Abstract and Introduction
Immunohistochemistry has recently emerged as a powerful ancillary tool for differentiating lung adenocarcinoma and squamous cell carcinoma—a distinction with important therapeutic implications. Although the most frequently recommended squamous marker p63 is extremely sensitive, it suffers from low specificity due to its reactivity in a substantial proportion of lung adenocarcinomas and other tumor types, particularly lymphomas. p40 is a relatively unknown antibody that recognizes ΔNp63—a p63 isoform suggested to be highly specific for squamous/basal cells. Here we compared the standard p63 antibody (4A4) and p40 in a series of 470 tumors from the archives of Memorial Sloan–Kettering Cancer Center and The Johns Hopkins Hospital, which included lung squamous cell carcinomas (n=81), adenocarcinomas (n=237), and large cell lymphomas (n=152). The p63 was positive in 100% of squamous cell carcinomas, 31% of adenocarcinomas, and 54% of large cell lymphomas (sensitivity 100%, specificity 60%). In contrast, although p40 was also positive in 100% of squamous cell carcinomas, only 3% of adenocarcinomas, and none of large cell lymphomas had p40 labeling (sensitivity 100%, specificity 98%). The mean percentage of p63 versus p40-immunoreactive cells in squamous cell carcinomas was equivalent (97 vs 96%, respectively, P=0.73). Rare adenocarcinomas with p40 labeling had reactivity in no more than 5% of tumor cells, whereas the mean (range) of p63-positive cells in adenocarcinomas and lymphomas was 26% (1–90%) and 48% (2–100%), respectively. In summary, p40 is equivalent to p63 in sensitivity for squamous cell carcinoma, but it is markedly superior to p63 in specificity, which eliminates a potential pitfall of misinterpreting a p63-positive adenocarcinoma or unsuspected lymphoma as squamous cell carcinoma. These findings strongly support the routine use of p40 in place of p63 for the diagnosis of pulmonary squamous cell carcinoma.
Until recently, the only clinically significant histological distinction for lung cancers was between small cell carcinoma and non-small cell carcinoma. With the introduction of novel targeted therapies that are used differentially in lung adenocarcinoma and squamous cell carcinoma—the two most common types of non-small cell lung carcinomas—the precise distinction of these tumor types has become imperative. For example, the small molecule EGFR inhibitors erlotinib and gefitinib are only indicated in non-small cell lung carcinomas with EGFR mutations, which occur almost exclusively in adenocarcinoma. Similarly, the recently identified EML4-ALK rearrangement, which predicts susceptibility to the targeted agent crizotinib, also occurs only in adenocarcinoma. In addition, the angiogenesis inhibitor bevacizumab and the folate antimetabolite pemetrexed are excluded from use in patients with squamous cell carcinoma due to the association with pulmonary hemorrhage or the lack of effectiveness, respectively. Finally, two therapeutically targetable alterations, DDR2 mutation and FGFR1 amplification, have recently been identified in pulmonary squamous cell carcinoma. These advances underlie the growing importance of accurate identification of non-small cell carcinoma subtype for assigning patients to appropriate 'histology-based' therapies and the triage of tissue for appropriate molecular studies.
Despite the clear need for pathologists to subclassify non-small cell carcinomas accurately, making the morphological distinction between squamous cell carcinoma and adenocarcinoma can be difficult. Distinguishing features, such as gland formation or keratinization, are not always evident in poorly differentiated carcinomas, particularly when the material for review is scant (eg, biopsy or cytology). Numerous immunohistochemical markers have been recently explored for their utility in distinguishing pulmonary squamous cell carcinoma and adenocarcinoma, and p63 has emerged as the 'front runner' of the squamous markers. Several studies have shown that p63 has an extremely high sensitivity (approaching 100%) for squamous cell carcinoma. However, the main limitation of p63 is low specificity due to its unexpected reactivity in 16–65% of lung adenocarcinomas. In the majority of p63-positive adenocarcinomas, expression is focal, but in a subset, it is strong and diffuse, approaching the extent typical of squamous cell carcinoma. Another important limitation of p63 as a 'squamous marker' is its unexpected expression in several other tumor types, particularly lymphomas, where reactivity has been reported in up to half of the cases. This is a particularly treacherous pitfall with drastic treatment implications, as large cell lymphoma may present as a solitary thoracic mass, and its epithelioid morphology may closely mimic non-small cell carcinoma. In this setting, strong expression of p63 could misclassify an unsuspected lymphoma as squamous cell carcinoma. Clearly, a marker of squamous differentiation that shares the sensitivity of p63, but is also highly specific, would be extremely useful.
p63 is normally expressed in the basal or progenitor cell layer of stratified epithelia (eg, squamous, urothelial, bronchial), basal cells of some glandular epithelia (eg, prostate), as well as myoepithelial cells of breast and salivary glands, trophoblasts and thymic epithelial cells. Tumors consistently positive for p63 include squamous cell carcinomas of lung and extra-pulmonary sites, urothelial, myoepithelial, trophoblastic, and thymic epithelial neoplasms—the malignant counterparts to cells normally expressing p63. The perplexing issue with p63 is its unexpected sporadic presence in various other tumor types, such as lung adenocarcinomas and lymphomas, mentioned above, as well as some sarcomas and various carcinomas, eg ovarian, endometrial, breast, and colorectal.
Although frequently thought of as a single molecule, p63 in fact consists of several variants (isoforms), which fall into two major groups—TAp63 and ΔNp63 (Figure 1). These isoforms differ in the structure of the N-terminal domain. TAp63 isoform contains a transactivation-competent 'TA' domain with homology to p53, which regulates expression of the growth-inhibitory genes. On the other hand, ΔNp63 isoform contains an alternative transcriptionally-inactive 'ΔN' domain, which is thought to antagonize the activity of TAp63 and p53. Therefore 'p63' is a 'two-in-one' family of opposing molecules: TA—a p53-like tumor suppressor and ΔN—an oncogene. It has been suggested primarily in laboratory studies that the predominant p63 isoform in basal/progenitor cells is specifically the ΔN variant, whereas the TA isoform has a wider tissue distribution. ΔNp63 is thought to function as a stem cell factor, responsible for maintaining cells in an uncommitted state with regenerative potential—a role that may be recapitulated in tumors derived from these cells. In line with this functional role, it was noted that the predominant p63 transcript in squamous cell carcinomas of lung and other sites is ΔNp63. As a corollary, these studies suggest that it is the TAp63 isoform that is responsible for the unexpected presence of p63 in certain tumors.
Diagram of p63 isoforms and antibody binding sites.
The p63 antibody that is routinely used in most pathology laboratories is 4A4. As shown in Figure 1, this antibody recognizes both TAp63 and ΔNp63 isoforms, and it is therefore a 'pan-p63' marker. Antibodies that distinguish different p63 isoforms, particularly the antibody designated p40 which recognizes exclusively ΔNp63 and not TAp63, have been available for several years. Surprisingly, p40 antibody is virtually unknown as a diagnostic marker in pathology, although its use has been reported in a several organ systems, including squamous cell carcinomas of head and neck, and esophagus, thymomas, urothelial carcinomas, and trophoblastic tumors. Recently, several studies have reported on the use of p40 for the distinction of lung squamous cell carcinoma and adenocarcinoma, suggesting that unlike p63 4A4 antibody, p40 antibody is highly squamous-specific. Here we sought to expand on these initial observations and to comprehensively compare the performance of p40 (antibody recognizing ΔNp63 only) versus p63/4A4 (antibody recognizing both ΔNp63 and TAp63 isoforms) in a large series of whole tissue sections of lung squamous cell carcinoma and adenocarcinoma (n=318). In addition, given the potential pitfall in differentiating unsuspected p63-positive large cell lymphomas from squamous cell carcinoma, we evaluated the reactivity of p40 versus p63 in various large cell lymphomas (n=152), specifically focusing on the types which may present as a solitary thoracic mass and feature epithelioid morphology (diffuse large B-cell lymphoma, mediastinal large B-cell lymphoma, anaplastic large cell lymphoma).