Research On Prognosis Prediction Of Breast Tumor-infiltrating Lymphocytes Using Multiphoton Microscopy

Multiphoton microscopy(MPM)based on second harmonic generation(SHG)and two-photon excited fluorescence(TPEF)is one of the most advanced medical optical imaging techniques.The interaction of the femtosecond laser with the intrinsic components of biological tissues can produce nonlinear optical effects such as fluorescence and harmonics,resulting in label-free access to the microstructure of the intrinsic components of the tissues.This method has the advantages of high sensitivity and high spatial resolution,low destruction of biological tissues,long effective observation time and deep imaging depth,etc.Therefore,MPM has been widely concerned in the field of medical pathological diagnosis,especially in the basic and applied research of tumor pathological diagnosis.The latest global cancer burden data in2020 showed that breast cancer has replaced lung cancer as the world’s largest cancer,with the highest incidence and mortality rates for women worldwide.The number of new cancers case and cancer deaths in China ranked first in the world,with the highest incidence of breast cancer.Studies have shown that the prognosis of breast cancer is closely related to the status of the tumor microenvironment.As an important component of tumor microenvironment,tumor-infiltrating lymphocytes(TILs)have been proved to play an important role on cancer prognosis.However,most traditional research methods have studied the impact of the proportion of TILs in the tumor microenvironment on the prognosis of breast cancer patients through haematoxylin-eosin(H&E)staining at present,and some studies have also shown that the distribution pattern of TILs in the tumor microenvironment affects the prognosis of cancer.At present,there are many researchers using MPM to study the prognosis of breast cancer,but there are few studies using MPM to study the impact of the spatial distribution pattern of TILs in the microenvironment on the prognosis,and to analyze the relationship between TILs and histological grade of breast cancer.Therefore,this paper conducts an in-depth study on the imaging of TILs in the tumor microenvironment of breast cancer using MPM,the main contents include:(1)First,ductal carcinoma in situ(DCIS)and three subtypes of invasive breast cancer are identified using MPM,namely invasive ductal carcinoma(IDC),invasive micropapillary carcinoma of the breast(IMPC)and invasive lobular carcinoma(ILC),and to explore the potential of MPM in the identification and diagnosis of invasive breast cancer.The results show that MPM can image the detailed microstructure and cellular morphology of invasive breast cancer tissue and can be used to identify and detect invasive breast cancer.(2)Second,multiphoton imaging technology is used to image the important components of the tumor microenvironment of invasive breast cancer,especially TILs.According to the relative spatial location of tumor cells,collagen fibers and TILs,three tumor infiltrating lymphocyte patterns(TILs1-3)different from the traditional definition of TILs are proposed,and established the TILs-score model to explore the relationship between TILsscore and the prognosis of breast cancer.The results of the statistical analysis indicate that the TILs-score is an independent prognostic factor for breast cancer and is more applicable to assessing the prognosis of the ER-positive subgroup of breast cancer patients.(3)Third,the prognostic value of TILs in the tumor microenvironment of invasive breast cancer is evaluated according to the method proposed by the International Immuno-Oncology Biomarker Working Group(TILs-WG),and the differences and similarities between the guideline method and the multiphoton method(TILs-MPM)are compared,then combined the two methods to establish a model to evaluate the prognostic value of TILs in breast cancer.The results show that TILs-WG model is more suitable for assessing the prognosis of breast cancer patients in the ER-negative subgroup,while TILs-MPM model is more suitable for assessing the prognosis of breast cancer patients in the ER-positive subgroup,and that the combination of the two models can improves the prognostic value of TILs in breast cancer.(4)Finally,we divided all patients into low risk group(Grade 1)and high risk group(Grade 2/3)according to histological grade.Exploring the relationship between TILs-score obtained from MPM images and histological grade of breast cancer,than compared the predictive ability of TILs-score model and clinical model.The results show that TILs-score is significantly correlated with histological grade and is an independent predictor of histological grade,the predictive power of TILs-score is comparable to that of the clinical model.