Quantitative second harmonic generation microscopy was used to investigate collagen organization in the fibrillar capsules of human benign and malignant thyroid nodules. We demonstrate that the combination of texture analysis and second harmonic generation images of collagen can be used to differentiate between capsules surrounding the thyroid follicular adenoma and papillary carcinoma nodules. Our findings indicate that second harmonic generation microscopy can provide quantitative information about the collagenous capsule surrounding both the thyroid and thyroid nodules, which may complement traditional histopathological examination.

Background: squamous cell carcinoma (SCC) is the second most common type of malignancy worldwide. Skin and mucosa of the head and neck areas are the most frequently affected. An aggressive behavior in SCC is not easily detected, and despite all efforts, mortality in these types of cancer did not show major improvements during recent decades. In this study, we aim to determine the role of histological features available through standard pathology assessment in SCC and their relation with tumor behavior and patients’ survival. Method: in a group of one hundred patients diagnosed with SCC involving the head and neck areas, we assessed the presence of four histological features (tumor/stroma ratio, immune infiltration at the front of invasion, tumor-budding activity, and tumor necrosis), their correlations with tumor type (mucosal or cutaneous), tumor clinicopathological characteristics, and their prognostic potential. Results: the comparison between histological features in cutaneous versus mucosal SCC reveals no significant differences for any of the four parameters assessed. We found significant correlations between tumor/stroma ratio and lymphatic metastasis (p = 0.0275), perineural invasion (p = 0.0006), and clinical staging (p = 0.0116). Immune infiltration at the front of invasion revealed similar correlations with lymph node involvement (p = 0.002), perineural invasion (p = 0.0138), and clinical staging (p = 0.0043). Tumor budding and tumor necrosis correlated with the size of the tumor (p = 0.0077 and p = 0.0004) and the clinical staging (p = 0.0039 and p = 0.0143). In addition, tumor budding was significantly correlated with perineural invasion (p = 0.0454). In mucosal SCC, patients with improved outcome revealed high values for the tumor/stroma ratio (p = 0.0159) and immune infiltration at the front of invasion (p = 0.0274). However, the multivariate analysis did not confirm their independent prognostic roles. Conclusions: extended histological assessments that include features such as tumor/stroma ratio, immune infiltration at the front of invasion, tumor budding, and tumor necrosis can be an easy, accessible method to collect additional information on tumor aggressiveness in skin and mucosa SCC affecting the head and neck areas.

Papillary carcinoma is the most prevalent type of thyroid cancer. Its diagnosis requires accurate and subjective analyses from expert pathologists. Here we propose a method based on the Hough transform (HT) to detect and objectively quantify local structural differences in collagen thyroid nodule capsules. Second harmonic generation (SHG) microscopy images were acquired on non-stained histological sections of capsule fragments surrounding the healthy thyroid gland and benign and tumoral/malignant nodules. The HT was applied to each SHG image to extract numerical information on the organization of the collagen architecture in the tissues under analysis. Results show that control thyroid capsule samples present a non-organized structure composed of wavy collagen distribution with local orientations. On the opposite, in capsules surrounding malignant nodules, a remodeling of the collagen network takes place and local undulations disappear, resulting in an aligned pattern with a global preferential orientation. The HT procedure was able to quantitatively differentiate thyroid capsules from capsules surrounding papillary thyroid carcinoma (PTC) nodules. Moreover, the algorithm also reveals that the collagen arrangement of the capsules surrounding benign nodules significantly differs from both the thyroid control and PTC nodule capsules. Combining SHG imaging with the HT results thus in an automatic and objective tool to discriminate between the pathological modifications that affect the capsules of thyroid nodules across the progressions of PTC, with potential to be used in clinical settings to complement current state-of-the-art diagnostic methods.

Polarization-resolved second harmonic generation microscopy is used to provide pixel-level angular distribution of collagen in thyroid nodule capsules. The pixel-level angular distribution is combined with textural analysis to quantify the collagen distribution in follicular adenoma (benign) and papillary thyroid carcinoma (malignant). Three second order nonlinear susceptibility tensor elements ratios, the collagen angular distribution and two parameters accounting for the collagen angular dispersion in different sized areas are extracted and corresponding images are computed in a pixel-by-pixel fashion. Subsequently, we show that texture analysis can be performed on these images to detect significant differences between the considered benign and malignant nodule capsules.