A method and kit for detecting RECQL4 truncated mutations based on dual-antibody immunohistochemistry

Abstract

This invention discloses a method and kit for detecting RECQL4 truncated mutations based on dual-antibody immunohistochemistry, relating to the field of molecular pathology detection. The method includes the following steps: obtaining hepatocellular carcinoma tissue samples from the subject; performing independent immunohistochemical staining on the tissue samples using a first antibody that specifically recognizes the N-terminal region of the RECQL4 protein and a second antibody that specifically recognizes the C-terminal region of the RECQL4 protein; determining the presence of truncated mutations in RECQL4 based on the colorimetric results of the first and second antibodies. This detection method and kit transform complex gene mutation detection into a protein immunoassay that is easily performed in routine pathology departments, enabling rapid screening and individualized treatment guidance for hyperrecurrent hepatocellular carcinoma patients in a routine clinical setting.

Description

Technical Field

[0001] This invention relates to molecular pathology detection technology, specifically to a method and kit for detecting RECQL4 truncated mutations based on dual-antibody immunohistochemistry. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in my country and globally. Radical surgical resection is currently an important treatment for early-stage HCC, but the postoperative recurrence rate is high, seriously affecting patients' long-term survival. Recent studies have found that some HCC patients experience multifocal recurrence within six months post-surgery, accompanied by portal vein tumor thrombosis and / or distant metastasis, presenting a unique recurrence pattern known as hyperrecurrent disease (HRD). Compared to ordinary recurrent patients, HRD patients have a significantly worse prognosis, suggesting that surgical treatment alone may not be the optimal choice for these patients, and more precise risk assessment and treatment decision-making methods need to be explored.

[0003] To elucidate the molecular mechanisms of HRD, the inventors performed whole-genome sequencing (WGS) on six HRD patients in a previous study, finding that the RECQL4 gene mutation had the highest frequency (mutated in 5 out of 6 cases). RECQL4 is a member of the DNA helicase family, involved in maintaining genomic stability. Functional experiments showed that these mutations were frameshift or premature termination mutations, leading to premature truncation or even complete deletion of the C-terminal domain of the RECQL4 protein, and the truncated mutants exhibited significantly higher oncogenicity than wild-type RECQL4 in vitro and in vivo. Mechanistic studies showed that mutant RECQL4 can promote liver cancer progression by activating the PI3K–AKT–mTOR signaling pathway, while the application of mTOR inhibitors (such as Sirolimus) can significantly inhibit the growth of this type of tumor. Therefore, RECQL4 truncated mutations are not only potential molecular markers but can also serve as therapeutic targets to guide precision intervention.

[0004] Currently, clinical screening for RECQL4 mutations in HCC patients mainly relies on gene sequencing (such as whole exome sequencing or targeted sequencing). Although this method is accurate, it has problems such as high cost, long cycle, high requirements for equipment and personnel skills, and difficulty in popularizing it in routine pathology departments, which limits its application in primary hospitals or large-scale screening.

[0005] In the existing technology, there are several patented solutions related to cancer prognosis prediction or liver disease detection. For example, publication number CN114512184B discloses a method, device, and application for predicting cancer efficacy and prognosis, which mainly analyzes the expression level or mutation status of tumor-related molecular markers and combines clinical information to assess efficacy and prognosis. However, this solution focuses on multi-factor comprehensive modeling and software algorithm analysis, still relies on high-throughput detection methods such as gene sequencing or quantitative PCR, and does not design a visualization detection method for the structural characteristics of RECQL4 truncated mutations. It cannot achieve rapid screening directly on routine pathological sections and lacks a direct correlation with the risk of postoperative hyperrecurrence (HRD).

[0006] For example, publication number CN112996928A discloses a method for detecting liver diseases, which mainly assists in the diagnosis and classification of liver diseases by detecting specific molecular markers or gene expression profiles. Although this method can be used to detect liver diseases, the markers and detection system involved are not designed for RECQL4 truncated mutations, nor does it provide an immunohistochemical detection strategy that can intuitively distinguish between full-length proteins and truncated mutants. Therefore, it cannot effectively identify protein function changes caused by C-terminal deletion and cannot be directly used for HRD risk assessment or to guide surgical / non-surgical treatment decisions.

[0007] Furthermore, existing immunohistochemical (IHC) tests mostly target single antigenic epitopes and cannot directly distinguish between full-length proteins and truncated mutants. Even though a few studies have attempted to detect the expression level of RECQL4, a dual-antibody interpretation system targeting its truncated mutation characteristics has not been established. Therefore, it is difficult to effectively screen high-risk HRD patients in routine pathological practice and to directly guide individualized treatment.

[0008] In summary, there is currently a lack of a simple, low-cost method that provides intuitive results and is highly consistent with gene testing, enabling rapid screening of RECQL4 truncated mutation status in HCC patients in a routine clinical setting, assessing their risk of postoperative hyperrecurrence, and guiding treatment decisions. Summary of the Invention

[0009] The purpose of this invention is to provide a RECQL4 truncated mutation detection method and kit based on dual antibody immunohistochemistry, so as to solve the problems of high cost, complex operation, and difficulty in promotion in routine pathology departments of existing gene sequencing technology, as well as the inability of traditional immunohistochemistry to distinguish between wild-type and mutant proteins.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a RECQL4 truncated mutation detection method based on dual-antibody immunohistochemistry, the detection method being used to screen for the risk of postoperative hyperrecurrence in hepatocellular carcinoma patients, or to guide treatment decisions for hepatocellular carcinoma patients, the detection method comprising the following steps:

[0011] S1: Obtain hepatocellular carcinoma tissue samples from the subject to be tested;

[0012] S2: Tissue samples were subjected to independent immunohistochemical staining using a first antibody that specifically recognizes the N-terminal region of the RECQL4 protein and a second antibody that specifically recognizes the C-terminal region of the RECQL4 protein, respectively.

[0013] S3: Determine whether RECQL4 has a truncation mutation based on the colorimetric results of the primary and secondary antibodies. The criteria for determination are as follows:

[0014] If the N-terminus is positive and the C-terminus is negative, then RECQL4 is determined to have a truncated mutation.

[0015] If both the N-terminus and C-terminus are positive, it is determined to be RECQL4 wild-type expression;

[0016] If both the N-terminus and C-terminus are negative, it is determined that RECQL4 is not expressed or is expressed at low levels.

[0017] Furthermore, the first antibody is an antibody targeting amino acid regions 150-350 of the RECQL4 protein, preferably the antibody with clone number ab188125. This region is located at the N-terminus of the RECQL4 protein and remains stable even after various common frameshift mutations. Selecting an antibody from this region as the capture antibody ensures effective binding and color development in truncated mutant proteins, avoiding false negatives caused by the complete loss of epitopes due to mutations.

[0018] Furthermore, the second antibody targets amino acid regions 853–1208 of the RECQL4 protein, preferably the antibody with clone number 17008-1-AP. This region is located at the C-terminus of the RECQL4 protein. When a frameshift or premature stop codon mutation occurs in the gene, the C-terminal domain of the translated protein is truncated or completely deleted. Therefore, an antibody targeting this region can serve as a reporter antibody to indicate the presence of the complete, full-length RECQL4 protein. If this antibody stains negative, while the N-terminal antibody stains positive, it suggests the presence of a truncated mutation that leads to the loss of the C-terminal domain.

[0019] Further, the immunohistochemical staining steps include: high-temperature, high-pressure antigen retrieval using EDTA (pH 9.0) buffer, blocking with goat serum, and incubation of both the primary and secondary antibodies overnight at 4°C. Specifically, after dewaxing and hydration, paraffin sections are placed in a retrieval chamber containing preheated EDTA (pH 9.0) antigen retrieval solution for high-temperature, high-pressure retrieval to fully expose the RECQL4 protein epitopes. After retrieval, the sections are allowed to cool naturally and washed with PBS. Subsequently, they are blocked at room temperature for 30 minutes with blocking buffer containing 3% goat serum and 0.1% Triton X-100 to block non-specific binding. Diluted antibodies (primary or secondary antibody) are added, and the sections are placed in a humidified chamber and incubated overnight at 4°C (approximately 12–16 hours). The next day, after thawing, a standard three-step or polymer method is used for secondary antibody incubation, chromogenic development (e.g., DAB), hematoxylin counterstaining, dehydration, clearing, and mounting. This procedure maximizes the balance between staining signal intensity and background noise.

[0020] Furthermore, the tissue samples were formalin-fixed and paraffin-embedded tissue sections.

[0021] Furthermore, the tissue samples are obtained through preoperative biopsy or postoperative surgical excision. Testing can be completed using small pieces of tissue obtained through preoperative biopsy, eliminating the need to wait for large specimens from surgical excision. This allows clinicians to obtain molecular information about whether a patient has a high risk of HRD (RECQL4 truncated mutation) before developing an initial treatment plan, enabling more precise individualized treatment decisions. For example, postoperative adjuvant therapy can be considered for high-risk patients, or non-surgical treatment options can be directly chosen, avoiding unnecessary surgical trauma and rapid postoperative recurrence.

[0022] Furthermore, the kit includes the aforementioned primary and secondary antibodies, as well as auxiliary reagents for immunohistochemical staining. These auxiliary reagents include, but are not limited to: antigen retrieval solution, blocking solution, antibody dilution solution, washing buffer (such as PBS), HRP-labeled secondary antibody, chromogenic substrate (such as DAB), hematoxylin staining solution, and mounting medium.

[0023] Furthermore, the kit includes a result interpretation diagram.

[0024] Furthermore, the kit also includes instructions, which specify sample processing methods, immunohistochemical staining procedures, result interpretation criteria, the association between RECQL4 truncated mutations and hepatocellular carcinoma hyperrecurrence, and recommendations for guiding treatment decisions based on test results, including whether surgical treatment or combined treatment with mTOR inhibitors is appropriate.

[0025] Compared with existing technologies, the present invention provides a RECQL4 truncated mutation detection method and kit based on dual-antibody immunohistochemistry. This method can intuitively and quickly determine whether RECQL4 has truncated mutations by recognizing the dual-antibody immunohistochemical staining pattern of the N-terminal and C-terminal domains of the RECQL4 protein in a routine pathological environment, without relying on expensive, complex and time-consuming gene sequencing. This method has both high sensitivity and high specificity, and can accurately screen the risk of postoperative hyperrecurrence (HRD) in hepatocellular carcinoma patients and guide individualized treatment decisions (such as avoiding simple surgical treatment or combining mTOR inhibitors). This overcomes the shortcomings of existing gene testing methods, such as poor accessibility and the inability of existing immunohistochemical protocols to distinguish between full-length and truncated mutants, and significantly improves clinical feasibility. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0027] Figure 1 This is a schematic diagram of the typical imaging features of hyperrecurrent HRD hepatocellular carcinoma patients targeted by this invention.

[0028] Figure 2 This is a survival curve comparison chart showing the prognostic differences between HRD patients and ordinary relapse patients involved in this invention.

[0029] Figure 3 This is a diagram showing the verification results of high-frequency RECQL4 gene mutations in HRD patients discovered by the present invention using whole-genome sequencing technology;

[0030] Figure 4 A comparative diagram of the gene structure patterns of the wild-type and truncated mutant RECQL4 proteins constructed in this invention;

[0031] Figure 5 This invention presents experimental results, validated in animal models, demonstrating the sensitivity of tumors carrying RECQL4 truncated mutations to mTOR inhibitor (Sirolimus) treatment. Figure 1 ;

[0032] Figure 6 This invention presents experimental results, validated in animal models, demonstrating the sensitivity of tumors carrying RECQL4 truncated mutations to mTOR inhibitor (Sirolimus) treatment. Figure 2 ;

[0033] Figure 7This is a schematic diagram of the immunohistochemical staining pattern interpretation criteria based on the N-terminal and C-terminal antibodies of the RECQL4 protein according to the present invention.

[0034] Figure 8 This is a graph showing the consistency verification and statistical analysis between the immunohistochemical detection method and gene sequencing results of this invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0036] This invention provides a method for detecting RECQL4 truncated mutations based on dual-antibody immunohistochemistry. The detection method includes the following steps:

[0037] S1: Obtain hepatocellular carcinoma tissue samples from the subject of the test. The tissue samples are formalin-fixed and paraffin-embedded tissue sections.

[0038] S2: Tissue samples were subjected to independent immunohistochemical staining using a first antibody that specifically recognizes the N-terminal region of the RECQL4 protein and a second antibody that specifically recognizes the C-terminal region of the RECQL4 protein, respectively.

[0039] S3: Determine whether RECQL4 has a truncation mutation based on the colorimetric results of the primary and secondary antibodies. The criteria for determination are as follows:

[0040] If the N-terminus is positive and the C-terminus is negative, then RECQL4 is determined to have a truncated mutation.

[0041] If both the N-terminus and C-terminus are positive, it is determined to be RECQL4 wild-type expression;

[0042] If both the N-terminus and C-terminus are negative, it is determined that RECQL4 is not expressed or is expressed at low levels.

[0043] 1. In one embodiment of the present invention, the first antibody is an antibody targeting amino acid regions 150-350 of the RECQL4 protein, preferably the antibody with clone number ab188125. This region is located at the N-terminus of the RECQL4 protein and remains stable even after various common frameshift mutations. Selecting an antibody targeting this region as a capture antibody ensures effective binding and color development in truncated mutant proteins, avoiding false negatives caused by complete loss of epitopes due to mutations.

[0044] 2. In one embodiment of the present invention, the second antibody is an antibody targeting amino acid regions 853-1208 of the RECQL4 protein, preferably the antibody with clone number 17008-1-AP. This region is located at the C-terminus of the RECQL4 protein. When a frameshift or premature stop codon mutation occurs in the gene, the C-terminal domain of the translated protein is truncated or completely deleted. Therefore, an antibody targeting this region can serve as a reporter antibody to indicate the presence of the complete, full-length RECQL4 protein. If this antibody staining is negative, while the N-terminal antibody staining is positive, it indicates the presence of a truncated mutation that leads to the loss of the C-terminal domain.

[0045] 3. In one embodiment of the present invention, the immunohistochemical staining steps include: high-temperature and high-pressure antigen retrieval using EDTA (pH 9.0) buffer, blocking with goat serum, and incubation of both the first and second antibodies overnight at 4°C. Specifically, the steps include: after dewaxing and hydration, paraffin sections are placed in a retrieval chamber containing preheated EDTA (pH 9.0) antigen retrieval solution for high-temperature and high-pressure retrieval to fully expose the epitopes of the RECQL4 protein. After retrieval, the sections are allowed to cool naturally and washed with PBS. Subsequently, they are blocked at room temperature for 30 minutes with blocking solution containing 3% goat serum and 0.1% Triton X-100 to block non-specific binding. Diluted antibodies (first or second antibody) are added, and the sections are placed in a humidified chamber and incubated overnight at 4°C (approximately 12-16 hours). The next day, after warming, the sections are incubated with secondary antibody, developed with a chromogenic agent (such as DAB), counterstained with hematoxylin, dehydrated, cleared, and mounted using a standard three-step method or polymer method. This process can maximize the balance between staining signal intensity and background noise.

[0046] 4. In one embodiment of the present invention, the tissue sample is a tissue specimen obtained through preoperative puncture biopsy or postoperative surgical resection. Testing can be completed using a small piece of tissue obtained through preoperative puncture biopsy, eliminating the need to wait for a large specimen from surgical resection. This allows clinicians to obtain molecular information about whether a patient has a high risk of HRD (RECQL4 truncated mutation) before developing an initial treatment plan, enabling more precise individualized treatment decisions. For example, postoperative adjuvant therapy can be considered for high-risk patients, or non-surgical treatment options can be directly selected, avoiding unnecessary surgical trauma and rapid postoperative recurrence.

[0047] This invention provides a RECQL4 truncated mutation detection kit based on dual-antibody immunohistochemistry. The kit includes the aforementioned first and second antibodies, as well as auxiliary reagents for immunohistochemical staining. Auxiliary reagents include, but are not limited to: antigen retrieval solution, blocking solution, antibody dilution solution, washing buffer (such as PBS), HRP-labeled secondary antibody, chromogenic substrate (such as DAB), hematoxylin staining solution, and mounting medium.

[0048] 5. In one embodiment of the present invention, the kit includes a result interpretation diagram. This diagram is an immunohistochemical staining reference illustration printed on a card included with the kit packaging, and the illustration includes at least:

[0049] RECQL4 truncated mutation positive staining image: A typical field of view showing tumor cells in hepatocellular carcinoma tissue that are positive for N-terminal antibodies but negative for C-terminal antibodies;

[0050] RECQL4 wild-type staining image: A typical field of view showing that both the N-terminus and C-terminus of tumor cells are positive for antibodies;

[0051] RECQL4 non-expression / low expression staining pattern: A typical field of view showing that both N-terminal and C-terminal antibodies of tumor cells are negative;

[0052] Negative control staining image: shows tissue areas that show only background staining or no specific staining.

[0053] 6. In one embodiment of the present invention, the kit further includes an instruction manual, which specifies the sample processing method, immunohistochemical staining procedure, result interpretation criteria, the association between RECQL4 truncated mutations and hepatocellular carcinoma hyperrecurrence, and recommendations for guiding treatment decisions based on the test results, including whether surgical treatment or combined treatment with mTOR inhibitors is appropriate.

[0054] As attached Figure 1 Appendix Figure 2 As shown:

[0055] Example 1: Clinical characteristics and prognostic verification of HRD patients:

[0056] A cohort of patients with pathologically confirmed hepatocellular carcinoma was selected, and their preoperative, 1-month postoperative, and 4-month postoperative MRI imaging data and follow-up data were collected. (See attached...) Figure 1 As shown, the HRD patient presented with a solitary tumor in the right lobe of the liver preoperatively (top image); MRI one month postoperatively showed complete tumor resection (middle image); and multifocal recurrence with tumor thrombus occurred four months postoperatively (bottom image). Kaplan-Meier survival curves were plotted by comparing survival data from a non-HRD patient cohort (see attached image). Figure 2 The results showed that the median survival of patients with HRD was significantly shorter than that of non-HRD patients (Log-rank test P<0.01), confirming that HRD is an independent risk factor for extremely poor postoperative prognosis in liver cancer.

[0057] As attached Figure 3 Appendix Figure 4 As shown:

[0058] Example 2: RECQL4 Mutation Spectrum and Functional Validation:

[0059] Whole-genome sequencing (WGS) was performed on tumor tissues from six HRD patients to analyze significantly mutated genes. (See attached image.) Figure 3 As shown, RECQL4 is the gene with the highest mutation frequency (the horizontal axis represents the number of mutations, and the vertical axis represents the gene name). Further cloning and sequencing revealed the types of RECQL4 mutations (see attached diagram). Figure 4 We constructed wild-type and mutant (e.g., G822fs and R962f mutant) RECQL4 expression vectors. In vitro functional experiments showed that mutant RECQL4 significantly enhanced the proliferation and invasion of liver cancer cells (P<0.05), verifying its strong oncogenicity.

[0060] As attached Figure 5 Appendix Figure 6 As shown:

[0061] Example 3: Validation of the sensitivity of mutant RECQL4 to mTOR inhibitors:

[0062] Hepatocellular carcinoma (HCC) cancer cell lines were constructed by knocking down endogenous RECQL4 and then overexpressing wild-type or mutant RECQL4. These lines were then subcutaneously inoculated into nude mice to create xenograft models. After tumor formation, mice were randomly divided into two groups, receiving either the mTOR inhibitor Sirolimus or PBS (5 doses) as a control. (See attached image.) Figure 5 Appendix Figure 6 As shown in the figure, the left-hand statistical graph shows that the tumor weight in the Sirolimus group was significantly lower than that in the PBS group (P<0.01); the right-hand statistical graph shows that the shrinkage rate of tumors with mutated RECQL4 was significantly higher in Sirolimus than in wild-type tumors (P<0.05), suggesting that RECQL4 truncated mutations can serve as a predictive biomarker for the efficacy of mTOR inhibitors.

[0063] As attached Figure 7 Appendix Figure 8 As shown:

[0064] Example 4: Establishment and Validation of the Double Antibody IHC Method:

[0065] Design a dual-antibody immunohistochemical detection protocol targeting the N-terminus (antibody ab188125) and C-terminus (antibody 17008-1-AP) of RECQL4. (See attached diagram) Figure 7 As shown, the horizontal axis represents the three staining results (N⁻ / C⁻, N⁺ / C⁻, N⁺ / C⁺), and the vertical axis represents the staining intensity of the two antibodies. IHC was performed on 32 liver cancer tissue samples, and the results were compared with those from targeted sequencing. (See attached image.) Figure 8 As shown, the consistency between IHC and sequencing results reached 97% (Kappa value = 0.92, P < 0.001), proving that the dual-antibody IHC method can replace gene sequencing and accurately identify RECQL4 truncated mutations.

[0066] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for detecting RECQL4 truncated mutations based on dual-antibody immunohistochemistry, wherein the method is used to screen for the risk of postoperative hyperrecurrence in hepatocellular carcinoma patients, or to guide treatment decisions for hepatocellular carcinoma patients, characterized in that... The detection method includes the following steps: S1: Obtain hepatocellular carcinoma tissue samples from the subject to be tested; S2: Tissue samples were subjected to independent immunohistochemical staining using a first antibody that specifically recognizes the N-terminal region of the RECQL4 protein and a second antibody that specifically recognizes the C-terminal region of the RECQL4 protein, respectively. S3: Determine whether RECQL4 has a truncation mutation based on the colorimetric results of the primary and secondary antibodies. The criteria for determination are as follows: If the N-terminus is positive and the C-terminus is negative, then RECQL4 is determined to have a truncated mutation. If both the N-terminus and C-terminus are positive, it is determined to be RECQL4 wild-type expression; If both the N-terminus and C-terminus are negative, it is determined that RECQL4 is not expressed or is expressed at low levels.

2. The RECQL4 truncated mutation detection method based on dual-antibody immunohistochemistry according to claim 1, characterized in that, The first antibody is an antibody that targets the 150-350 amino acid region of the RECQL4 protein.

3. The RECQL4 truncated mutation detection method based on dual-antibody immunohistochemistry according to claim 1, characterized in that, The second antibody is an antibody that targets amino acid regions 853-1208 of the RECQL4 protein.

4. The RECQL4 truncated mutation detection method based on dual-antibody immunohistochemistry according to claim 1, characterized in that, The immunohistochemical staining steps include: high-temperature and high-pressure antigen retrieval using EDTA buffer, blocking with goat serum, and incubating both the first and second antibodies overnight at 4°C.

5. The RECQL4 truncated mutation detection method based on dual-antibody immunohistochemistry according to claim 1, characterized in that, The tissue sample was a formalin-fixed, paraffin-embedded tissue section.

6. The RECQL4 truncated mutation detection method based on dual-antibody immunohistochemistry according to claim 1, characterized in that, The tissue samples are obtained through preoperative puncture biopsy or postoperative surgical excision.

7. A RECQL4 truncated mutation detection kit based on dual-antibody immunohistochemistry, characterized in that, The kit includes the first antibody and the second antibody as described in any one of claims 1-3, as well as auxiliary reagents for immunohistochemical staining.

8. The RECQL4 truncated mutation detection kit based on dual antibody immunohistochemistry according to claim 7, characterized in that, The kit includes an instruction manual that specifies sample processing methods, immunohistochemical staining procedures, result interpretation criteria, the association between RECQL4 truncated mutations and hepatocellular carcinoma hyperrecurrence, and recommendations for guiding treatment decisions based on test results, including whether surgical treatment or combined treatment with mTOR inhibitors is appropriate.

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