CN117604110B - Biomarker for breast cancer diagnosis and prognosis and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to a biomarker for diagnosing and prognosis judging breast cancer and application thereof. More specifically, the present invention relates to a biomarker for diagnosis and prognosis of breast cancer, in particular HER2 low expressing breast cancer, said biomarker being SHCBP1. The inventors have found that SHCBP1 is expressed at significantly higher levels in breast cancer patients than in healthy humans. More particularly, the inventors have found that high levels of expression of SHCBP1 are dependent on the expression of HER2 in breast cancer patients, even low levels of expressed HER2 may cause high levels of expression of SHCBP1. High levels of SHCBP1 expression can therefore be used as markers for HER2 expression for diagnosis of HER2 low expressing breast cancer.

Description

Biomarker for breast cancer diagnosis and prognosis and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a biomarker for diagnosing and prognosis judging breast cancer and application thereof.

Background

Improving the accuracy and the high efficiency of breast cancer treatment has become the medical target of the new era. HER2 is an important target for accurate treatment of breast cancer, and recently, with rapid development of targeted drugs and novel Antibody Drug Conjugates (ADCs), HER2 low expression and heterogeneity are hot spot problems of current interest.

The expression of HER2 in breast cancer is divided into HER2 negative (HER 2-) and HER2 positive (her2+), with hr+her2-breast cancer being the most common breast cancer subtype, by using Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH). IHC results from HER2 detection were classified into IHC 0, IHC1+, IHC2+ and IHC3+ according to the 2018 ASCO/CAP guidelines, based on staining intensity and percentage of intact cell membrane staining number; wherein IHC 0 represents an incomplete cell membrane staining without staining or weakly staining and the stained tumor cells are 10% or less; ihc1+ represents weak stained incomplete cell membrane staining and stained tumor cells are greater than 10%; IHC2+ represents weak to moderate intact cell membrane staining, and stained tumor cells are greater than 10%; ihc3+ represents highly intact cell membrane staining, and stained tumor cells are greater than 10%. HER2 positive includes ihc2+ and fish+ and ihc3+, HER2 negative includes IHC 0 or ihc1+ or ihc2+ and FISH negative. Whereas HER2 negative can be further divided into HER2low and HER2-0 based on IHC and FISH detection results. HER2low is defined as: IHC1+ or IHC2+ and FISH negative; HER2-0 is defined as: IHC 0.

Breast cancer HER2 positivity accounts for approximately 15-20% of all breast cancer cases. However, as the intensive research studies have found that HER2 expression levels are a continuous process, even in HER2 ihc3+ patients, the mRNA levels may be quite different. Thus, HER2 alone is positive or negative and does not meet clinical treatment requirements. With the rapid development of ADC drugs, the definition of HER2 low expression is proposed, i.e. HER2 ihc1+ or 2+ and no HER2 amplification. If the breast cancers are proportioned, the breast cancers of HER2 are not really detected, and about 30-40% of the total breast cancers are left, and about 45-55% of the breast cancers are left, which is called 'HER 2 low expression breast cancers'.

The american pathologist association (CAP) uses a tissue chip tableting method to statistically analyze the results of HER2 expression compartment interstitial control in 2019-2020, which shows that the HER2 positive or negative determination consistency is as high as 90% or more, but the consistency between 0 and 1+ is less than 70%. Subsequently, 18 pathologists with more than 5 years of working experience performed HER2 expression interpretation on 170 scan slices, with 0 and 1+ being only 26% identical, far less than 2+ and 3+58% identical. The results of the study demonstrate that pathologists still have identifying deficiencies in HER2 low expression, especially the accurate differentiation of 1+ from 0.

Therefore, although ADC drugs have made significant progress in the field of breast cancer treatment targeting HER2, there is still a worrying hidden danger behind it, and in-study patients, HER2 detection follows a dichotomy of HER2 expression, and no specific detection mode and standard are proposed for HER2 low expression. Thus, when the detection method does not exactly match a potentially benefited patient, it may result in some of the patients who would benefit losing medication opportunities (false negatives) or in poor benefited patients taking medication (false positives).

Thus, there is an urgent need in the art for methods and methods for HER2 low expression detection that are effective in improving the consistency of HER2 low expression interpretation.

Circulating tumor cells are a subset of tumor cells that shed from a primary tumor or metastatic tumor and are released into the blood circulation. Recent studies have found that, on the one hand, circulating tumor cells may appear in the peripheral blood of patients very early in tumorigenesis, which aids in early diagnosis of cancer. On the other hand, these circulating tumor cells can also be used to predict prognosis in cancer patients, and the discovery of circulating tumor cells often predicts recurrence or metastasis of a tumor, which also suggests poor prognosis in patients. How to use circulating tumor cells for diagnosis or prognosis of cancer, especially specific cancers such as breast cancer, is also an important direction in our future in the search of circulating tumor cell lines. A great benefit of using circulating tumor cells for diagnosis or prognosis is that it can effectively replace tumor biopsies, which is a good surrogate indicator for those patients who cannot take a pathological tissue biopsy, and can help clinicians to dynamically monitor and determine the biological characteristics of cancer in real time. However, due to the scarcity of circulating tumor cells, the use thereof as a means of diagnosing cancer, particularly specific cancers such as breast cancer, presents challenges, and not all cancer-related markers can be detected in circulating tumor cells.

Therefore, it is of great clinical value to find biomarkers suitable for diagnosis by means of circulating tumor cells.

Disclosure of Invention

To solve the above problems, the present inventors have found that SHCBP a has significantly higher expression levels in breast cancer patients than in healthy people. More particularly, the inventors have found that high levels of expression of SHCBP1 are dependent on the expression of HER2 in breast cancer patients, even low levels of expressed HER2 may cause high levels of expression of SHCBP 1. High levels of SHCBP1 expression can therefore be used as markers for HER2 expression for diagnosis of HER2 low expressing breast cancer.

Accordingly, the present invention provides a biomarker for diagnosis of breast cancer, wherein the breast cancer is HER2 low expressing breast cancer and the biomarker is SHCBP1.

In other aspects, the invention also provides a biomarker for prognosis of breast cancer, wherein the breast cancer is HER2 low expressing breast cancer and the biomarker is SHCBP a 1.

In other aspects, the invention also provides the use of an agent that detects SHCBP a expression in the manufacture of a tool for diagnosis of breast cancer, wherein the breast cancer is HER2 low expressing breast cancer.

In other aspects, the invention also provides the use of an agent that detects SHCBP a expression in the manufacture of a tool for prognosis determination of breast cancer, wherein the breast cancer is HER2 low expressing breast cancer.

Further, the diagnosis of breast cancer comprises the steps of:

(1) Collecting a sample of a test subject, and collecting a control sample;

(2) Detecting and comparing the expression level of SHCBP1 in the sample of the test subject and the control sample;

(3) Detecting the expression level of HER2 in a sample of a test subject by In Situ Hybridization (ISH);

If step (3) ISH detection is negative and the expression level of SHCBP1 in the sample of the test subject is increased compared to the expression level of SHCBP1 in the control sample, diagnosing that the test subject has HER 2-underexpressing breast cancer.

Further, the control sample is derived from healthy tissue of a healthy population or test subject.

Further, the prognosis of breast cancer comprises the following steps:

(1) Collecting samples of a pre-breast cancer patient as a group to be tested, and taking the samples of the pre-breast cancer patient as a control group;

(2) Detecting and comparing the expression level of SHCBP1 in the samples of the test group and the control group;

If the expression level of SHCBP1 in the test group sample is lower than the expression level of SHCBP1 in the control group sample, the prognosis of the test group is judged to be good.

As used herein, the subject includes a mammal, preferably a primate mammal, more preferably a human.

As used herein, a sample of the test subject includes a clinical biological sample of the subject, including, but not limited to, one or more of serum, plasma, whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, tumor tissue, biopsy samples, circulating tumor cells, circulating tumor DNA, or urine shed cells. In a preferred embodiment, the sample of the test subject comprises breast tissue of the test subject, such as a breast biopsy sample, and the control sample is derived from breast tissue of a healthy subject, such as a breast biopsy sample, or healthy tissue of the test subject, such as a paracancerous tissue. In a preferred embodiment, the sample of the test subject is a circulating tumor cell.

As used herein, the samples of the prognostic and pre-breast cancer patients include clinical biological samples of the subject, including, but not limited to, one or more of serum, plasma, whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, tumor tissue, biopsy samples, circulating tumor cells, circulating tumor DNA, or urine shed cells. In a preferred embodiment, the sample of the prognostic and pre-breast cancer patient comprises breast tissue of the subject to be tested, such as a breast biopsy sample. In a preferred embodiment, the sample of the prognostic and pre-breast cancer patient is circulating tumor cells.

As used herein, the reagent for detecting the expression of SHCBP1 in a sample of a test subject is not particularly limited and is a reagent for detecting the expression of SHCBP1 at the mRNA or protein level in a sample of a subject, which is well known and readily available to those skilled in the art. For example, reagents for detecting expression of SHCBP1 in a subject sample may include corresponding reagents for real-time fluorescent quantitative PCR, enzyme-linked immunosorbent assay (ELISA), protein/peptide fragment chip detection, chemiluminescence, immunoblotting, microbead immunodetection, microfluidic immunodetection.

The beneficial effects of the invention are that

According to the invention, SHCBP is selected as a diagnosis and prognosis marker of HER2 low-expression breast cancer, so that the problem of poor judgment consistency rate of HER2 IHC 0 and 1+ in the prior art is perfectly solved. The inventors have found that there is a certain correlation between the increase in SHCBP1 expression level and HER2 expression, and from the results of example 2 of the present invention, it is clear that the expression level of SHCBP1 is significantly increased even at lower HER2 expression levels, and therefore the expression level of SHCBP1 is very suitable as a measure of low HER2 expression levels. Thus, the present inventors have found that it is possible to determine that a subject is HER 2-low expressing breast cancer, as long as an increase in SHCBP1 expression level is detected and the ISH detection result of HER2 is negative, which has not been found by the past studies. In addition, the present invention also finds that diagnosis and prognosis of breast cancer can be performed by harvesting circulating tumor cells from a subject and detecting the expression level SHCBP therein.

Drawings

Figure 1 shows high expression of SHCBP1 in HER 2-low expressing breast cancer and HER 2-positive breast cancer tissue samples (part a in figure 1 is at the mRNA level and part B in figure 1 is at the protein level).

Figure 2 shows high expression of SHCBP1 in circulating tumor cells of HER 2-low expressing breast cancer and HER 2-positive breast cancer patients.

Figure 3 shows the verification of HER2 expression levels in T47D cells, ZR-75-1 cells and BT474 cells and changes in HER2 expression levels after HER2 knockout.

FIG. 4 shows the change in expression levels of SHCBP1 before and after HER2 knockout in T47D cells, ZR-75-1 cells, and BT474 cells.

FIG. 5 shows the results of a test of SHCBP expression versus tumor size.

Figure 6 shows the test results of SHCBP high expression versus pathological grading of HER2 low expressing breast cancer patients.

Fig. 7 shows the test results of SHCBP for high expression versus HER2 low expression breast cancer metastasis.

Figure 8 shows the test results of SHCBP on the level of expression versus overall survival of HER 2-low expressing breast cancer patients.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Example 1: SHCBP1 high expression in breast cancer

Clinical 138 breast cancer tissue samples and 24 paracancestral normal tissue samples are collected, tissue chips are made, HER2 expression levels in the breast cancer tissue samples are detected through In Situ Hybridization (ISH), and typing is carried out according to the HER2 expression levels, so that 50 HER2 IHC0+ breast cancer tissue samples, 58 HER2 IHC1+ or IHC2+ and ISH negative breast cancer tissue samples (namely, so-called HER2 low-expression breast cancer) and 30 HER2 positive (namely, IHC2+ and ISH positive and IHC3+) breast cancer tissue samples are obtained.

MRNA and protein levels of SHCBP were measured by RT-qPCR and Western Blot methods, respectively, using TRIzol (15596018, invitrogen) to extract mRNA and protein from the above-described breast cancer tissue sample and paracancerous normal tissue sample, respectively. The results are shown in fig. 1, which shows that SHCBP1 expression levels were significantly increased in both HER2 ihc1+, 2+ and 3+ breast cancer tissue samples, whereas the increase was not significant in HER2 ihc0+ breast cancer tissue samples, suggesting that SHCBP1 expression levels could be used to distinguish HER2 negative breast cancer from HER2 low expressing breast cancer.

Example 2: detection of SHCBP1 expression levels in circulating tumor cells of breast cancer patients

1) Respectively extracting 10mL of venous blood of HER2 IHC0+ breast cancer, HER2 IHC1+ or IHC2+ breast cancer with ISH negative (namely, so-called HER2 low-expression breast cancer) and HER2 positive (namely, IHC2+ breast cancer with ISH positive and IHC3+) breast cancer patients in an ACD anticoagulation tube, and conventionally centrifuging and separating plasma for later use;

2) Enrichment and separation of CTC cells in plasma comprises the following specific steps: extracting a single cell layer in the blood plasma by adding a sample density separating liquid (CYTELLIGEN) into the blood plasma, then adding immune cell removing magnetic beads to remove CD45 ⁺ immune cells in the extracted single cell layer, and concentrating and enriching CTC in the single cell layer by differential enrichment;

3) The enriched CTC cells were harvested by centrifugation and 1ml of RNA lysate was added to the enzyme-free EP tube; 200ul of chloroform is added into an EP tube, vigorously oscillated for 15 seconds, and kept still at room temperature for 3 minutes, and repeated for 3 times; centrifuging at 12000 Xg and 4 ℃ for 15min; adding the upper water phase into a new enzyme-free EP pipe, adding equal volume of isopropanol into the EP pipe, reversing, mixing uniformly, and standing for 10min; centrifuging at 12000 Xg and 4 ℃ for 15min; the EP tube liquid was discarded, 1ml of 75% ethanol was added, and the EP tube was shaken; centrifuging at 12000 Xg and 4 ℃ for 5min; discarding the supernatant, and standing at room temperature for drying; adding a proper amount of DEPC water to dissolve RNA; the purity and concentration of RNA was measured and the expression of SHCBP1 in CTC cells was measured by RT-qPCR and compared to the expression of SHCBP1 in cells harvested from normal pancreatic tissue, as shown in figure 2, which demonstrates that abnormally high expression levels of SHCBP1 can be detected in CTC cells of HER2 ihc1+, 2+ and 3+ breast cancer patients.

Example 3: high expression of SHCBP1 in breast cancer is dependent on HER expression

RNA was extracted using T47D cells (HER 2 negative breast cancer cells), ZR-75-1 cells (HER 2 low expressing breast cancer cells) and BT474 cells (HER 2 positive breast cancer cells), and the HER2 expression level was verified by RT-qPCR and SHCBP expression levels were detected. The results are shown in FIG. 3, which shows that SHCBP a has increased expression levels in both BT474 cells and ZR-75-1 cells compared to T47D cells.

HER2 gene (hHer 2 grna_f: CACCGCGGCACAGACAGTGCGCGTC; hHer 2grna_r: AAACGACGCGCACTGT CTGTGCCGC) was knocked out in BT474 cells and ZR-75-1 cells using CRISPR editing techniques, RNA was extracted, HER2 expression was verified using RT-qPCR and SHCBP1 expression levels were detected. The results are shown in figure 4, which shows that the expression level of SHCBP a is no longer elevated after HER2 knockout, indicating that high expression of SHCBP a in breast cancer is dependent on HER expression, and thus the expression level of SHCBP a can reflect HER expression levels and can be used to diagnose HER low expressing breast cancer.

As can be seen from a comparison of the results of fig. 3 and fig. 4, the expression level of SHCBP1 was significantly increased even at lower HER2 expression levels, and thus the expression level of SHCBP1 was very suitable as a measure of low HER2 expression levels.

Example 4: SHCBP1 relation to clinical prognosis of breast cancer

Based on SHCBP expression levels determined in example 1, the relationship between SHCBP1 and tumor size, pathological grading and metastasis of HER2 low expressing breast cancer patients was statistically analyzed, and the results are shown in fig. 5 and 6.

FIG. 5 shows the results of a test of the relationship between SHCBP and tumor size, and FIG. 6 shows the results of a test of the relationship between SHCBP high expression and HER2 low expression breast cancer patient pathology, it can be seen that the higher the SHCBP1 expression, the larger the tumor in the patient; it was also found that: in cases of grade I, grade II and grade III of HER2 low expressing breast cancer patients, SHCBP1 expression in the corresponding breast cancer patients showed an increasing trend as grade I, grade II and grade III levels increased, indicating: SHCBP1 and breast cancer patients' tumor size and pathological grading appear to be positively correlated.

The inventors performed statistical analysis based on the presence or absence of metastasis and the number of lymph node metastases in the obtained HER 2-low-expression breast cancer patients, and the results are shown in fig. 7: it can be seen that of the HER2 low expressing breast cancer patients with metastasis, 78.2% exhibited SHCBP1 high expression, and of the HER2 low expressing breast cancer patients without metastasis, 52.4% exhibited SHCBP1 high expression, which also suggests that SHCBP1 high expression and HER2 low expressing breast cancer metastasis may exhibit a high positive correlation.

Finally, the inventors analyzed the relationship between the level of SHCBP1 expression and the overall survival of patients with HER 2-low expressing breast cancer. As shown in fig. 8, it can be seen that the overall survival rate was significantly lower for five years in the SHCBP1 High (SHCBP 1 High) HER2 Low expressing breast cancer patient group than in the SHCBP1 Low expressing (SHCBP Low) breast cancer patient group. This illustrates: SHCBP1 high expression results in a poor prognosis for patients with HER2 low expression breast cancer.

It should be noted that while the present invention has been illustrated in the drawings and described in connection with the preferred embodiments thereof, it is to be understood that the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but are to be construed as providing a full breadth of the disclosure. The above-described features are further combined with each other to form various embodiments not listed above, and are considered to be the scope of the present invention described in the specification; further, modifications and variations of the present invention may be apparent to those skilled in the art in light of the foregoing teachings, and all such modifications and variations are intended to be included within the scope of this invention as defined in the appended claims.

Claims (4)

1. Use of an agent that detects SHCBP1 expression in the manufacture of a tool for diagnosis of breast cancer, wherein the breast cancer is HER2 low expressing breast cancer;

The definition of HER2 low expression is: HER2 ihc1+, or HER2 ihc2+ without HER2 amplification.

2. The use according to claim 1, wherein the diagnosis of breast cancer comprises the steps of:

(1) Collecting a sample of a test subject, and collecting a control sample;

The control sample is derived from healthy tissues of healthy people or a to-be-detected subject, and the sample of the to-be-detected subject is one or more of serum, plasma, whole blood, pus, organs, tumor tissues, circulating tumor cells and circulating tumor DNA;

(2) Detecting and comparing the expression level of SHCBP1 in the sample of the test subject and the control sample;

(3) Detecting the expression level of HER2 in a sample of a test subject by In Situ Hybridization (ISH);

If step (3) ISH detection is negative and the expression level of SHCBP1 in the sample of the test subject is increased compared to the expression level of SHCBP1 in the control sample, diagnosing that the test subject has HER 2-underexpressing breast cancer.

3. Use of an agent that detects SHCBP1 expression in the manufacture of a tool for prognosis of breast cancer, wherein the breast cancer is HER2 low expressing breast cancer;

The definition of HER2 low expression is: HER2 ihc1+, or HER2 ihc2+ without HER2 amplification.

4. The use according to claim 3, wherein the prognosis of breast cancer comprises the steps of:

(1) Collecting samples of a pre-breast cancer patient as a group to be tested, and taking the samples of the pre-breast cancer patient as a control group;

the samples of the prognosis and pre-breast cancer patients are one or more of serum, plasma, whole blood, pus, organs, tumor tissues, circulating tumor cells and circulating tumor DNA;

(2) Detecting and comparing the expression level of SHCBP1 in the samples of the test group and the control group;

If the expression level of SHCBP1 in the test group sample is lower than the expression level of SHCBP1 in the control group sample, the prognosis of the test group is judged to be good.