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. 2019 May 24;9(1):7833.
doi: 10.1038/s41598-019-44287-z.

Method for diagnosing neoplastic lesions by quantitative fluorescence value

Ayaka Kosugi  1   2   3 , Masataka Kasahara  3 , Longqiang Yang  3 , Aki Nakamura-Takahashi  3 , Takahiko Shibahara  2 , Taisuke Mori  4
Affiliations

Method for diagnosing neoplastic lesions by quantitative fluorescence value

Ayaka Kosugi et al. Sci Rep. .

Abstract

Fluorescence visualization devices (FVs) are useful for detecting malignant lesions because of their simple and noninvasive application. However, their quantitative application has been challenging. This study aimed to quantitatively and statistically evaluate the change in fluorescence intensity (FI) during the progression from normal epithelium to squamous cell carcinoma using a reproducible animal tongue carcinogenesis model. To establish this model, rats were treated with 50 ppm 4-Nitroquinoline 1-oxide (4NQO) in their drinking water for 10, 15, and 20 weeks. After 4NQO administration, each rat tongue was evaluated by gross observation, histology, and FI measurements. Fluorescence images were captured by FV, and ImageJ was used to measure FI, which was analyzed quantitatively and statistically. The establishment of a reproducible tumor progression model was confirmed, showing precancerous lesions (low-grade dysplasia [LGD]), early cancers (high-grade dysplasia/carcinoma in situ [HGD/CIS]), and advanced cancers (Cancer). This carcinogenesis model was quantitatively evaluated by FI. The FI of LGD stage was 54.6, which was highest intensity of all groups. Subsequently, the HGD/CIS and Cancer stages showed decreased FI (HGD/CIS: 46.1, Cancer: 49.1) and manifested as dark spots. This result indicates that FI had more variation and a wider range with increasing tumor progression. We demonstrated that FI migration and an uneven distribution are consistent with tumor progression. Since each step of tumor progression occurs reproducibly in this animal model, statistical evaluation was possible. In addition, tumor progression can be monitored by this new FI analysis method in humans.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Experimental design for fluorescence visualization. Flow chart of the experimental design. (A) Method for recording fluorescence images. (B) The focal length of the camera to the floor was 5 cm. Gross observation (C) and fluorescence images (D) of a normal rat tongue are shown. The tongue contains a “half-moon structure” (yellow arrow head). The measuring sight of the anterior tongue corresponded to 200 pixels (white arrow, A: tongue apex, R: tongue root, scale bar: 10 mm).
Figure 2
Figure 2
Accurate reproduction of tongue carcinogenesis. Each panel shows four images: gross observation, fluorescence image, hematoxylin and eosin (H&E) stain, and surface plot for a control tongue (A), low-grade dysplasia (B), high-grade dysplasia/carcinoma in situ (C), and cancer (D). For the gross observation and fluorescence images, scale bars indicate 10 mm. For H&E staining, the magnification was ×100, and scale bars indicate 250 μm. Surface plots show three-dimensional graphs of fluorescence intensities.
Figure 3
Figure 3
The analysis of the intensity elements. (A) Graphs show the mean fluorescence intensity of each group. The vertical axis shows fluorescence intensity (G-value). All experimental groups had significantly higher intensity than Control, while the intensity of high-grade dysplasia/carcinoma in situ (HGD/CIS) and Cancer was significantly darker than that of low-grade dysplasia (LGD). However, HGD/CIS was not significantly darker than cancer (p < 0.05, Dunnett’s test). (B) Changes in fluorescence intensity and distribution between the groups. The pixels showing fluorescence were plotted to examine their distribution. (C) Representative example of fluorescence measurements at each pixel (black line) and the mean inclination line (y = ax + b, red line). The inclination line indicated the minimum distance from the measured intensity for all pixels. The horizontal axis indicates the distance from the tongue apex (A) to tongue root (R), while the vertical axis shows the fluorescence intensity (G-value). (D) The corresponding bar graph shows the average slopes of the linear approximations, separated by whether they showed positive (+) or negative (−) slopes. Error bars indicate standard deviation.
Figure 4
Figure 4
Practical application to human tongue cancer and relationship between the schema of tumor progression and fluorescence intensity transition. Gross observation (A) and fluorescence images (B) of a tongue cancer patient are shown. Measurement were taken at four sites (a: Control, b: low-grade dysplasia [LGD], c: high-grade dysplasia/carcinoma in situ [HGD/CIS], d: Cancer). The white line indicates 50 pixels. The mean fluorescence intensity (C) is shown, along with the scattering of fluorescence intensity (D). Significant differences were observed for all sites except between control and LGD (p < 0.05, Dunnett’s test). The transition of fluorescence intensity with the progression from normal epithelium to cancer (upper diagram) and tumor progression are illustrated (lower diagram) (E).
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