic burden associated with treatment being greatest.

Colonoscopy is the current reference method for CRC screening, suggesting once every 10 years in the average risk population 50 years or older. The ability of colonoscopy to detect cancerous and precancerous lesions by direct visualization has been demonstrated in several large cohort studies. Colonoscopy detects CRC more than 95% sensitive, while it detects advanced adenomas (above 10 mm in diameter) at 88% -98% sensitive. Case control studies indicate that colonoscopy can reduce CRC incidence by 53% -72% and CRC-related mortality by 31%. One of the greatest advantages of colonoscopy is the ability to remove pre-cancerous lesions and small cancerous lesions at the time of detection. It also detects the proximal and distal ends of the colon. Drawbacks associated with colonoscopy include the invasiveness of the testing procedure, the required bowel preparation, the use of sedatives or anesthetics, the need for post-testing care. Intestinal preparation is often an uncomfortable and time consuming procedure, requires temporary changes in medications and diets, and requires the use of cleansers. There is a risk of intestinal perforation during colonoscopy and a risk of post-colonoscopy bleeding, especially in post-polypectomy patients. These drawbacks result in low compliance with patient colonoscopy. Furthermore, this examination is highly dependent on the technique of the endoscopist to visualize and remove lesions, especially in the proximal colon, which may be more difficult to find. Flat or sessile polyps can be particularly difficult to detect, requiring more expertise.

CT colonography detects the sensitivity of CRC and advanced adenomas in daily clinical practice, 90% for lesions larger than 10mm and 78% for lesions larger than 6 mm. It is mainly used for patients who cannot perform adequate optical examination of the colon due to other complications or structural problems, which are not suitable for colonoscopy. CT colonography has the advantage of visually inspecting the entire colon, but is semi-invasive. For better visualization, the colon needs to be inflated with air; while this is uncomfortable for many patients, it does not require sedation. Drawbacks of CT colonography include unpleasant bowel preparation, similar to colonoscopy, and the use of ionizing radiation, which presents additional safety issues and costs. This screening method is highly dependent on the technical expertise of the radiologist to interpret the examination results.

Guaiac Fecal Occult Blood Test (FOBT) and Fecal Immunochemical Test (FIT) aim to detect hemoglobin as a marker of occult blood in fecal matter. Most FOBT/FIT studies are observational, which may overestimate their sensitivity. In a study with colonoscopy as a reference, the sensitivity of FIT to detect CRC and precancerous lesions was 71% -75% and 27% -29%, respectively, higher than that of FOBT 33% -75% and 11% -25%. The specificity of FOBT (98% -99%) is higher than FIT (94% -95%). FOBT has been shown to reduce CRC mortality by 15% -33%. FOBT and FIT assays should be repeated annually without additional screening methods. FOBT and FIT can be performed at home, are noninvasive, and do not require extensive intestinal preparation. Early lesions bleed less frequently, may lead to false negative detection results, and therefore these examinations are more likely to detect more mature lesions and miss pre-cancerous lesions. Furthermore, dietary intake of vitamin C leads to false negative results, as it inhibits the peroxidase activity of FOBT, while dietary hemoglobin intake of red meat leads to false positive results of FOBT. Although FIT kits only require one test, some FOBT tests require testing in duplicate or triplicate. In one study with over 100 ten thousand age-screened participants, compliance with FOBT per year was found to be 42.1% in men and 42.9% in women. Also, those who are positive for detection have a low follow-up rate for colonoscopy, thus affecting the value of FOBT/FIT screening.

Multi-target fecal DNA (mt-sDNA) detection is a non-invasive screening method aimed at detecting abnormal DNA and occult blood in fecal samples. The test was aimed at detecting ten DNA-based markers and human hemoglobin in feces. The results of the assays for detecting DNA and hemoglobin biomarkers are then combined into a diagnostic algorithm to produce negative or positive detection results. In a blind, cross-sectional screening study, the study included at least 10,000 asymptomatic participants older than 50 years of age and at average risk who had the results of FIT and colonoscopy.

In this study, the mt-sDNA test had a sensitivity of 92.3% whereas the FIT test had a sensitivity of 73.8%. mt-sDNA detection shows a higher sensitivity for detection of precancerous lesions, detecting 42.4% of advanced adenomatous subjects, 69.2% of highly atypical hyperplastic precancerous lesion subjects, and 42.4% of sessile serrated polyp subjects. Limitations of this method include the need for broader clinical study evidence for the test, which, although it has higher sensitivity, is somewhat less specific and at the same time costly to detect. Therefore, according to the current state of the Chinese medical system, the use of blood as a sample for early tumor screening is more feasible.

Circulating tumor DNA (ctDNA), also known as cell-free DNA, is a DNA sequence derived from apoptotic tumor cells detected in the circulatory system. The DNA fragment with the variation or mutation found in cancer cells may be a specific marker for a tumor, such as Septin9 methylation detection for colorectal cancer detection. However, the number of mutant DNA fragments found in plasma varies with the tumor type. ctDNA levels may indicate overall tumor burden (higher levels associated with larger or more advanced tumors), but some advanced tumor patients have no detectable ctDNA. Furthermore, for some specific tumor types, it is not clear why some patients have detectable ctDNA and others do not. The number of ctDNA fragments can be as low as 1 site mutant per 1ml of plasma, thus requiring the use of digital genomic techniques of next generation sequencing and massively parallel sequencing to amplify the mutant ctDNA fragments. These systems require identification of whether the mutated fragment of DNA is from random errors of DNA polymerase in cancer cells or normal cells.

Tumor autoantibodies (TAAs) can be stably present at high levels under induction of the immune cascade, despite lower levels of the corresponding antigen. Therefore, autoantibodies have been proposed as biomarkers for early detection of cancer. In recent years autoantibodies against TAA have been reported in CRC patients, but only through a series of studies on a small cohort of people, and no high throughput screening has been performed in large numbers of samples, especially against advanced adenomas, a class of advanced precancerous patient populations. Analysis of binding to autoantibodies most autoantibodies associated with CRC tend to have low sensitivity and high specificity. The diagnostic capability is limited when used alone, and higher specificity and significantly increased sensitivity can be ensured when used in combination. Also, when the same or similar autoantibodies are used in combination in a study, the diagnostic ability exhibited in different studies varies. This may be due to differences in the sample size, the procedure of sample preparation, and the method of testing used.

Thus, there is a great need in the art for a novel tumor early detection marker or method that can be conveniently, inexpensively, and sensitively used for early diagnosis and/or prognosis evaluation of colorectal cancer.

Disclosure of Invention

The invention provides a tumor autoantigen/antibody combination for early detection of colorectal cancer and application thereof.

In a first aspect of the invention there is provided the use of an autoantibody diagnostic reagent for the preparation of a diagnostic reagent or kit for (a) diagnosing the risk of developing colorectal cancer; and/or (b) prognostic evaluation of colorectal cancer;

wherein the diagnostic reagent is for detecting the level of the autoantibody and the autoantibody is selected from the group consisting of:

(A) Any one of antibodies selected from A1 to A8, or a combination thereof: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2; (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (A7) Anti-DAL-1; (A8) Anti-PARP1.

In another preferred embodiment, the autoantibody comprises at least 2 selected from A1 to A8.

In another preferred embodiment, the autoantibodies comprise a combination of one or more autoantibodies selected from A1 to A8 with B1.

In another preferred embodiment, there is provided the use of an autoantibody diagnostic reagent for the preparation of a diagnostic reagent or kit for (a) diagnosing the risk of developing colorectal cancer; and/or (b) prognostic evaluation of colorectal cancer;

wherein the diagnostic reagent is for detecting the level of the autoantibody and the autoantibody comprises a combination of:

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2.

In another preferred embodiment, the autoantibody further comprises any antibody selected from the group consisting of: (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (A7) Anti-DAL-1; (A8) Anti-PARP1.

In another preferred embodiment, the autoantibody further comprises: (B1) Anti-P53.

In another preferred embodiment, the autoantibody comprises a combination of: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2; (A4) Anti-COPB1; (A5) Anti-PEBP1 (A6) Anti-ATP4A; and (B1) Anti-P53.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53 and (A3) Anti-FGFR2.

In another preferred embodiment, the autoantibody combination is: (A4) Anti-COPB1, (A2) Anti-PBRM1, (A8) Anti-PARP1 and (A1) Anti-PSMC1.

In another preferred embodiment, the autoantibody combination is: (A7) Anti-DAL-1, (A6) Anti-ATP4A and (A5) Anti-PEBP1.

In another preferred embodiment, the autoantibody combination is: (A6) Anti-ATP4A, (A7) Anti-DAL-1 and (A8) Anti-PARP1.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53, (A3) Anti-FGFR2 and (A4) Anti-COPB1.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53, (A3) Anti-FGFR2, (A4) Anti-COPB1 and (A2) Anti-PBRM1.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53, (A3) Anti-FGFR2, (A4) Anti-COPB1, (A2) Anti-PBRM1 and (A8) Anti-PARP1.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53, (A3) Anti-FGFR2, (A4) Anti-COPB1, (A2) Anti-PBRM1, (A8) Anti-PARP1, and (A1) Anti-PSMC1.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53, (A3) Anti-FGFR2, (A4) Anti-COPB1, (A2) Anti-PBRM1, (A8) Anti-PARP1, (A1) Anti-PSMC1 and (A5) Anti-PEBP1.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53, (A3) Anti-FGFR2, (A4) Anti-COPB1, (A2) Anti-PBRM1, (A8) Anti-PARP1, (A1) Anti-PSMC1, (A5) Anti-PEBP1 and (A7) Anti-DAL 1.

In another preferred embodiment, the autoantibody combination is: (B1) Anti-P53, (A3) Anti-FGFR2, (A4) Anti-COPB1, (A2) Anti-PBRM1, (A8) Anti-PARP1, (A1) Anti-PSMC1, (A7) Anti-DAL-1, (A6) Anti-ATP4A and (A5) Anti-PEBP1.

In another preferred embodiment, the diagnostic reagent for an autoantibody is an antigenic protein.

In another preferred embodiment, the antigenic protein is selected from the group consisting of:

(C) Any antigen selected from the group consisting of C1 to C9, or a combination thereof: (C1) PSMC1; (C2) PBRM1; (C3) FGFR2; (C4) COPB1; (C5) PEBP1; (C6) ATP4A; (C7) DAL-1; (C8) PARP1; (C9) P53.

In another preferred embodiment, the antigenic proteins are: (C9) P53 and (C3) FGFR2.

In another preferred embodiment, the antigenic proteins are: (C4) COPB1, (C2) PBRM1, (C8) PARP1, and (C1) PSMC1.

In another preferred embodiment, the antigenic proteins are: (C7) DAL-1, (C6) ATP4A, and (C5) PEBP1.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2 and (C4) COPB1.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2, (C4) COPB1 and (C2) PBRM1.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2, (C4) COPB1, (C2) PBRM1 and (C8) PARP1.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2, (C4) COPB1, (C2) PBRM1, (C8) PARP1, and (C1) PSMC1.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2, (C4) COPB1, (C2) PBRM1, (C8) PARP1, (C1) PSMC1, and (C5) PEBP1.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2, (C4) COPB1, (C2) PBRM1, (C1) PSMC1, (C5) PEBP1, and (C6) ATP4A.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2, (C4) COPB1, (C2) PBRM1, (C8) PARP1, (C1) PSMC1, (C5) PEBP1, and (C7) DAL-1.

In another preferred embodiment, the antigenic proteins are: (C9) P53, (C3) FGFR2, (C4) COPB1, (C2) PBRM1, (C8) PARP1, (C1) PSMC1, (C5) PEBP1, (C7) DAL-1, and (C6) ATP4A.

In a second aspect of the invention, there is provided a kit comprising a detection reagent for detecting an autoantibody,

wherein the autoantibody is selected from the group consisting of:

(A) A combination of two or more autoantibodies selected from A1 to A8:

（A1）Anti-FGFR2；（A2）Anti-COPB1；（A3）Anti-PBRM1；（A4）Anti-PARP1；（A5）Anti-PSMC1；（A6）Anti-DAL-1；（A7）Anti-ATP4A；（A8）Anti-PEBP1。

in another preferred embodiment, a kit is provided, the kit comprising a detection reagent for detecting an autoantibody,

wherein the autoantibody comprises a combination of:

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2.

In another preferred embodiment, the kit is for detection using the following method: enzyme-linked immunosorbent assay (ELISA), protein/peptide fragment chip detection, chemiluminescence, immunoblotting, microbead immunodetection, microfluidic immunization, or combinations thereof.

In another preferred embodiment, the kit detects autoantibodies by antigen-antibody reaction.

In a third aspect of the present invention, there is provided a detection method comprising the steps of:

(a) Providing a detection sample;

(b) Detecting the level of autoantibodies in the detection sample, denoted Y1; and

(c) Comparing the autoantibody level to a control reference value Y0;

wherein the autoantibody is selected from the group consisting of:

(A) Any one of antibodies selected from A1 to A8, or a combination thereof: (A1) Anti-FGFR2; (A2) Anti-COPB1; (A3) Anti-PBRM1; (A4) Anti-PARP1; (A5) Anti-PSMC1; (A6) Anti-DAL-1; (A7) Anti-ATP4A; (A8) Anti-PEBP1;

if the detection result of the autoantibodies of the detected object meets the following conditions, the colorectal cancer occurrence risk of the object is indicated to be high:

when the level of a certain autoantibody is higher than a reference value or a standard value Y0, the colorectal cancer occurrence risk of the detection object is indicated to be high.

In another preferred embodiment, there is provided a detection method including the steps of:

(a) Providing a detection sample;

(c) Comparing the autoantibody level to a control reference value Y0;

wherein the autoantibody comprises the following combination:

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2;

In another preferred embodiment, the test sample is selected from the group consisting of: whole blood, serum, plasma, tissue, cells, interstitial fluid, cerebrospinal fluid, urine, or combinations thereof.

In another preferred embodiment, the test sample is selected from the group consisting of: whole blood, serum, plasma, or a combination thereof.

In another preferred embodiment, the test sample is from a mammal, preferably a primate mammal, more preferably a human.

In a fourth aspect of the present invention, there is provided a colorectal cancer risk judging apparatus, the apparatus comprising:

(a) The input module is used for inputting autoantibody data of a certain object;

wherein the autoantibody is selected from the group consisting of:

(b) The processing module is used for: comparing the input autoantibody level Y1 with a control reference value Y0 by the processing module so as to obtain a judging result, wherein when the comparing result meets the judging condition, the object is prompted to have high colorectal cancer risk; otherwise, the subject is prompted that the colorectal cancer risk is not high;

(c) And the output module is used for outputting the judging result.

In another preferred embodiment, there is provided a colorectal cancer risk judging apparatus including:

wherein said autoantibody comprises the following combination:

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2;

(c) And the output module is used for outputting the judging result.

In a fifth aspect of the invention there is provided the use of a diagnostic reagent of an autoantibody-antigen combination for the preparation of a diagnostic reagent or kit for the combined diagnosis of the risk of occurrence of colorectal cancer;

wherein the autoantibody-antigen combination comprises:

an autoantibody selected from the group consisting of:

(A) Any one of antibodies selected from A1 to A8, or a combination thereof: (A1) Anti-FGFR2; (A2) Anti-COPB1; (A3) Anti-PBRM1; (A4) Anti-PARP1; (A5) Anti-PSMC1; (A6) Anti-DAL-1; (A7) Anti-ATP4A; (A8) Anti-PEBP1; and

a tumor antigen (or tumor marker) selected from the group consisting of:

(D) Any antigen selected from D1 to D5, or a combination thereof: (D1) CEA; (D2) CA199; (D3) CA125; (D4) CA50; (D5) CA153.

In another preferred embodiment, there is provided the use of a diagnostic agent of an autoantibody-antigen combination for the preparation of a diagnostic agent or kit for the combined diagnosis of the risk of occurrence of colorectal cancer;

wherein the autoantibody-antigen combination comprises:

an autoantibody selected from the group consisting of:

(H1) Antibody combinations of A1-A3: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2;

(H2) Any one antibody selected from the group consisting of: (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (A7) Anti-DAL-1; (A8) Anti-PARP1; and

(H3) Autoantibodies (B1) Anti-P53; and

a tumor antigen selected from the group consisting of:

In another preferred embodiment, the tumor antigen is: (D1) CEA, (D2) CA199, or a combination thereof.

In another preferred embodiment, the autoantibody further comprises:

(B) Combinations of one or more autoantibodies selected from A1 to A8 with B1

Wherein, B1 is: (B1) Anti-P53.

In another preferred embodiment, the autoantibody is: igA, igM, igG, or a combination thereof.

In another preferred embodiment, the autoantibody-antigen combination is: (A3) Anti-FGFR2; (A4) Anti-COPB1; (A2) Anti-PBRM1; (A1) Anti-PSMC1; (A6) Anti-ATP4A; (A5) Anti-PEBP1; (B1) Anti-P53 and (D1) CEA.

In another preferred embodiment, the autoantibody-antigen combination is: (A3) Anti-FGFR2; (A4) Anti-COPB1; (A2) Anti-PBRM1; (A1) Anti-PSMC1; (A6) Anti-ATP4A; (A5) Anti-PEBP1; (B1) Anti-P53 and (D2) CA199.

In another preferred embodiment, the autoantibody-antigen combination is: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2; (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (B1) Anti-P53; (D1) CEA and (D2) CA199.

In another preferred embodiment, the antigenic protein is selected from the group consisting of: (C) any antigen selected from the group C1 to C9, or a combination thereof: (C3) FGFR2; (C4) COPB1; (C2) PBRM1; (C8) PARP1; (C1) PSMC1; (C7) DAL-1; (C6) ATP4A; (C5) PEBP1; (C9) P53.

In another preferred embodiment, the diagnostic reagent for tumor antigen is an antibody:

In another preferred embodiment, the antibody is selected from the group consisting of: (E) any one antibody selected from the group E1 to E5, or a combination thereof: (E1) Anti-CEA; (E2) Anti-CA199; (E3) Anti-CA 125; (E4) Anti-CA50 and (E5) Anti-CA153.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a scatter plot of the horizontal distribution of 9 autoantibodies (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PARP1, anti-PSMC1, anti-DAL-1, anti-ATP4A and Anti-PEBP 1) in a training cohort tumor combined control group.

FIG. 2 shows a scatter plot of the horizontal distribution of 9 autoantibodies (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PARP1, anti-PSMC1, anti-DAL-1, anti-ATP4A and Anti-PEBP 1) in a control group of tumor combinations in the validation queue.

FIG. 3 shows ROC curves for the molecular combinations of the invention (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PSMC1, anti-PEBP1 and Anti-ATP 4A) at about the maximum value of the dengue index (under ideal conditions) in a training cohort with healthy physical examination population controls.

FIG. 4 shows ROC curves of the molecular combinations of the invention (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PSMC1, anti-PEBP1 and Anti-ATP 4A) at about the maximum value of the dengue index (under ideal conditions) in a validation cohort with healthy physical examination population controls.

FIG. 5 shows ROC curves for the molecular combinations of the invention (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PSMC1, anti-PEBP1 and Anti-ATP 4A) at about the maximum of the dengue index (under ideal conditions) in the case of antigen-to-antibody binding assays, as compared to healthy human populations.

FIG. 6 shows ROC curves of the detection capacity of the antibody and antigen combination detection model of the present invention (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PSMC1, anti-PEBP1 and Anti-ATP4A, CEA and CA 199) for colon cancer.

FIG. 7 shows ROC curves of the combined detection of antibodies and antigens of the invention (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PSMC1, anti-PEBP1 and Anti-ATP4A, CEA and CA 199) for the detection of rectal cancer.

FIG. 8 shows ROC curves of the detection capacity of the combined antibody and antigen detection model of the present invention (Anti-P53, anti-FGFR2, anti-COPB1, anti-PBRM1, anti-PSMC1, anti-PEBP1 and Anti-ATP4A, CEA and CA 199) for early colorectal cancer.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly found a highly sensitive and highly specific diagnostic and prognostic autoantibody (or autoantibody against a target antigen) for colorectal cancer. The number of autoantibodies for colorectal cancer diagnosis and prognosis is 9 in total, and methods and kits for colorectal cancer occurrence risk and prognosis evaluation are correspondingly developed. The autoantibody can be combined with autoantigens, so that the occurrence risk and prognosis effect of colorectal cancer can be judged, and early colorectal cancer patients can be detected with higher sensitivity and specificity. The present invention has been completed on the basis of this finding.

Experimental operation or definition

The present invention relates to the following experimental operations or definitions. It should be noted that the present invention may also be practiced using other techniques conventional in the art and is not limited to the following experimental procedures.

Preparation of recombinant antigen proteins

The cDNA fragment of the tumor antigen was cloned into PET28 (a) expression vector containing the 6XHIS tag. At the N-or C-terminus of the antigen, streptavidin or the like (biotin-binding tag protein) is introduced. The obtained recombinant expression vector is transformed into escherichia coli for expression. The protein expressed by the supernatant was purified by Ni-NTA affinity column and ion column. When the protein is expressed in inclusion bodies, the protein is denatured by 6M guanidine hydrochloride, renaturated and folded in vitro according to a standard method, and then purified by a Ni-NTA affinity column through a 6XHIS tag, so that antigen protein is obtained.

(II) preparation and preservation of serum or plasma

Colorectal cancer patient serum or plasma is collected when the patient is initially diagnosed with colorectal cancer and has not received any radiotherapy or chemotherapy or surgical treatment. Plasma or serum was prepared according to standard clinical procedures and stored in a-80 ℃ refrigerator for long periods of time.

(III) ELISA detection

The concentration of autoantibody markers in the sample was quantified by enzyme-linked immunosorbent assay (ELISA). The purified tumor antigen is immobilized to the microwell surface by its tag streptavidin or the like. Microwells were pre-coated with biotin-labeled Bovine Serum Albumin (BSA). Serum or plasma samples were diluted 1:110 fold with phosphate buffer and reacted by adding microwells (50 ml/well). After washing unbound serum or plasma components with wash solution, horseradish peroxidase (HRP) -conjugated anti-human IgG was added to each well for reaction. Then, TMB (3, 3', 5' -tetramethylbenzidine) as a reaction substrate was added for color development. Stop solution (1N HCl) was added and absorbance was measured at 450nm using a microplate reader (OD). In this case, the amount of enzyme carried on the solid support is positively correlated with the amount of the test substance in the specimen, and the enzyme catalyzes the substrate to be a colored product. Qualitative or quantitative determination of the autoantibody is performed according to the degree of color reaction. Serum autoantibody concentrations were quantified using a standard curve.

The concentration of the antigen marker in the sample is quantified by a sandwich enzyme-linked immunosorbent assay. Connecting the specific antibody with a solid phase carrier to form a solid phase antibody, and washing to remove unbound antibody and impurities; and (3) adding a sample to be tested, namely diluting a serum or plasma sample by 1:110 times by using phosphate buffer, adding micropores to react (50 ml/hole), enabling the sample to contact and react with the solid-phase antibody for a period of time, and combining the antigen in the sample with the antibody on the solid-phase carrier to form a solid-phase antigen complex. Washing to remove other unbound material. Horseradish peroxidase (HRP) -conjugated anti-human IgG was added for reaction. Then, TMB (3, 3', 5' -tetramethylbenzidine) as a reaction substrate was added for color development. Stop solution (1N HCl) was added and absorbance was measured at 450nm using a microplate reader (OD). The amount of enzyme carried on the solid support is now positively correlated with the amount of test substance in the sample. The enzyme in the sandwich complex catalyzes the substrate to a colored product. Qualitative or quantitative determination of the antigen is performed according to the degree of color reaction.

(IV) threshold value of autoantibody and antigen protein (cutoff value)

The cutoff values for autoantibody and antigen levels were defined as being equal to the average of the healthy control cohorts in the control group (the group of people confirmed to have no cancer by physical examination) plus 2 Standard Deviations (SDs).

Fifth, positive and negative judgment of single autoantibody and antigen protein

For each autoantibody and antigen protein assay, positive reaction is defined as quantifying the level of autoantibody or antigen protein in the sample, and then comparing it with a cutoff value, which is not less than the cutoff value, positive; accordingly, a negative response is defined as < cutoff value negative.

Positive determination of autoantibody and/or antigen protein combinations

Since the single autoantibody and/or single antigen protein has a low positive rate, the result of the analysis is combined with a plurality of autoantibodies and/or a plurality of antigen proteins to determine the predictive effect in order to increase the positive rate of detection of the autoantibody and/or antigen protein. The rules are: (1) Detecting a plurality of autoantibodies in a sample, and judging that the combined result of the antibodies is positive as long as one or more autoantibodies show positive; and if all the autoantibodies are negative, the judgment result is negative. (2) Detecting a plurality of antigen proteins in a sample, and judging that the result is positive as long as one or more antigen proteins are positive; and if all antigen proteins are negative, the judgment result is negative. (3) Detecting a plurality of autoantibodies and a plurality of antigen proteins in a sample at the same time, and judging that the result is positive as long as one or more autoantibodies and/or antigen proteins are positive; and if all antibodies and antigen proteins are negative, the judgment result is negative.

(seventh) statistical analysis method

Both groups were statistically analyzed using GraphPad Prism v.6 (GraphPad Prism software, san diego, california) and IBM SPSS Statistics for Windows (IBM, new york) using the Mann-Whitney U test. In analyzing the relationship between each parameter, a Spearman correlation analysis was performed.

Eighth) sensitivity and specificity determination

Sensitivity: among all cases diagnosed with the gold standard, the cases in which the detection results of autoantibodies, autoantibody combinations, antigen proteins, antigen protein combinations, and combinations of autoantibodies and antigen proteins were positive account for the proportion of all cases.

Specificity: among all subjects diagnosed with no disease by gold standard, the subjects whose detection results of autoantibodies, autoantibody combinations, antigen proteins, antigen protein combinations, and combinations of autoantibodies and antigen proteins were negative were the proportion of all subjects.

CEA, CA125, CA199, CA50 and CA153

Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker, can reflect the existence of various tumors to people, and is a better tumor marker for judging the curative effect, disease development, monitoring and prognosis estimation of colorectal cancer, breast cancer and lung cancer, but has limited specificity and sensitivity in application.

CA125 is a tumor marker, a glycoprotein tumor-associated antigen. It is present in the epithelial cells of ovarian tumors and when patients have epithelial ovarian and endometrial cancers, the serum CA125 level can be significantly elevated. However, CA125 also has a positive response to other tumors, such as cervical, breast, pancreatic, cholangiocarcinoma, liver, gastric, colorectal, lung, and some benign lesions.

CA19-9 is a mucin type glycoprotein tumor marker, is a glycolipid on a cell membrane, and is a gastrointestinal tumor-related antigen existing in blood circulation. It has high positive rate in pancreatic cancer, colorectal cancer, gallbladder cancer, cholangiocarcinoma, liver cancer and gastric cancer. The overgrowth is manifested by chronic pancreatitis, cholelithiasis, liver cirrhosis, renal insufficiency, diabetes, etc.

CA50 is a specific glycoprotein antigen secreted by tumor cells. The increase in CA50 is generally seen in breast cancer, but also in other adenocarcinomas such as lung cancer, gastrointestinal tumors, etc. Clinically, it is also seen that some benign diseases, such as gastrointestinal inflammation, can cause a slight increase or transient increase in CA 50. CA153 is a breast cancer related antigen and has certain value for diagnosis and postoperative follow-up of breast cancer. However, other tumors, such as lung cancer, kidney cancer, colon cancer, pancreatic cancer, ovarian cancer, liver cancer, etc., may also be elevated to varying degrees.

Detection method

Based on the elevated levels of autoantibodies in blood, plasma, serum, etc. of colorectal cancer patients, the present invention also provides corresponding methods and methods for diagnosing colorectal morbidity risk.

The autoantibodies provided by the invention comprise 9 autoantibodies selected from the group consisting of: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2; (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (A7) Anti-DAL-1; (A8) Anti-PARP1; (B1) Anti-P53.

The present invention relates to diagnostic assays for quantitative and positional detection of human autoantibody levels. Such tests are well known in the art. The level of autoantibodies detected in the assay can be used to diagnose (including aiding in the diagnosis of) the risk of colorectal cancer occurrence, and/or prognosis of colorectal cancer.

One preferred method is to quantitatively detect autoantibodies.

Preferably, one method of detecting the presence or absence of autoantibodies in a sample is by use of a specific antigen, which comprises: contacting the sample with an antigen protein specific antibody; observing whether an antibody complex is formed, the formation of an antibody complex indicates the presence of autoantibodies in the sample.

The autoantibodies of the invention are useful in the diagnosis of colorectal cancer. The antigen protein of the autoantibody can be immobilized on a protein chip for detecting autoantibody white in a sample.

Based on the studies of the present invention, there was a significant rise in the autoantibody levels of the present invention in colorectal cancer patients. Thus, the autoantibodies of the invention are useful as markers for detecting or diagnosing (especially aiding in the diagnosis and/or early diagnosis) the risk of colorectal cancer occurrence. In the detection, an increased risk of colorectal cancer occurrence is considered if the ratio of the autoantibody level Y1 to the corresponding level Y0 in the normal population (Y1/Y0) is not less than 1.5, preferably not less than 2, more preferably not less than 3.

In addition, the inventors have unexpectedly found that the preferred autoantibody combination of the invention in combination with a tumor autoantigen is effective in improving the detection rate of colorectal cancer.

Detection kit

Based on the correlation of the autoantibodies of the invention with colorectal cancer risk and prognosis, the autoantibodies of the invention can be used as diagnostic markers of colorectal cancer occurrence, and/or as prognostic evaluation markers of colorectal cancer.

The autoantibodies provided by the invention comprise 9 autoantibodies selected from the group consisting of: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2; (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (A7) Anti-DAL-1; (A8) Anti-PARP1 and (B1) Anti-P53.

The invention also provides a kit for diagnosing colorectal cancer, which contains a detection reagent for detecting the autoantibody. Preferably, the kit contains an antigen of the autoantibody of the invention, or an active fragment thereof.

In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used for diagnosing a risk of developing colorectal cancer and/or for assessing a prognosis of colorectal cancer.

The invention has the main advantages that:

(1) Simple and convenient operation, less blood consumption and low cost.

(2) The autoantibody composition provided by the invention is used as a tumor marker in serum or plasma of an early colorectal cancer patient, and has high sensitivity and specificity. The sensitivity in training trains can reach 65.79% and the specificity can reach 86.25%.

(3) After the autoantibody is combined with CEA and CA199, the sensitivity and specificity of the antigen can be obviously improved for rectal cancer or colon cancer. The sensitivity can reach more than 70%, the specificity can reach more than 90%, especially the sensitivity of early colorectal cancer can reach more than 85%, and the specificity is more than 95%.

(4) The fecal detection is mostly aimed at occult blood, but not malignant lesions, and the detection result is easily influenced by factors such as food, so the specificity of the autoantibody combination is stronger than that of fecal self-detection.

(5) The methylation detection has low standardization degree, is greatly influenced by tumor load, has high cost and is not beneficial to large-scale screening; the autoantibody detection method is low in operation difficulty and cost and is easy to develop in primary hospitals.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: the conditions described in the laboratory Manual (NewYork: cold spring harbor laboratory Press, 1989) or according to the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1 sensitivity and specificity detection of Single autoantibodies in training cohorts

This example included 90 healthy physical examination populations and 45 colorectal cancer patients as training cohorts for screening of autoantibody markers. The healthy physical examination population is from not less than 3 different physical examination centers. All colorectal cancer patient serum was collected when the patient was diagnosed as colorectal cancer not receiving any radiotherapy and surgery treatment, and stored in a-80 ℃ refrigerator. Training cohort colorectal cancer patient information is presented in table 1.

TABLE 1 training of colorectal cancer patient characteristics

The colorectal cancer antigen is coated on the surface of a 96-well plate after being expressed and purified, and then reacts with colorectal cancer serum diluted by 1:110 times or serum of a physical examination control group, then reacts with an anti-human IgG antibody-HRP horseradish catalase, then carries out color reaction, and detects with an enzyme-labeled instrument OD450nm wavelength. Table 2 shows the sensitivity and specificity of individual autoantibodies in training cohort serum samples as colorectal cancer markers.

The horizontal distribution scatter plots of the above 9 autoantibodies in the training cohort tumor group and control group are shown in figure 1. Due to the difference of the immune system of tumor patients and the diversity of tumor generation mechanisms, the distribution sensitivity of single tumor autoantibodies in tumor patients is low.

Statistical analysis of the level distribution of autoantibodies in tumor and control groups using Mann Whitney test revealed that the level distribution of antibodies against P53, COPB1, PBRM1 and PSMC1 was significantly different in the training-cohort tumor and control groups (P < 0.05), and that other antibody molecules also had an upward trend in the colorectal cancer group.

Example 2 sensitivity and specificity detection of Single autoantibodies in validation cohorts

This example included another independent group of populations, of which 47 healthy physical examination populations and 45 colorectal cancer patients, were used as a validation cohort for screening for autoantibody markers. The healthy physical examination population is from not less than 3 different physical examination centers.

All colorectal cancer patient serum was collected when the patient was diagnosed as colorectal cancer not receiving any radiotherapy and surgery treatment, and stored in a-80 ℃ refrigerator. Verification cohort colorectal cancer patient information is presented in table 3.

Similarly, colorectal cancer antigens are coated on the surface of a 96-well plate after expression and purification, are subjected to reaction with colorectal cancer serum diluted 1:110 times or serum of a physical examination control group after sealing, are subjected to reaction with anti-human IgG antibody-HRP horseradish catalase, and then are subjected to color development reaction, and are detected by using an enzyme-labeled instrument OD450nm wavelength. The sensitivity and specificity of individual autoantibodies as colorectal cancer markers in pooled serum samples are validated as shown in table 4.

The horizontal distribution scatter plots of the above 9 autoantibodies in the tumor group and control group of the validation cohort are shown in fig. 2. Due to the difference of the immune system of tumor patients and the diversity of tumor generation mechanisms, the distribution sensitivity of single tumor autoantibodies in tumor patients is low.

Statistical analysis of the level distribution of autoantibodies in tumor and control groups using Mann Whitney test found that antibodies against P53, FGFR2, PARP1 and PSMC1 were significantly different in the level distribution of the validation cohort tumor and control groups (P < 0.05), and other antibody molecules also had an upward trend in the colorectal cancer group.

EXAMPLE 3 selection of autoantibody combinations

According to the detection condition of single candidate autoantibodies in a training queue crowd, the invention selects the candidate antibody with the specificity of more than 95 percent, combines the independent positive contribution of the antibody (namely, excludes the candidate molecules with high overlapping positive detection rate) on the premise of ensuring high specificity, furthest ensures that a detection model covers more colorectal cancer patients, forms different autoantibody combinations and uses corresponding detection reagents to detect the colorectal cancer patients. The results are shown in tables 5 and 6.

The sensitivity of colorectal cancer autoantibody combinations is shown in table 5.

It can be seen from the above table that the detection of anti-PARP1 and anti-DAL-1 does not increase the number of detections in the colorectal cancer patient population, and therefore they do not contribute to the molecular combination, so they are excluded from the colorectal cancer autoantibody detection combination.

After that, the present inventors continued to examine the specificity of the non-excluded molecular combinations as shown in table 6.

Based on the principle that the sum of the sensitivity and the specificity of different molecular combinations is maximum, the inventor selects anti-P53+anti-FGFR2+anti-COPB1+anti-PBRM1+anti-PSMC1+anti-PEBP1+anti-ATP4A as the optimal molecular combination, wherein the sensitivity is 53.33% and the specificity is 85.56%.

Example 4 subject working characteristics (ROC) analysis of the autoantibody combinations of the invention in training cohorts

The inventors further analyzed the screening ability of the antibody combination of the invention (anti-p53+ anti-fgfr2+ anti-copb1+ anti-pbrm1+ anti-psmc1+ anti-pebp1+ anti-ATP 4A) in a training queue for colorectal cancer patients using ROC curves.

As a result, as shown in FIG. 3, the sensitivity of the molecular combination of the present invention reached 65.79% at about the maximum value of the dengue index (under ideal conditions) in the case of the healthy physical examination population, the specificity was 86.25%, and the area under the curve was 0.8003.

Example 5 subject working characteristics (ROC) analysis of autoantibody combinations of the invention in validation cohorts

The inventors further analyzed the screening ability of the antibody combination of the invention (anti-p53+ anti-fgfr2+ anti-copb1+ anti-pbrm1+ anti-psmc1+ anti-pebp1+ anti-ATP 4A) in a validation cohort for colorectal cancer patients using ROC curves.

As a result, as shown in FIG. 4, the sensitivity of the molecular combination of the present invention reached 53.66% at about the maximum value of the dengue index (under ideal conditions), at which time the specificity was 88.37% and the area under the curve was 0.7856, as compared with the healthy physical examination population.

EXAMPLE 6 screening of patients with colorectal cancer in combination with the clinically common tumor antigen markers CEA and CA199 autoantibodies

The biomarkers related to colorectal cancer which are clinically adopted at present are CEA and CA199, and the inventor of the invention also detects the two tumor antigen markers for patient groups in a verification queue. And combining the two antigen markers with the established antibody combination to establish a colorectal cancer detection model of the antibody and antigen collocation.

As shown in Table 7, the sensitivity of colorectal cancer screening was further improved after CEA (threshold 5. Mu.g/L) and CA199 (threshold 40 kU/L) were combined with the antibodies.

The inventors further performed ROC analysis on the antibody and antigen combination (anti-p53+anti-fgfr2+anti-copb1+anti-pbrm1+anti-psmc1+anti-pebp1+anti-atp4a+cea+ca 199) in the validation cohort.

The results are shown in FIG. 5. In the case of antigen-antibody binding assays, the sensitivity of the molecule combination of the invention reached 70.00% at about the maximum value of the dengue index (under ideal conditions), with a specificity of 90.70% and an area under the curve of 0.8814, as compared to healthy physical examination populations. This further improves the screening ability of the colorectal cancer detection model of the present invention to patients.

Example 7 analysis of the ability of the antibody and antigen Combined detection model of the invention to detect intestinal cancer at different locations

The inventors of the present invention classified the incidence of intestinal cancer in the subject patient, and classified them into colon cancer and rectal cancer. For different types of patients, their serum test data were analyzed. The antibody and antigen combined detection model (anti-P53+anti-FGFR2+anti-COPB1+anti-PBRM 1+

anti-PSMC1+anti-PEBP 1+anti-ATPase+CEA+CA 199) and analyzed for their ability to detect colon and rectum cancer.

As shown in the results of FIGS. 6-7, the detection sensitivity of the detection model of the present invention for colon cancer was 73.33% at the maximum about Density index, the specificity was 91.25%, and the area under the curve was 0.8694; the sensitivity of detection for rectal cancer was 77.50%, the specificity at this time was 93.75%, and the area under the curve was 0.8681.

From the ROC analysis results, the detection model has similar colorectal cancer detection capability on different positions, and has no obvious preference on colorectal cancer detection on different positions.

Example 8 the antibody and antigen combination test model of the present invention has ideal detection ability for early colorectal cancer

The inventors of the present invention classified colorectal cancer pathological stage of the subject patients, which included 33 early (stage I and stage II) patients. Aiming at the serum detection data of the human body, the antibody and antigen combined detection model (anti-P53+anti-FGFR2+anti-COPB1+anti-PBRM1+anti-PSMC 1+

anti-PEBP1+ anti-ATPase + CEA + CA 199) and its ability to detect early colorectal cancer was analyzed.

As a result, as shown in FIG. 8, the detection sensitivity of the detection model of the present invention to early colorectal cancer was 88.89% at the maximum about dengue index, the specificity was 97.30%, and the area under the curve was 0.9505.

From the results of the ROC analysis described above, it can be seen that the detection model of the present invention has an ideal detection capability for early colorectal cancer.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. Use of an autoantibody diagnostic reagent for the preparation of a diagnostic reagent or kit for (a) diagnosing the risk of developing colorectal cancer; and/or (b) prognostic evaluation of colorectal cancer;

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2.

2. The use of claim 1, wherein the autoantibody further comprises any antibody selected from the group consisting of: (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (A7) Anti-DAL-1; (A8) Anti-PARP1.

3. The use of claim 1, wherein the autoantibody further comprises: (B1) Anti-P53.

4. The use of claim 1, wherein the autoantibody comprises a combination of: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2; (A4) Anti-COPB1; (A5) Anti-PEBP1 (A6) Anti-ATP4A; and (B1) Anti-P53.

5. A kit comprising a detection reagent for detecting an autoantibody,

wherein the autoantibody comprises a combination of:

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2.

6. A method of detection comprising the steps of:

(a) Providing a detection sample;

(c) Comparing the autoantibody level to a control reference value Y0;

wherein said autoantibody to the target antigen comprises the following combination:

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2;

7. The assay of claim 6, wherein the test sample is selected from the group consisting of: whole blood, serum, plasma, or a combination thereof.

8. A colorectal cancer risk judging apparatus, characterized in that the apparatus comprises:

wherein said autoantibody comprises the following combination:

(A1) Anti-PSMC1, (A2) Anti-PBRM1 and (A3) Anti-FGFR2;

(b) The processing module compares the input autoantibody level Y1 with a control reference value Y0 so as to obtain a judging result, wherein when the comparing result meets the judging condition, the object colorectal cancer risk is prompted to be high; otherwise, the subject is prompted that the colorectal cancer risk is not high;

(c) And the output module is used for outputting the judging result.

9. Use of a diagnostic reagent of an autoantibody-antigen combination for the preparation of a diagnostic reagent or kit for the combined diagnosis of the risk of occurrence of colorectal cancer;

wherein the autoantibody-antigen combination comprises:

an autoantibody selected from the group consisting of:

(H3) Autoantibodies (B1) Anti-P53; and

a tumor antigen selected from the group consisting of:

10. The use of claim 9, wherein the autoantibody-antigen combination is: (A1) Anti-PSMC1; (A2) Anti-PBRM1; (A3) Anti-FGFR2; (A4) Anti-COPB1; (A5) Anti-PEBP1; (A6) Anti-ATP4A; (B1) Anti-P53; (D1) CEA and (D2) CA199.