Rapid label-free detection of early-stage lung adenocarcinoma and tumor boundary via multiphoton microscopy.

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths in China. Rapid and precise evaluation of tumor tissue during lung cancer surgery can reduce operative time and improve negative-margin assessment, thus increasing disease-free and overall survival rates. This study aimed to explore the potential of label-free multiphoton microscopy (MPM) for imaging adenocarcinoma tissues, detecting histopathological features, and distinguishing between normal and cancerous lung tissues. We showed that second harmonic generation (SHG) signals exhibit significant specificity for collagen fibers, enabling the quantification of collagen features in lung adenocarcinomas. In addition, we developed a collagen score that could be used to distinguish between normal and tumor areas at the tumor boundary, showing good classification performance. Our findings demonstrate that MPM imaging technology combined with an image-based collagen feature extraction method can rapidly and accurately detect early-stage lung adenocarcinoma tissues.

AutoFibroNet: A deep learning and multi-photon microscopy-derived automated network for liver fibrosis quantification in MAFLD.

BACKGROUND: Liver fibrosis is the strongest histological risk factor for liver-related complications and mortality in metabolic dysfunction-associated fatty liver disease (MAFLD). Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) is a powerful tool for label-free two-dimensional and three-dimensional tissue visualisation that shows promise in liver fibrosis assessment. AIM: To investigate combining multi-photon microscopy (MPM) and deep learning techniques to develop and validate a new automated quantitative histological classification tool, named AutoFibroNet (Automated Liver Fibrosis Grading Network), for accurately staging liver fibrosis in MAFLD. METHODS: AutoFibroNet was developed in a training cohort that consisted of 203 Chinese adults with biopsy-confirmed MAFLD. Three deep learning models (VGG16, ResNet34, and MobileNet V3) were used to train pre-processed images and test data sets. Multi-layer perceptrons were used to fuse data (deep learning features, clinical features, and manual features) to build a joint model. This model was then validated in two further independent cohorts. RESULTS: AutoFibroNet showed good discrimination in the training set. For F0, F1, F2 and F3-4 fibrosis stages, the area under the receiver operating characteristic curves (AUROC) of AutoFibroNet were 1.00, 0.99, 0.98 and 0.98. The AUROCs of F0, F1, F2 and F3-4 fibrosis stages for AutoFibroNet in the two validation cohorts were 0.99, 0.83, 0.80 and 0.90 and 1.00, 0.83, 0.80 and 0.94, respectively, showing a good discriminatory ability in different cohorts. CONCLUSION: AutoFibroNet is an automated quantitative tool that accurately identifies histological stages of liver fibrosis in Chinese individuals with MAFLD.

Prognostic value of tumor necrosis based on the evaluation of frequency in invasive breast cancer.

BACKGROUND: Tumor necrosis (TN) was associated with poor prognosis. However, the traditional classification of TN ignored spatial intratumor heterogeneity, which may be associated with important prognosis. The purpose of this study was to propose a new method to reveal the hidden prognostic value of spatial heterogeneity of TN in invasive breast cancer (IBC). METHODS: Multiphoton microscopy (MPM) was used to obtain multiphoton images from 471 patients. According to the relative spatial positions of TN, tumor cells, collagen fibers and myoepithelium, four spatial heterogeneities of TN (TN1-4) were defined. Based on the frequency of individual TN, TN-score was obtained to investigate the prognostic value of TN. RESULTS: Patients with high-risk TN had worse 5-year disease-free survival (DFS) than patients with no necrosis (32.5% vs. 64.7%; P < 0.0001 in training set; 45.8% vs. 70.8%; P = 0.017 in validation set), while patients with low-risk TN had a 5-year DFS comparable to patients with no necrosis (60.0% vs. 64.7%; P = 0.497 in training set; 59.8% vs. 70.8%; P = 0.121 in validation set). Furthermore, high-risk TN "up-staged" the patients with IBC. Patients with high-risk TN and stage I tumors had a 5-year DFS comparable to patients with stage II tumors (55.6% vs. 62.0%; P = 0.565 in training set; 62.5% vs. 66.3%; P = 0.856 in validation set), as well as patients with high-risk TN and stage II tumors had a 5-year DFS comparable to patients with stage III tumors (33.3% vs. 24.6%; P = 0.271 in training set; 44.4% vs. 39.3%; P = 0.519 in validation set). CONCLUSIONS: TN-score was an independent prognostic factor for 5-year DFS. Only high-risk TN was associated with poor prognosis. High-risk TN "up-staged" the patients with IBC. Incorporating TN-score into staging category could improve its performance to stratify patients.

Combining the guidelines and multiphoton imaging methods to improve the prognostic value of tumor-infiltrating lymphocytes in breast cancer.

Multiphoton microscopy (MPM) was introduced to label-freely obtain tumor-infiltrating lymphocytes (TILs) images from a total of 611 patients, and the prognostic value of TILs in breast cancer was assessed by the MPM method (TILs-MPM) and guidelines method proposed by the International Immuno-Oncology Biomarker Working Group (TILs-WG), respectively. Moreover, the clinical (CLI) model, TILs-WG + TILs-MPM model, and full model (CLI + TILs-WG + TILs-MPM) were developed to investigate the prognostic value of TILs. The results show that TILs-WG performs better in estrogen receptor (ER)-negative subgroup, and TILs-MPM is comparable with TILs-WG in the ER-negative subgroup, but has the best performance in the ER-positive subgroup. Furthermore, the TILs-WG + TILs-MPM model can significantly improve the prognostic power compared with the TILs-WG model, and the full model has excellent performance, with high area under the curve (AUC) and hazard ratio (HR) in both ER-positive, ER-negative subgroups, and the complete cohort. Our results suggest that the combination of TILs-WG with TILs-MPM model can greatly improve the prognostic value of TILs.

Label-free detection of invasive micropapillary carcinoma of the breast using multiphoton microscopy.

Invasive micropapillary carcinoma of the breast (IMPC) is a rare form of breast cancer with unique histological features, and is associated with high axillary lymph node metastasis and poor clinical prognosis. Thus, IMPC should be diagnosed in time to improve the treatment and management of patients. In this study, multiphoton microscopy (MPM) is used to label-free visualize the morphological features of IMPC. Our results demonstrate that MPM images are well in agreement with hematoxylin and eosin staining and epithelial membrane antigen staining, indicating MPM is comparable to traditional histological analysis in identifying the tissue structure and cell morphology. Statistical analysis shows significant differences in the circumference and area of the glandular lumen and cancer nest between the different IMPC cell clusters with complete glandular lumen morphology, and also shows difference in collagen length, width, and orientation, indicating the invasive ability of different morphologies of IMPC may be different.

Intratumor graph neural network recovers hidden prognostic value of multi-biomarker spatial heterogeneity.

Biomarkers are indispensable for precision medicine. However, focused single-biomarker development using human tissue has been complicated by sample spatial heterogeneity. To address this challenge, we tested a representation of primary tumor that synergistically integrated multiple in situ biomarkers of extracellular matrix from multiple sampling regions into an intratumor graph neural network. Surprisingly, the differential prognostic value of this computational model over its conventional non-graph counterpart approximated that of combined routine prognostic biomarkers (tumor size, nodal status, histologic grade, molecular subtype, etc.) for 995 breast cancer patients under a retrospective study. This large prognostic value, originated from implicit but interpretable regional interactions among the graphically integrated in situ biomarkers, would otherwise be lost if they were separately developed into single conventional (spatially homogenized) biomarkers. Our study demonstrates an alternative route to cancer prognosis by taping the regional interactions among existing biomarkers rather than developing novel biomarkers.

Collagen signature as a novel biomarker to predict axillary lymph node metastasis in breast cancer using multiphoton microscopy.

Accurate identification of axillary lymph node (ALN) status is crucial for tumor staging procedure and decision making. This retrospective study of 898 participants from two institutions was conducted. The aim of this study is to evaluate the diagnostic performance of clinical parameters combined with collagen signatures (tumor-associated collagen signatures [TACS] and the TACS corresponding microscopic features [TCMF]) in predicting the probability of ALN metastasis in patients with breast cancer. These findings suggest that TACS and TCMF in the breast tumor microenvironment are both novel and independent biomarkers for the estimation of ALN metastasis. The nomogram based on independent clinical parameters combined with TACS and TCMF yields good diagnostic performance in predicting ALN status.

Computer-assisted quantification of tumor-associated collagen signatures to improve the prognosis prediction of breast cancer.

BACKGROUND: Collagen fibers play an important role in tumor initiation, progression, and invasion. Our previous research has already shown that large-scale tumor-associated collagen signatures (TACS) are powerful prognostic biomarkers independent of clinicopathological factors in invasive breast cancer. However, they are observed on a macroscale and are more suitable for identifying high-risk patients. It is necessary to investigate the effect of the corresponding microscopic features of TACS so as to more accurately and comprehensively predict the prognosis of breast cancer patients. METHODS: In this retrospective and multicenter study, we included 942 invasive breast cancer patients in both a training cohort (n = 355) and an internal validation cohort (n = 334) from one clinical center and in an external validation cohort (n = 253) from a different clinical center. TACS corresponding microscopic features (TCMFs) were firstly extracted from multiphoton images for each patient, and then least absolute shrinkage and selection operator (LASSO) regression was applied to select the most robust features to build a TCMF-score. Finally, the Cox proportional hazard regression analysis was used to evaluate the association of TCMF-score with disease-free survival (DFS). RESULTS: TCMF-score is significantly associated with DFS in univariate Cox proportional hazard regression analysis. After adjusting for clinical variables by multivariate Cox regression analysis, the TCMF-score remains an independent prognostic indicator. Remarkably, the TCMF model performs better than the clinical (CLI) model in the three cohorts and is particularly outstanding in the ER-positive and lower-risk subgroups. By contrast, the TACS model is more suitable for the ER-negative and higher-risk subgroups. When the TACS and TCMF are combined, they could complement each other and perform well in all patients. As expected, the full model (CLI+TCMF+TACS) achieves the best performance (AUC 0.905, [0.873-0.938]; 0.896, [0.860-0.931]; 0.882, [0.840-0.925] in the three cohorts). CONCLUSION: These results demonstrate that the TCMF-score is an independent prognostic factor for breast cancer, and the increased prognostic performance (TCMF+TACS-score) may help us develop more appropriate treatment protocols.

Nomogram model combining macro and micro tumor-associated collagen signatures obtained from multiphoton images to predict the histologic grade in breast cancer.

The purpose of this study is to develop and validate a new nomogram model combining macro and micro tumor-associated collagen signatures obtained from multiphoton images to differentiate tumor grade in patients with invasive breast cancer. A total of 543 patients were included in this study. We used computer-generated random numbers to assign 328 of these patients to the training cohort and 215 patients to the validation cohort. Macroscopic tumor-associated collagen signatures (TACS1-8) were obtained by multiphoton microscopy at the invasion front and inside of the breast primary tumor. TACS corresponding microscopic features (TCMF) including morphology and texture features were extracted from the segmented regions of interest using Matlab 2016b. Using ridge regression analysis, we obtained a TACS-score for each patient based on the combined TACS1-8, and the least absolute shrinkage and selection operator (LASSO) regression was applied to select the most robust TCMF features to build a TCMF-score. Univariate logistic regression analysis demonstrates that the TACS-score and TCMF-score are significantly associated with histologic grade (odds ratio, 2.994; 95% CI, 2.013-4.452; P < 0.001; 4.245, 2.876-6.264, P < 0.001 in the training cohort). The nomogram (collagen) model combining the TACS-score and TCMF-score could stratify patients into Grade1 and Grade2/3 groups with the AUC of 0.859 and 0.863 in the training and validation cohorts. The predictive performance can be further improved by combining the clinical factors, achieving the AUC of 0.874 in both data cohorts. The nomogram model combining the TACS-score and TCMF-score can be useful in differentiating breast tumor patients with Grade1 and Grade2/3.

Prognostic value of tumour-infiltrating lymphocytes based on the evaluation of frequency in patients with oestrogen receptor-positive breast cancer.

PURPOSE: We investigate the prognostic value of tumour-infiltrating lymphocytes (TILs) based on the evaluation of the present frequency in patients with breast cancer rather than that of the density proposed in previous research. METHODS: Multiphoton microscopy (MPM) was introduced to label-freely obtain TIL images from a total of 564 patients, and then TILs were redefined as TILs-1 to TILs-3 from MPM images according to the relative positions between TILs, tumour cells and collagen fibres. More seminally, a new method, which was based on the present frequency of TILs-1 to TILs-3, was presented for assessing the predictive ability of TILs, and then a tumour-infiltrating lymphocytes score (TILs-score) for each patient was obtained by ridge regression analysis. RESULTS: Data results from Cox proportional hazards regression analysis showed that the TILs-score was an independent prognostic factor for both disease-free survival (DFS) and overall survival (OS) in the complete cohort (n = 564), oestrogen receptor (ER)-positive subgroup (n = 352) and ER-negative subgroup (n = 212), but was more suitable for the ER-positive subgroup. Furthermore, the nomogram model combining the TILs-score with independent clinical factors further improved the predictive ability for the ER-positive subgroup: area under the curve (AUC) at 5-year DFS (OS) and hazard ratio (HR) for DFS (OS) in the training cohort increase from 0.735 (0.785) to 0.814 (0.830) and from 3.156 (5.845) to 4.643 (7.006), respectively, and in the validation cohort from 0.749 (0.748) to 0.804 (0.830) and from 3.104 (3.701) to 3.729 (5.132), respectively. CONCLUSION: The TILs-score is an independent prognostic factor and displays a strong prognostic value for ER-positive breast cancer. To our knowledge, this is the first time to use MPM for studying the prognostic value of TILs in breast cancer.

Large-scale tumor-associated collagen signatures identify high-risk breast cancer patients.

The notion of personalized medicine demands proper prognostic biomarkers to guide the optimal therapy for an invasive breast cancer patient. However, various risk prediction models based on conventional clinicopathological factors and emergent molecular assays have been frequently limited by either a low strength of prognosis or restricted applicability to specific types of patients. Therefore, there is a critical need to develop a strong and general prognosticator. Methods: We observed five large-scale tumor-associated collagen signatures (TACS4-8) obtained by multiphoton microscopy at the invasion front of the breast primary tumor, which contrasted with the three tumor-associated collagen signatures (TACS1-3) discovered by Keely and coworkers at a smaller scale. Highly concordant TACS1-8 classifications were obtained by three independent observers. Using the ridge regression analysis, we obtained a TACS-score for each patient based on the combined TACS1-8 and established a risk prediction model based on the TACS-score. In a blind fashion, consistent retrospective prognosis was obtained from 995 breast cancer patients in both a training cohort (n= 431) and an internal validation cohort (n = 300) collected from one clinical center, and in an external validation cohort (n = 264) collected from a different clinical center. Results: TACS1-8 model alone competed favorably with all reported models in predicting disease-free survival (AUC: 0.838, [0.800-0.872]; 0.827, [0.779-0.868]; 0.807, [0.754-0.853] in the three cohorts) and stratifying low- and high-risk patients (HR 7.032, [4.869-10.158]; 6.846, [4.370-10.726], 4.423, [2.917-6.708]). The combination of these factors with the TACS-score into a nomogram model further improved the prognosis (AUC: 0.865, [0.829-0.896]; 0.861, [0.816-0.898]; 0.854, [0.805-0.894]; HR 7.882, [5.487-11.323]; 9.176, [5.683-14.816], and 5.548, [3.705-8.307]). The nomogram identified 72 of 357 (~20%) patients with unsuccessful 5-year disease-free survival that might have been undertreated postoperatively. Conclusions: The risk prediction model based on TACS1-8 considerably outperforms the contextual clinical model and may thus convince pathologists to pursue a TACS-based breast cancer prognosis. Our methodology identifies a significant portion of patients susceptible to undertreatment (high-risk patients), in contrast to the multigene assays that often strive to mitigate overtreatment. The compatibility of our methodology with standard histology using traditional (non-tissue-microarray) formalin-fixed paraffin-embedded (FFPE) tissue sections could simplify subsequent clinical translation.

Visualization of lymphatic vascular invasion in breast cancer by multiphoton microscopy.

Lymphatic vascular invasion (LVI) is regarded as one of the independent factors which affect the prognosis of breast cancer. Once LVI is formed, it indicates the tumor has metastasized or has the possibility of metastasis. In this work, multiphoton microscopy (MPM), which relies on the two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was applied to identify the typical morphology of LVI and also visualize the histological features of LVI. Furthermore, the pixel density of collagen fibers was extracted as a quantitative parameter to differentiate LVI from the ductal carcinoma in situ (DCIS). By comparing with the corresponding H&E-stained images, it was confirmed that MPM can be used as an auxiliary tool for pathologists to diagnose LVI, and has a possibility for the application in clinical examination.

Label-Free Identification of Early Stages of Breast Ductal Carcinoma via Multiphoton Microscopy.

Breast cancer can be cured by early diagnosis. Appropriate and effective clinical treatment benefits from accurate pathological diagnosis. However, due to the lack of effective screening and diagnostic imaging methods, early stages of breast cancer often progress to malignant breast cancer. In this study, multiphoton microscopy (MPM) via two-photon excited fluorescence combined with second-harmonic generation was used for identifying the early stages of breast ductal carcinoma. The results showed differences in both cytological features and collagen distribution among normal breast tissue, atypical ductal hyperplasia, low-grade ductal carcinoma in situ, and high-grade ductal carcinoma in situ with microinvasion. Furthermore, three features extracted from the MPM images were used to describe differences in cytological features, collagen density, and basement membrane circumference in the early stages of breast ductal carcinoma. They revealed that MPM has the ability to identify early stages of breast ductal carcinoma label-free, which would contribute to the early diagnosis and treatment of breast cancer. This study may provide the groundwork for the further application of MPM in the clinic.

Label-Free Imaging of Blood Vessels in Human Normal Breast and Breast Tumor Tissue Using Multiphoton Microscopy.

Blood vessels are the important components of the circulatory systems that transport blood throughout the human body and maintain the homeostasis of physiological tissues. Pathologically, blood vessels are often affected by diseases, leading to the formation of unstable, irregular, and hyperpermeable blood vessels. In the tumor microenvironment, abnormal leakage of tumor blood vessels is related to the histological grade and malignant potential of tumors and may also facilitate metastasis of cancer. Visual diagnosis of blood vessels is very important for us to understand the occurrence and development of diseases. Multiphoton microscopy (MPM) is a potential label-free diagnostic tool based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). MPM can effectively observe the morphological changes of biological tissues at the molecular and cellular levels. In this work, we demonstrate that label-free MPM can be used to visualize the microstructure of blood vessels in human normal breast and breast tumor tissue. Moreover, MPM can monitor the changes of blood vessels in tumor microenvironment. These results show that the MPM will become a promising technique for clinicians to study the properties of the microstructure of the blood vessels.

Immunoassay for LMP1 in nasopharyngeal tissue based on surface-enhanced Raman scattering.

BACKGROUND: Previous studies have shown that Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is closely associated with the occurrence and development of nasopharyngeal carcinoma, and can be used as a tumor marker in screening for the disease. Here we report a new methodology based on highly specific and sensitive surface-enhanced Raman scattering (SERS) technology to detect LMP1 in nasopharyngeal tissue sections directly with no need of tedious procedures as with conventional immunohistochemistry methods. METHODS: LMP1-functionalized 4-mercaptobenzoic acid (4-MBA)-labeled Au/Ag core-shell bimetallic nanoparticles were prepared first and then applied for analyzing LMP1 in formalin-fixed paraffin-embedded nasopharyngeal tissue sections obtained from 34 cancer patients and 20 healthy controls. SERS spectra were acquired from a 25 × 25 spot square area on each tissue section and used to generate SERS images. RESULTS: Data from SERS spectra and images show that this new SERS-based immunoassay detected LMP1 in formalin-fixed paraffin-embedded nasopharyngeal tissue sections with high sensitivity and specificity. The results from the new LMP1-SERS probe method are superior to those of conventional immunohistochemistry staining for LMP1, and in excellent agreement with those of in situ hybridization for EBV-encoded small RNA (EBER). CONCLUSION: This new SERS technique has the potential to be developed into a new clinical tool for detection and differential diagnosis of nasopharyngeal carcinoma as well as for predicting metastasis and immune-targeted treatment of nasopharyngeal carcinoma.

[Preliminary study on the surface enhanced Raman spectrum of tea].

In the present paper, taking Tiegunyin tea as an example, surface enhanced Raman scattering (SERS) spectrum of tea was obtained, the effects of different active substrates were explored from the aspects of enhanced factor, background noise, repeatability, and signal to noise ratio (SNR), while the impact of adsorption time on the measurement was also discussed. The results show that it is feasible to obtain the SERS spectrum of tea, and Raman signal of SERS spectrum was greatly enhanced compared with that of regular Raman spectrum. The active substrate of silver colloid prepared by the reduction of silver nitrate and trisodium citrate has a better enhanced effect, while different adsorption times have no direct influences on the SERS measurement. The method based on SERS firstly proposed in this paper may provide an alternative method for the discriminant analysis and quality identification of tea.

Study on gastric cancer blood plasma based on surface-enhanced Raman spectroscopy combined with multivariate analysis.

A surface-enhanced Raman spectroscopy (SERS) method combined with multivariate analysis was developed for non-invasive gastric cancer detection. SERS measurements were performed on two groups of blood plasma samples: one group from 32 gastric patients and the other group from 33 healthy volunteers. Tentative assignments of the Raman bands in the measured SERS spectra suggest interesting cancer-specific biomolecular changes, including an increase in the relative amounts of nucleic acid, collagen, phospholipids and phenylalanine and a decrease in the percentage of amino acids and saccharide in the blood plasma of gastric cancer patients as compared with those of healthy subjects. Principal components analysis (PCA) and linear discriminant analysis (LDA) were employed to develop effective diagnostic algorithms for classification of SERS spectra between normal and cancer plasma with high sensitivity (79.5%) and specificity (91%). A receiver operating characteristic (ROC) curve was employed to assess the accuracy of diagnostic algorithms based on PCA-LDA. The results from this exploratory study demonstrate that SERS plasma analysis combined with PCA-LDA has tremendous potential for the non-invasive detection of gastric cancers.

[Raman spectral analysis of theanine].

The L-theanine was tested using confocal Raman microscopy. Obvious Raman bands were showed in the range of 250 -1 700 and 2 800-3 000 cm(-1). The Raman bands were assigned with a preliminary analysis and the characteristic vibrational modes were gained in different range of wave numbers. Eight strong Raman bands were observed in the Raman spectra at 321, 900, 938, 1 153, 1 312, 1 358, 1 454 and 1 647 cm(-1), respectively. They are the characteristic Raman bands of L-theanine. The results showed that Raman spectroscopy might be a new kind of precise, direct and fast detecting method for theanine.