Image analysis of the nuclear characteristics of emerin protein and the correlation with nuclear grooves and intranuclear cytoplasmic inclusions in lung adenocarcinoma

Abstract

Nuclear size and shape are important components in the diagnosis of pathological specimens. However, a qualitative evaluation is typically applied rather than a quantitative evaluation technique. In the present study, we sought to evaluate the nuclear morphological characteristics of lung adenocarcinoma using whole-slide imaging (WSI) and computer-assisted image analysis (IA). We evaluated the nuclear characteristics of 106 cases of surgically resected lung adenocarcinoma according to Feulgen staining and immunohistochemistry (IHC) for the inner nuclear membrane protein emerin. According to the Feulgen reaction, although the nuclear area (size) of the carcinoma cells was correlated with the nuclear perimeter (NP) (R=0.8973), the nuclear staining intensity of carcinoma cells was not correlated with the nuclear area. Using emerin IHC, we used IA software that was able to detect both the NP and the emerin-stained nuclear membrane length (ENML) in the nucleus, and found that the more nuclei exhibited a longer ENML relative to the NP, the more nuclear grooves and intranuclear cytoplasmic inclusions were present. In addition, the nuclear area was correlated with the percentage of nuclei that had a longer ENML compared to the NP against the total nuclei (R=0.7759). Furthermore, the emerin low expression group showed an enlarged nuclear area (P=0.0264), elongated NP (P=0.0091), and lower shape factor (P=0.0486) compared with the normal emerin expression group. Our data indicated the usefulness of WSI and IA for pathological specimen analysis. In addition, this study is the first to report that the low expression of emerin in cancer cell results in an oval shape of nuclei and nuclear enlargement in clinical samples.

Figure 1.

Flow chart of the analysis in this study shows the Feulgen reaction and emerin IHC. IHC, immunohistochemistry.

Figure 2.

(A-C) Representative nuclear overlapping and the effect of specimen thickness on nuclear overlapping and (D and E) representative data from the correlation analysis for nuclear size, NP, or staining intensity of specimens stained by the Feulgen reaction. Effect of section thickness is shown in case no. 1: (A) 1-µm-thick, (B) 2-µm-thick and (C) 3-µm-thick. (D) Correlation of nuclear area (Avg) and NP (Avg), and correlation of (E) nuclear area (Avg) and staining intensity (Avg). For A-C, images were captured using VS (×40 objective lens). NP, nuclear perimeter; VS, virtual slide scanner.

Figure 3.

(A-D) Representative effect of specimen thickness in the nuclear analysis of emerin-stained specimens after image analysis and (E-G) representative data of the correlation analysis for nuclear size, NP or ENML for specimens stained by immunohistochemistry (IHC) for emerin. Effect of section thickness is shown in case no. 14: (A and B) 1-µm-thick, (C and D) 2-µm-thick. NP as detected by our protocol is indicated in red, while the emerin-stained membrane-traced line is indicated in green (B and D). (E-G) Correlation of nuclear area (Avg), NP (Avg), and ENML. Emerin in the x-axis in (G) stands for ENML (emerin-stained nuclear membrane length). Images in A-D were captured using VS (×40 objective lens). NP, nuclear perimeter; VS, virtual slide scanner.

Figure 4.

A representative case with predominantly smooth nuclei (case no. 24) (A and C) and a case with eccentric nuclei (case no. 97) (B and D) as shown by emerin immunohistochemistry (IHC). In B and D, the NP is indicated in red and the ENML traced by the software is shown in green. Avg data of NP and ENML (emerin) and NP minus ENML is shown at the bottom of the figure. All images were captured by a VS (×40 objective lens). NP, nuclear perimeter; ENML, emerin-stained nuclear membrane length; VS, virtual slide scanner.

Figure 5.

(A and C) Representative nuclear groove and intracytoplasmic inclusion, and (B and D) the relationship among nuclear grooves and intracytoplasmic inclusions, and a high nuclear deformity rate among all nuclei (NP minus the ENML-negative nuclei ratio out of all nuclei) in emerin-stained specimens. (E) The correlation between nuclear area and high nuclear deformity rate (HNDR) out of all nuclei. (A and C) A representative nuclear groove is seen as a ‘coffee bean shape’, indicated by arrows in A. A representative intracytoplasmic inclusion is indicated by an arrow in C. On the left is an H&E-stained section, whereas the right panel shows emerin IHC (×100 objective lens). (B and D) On the x-axis, a high nuclear deformity rate out of all nuclei was categorized for every 5%. The total number of cases in each category is shown in the gray bar. The percentage of cases with nuclear grooves (NGs) (B) or intranuclear cytoplasmic inclusions (ICIs) (D) out of all cases in each category is shown in the line plot. (E) Correlation between nuclear area and the rate of HNDN. NP, nuclear perimeter; ENML, emerin-stained nuclear membrane length.

Figure 6.

Comparison of cases with low emerin expression (n=34) and cases with positive emerin expression (n=54). (A) Nuclear area, (B) NP, (C) shape factor, and (D) staining intensity of each group were analyzed by Student's t-test. NP, nuclear perimeter.