CLDN15 is a novel diagnostic marker for malignant pleural mesothelioma

Abstract

Malignant mesothelioma is a cancer with a poor survival rate. It is difficult to diagnose mesotheliomas because they show a variety of histological patterns similar to those of various other cancers. However, since currently used positive markers for mesotheliomas may show false positives or false negatives, a novel mesothelial positive marker is required. In the present study, we screened 25 claudins and found that claudin-15 is expressed in the mesothelial cells. We made new rat anti-human claudin-15 (CLDN15) monoclonal antibodies that selectively recognize CLDN15, and investigated whether CLDN15 is a good positive marker for malignant pleural mesotheliomas (MPMs) using MPM tissue samples by immunohistochemistry and semi-quantification of the expression level using an immunoreactive score (IRS) method. Of 42 MPM samples, 83% were positive for CLDN15. The positive ratio was equal to or greater than other positive markers for MPMs including calretinin (81%), WT-1 (50%), and D2-40 (81%). In 50 lung adenocarcinoma sections, four cases were positive for CLDN15 and the specificity (92%) was comparable with other markers (90-100%). Notably, CLDN15 was rarely detected in 24 non-mesothelial tumors in the tissue microarray (12/327 cases). In conclusion, CLDN15 can be used in the clinical setting as a positive marker for MPM diagnosis.

Figure 1

Claudin-15 is the major constituent of mesothelial tight junctions. a RT-PCR of mouse pleura and peritoneum using specific primers for claudins. Note that claudin-1, -5, -10b, -12 and -15 are expressed in the mesothelial tissues and that tight junction markers occludin and tricellulin are also expressed. Genomic DNA and samples prepared without reverse transcriptase were used as positive and negative controls, respectively. b Real-time RT-PCR of claudins. The relative expression levels were calculated using purified DNA fragments of each claudin as a standard. Note that claudin-15 is most abundantly expressed in both the pleura and peritoneum samples. c Immunofluorescence staining of mesothelial tissues. Visceral and parietal pleura, visceral and parietal peritoneum, pericardium, and tunica vaginalis were stained for claudin-15 (green), HSPG (basement membrane marker, red) and DAPI (DNA, blue). Note that claudin-15 is localized at cell–cell junctions of mesothelial cells.

Figure 2

Establishment of a novel anti-CLDN15 mAb suitable for IHCs. a Schematic representation of the domain structure of human CLDN15 protein. The region corresponding to the peptide used for the immunization is shown in orange. b Amino-acid sequence of the cytoplasmic tail of CLDN15 and alanine mutants used in this study. Alignment with other CLDNs is also shown. c IHC of HEK293T cells expressing CLDNs. Note that 2C11 specifically stains only CLDN15-expressing cells. d Epitope analysis of 2C11. Note that 2C11 signal is lost in mutants 9–19, indicating that the epitope is ²¹⁹FGKYGRNA²²⁶ of CLDN15. e Amino-acid sequences of CDRs of 2C11.

Figure 3

CLDN15 is expressed in MPM tissues. a Typical staining patterns of anti-CLDN15 (2C11) mAb exhibiting strong to weak staining intensities. Scale bar, 50 µm. b IHC of epithelioid-, biphasic- and sarcomatoid-type MPMs using anti-CLDN15 (2C11), anti-calretinin, anti-D2-40 and anti-WT1 antibodies. “E” and “S” in the HE staining panel of the biphasic-type MPM indicate the epitheliod-type and sarcomatoid-type cell regions, respectively. c IHC of lung adenocarcinomas using anti-CLDN15 (2C11) mAb. d Distribution of IRS scores in MPMs and lung adenocarcinomas. e–g IHC of lung squamous cell carcinomas (e), chest wall fibrosarcoma (f) and abdominal wall fibrosarcoma (g) using anti-CLDN15 (2C11) mAb.