A fine decision tree consisted of CK5/6, IMP3 and TTF1 for cytological diagnosis among reactive mesothelial cells, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion

Abstract

The utility of combination with CK5/6, IMP3 and TTF1 to differentiate among reactive mesothelial cells (RMs), metastatic adenocarcinoma of lung (LAC) and non-lung (NLAC) origin was investigated by using immunocytochemistry (ICC) and conventional PCR (C-PCR) in pleural effusion. A total of 108 cell blocks (32 RMs, 51 LAC and 25 NLAC were evaluated by ICC for CK5/6, IMP3 and TTF1 protein expression. In addition, we further performed C-PCR for amplification of CK5/6, IMP3 and TTF1 DNA from 28 specimens (9 MAC and 7 RMs, 6 LAC and 6 NLAC) for molecular diagnosis. CK5/6 staining was observed in the majority of reactive specimens (78.1%) and was rare in adenocarcinoma cells (14.5%), whereas the opposite was true for IMP3 and TTF1. We found a high frequency of TTF1 positivity (76.5%) in LAC, but not in NLAC (4.0%); while there was no significant difference of IMP3 expression in LAC (88.2%) and NLAC (88.0%). The 487 bp DNA fragments of IMP3 was expected to be amplified in 6/9 of adenocarcinoma cases showed negative in ICC; and the 394 bp DNA fragments of CK5/6 was also expected to be amplified in 4/7 of RMs cases showed negative in ICC. Consistent with ICC results, there was significant difference of TTF1 expression in the LAC and NLAC compared with IMP3 expression. The combination with CK5/6, IMP3 and TTF1 immunostaining appears to be useful to improve the accuracy of cytological diagnoses between RMs, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion. In addition, C-PCR may act as a useful supplemental approach for ICC, especially negative cases in ICC for differential cytological diagnosis.

Keywords: CK5/6; IMP3; PCR; TTF1; adenocarcinoma; pleural effusion.

Figure 1

Reactive mesothelial cells (RMs) are shown in Pleural Effusion using by (A) HE, (B) CK5/6 ICC stain, (C) IMP3 ICC stain; Metastatic adenocarcinoma (MAC) cells are shown in pleural effusion using by (D) HE, (E) CK5/6 ICC stain, (F) IMP3 ICC stain. HE, Hematoxylin & Eosin staining; ICC, Immunocytochemistry. (×400).

Figure 2

Metastatic lung adenocarcinoma (LAC) cells are shown in pleural effusion using by (A) HE, (B) IMP3 ICC stain, (C) TTF1 ICC stain; Metastatic adenocarcinoma of non-lung (NLAC) origin are shown in pleural effusion using by (D) HE, (E) IMP3 ICC stain, (F) TTF1 ICC stain. HE, Hematoxylin & Eosin staining; ICC, Immunocytochemistry. (×400).

Figure 3

Conventional PCR amplified products of CK5/6, IMP3, TTF1 DNA in paraffin-embedded cell blocks of patients with reactive mesothelial cells (RMs), metastatic adenocarcinoma of lung (LAC) and non-lung (NLAC) origin in 2% agarose gel. A. IMP3 expression in 6/9 of MAC showed negative in ICC; B. CK5/6 expression in 4/7 of RMs showed negative in ICC; C. IMP3 expression in 6/6 of LAC and 6/6 of NLAC; D. TTF1 expression in 6/6 of LAC and 0/6 of NLAC. M, DNA Maker (DL1000); A1-A6, Paraffin-embedded MAC cell blocks; R1-R6, Paraffin-embedded RMs cell blocks; N, Negative control.

Figure 4

Decision tree for the cytologic diagnosis of reactive mesothelial cells (RMs), metastatic adenocarcinoma of lung (LAC) and non-lung (NLAC) origin by using CK5/6, IMP3 and TTF1 immunocytochemistry (ICC) and conventional PCR (C-PCR) in pleural effusion. HE, Hematoxylin & Eosin staining.