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. 2023 Apr;14(12):1089-1097.
doi: 10.1111/1759-7714.14853. Epub 2023 Mar 16.

CALML5 is a novel diagnostic marker for differentiating thymic squamous cell carcinoma from type B3 thymoma

Koichiro Kanamori  1   2 , Kentaro Suina  1   2 , Takehito Shukuya  1   2 , Fumiyuki Takahashi  1   2 , Takuo Hayashi  3 , Kieko Hara  3 , Tsuyoshi Saito  3 , Yoichiro Mitsuishi  1   2 , Shoko Sonobe Shimamura  1   2 , Wira Winardi  1   2 , Ken Tajima  1   2 , Ryo Ko  1   2 , Tomoyasu Mimori  1   2 , Tetsuhiko Asao  1   2 , Masayoshi Itoh  4   5 , Hideya Kawaji  4   6   7 , Yoshiyuki Suehara  8 , Kazuya Takamochi  9 , Kenji Suzuki  9 , Kazuhisa Takahashi  1   2
Affiliations

CALML5 is a novel diagnostic marker for differentiating thymic squamous cell carcinoma from type B3 thymoma

Koichiro Kanamori et al. Thorac Cancer. 2023 Apr.

Abstract

Background: Thymic squamous cell carcinoma and type B3 thymoma are primary neoplasms of the anterior mediastinum that are sometimes difficult to differentiate from one another histologically. However, only a few immunohistochemical markers are available for the differential diagnosis. The purpose of this study was to discover a novel marker for differentiating between thymic squamous cell carcinoma and type B3 thymoma.

Methods: We used histological samples of thymic carcinomas (n = 26) and type B3 thymomas (n = 38) which were resected between 1986 and 2017. To search for candidates of differential markers, gene expression levels were evaluated in samples using promoter analysis by cap analysis of gene expression (CAGE) sequencing.

Results: Promoter level expression of CALML5 genes was significantly higher in thymic carcinomas than in type B3 thymomas. We further validated the results of the CAGE analysis in all 26 thymic carcinomas and 38 type B3 thymomas by immunohistochemistry (IHC). CALML5 was strongly expressed in the cytoplasm in 19 of 26 cases with thymic carcinoma, whereas positivity at the protein level was shown in two of 38 type B3 thymomas. Thus, the sensitivity (73.1%) and specificity (94.7%) of CALML5 as markers for immunohistochemical diagnosis of thymic carcinoma were extremely high.

Conclusion: We identified CALML5 as a potential marker for differentiating thymic squamous cell carcinoma from type B3 thymoma. It is assumed that future clinical use of CALML5 may improve the diagnostic accuracy of differentiating between these two diseases.

Keywords: CALML5; immunohistochemistry; thymic carcinoma; thymoma.

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Conflict of interest statement

The authors have no conflicts of interest directly relevant to the content of this article.

Figures

FIGURE 1
FIGURE 1
Promoter‐level expression analyses between thymic squamous cell carcinoma and type B3 thymoma. The log2‐fold change in the average expression levels in count per million (CPM) (x‐axis) is plotted against the −log10 adjusted p‐value. Individual dots represent the activities of individual promoters, and the gray dots indicate an adjusted p‐value of 0.01 or higher, and black dots indicate an adjusted p‐value of less than 0.01, and blue dots indicates known markers, and red dot indicates a novel candidate.
FIGURE 2
FIGURE 2
Promoter activity and mRNA expression levels of novel candidates and known markers. (a) The promoter activities of two novel candidates and three known markers for thymic squamous cell carcinoma in scatter plots with cap analysis of gene expression (CAGE) data. (b) The mRNA expression levels of novel candidates and known markers for thymic squamous cell carcinoma with the Cancer Genome Atlas (TCGA) data are shown as bar graphs (mean with standard error of the mean). CAGE, cap analysis of gene expression; CPM, count per million; THYM, type B3 thymoma; TPM, transcripts per million; TSQCC, thymic squamous cell carcinoma. *p < 0.05, **p < 0.005.
FIGURE 3
FIGURE 3
Immunohistochemistry (IHC) for thymic carcinoma and type B3 thymoma with CALML5, CD5, c‐kit, and GLUT‐1. (a–e) A case of thymic carcinoma. (a) Hematoxylin and eosin (H&E). (b) CALML5 is expressed in the cytoplasm and the nuclei. (c) CD5 is expressed in the membrane. (d) C‐kit is expressed in the membrane. (e) GLUT‐1 is expressed in the membrane. (f–j) A case of type B3 thymoma. (f) H&E. The tumor cells are (g) negative for CALML5, (h) negative for CD5, (i) negative for c‐kit, and (j) negative for GLUT‐1. Scale bar is 100 μm. IHC, immunohistochemistry; TdT, terminal deoxynucleotidyl transferase; THYM, type B3 thymoma; TSQCC, thymic squamous cell carcinoma.
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