Yinghong Yang

Layer-resolved colorectal tissues using nonlinear microscopy

In this work, multiphoton microscopy (MPM), based on the nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to evaluate the feasibility of using MPM to distinguish layers of the bowel wall. It was found that MPM has the ability to identify the four-layer microstructures of colorectal tissues including mucosa, submucosa, muscularis propria, and serosa as there are many intrinsic signal sources in each layer. Our results also showed the capability of using the quantitative analyses of MPM images for quantifying some feature parameters including the nuclear area, nuclear-to-cytoplasmic ratio, and optical redox ratio. This work demonstrates that MPM has the potential in noninvasively monitoring the development and progression of colorectal diseases and then guiding effective treatment.

Jadassohn-Pellizzari anetoderma: Study of multiphoton microscopy based on two-photon excited fluorescence and second harmonic generation

Anetoderma is a rare skin disease with loss of dermal elastic tissue resulting in clinically localized areas of flaccid or herniated sack-like skin. In this study, we report a case of Jadassohn-Pellizzari anetoderma, in a 21-year-old Chinese female with an 18-year history of progressively generalized wrinkled skin lesions. Multiphoton microscopy based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was firstly employed to investigate the pathological process from unaffected skin to the erythematous phase and finally with affected skin of this case. The results showed that the normal elastic fibers in unaffected skin were almost completely absent in erythematous skin tissue, then replaced by a lot of elastic fibers with granular morphology in affected skin, which was consistent with the histopathological results. The obvious changes in collagen fibers and the occurrence of inflammatory cell infiltration in erythematous tissue suggested that the variations of these two components were also the main pathogenesis of anetoderma, except for the deficiency of elastic fibers. Based on these data, we demonstrated that multiphoton microscopy was a promising tool for non-invasive investigation of the pathology of anetoderma at nearly histological resolution, and has potential for observing the dermatological dynamic processes for living specimens because it is based on the intrinsic signals of tissue components.

Identifying and quantifying the stromal fibrosis in muscularis propria of colorectal carcinoma by multiphoton microscopy

The examination of stromal fibrosis within colorectal cancer is overlooked, not only because the routine pathological examinations seem to focus more on tumour staging and precise surgical margins, but also because of the lack of efficient diagnostic methods. Multiphoton microscopy (MPM) can be used to study the muscularis stroma of normal and colorectal carcinoma tissue at the molecular level. In this work, we attempt to show the feasibility of MPM for discerning the microstructure of the normal human rectal muscle layer and fibrosis colorectal carcinoma tissue practicably. Three types of muscularis propria stromal fibrosis beneath the colorectal cancer infiltration were first observed through the MPM imaging system by providing intercellular microstructural details in fresh, unstained tissue samples. Our approach also presents the capability of quantifying the extent of stromal fibrosis from both amount and orientation of collagen, which may further characterize the severity of fibrosis. By comparing with the pathology analysis, these results show that the MPM has potential advantages in becoming a histological tool for detecting the stromal fibrosis and collecting prognosis evidence, which may guide subsequent therapy procedures for patients into good prognosis.

Assessment of Tumor Invasion Depth in Colorectal Carcinoma Using Multiphoton Microscopy

Assessment of tumor invasion depth prior to making therapeutic decisions in colorectal carcinoma is crucial for both the patient and the physician. In this paper, multiphoton microscopy (MPM) was used to simultaneously label freely image loose areolar connective tissue in the submucosa and intramuscular septa in the muscularis propria to perform assessment of colorectal carcinoma invasion depth. The results indicated that MPM can accurately exhibit whether colorectal carcinoma invades into the submucosa or the muscularis propria. Collagen content alteration and the presence of dirty necrosis can be extracted to serve as quantitatively intrinsic biomarkers for reflecting collagen degradation, the occurrence of desmoplastic reaction, and breakdown of cancer cells, which are tightly related to the prognosis of colorectal carcinoma. With the development and clinical applications of the multiphoton endoscope, in vivo histological-like diagnosis of tumor invasion depth may become its main application in the field of colorectal carcinoma and lead to faster and improved therapeutic decision making in the clinics.

Pilot study of intense neoadjuvant chemoradiotherapy for locally advanced rectal cancer: retrospective review of a phase II study

AIMS AND BACKGROUND Locally advanced rectal adenocarcinoma is typically treated with neoadjuvant chemoradiotherapy and surgery. We assessed the effect of an additional cycle of capecitabine/oxaliplatin chemotherapy before surgery in 57 patients with T3/4, N+/- or T1/2, N+ rectal cancer. MATERIALS AND STUDY DESIGN: Radiotherapy (total dose, 50.4 Gy) was combined with three cycles of chemotherapy (two cycles concomitant with radiotherapy), and each cycle consisted of oxaliplatin (130 mg/m2 on day 1) and capecitabine (825 mg/m2, twice per day from day 1 to day 14) for 21 days. In addition to assessing the safety of this treatment, the primary endpoint was pathological complete response (pCR). The secondary endpoint was the change in primary tumor and node stage from pre-treatment to post-surgery. RESULTS Eleven patients (19%) experienced complete tumor regression and 23 patients (40%) experienced tumor regression grade 3. Tumor down-staging occurred in 31 patients (54.4%) and down-staging of nodes occurred in 25 patients (43.9%). There was a significant difference in tumor stage between pre-treatment and post-surgery (P <0.001). Patients with less advanced N stages had significantly better recurrence-free survival but similar metastasis-free survival and overall survival. Tumor regression grade was not associated with overall survival, recurrence-free survival or metastasis-free survival. The most common adverse events were pulmonary infection (n = 6, 10.5%) and intestinal obstruction (n = 6, 10.5%): CONCLUSIONS. An additional cycle of chemotherapy given after chemoradiotherapy and before surgery provided good efficacy and had a satisfactory safety profile in patients with locally advanced rectal cancer.

DISTINGUISHING BETWEEN HYPERPLASTIC AND ADENOMATOUS POLYPS AND NORMAL COLONIC MUCOSA BY USING MULTIPHOTON LASER SCANNING MICROSCOPY

Precisely distinguishing between hyperplastic and adenomatous polyps and normal human colonic mucosa at the cellular level is of great medical significance. In this work, multiphoton laser scanning microscopy (MPLSM) was used to obtain the high-contrast images and the morphological characteristics from normal colonic mucosa, hyperplastic polyps and tubular adenoma. By integrating the length and area measurement tools and computing tool, we quantified the difference of crypt morphology and the alteration of nuclei in normal and diseased human colonic mucosa. Our results demonstrated that the morphology of crypts had an obvious tendency to cystic dilatation or elongated in hyperplastic polyps and tubular adenoma. The content and number of mucin droplets of the scattered goblet cells had a piecemeal reduction in hyperplastic polyps and a large decrease in tubular adenoma. The nuclei of epithelial cells might be elongated and pseudostratified, but overt dysplasia was absent in hyperplastic polyps. Nevertheless, the nuclei showed enlarged, crowded, stratified and a rod-like structure, with loss of polarity in tubular adenoma. These results suggest that MPLSM has the capacity to distinguish between hyperplastic and adenomatous polyps and normal human colonic mucosa at the cellular level.

Monitoring changes of tumor microenvironment in colorectal submucosa using multiphoton microscopy

Recently, targeting tumor microenvironment has become a novel approach for cancer therapy. Collagen is one of important components of tissue microenvironment, and has been considered as a new visible target for cancer therapy. In this work, multiphoton microscopy (MPM) was used to monitor the changes of collagen in tumor microenvironment during tumor progression. It was found that MPM facilitates imaging of tumor cells and collagen. MPM images in different tumor microenvironment during tumor progression shows obvious increase in cell number and collagen degration. In addition, the quantitative analysis of collagen content and orientation index in tumor microenvironment shows significant alteration during tumor progression. These results suggest that MPM has the ability to monitor the changes of collagen morphology in tumor microenvironment and quantify content and orientation index of collagen during tumor progression. Therefore this technique is a powerful imaging tool for the investigation of targeting tumor microenvironment for cancer therapy.

Pretreatment Tumor Thickness as a Predictor of Pathologic Complete Response to Neoadjuvant Chemoradiation Therapy for Stage II/III Rectal Adenocarcinoma.

Objectives: To evaluate pretreatment tumor thickness in predicting pathologic complete response (pCR) of stage II/III rectal adenocarcinoma to neoadjuvant chemoradiation (chemoradiotherapy [CRT]). Methods: We retrospectively analyzed 185 patients who were diagnosed with stage II or III rectal adenocarcinoma from January 2011 to July 2013 and treated with neoadjuvant intensity-modulated radiation therapy (45 Gy in 1.8-Gy fractions to pelvis and 50 Gy in 2-Gy fractions to rectal tumor as an integrated boost) or 3 dimensionally conformal radiation therapy (45 Gy in 1.8-Gy fractions to pelvis followed by an additional 5.4-Gy to rectal tumor) concurrently with two 3-week cycles of chemotherapy (oxaliplatin 130 mg/m2 on day 1 and capecitabine 825 mg/m2, twice per day from day 1 to 14, cycle 2 starts on week 4). One week after CRT, 36% patients received 1 more cycle of the above chemotherapy and 55% received 1 to 2 cycles of FOLFOX6. Tumor response was categorized as pCR and non-pCR. Tumor thickness measured on magnetic resonance imaging was collected. A multivariate logistic regression model was used to evaluate the association of potential predictors and pCR. Results: Thirty-eight patients (20.5%) reached pCR. Multivariate analysis found the pretreatment tumor thickness to be associated with higher probability of pCR after adjusting for radiation therapy-surgery interval time and pretreatment carcino-embryonic antigen level. The pretreatment carcino-embryonic antigen level was associated with pCR in the univariate analysis but lost the association in the multivatiate model. The pretreatment T or N stage, tumor volume, distance from tumor to anal verge, craniocaudal length of tumor, radiation therapy technique, and patient age and sex were not associated with pCR. Conclusions: We concluded that pretreatment tumor thickness is an independent predictor for pCR of stage II/III rectal adenocarcinoma to the neoadjuvant CRT.

Label-free visualization of collagen in submucosa as a potential diagnostic marker for early detection of colorectal cancer

The collagen signature in colorectal submucosa is changed due to remodeling of the extracellular matrix during the malignant process and plays an important role in noninvasive early detection of human colorectal cancer. In this work, multiphoton microscopy (MPM) was used to monitor the changes of collagen in normal colorectal submucosa (NCS) and cancerous colorectal submucosa (CCS). What’s more, the collagen content was quantitatively measured. It was found that in CCS the morphology of collagen becomes much looser and the collagen content is significantly reduced compared to NCS. These results suggest that MPM has the ability to provide collagen signature as a potential diagnostic marker for early detection of colorectal cancer.

Visualization of dermal alteration in skin lesions with discoid lupus erythematosus by multiphoton microscopy

Discoid lupus erythematosus (DLE) is a chronic dermatological disease which lacks valid methods for early diagnosis and therapeutic monitoring. Considering the collagen and elastin disorder due to mucin deposition of DLE, multiphoton microscopy (MPM) imaging techniques were employed to obtain high-resolution collagen and elastin images from the dermis. The content and distribution of collagen and elastin were quantified to characterize the dermal pathological status of skin lesions with DLE in comparison with normal skin. Our results showed a significant difference between skin lesions with DLE and normal skin in terms of the morphological structure of collagen and elastin in the dermis, demonstrating the possibility of MPM for noninvasively tracking the pathological process of DLE even in its early stages and evaluating the therapeutic efficacy at the molecular level.