Abstract
Background: DeltaNp63 is an isoform of the p53 homolog p63, which lacks an amino-terminal transactivation domain. The aim of this study was to detect the deltaNp63 expression in the squamous carcinoma component of adenosquamous carcinoma and evaluate its usefulness as a specific squamous carcinoma marker. Patients and Methods: Immunohistochemistry was used to analyze the protein expression of deltaNp63 and high molecular weight cytokeratin in paraffin-embedded tumor samples from 17 patients with well-characterized adenosquamous carcinoma. Results: Of 17 cases, 13 (76.5%) and 14 (82.4%) cases showed positive staining for deltaNp63 and HMWCK in the tumor cells, respectively. It was easy to discriminate the squamous carcinoma and adenocarcinoma components in all tumors. Interestingly, positive expression of deltaNp63 was detected in one case with a negative expression of HMWCK. Conclusion: These findings indicated that the deltaNp63 status was useful for distinguishing squamous carcinoma from adenocarcinoma in formalin-postfixed adenosquamous carcinoma specimens.
The essential histological diagnosis for lung cancer has been its classification into small cell carcinoma (SCLC) or non-small cell carcinoma (NSCLC) because treatment did not differ between the variants. However, several drugs have become available with indications dependent on the precise definition of tumor subtype, in relation to both the efficacy and safety for specific histology. For example, the epidermal growth factor receptor – tyrosine kinase inhibitor (EGFR–TKI) such as gefitinib have superior efficacy in patients harboring a somatic mutation, which is usually observed in only adenocarcinoma (AD) (1). Squamous carcinoma (SQ) histology is now a contraindication for bevacizumab due to fatal adverse events (2). Pemetrexed is also contraindicated in SQ with no significant efficacy, but has superior efficacy in non-SQ over standard platinum-doublet chemotherapy (3). Therefore, the dichotomization of the NSCLC subtype into SQ and AD is increasingly required for both diagnostic meaning and therapeutic selection. Furthermore, the therapeutic strategy for adenosquamous carcinoma (ADSQ) remains unclear. ADSQs are the most suitable research object to determine a useful marker because these tumors include both SQ and AD components (4).
The tumor suppressor p53 and its two homolog p63 and p73, form a family of proteins (5). P63 and p73 show a much greater molecular complexity than p53 because they are both expressed as multiple alternatively spliced C-terminal isoforms, and as an N-terminally deleted form (6). In addition, several other factors, such as post-translational modifications, result in the subtle modulation of their biological effects (7). P63 knockout animals showed defects in epidermal development, suggesting a role of p63 in regulating differentiation in epithelial cells (8). P63 gene amplification and protein overexpression are more often detected in a variety of carcinomas of the head and neck (9), lung (10), and skin (11). Therefore, p63 may play an important role in tumor progression. On the other hands, the overexpression of DeltaNp63, which is a different isoform due to alternative splicing (12), often enhances the oncogenic growth of squamous cell carcinomas (9). Traditionally, p63 has been used as an immunomarker for SQ on tissue sections (13). However, an immunohistochemical (IHC) examination of the expression of p63 used a nonspecific antibody, which reacts not only with the transactivating domain (TA)-p63, but also with deltaNp63 (14, 15). Furthermore, the positive expression of deltaNp63 on SQ was significantly higher than on AD (5).
This study attempted to identify a specific marker to detect only the SQ component of ADSQs using a commercially available antibody. This study investigated the ability of deltaNp63 to discriminate the histology in NSCLC and compared it to high molecular weight cytokeratin (HMWCK), which is known to be a differential marker for SQ and AD (16).
Patients and Methods
Patient samples. The characteristics and clinicopathological factors were evaluated retrospectively. One thousand and one hundred thirty-four patients with primary lung cancer underwent a surgical resection between June 1995 to March 2010 at the Second Department of Surgery at University of Occupational and Environmental Health, Kitakyushu, Japan. The established criteria classified an ADSQ as a tumor containing at least 20% of AD and SQ each (4). Specimens from each of these patients were reviewed critically by one (S.Y.) pathologist. This study evaluated 17 cases (1.5%) with ADSQ. The characteristics of the patients with ADSQ are shown in Table I. Patients included 6 males and 11 females with a mean age of 68.5 years, ranging in age from 48 to 86 years. Institutional Review Board-approved informed consent was obtained from all patients for the use of tumor tissue specimens collected at the time of tumor resection.
Immunohistochemical (IHC) staining in paraffin-embedded tumor samples. All specimens were stained with hematoxylin-eosin (H-E) for the histological diagnosis. IHC staining was conducted using serial sections from the same paraffin-embedded blocks as previously described (17, 18). Briefly, all tissue specimens were formalin-fixed and processed similarly, according to standard histological practices. The sections were briefly immersed in citrate buffer [0.01 mol/l citric acid (pH 6.0)] and were incubated for two 10-minute periods at 121°C in a high-pressure sterilization oven for antigen retrieval. They were then incubated with anti-deltaNp63 (PC373, Calbiochem, Darmstadt, Germany) diluted at 1:3000, in phosphate-buffered saline for 60 minutes at 4°C. They were also incubated with anti-HMWCK (34beta E12; keratin-903; Enzo Life Science, Inc, NY, USA) diluted at 1:40, in phosphate-buffered saline for 60 minutes at room temperature. Thereafter, IHC staining of deltaNp63 and HMWCK was performed by the labeled polymer method using Histofine Simple Stain MAX-PO kit (Nichirei, Tokyo, Japan) and EnVision kit (DAKO, Tokyo, Japan) according to the manufacturer's instructions, respectively (18, 19). Negative controls were processed by exclusion of the primary antibody.
Evaluation of the stained specimens. IHC detected the protein expression in each specimen, then the stained cancer cells were evaluated by a combination of proportional and intensity score because the tumor samples showed various degrees of staining intensity and different numbers of positive cells, (20). Initially, 4 degrees of a proportional score (PS) for the positively staining cells were assigned according to the frequency of positive tumor cells (0, none; 1, <25%; 2, 25-50%; and 3, >50%). Thereafter, 4 degrees of an intensity score (IS) were assigned according to the intensity of the staining (none, weak, intermediate, and strong). Normal epithelial cells of the basal of the bronchus showed intense nuclear staining of deltaNp63, which was used as an internal positive control of intensity 2 (20). The slides were independently examined by two of the investigators (H.U. and S.Y.) who were blinded to the clinicopathological data. When a discrepancy was found between the two investigators, a consensus was reached via their simultaneous examination using a double-headed microscope.
Summary of immunostaining in ADSQ tumors.
Results
Figure 1A and C show the H-E staining for the tumor cells. The typical appearance of staining for deltaNp63 is shown in Figure 1B and D. DeltaNp63 expression only clearly stained the nuclei in the SQ component deep brown, whereas HMWCK expression was specifically detected in the cytoplasm. It was easy to discriminate the SQ in ADSQ. DeltaNp63- and HMWCK-expressing tumor cells were observed in 13 (76.5%) and 14 (82.4%) out of 17 cases, respectively. Interestingly, positive expression of deltaNp63 was detected in a case with a negative expression of HMWCK. The opposite was equally true.
Discussion
The discrimination of tumor histology is important for selecting the modality of therapy. Therefore, an accurate histological diagnosis is imperative and a specific marker is needed to detect the SQ component. The incidence of the positive expression of deltaNp63 is significantly higher in SQ than AD (5). Most of the SQ component of ADSQ showed positive expression of deltaNp63 in spite of the genetic intratumoral heterogeneity (21). Therefore, deltaNp63 is a useful marker for its histopathologic diagnosis. Surprisingly, positive expression of deltaNp63 was detected in a case with negative expression of cytokeratin. Nicholson et al. pointed out low the specificity for SQ of 34beta E2 antibody, though HMWCK is useful as a differential marker for SQ (22). DeltaNp63 and HMWCK antibody might compensate each other as an SQ marker. Why is deltaNp63 localized in the SQ component rather than AD? Boldrup et al. reported elevated CD44, which is a marker of cancer stem cells, expression in the clones overexpressing deltaNp63 (12). In fact, deltaNp63 acts as a dominant-negative reagent toward tumor suppressor p53, thus indicating its oncogenic role (23). Higashikawa et al. reported that the epithelial–mesenchymal transition (EMT) is accompanied by a down-regulation of deltaNp63 (25). Cancer cells form a new focus through the mesenchymal–epithelial transition (MET) (26). Therefore, cancer cells in the SQ component might have a more aggressive biological behavior than that of the AD component. In fact, the survival rate for patients with ADSQ is statistically worse than for those with SQ and AD (27). The triangle of relationships among p53 family, EGFR, and EMT might be key in lung cancer translational research (28-30).
A: Representative low-power view of H-E staining of ADSQ (original magnification ×40). B: DeltaNp63-expressing tumor cells with deep brown-stained nuclei are shown for the same specimen and alignment as shown in A. C: Representative high-power view of H-E staining of ADSQ (original magnification ×200). D: DeltaNp63 IHC staining in the same specimen and alignment as shown in C.
No firm conclusion can be drawn from the result of this study because it observed only a small number of patients analyzed at a single institution; nonetheless, the current results apparently indicate that deltaNp63 status is useful as a specific marker of SQ. Further investigation is necessary to determine the clinical benefit of the biomarker in a larger cohort of patients and in prospective studies.
Acknowledgements
We thank Yukiko Koyanagi and Kanako Sasaki and for valuable technical assistance. This work was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
- Received September 13, 2010.
- Revision received October 25, 2010.
- Accepted October 26, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
