Application of lymphocyte CD44 in the preparation of products for screening early-stage NSCLC advantage populations for SBRT or surgery

Abstract

The present invention discloses the application of lymphocyte CD44 in the preparation of a product for screening an early-stage NSCLC advantage population to receive SBRT or surgery, and belongs to the field of medicine. The present invention uses puncture tissue of early-stage NSCLC patients before SBRT to perform DSP sequencing analysis, and analyzes the tumor area, leukocyte area, lymphocyte area, macrophage area and matrix area. Based on the patient's recurrence-free survival, overall survival and recurrence situation, an analysis is performed, and only one marker, CD44 in the lymphocyte area, is screened out, which is related to SBRT recurrence and poor survival. Further experiments have drawn the following conclusions: patients with high expression of lymphocyte CD44 receive surgical treatment, and patients with low expression of lymphocyte CD44 receive SBRT. The present invention provides a scientific basis for the optimization of SBRT or surgery for early-stage NSCLC, and has important research value and clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of lymphocyte CD44 in preparing a product for screening an early-stage NSCLC advantage population to receive SBRT or surgery. Background Art

[0002] In recent years, due to the promotion of low-dose computed tomography (CT) cancer screening, the discovery of early non-small cell lung cancer (NSCLC) cases has increased year by year. Lobectomy with mediastinal lymph node dissection is the standard treatment for operable patients, while stereotactic body radiotherapy (SBRT) has become the first choice for patients who are medically inoperable or refuse surgery.

[0003] Even for patients with operable early-stage NSCLC, multiple studies have shown that SBRT is not inferior to surgery. Two single-arm prospective studies of operable patients, RTOG 0618 and JCOG 04013, showed that SBRT had better overall survival (OS) compared with previous surgical results. A pooled analysis of two independent phase III trials, STARS and ROSEL, compared the survival of patients with operable stage I NSCLC with SBRT and surgery, and the results showed that the overall survival rate of SBRT was better than that of lobectomy (3-year overall survival rate 95% vs. 79%, p = 0.037). The long-term results of the revised STARS trial showed that for patients with operable stage IA NSCLC, SBRT was not inferior to surgery in terms of OS and progression-free survival (PFS).

[0004] Given these recent findings, some patients who are operable for SBRT may be better candidates for surgery and some patients with operable early-stage NSCLC (ES-NSCLC) may be better candidates for SBRT. Given the lack of a clear understanding of the biology of this population, it is critical to leverage biology to identify the ES-NSCLC population that may benefit more from specific treatment modalities. Summary of the invention

[0005] The purpose of the present invention is to provide an application of lymphocyte CD44 in the preparation of a product for screening a favorable population of early NSCLC to receive SBRT or surgery, so as to solve the problems existing in the above-mentioned prior art. Lymphocyte CD44 is used as a protein marker, and its expression level is detected to guide clinical decision-making, and screen a favorable population of early NSCLC to receive SBRT or surgery, which has important scientific research value and clinical significance.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of lymphocyte CD44 as a protein marker in preparing a product for screening a preponderance of early non-small cell lung cancer patients for receiving stereotactic radiotherapy or surgery.

[0008] The present invention also provides the use of a detection reagent for detecting the expression level of lymphocyte CD44 in the preparation of a product for screening a predominant population of early non-small cell lung cancer for stereotactic radiotherapy or surgery.

[0009] Preferably, the product comprises a test kit and a DSP chip.

[0010] Preferably, the patients with early non-small cell lung cancer with high expression of lymphocyte CD44 are the advantageous population for receiving surgical treatment, and the patients with early non-small cell lung cancer with low expression of lymphocyte CD44 are the advantageous population for receiving stereotactic radiotherapy.

[0011] The present invention also provides the use of lymphocyte CD44 in screening drugs for reversing stereotactic radiotherapy resistance in patients with early non-small cell lung cancer.

[0012] Preferably, the early stage non-small cell lung cancer patients include patients whose lymphocytes highly express CD44 and who are unable to receive surgical treatment.

[0013] Preferably, the lymphocyte CD44 is CD44 in tumor tissue of patients with early non-small cell lung cancer.

[0014] The present invention discloses the following technical effects:

[0015] The present invention uses digital spatial analysis (DSP) technology to screen protein markers based on the patient's recurrence-free survival (RFS), overall survival and recurrence status. It is found that in early NSCLC patients receiving SBRT treatment, high expression of CD44 in lymphocytes is associated with a higher recurrence rate, worse recurrence-free survival and overall survival. CD44 in the lymphocyte region is the only protein in the intersection of protein markers that can predict recurrence-free survival, overall survival and recurrence status. Further multi-color immunofluorescence verification found that patients with lower expression of CD44 in lymphocytes have longer recurrence-free survival and overall survival after SBRT, while patients with higher expression of CD44 have better recurrence-free survival after surgery, indicating that patients with high expression of lymphocyte CD44 are preferred to receive surgical treatment, and patients with low expression of lymphocyte CD44 are preferred to receive SBRT treatment. The present invention provides a protein-level basis for the treatment selection of early non-small cell lung cancer that is operable.

[0016] The present invention lays a foundation for exploring the molecular mechanism of SBRT resistance caused by lymphocyte CD44, designing drugs targeting this target and action pathway, and reversing SBRT resistance, especially providing a treatment option for patients with high expression of lymphocyte CD44 who cannot undergo surgical treatment.

[0017] The present invention can formulate a treatment plan for patients by testing the puncture tissue or blood tissue of patients with early non-small cell lung cancer before treatment, based on the expression of CD44 in tumor tissue or circulating tumor lymphocytes. The present invention provides treatment options for the first-diagnosed operable early NSCLC, and has high scientific research and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 A flowchart for screening protein markers for the early-stage NSCLC advantage population to receive SBRT or surgery in the present invention;

[0020] Figure 2 This is a verification analysis of the operation process and technical reliability of DSP technology; A is the operation flow chart of DSP technology, B is the staining effect chart, and C is the efficiency chart of detection and sequencing;

[0021] Figure 3 Figure 3 Comparison of survival between SBRT and surgery patients based on lymphocyte CD44 expression; A and B are the Kaplan-Meier RFS and OS curves between the SBRT group and the surgery group before propensity matching, respectively; C and D are the RFS and OS curves between the SBRT group and the surgery group for patients with low lymphocyte CD44 expression, respectively; E and F are the RFS and OS curves between the SBRT group and the surgery group for patients with high lymphocyte CD44 expression, respectively; G and H are the results of the correlation analysis between the spatial distribution of lymphocyte CD44 and clinical outcomes in the SBRT and surgery cohorts, scale bar 100 μm. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The tumor immune microenvironment (TIME) is increasingly recognized to play a crucial role in predicting SBRT efficacy. However, TIME is complex, characterized by diverse cell types and complex spatial distribution. Identifying key subpopulations that affect SBRT efficacy requires the use of precise sequencing technology. Therefore, the inventors attempted to discover new biomarkers of SBRT resistance using a spatially informed high-complexity discovery tool called digital spatial analysis (DSP). The Nanostring GeoMX DSP system is able to simultaneously detect multiple proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections in a quantitative and spatially resolved manner. Next, candidate predictors were further validated by multiplex immunofluorescence (mIF). Unlike traditional single-color immunohistochemistry (IHC), mIF can simultaneously detect multiple protein markers in one tissue section to identify multiple cell phenotypes. Therefore, DSP technology can be used as a discovery tool to find spatially resolved protein markers of infiltrating immune cells, stromal cells, and tumor cells associated with SBRT resistance in early-stage NSCLC, and then used to screen the early-stage NSCLC advantage population for SBRT or surgical treatment. The flowchart for screening protein markers is shown in Figure 1 .

[0028] The above technical solution is further described below with reference to specific embodiments.

[0029] Example 1

[0030] 1. Patients and Cohorts

[0031] The present invention included three cohorts, including a SBRT discovery cohort, a SBRT validation cohort, and a surgery cohort.

[0032] The SBRT discovery cohort included 44 patients with early-stage NSCLC (T1-2N0) who received first-line SBRT between 2015 and 2020 and had complete pre-SBRT tumor biopsy specimens. The SBRT validation cohort included 52 patients with early-stage NSCLC (T1-2N0) who received first-line SBRT between 2019 and 2022. All patients enrolled in the two SBRT cohorts had early-stage NSCLC (T1-2N0), complete pre-treatment puncture biopsy tissue for DSP testing, and complete clinical data and follow-up records. SBRT patients without pathological tissue, patients with tissue too small for DSP testing, and patients with incomplete clinical data and follow-up records were excluded. The surgical cohort included 62 patients with early-stage NSCLC (T1-2N0) who underwent lobectomy and mediastinal lymph node dissection. Patients enrolled in the surgical cohort had complete surgical tissue for DSP testing, complete clinical data and follow-up records.

[0033] Clinical pathological data and hematological parameters were collected from all patients. Complete blood counts, including absolute lymphocyte counts before treatment (pre-ALC) and absolute lymphocyte counts after treatment (post-ALC), absolute neutrophil counts (ANC), absolute monocyte counts (AMC), and platelet counts were systematically recorded. All patients underwent follow-up with CT or positron emission tomography (PET-CT) 2-3 months after radiotherapy or 1 month after lobectomy. Follow-up CT was performed every 3-6 months for the first 2 years and every 6 months thereafter.

[0034] 2. Spatial proteomic analysis

[0035] In the SBRT discovery cohort, two consecutive FFPE whole tissue sections (88 in total) from 44 patients were selected for morphological staining using the DSP technique (see the experimental procedure for details). Figure 2 A). The details are as follows:

[0036] Slides were first dewaxed and then subjected to an antigen retrieval procedure, after which they were incubated overnight with fluorescently labeled antibodies (morphological markers) as well as unique photocleavable oligonucleotide-conjugated antibodies. In the SBRT cohort, two consecutive tissue sections were stained with fluorescently labeled morphological markers (e.g., epithelial marker pan-cytokeratin [panCK], lymphocyte marker CD3, leukocyte marker CD45, macrophage marker CD68, cancer-associated fibroblast [CAF] marker α-smooth muscle actin [α-SMA], and nuclear stain SYTO 13). One of the sections was stained with CD3, CD45, panCK, and SYTO 13 to analyze tumor cells, all immune cells, and lymphocytes. The other section was stained with α-SMA, CD68, panCK, and SYTO13 to analyze macrophages and stromal cells. An average of 12 regions of interest (ROIs) were selected per patient based on the fluorescently stained slides. The DSP staining panel includes the immune cell analysis core, pan-tumor module, immune cell typing module, IO (Immuno-Oncology) drug target module, immune activation status module, cell death module and PI3K-AKT module, with a total of 70 proteins and 6 internal reference proteins (3 internal reference proteins and 3 background proteins). Once the staining steps are completed, the slides are loaded into the GeoMx DSP instrument (NanoString) and scanned to produce digital immunofluorescence images of the tissue. In order to more accurately select the area of ​​interest, each tissue section is stained with hematoxylin and eosin (HE) to select representative tumor areas. Circular ROIs or other shaped ROIs are then depicted on the entire tissue fluorescence image. Thereafter, in order to obtain protein measurements of compartment-specific regions of interest (AOIs), each ROI is divided into one or two molecularly defined tissue regions by co-localization of fluorescent markers. As Figure 2 As shown in B and C, in the SBRT cohort, 5 regions were generated: tumor region (panCK + ), leukocyte area (CD45 + ), lymphocyte area (CD3 + ), macrophage region (CD68 + ) and matrix region (α-SMA + Subsequently, oligonucleotide tag sequences in different spatial regions are released under UV irradiation, collected by microcapillary aspiration and deposited into a 96-well plate, hybridized into a 4-color 6-label optical barcode, and finally digitally quantified on the nCounter platform.

[0037] 3. Multiplex immunofluorescence (mIF) staining

[0038] The potential biomarkers obtained from the above DSP analysis were further validated by mIF experiments.

[0039] The present invention used tissue sections from SBRT and surgical cohorts for multiple immunofluorescence staining, with the staining panel including CD3, CD44, panCK, and DAPI. After staining, the Vectra Polaris imaging system (Akoya Bioscience) was used to obtain multispectral imaging and Qupath software was used to quantify the fluorescence signal. + Tumor cell region and CD3 + In the T lymphocyte area, CD44 was calculated by binarizing the CD3 signal. + Fluorescence intensity of the T lymphocyte area.

[0040] Mean fluorescence intensity (MFI) of CD44 = total fluorescence intensity of target / CD3 + T lymphocyte area

[0041] 4. Data processing

[0042] To adjust for system-related and experimental biases and to offset the effect of ROI size variation, the raw digital counts of the corresponding barcodes were first normalized to an internal spike control. ROI inclusion criteria were limited to a minimum surface area of ​​1.6 × 10 3 μm 2 , with a minimum number of nuclei of 20. The quality control (QC) step generates normalized positive factors from individual ROIs. Any ROI that results in a normalized positive factor higher than 3 or lower than 0.3 is excluded from downstream analysis. Finally, QC criteria were used for downstream analysis of the CD3 region of 32 patients, the CD45 region of 41 patients, the CD68 region of 40 patients, the α-SMA region of 37 patients, and the tumor region of 40 patients. The QC-qualified ROI count files were then normalized using the geometric mean of three negative isotype controls (mouse immunoglobulin G [IgG] 1, mouse IgG2a, and rabbit IgG). Before comparison and plotting, the normalized data were log-transformed, centered or not centered around the median. For further statistical analysis, the normalized values ​​obtained from the ROIs of each region for each patient were averaged.

[0043] 5. Statistical analysis

[0044] The median of the DSP value or MFI value of lymphocyte CD44 was used as the cut-off value to define patients into high-expression and low-expression groups. All statistical tests were two-sided, and p<0.05 was considered statistically significant. Due to the limited number of probes and patients, the p value was not adjusted. Fisher's exact test was used to calculate the differences in patient characteristics among the groups. The distribution of panCK, CD45, CD3, CD68, and α-SMA in the five regions was compared using the Kruskal-Wallis test. For differential expression analysis, the nonparametric Mann-Whitney U test was used. Follow-up time was calculated from diagnosis to death or last follow-up using the inverse Kaplan-Meier method. Overall survival was calculated from the date of diagnosis to the date of death from any cause, and if alive, it was censored at the last follow-up. Relapse-free survival was calculated from the date of the last treatment to the date of the first relapse or death, and if alive without relapse, it was censored at the last follow-up. Relapse was defined as new or progressive disease confirmed by imaging or biopsy. Survival curves were calculated according to Kaplan-Meier analysis and compared using the log-rank test. Univariate and multivariate Cox regression models were used to compare OS and RFS between the two groups after adjusting for age, Karnofsky performance status (KPS), tumor stage, tumor site, and histology as covariates. Univariate and multivariate binary logistic regression models were used to identify predictors of treatment recurrence. Nearest-neighbor propensity score matching (PSM) (caliper = 0.01) was performed according to age, KPS, tumor stage, tumor site, and histology in a 1:1 ratio. All data were processed and analyzed in DSP analysis software and R version 4.2.1 with associated software packages. Univariate and multivariate survival analyses were performed using the R packages survival and survminer.

[0045] 6. Results and Analysis

[0046] Pre-treatment biopsy tissues of 44 patients with early-stage NSCLC who received first-line SBRT (discovery cohort) were selected and morphological staining was performed using GeoMx digital spatial analysis (DSP) technology, including tumor (pan-CK+), leukocytes (CD45+), lymphocytes (CD3 + ), macrophages (CD68 + ) and matrix (α-SMA +). Then the region of interest was circled, and DSP was used to analyze 70 proteins in the five spatial cell compartments. Using the 70 proteins in the five regions as parameters, univariate and multivariate Cox regression models were used to screen markers for predicting RFS and OS, and univariate and multivariate binary logistic regression models were used to determine markers for predicting recurrence. The results showed that CD44 in the lymphocyte region was the only one screened out by the intersection of markers for RFS, OS, and recurrence status. In patients with early-stage NSCLC treated with SBRT, high expression of CD44 in lymphocytes was associated with a higher recurrence rate and worse recurrence-free survival and overall survival.

[0047] The same conclusion was reached by multicolor immunofluorescence (PANCK / CD3 / CD44 / DAPI) validation using films of 44 SBRT patients. At the same time, the pre-treatment puncture tissues of 52 patients with early-stage NSCLC who received SBRT were selected as validation cohort 1, and the same conclusion was reached. Tissues of 62 patients with early-stage NSCLC who underwent surgery (validation cohort 2) were selected for lymphocyte CD44 staining analysis. The results showed that high expression of CD44 in lymphocytes was associated with longer recurrence-free survival and overall survival, indicating that lymphocyte CD44 is a good predictive marker for surgical patients.

[0048] Afterwards, the SBRT cohort and the surgical cohort were subjected to propensity matching score analysis. On the basis of ensuring the consistency of the baseline characteristics of the two groups of patients, the comparative analysis results of the SBRT cohort and the surgical cohort showed that there was no significant difference in OS and RFS between the SBRT group and the surgical group before propensity matching (see Figure 3 In patients with low expression of lymphocyte CD44, the RFS curves of the SBRT group and the surgery group are shown in Figure 2A and B. Figure 3 C and OS curves are shown in Figure 3 In D, by using the median cutoff point, it was found that patients with low lymphocyte CD44 expression who received SBRT had better RFS and OS than those who received surgery. In patients with high lymphocyte CD44 expression, the RFS curves of the SBRT group and the surgery group are shown in Figure 3 E and OS curves are shown in Figure 3 Middle F, the results showed that patients with high expression of lymphocyte CD44 in the surgical cohort showed better RFS compared with the SBRT cohort, but there was no significant difference in OS between the two treatment groups. The correlation analysis between the spatial distribution of lymphocyte CD44 and clinical outcomes in the SBRT and surgical cohorts is shown in Figure 3 In G and H, the results showed that patients with low expression of lymphocyte CD44 were favorable candidates for SBRT, while patients with high expression of lymphocyte CD44 were favorable candidates for surgery.

[0049] In summary, the above experimental analysis found that patients with lower CD44 expression in lymphocytes had a longer recurrence-free survival after SBRT, while patients with higher CD44 expression had a better recurrence-free survival after surgery.

[0050] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of lymphocyte CD44 as a protein marker in the preparation of a product for screening a predominant population of early non-small cell lung cancer for stereotactic radiotherapy or surgery, characterized in that: The lymphocyte CD44 is CD44 in the tumor tissue of patients with early non-small cell lung cancer.

2. Use of a detection reagent for detecting the expression level of lymphocyte CD44 in the preparation of a product for screening a predominant population of early non-small cell lung cancer for stereotactic radiotherapy or surgery, characterized in that: The lymphocyte CD44 is CD44 in the tumor tissue of patients with early non-small cell lung cancer.

3. The use according to claim 1 or 2, characterized in that: The product includes a test kit and a DSP chip.

4. The use according to claim 1 or 2, characterized in that: The patients with early non-small cell lung cancer with high expression of lymphocyte CD44 are the advantageous population for receiving surgical treatment, and the patients with early non-small cell lung cancer with low expression of lymphocyte CD44 are the advantageous population for receiving stereotactic radiotherapy.

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