European Journal of Cardiovascular Medicine

Lung cancer is one of the most prevalent and deadly malignancies worldwide, with varying histopathological subtypes that significantly influence treatment decisions and patient outcomes. Among these subtypes, squamous cell carcinoma (SCC) and adenocarcinoma (ADC) are two major types that differ in their histological features, molecular profiles, and therapeutic responses. ^[1] Effective differentiation between SCC and ADC is crucial for guiding appropriate treatment strategies, as they may respond differently to targeted therapies and chemotherapy regimens. ^[2]

Immunohistochemistry (IHC) has emerged as a valuable tool in the diagnostic workup of lung cancers, offering insights into the differentiation of these tumor types through the expression of specific biomarkers. Two commonly used markers in this context are P40 and P63, which belong to the p53 family of nuclear proteins. ^[3] While both markers are predominantly associated with squamous differentiation, their utility and specificity in distinguishing SCC from ADC in lung cancer have been subjects of debate and investigation. ^[4]

This comparative analysis aims to evaluate the diagnostic performance of P40 and P63 immunomarkers in differentiating SCC from ADC in lung cancer specimens. ^[5] By systematically reviewing the existing literature and comparing their sensitivity, specificity, and overall diagnostic accuracy, this study seeks to provide evidence-based recommendations for their clinical application.

By critically examining the evidence surrounding P40 and P63 as immunomarkers for the differentiation of SCC and ADC in lung cancer, this comparative analysis aims to contribute to the refinement of diagnostic practices and ultimately improve patient care and outcomes in clinical settings.

This is a prospective study was conducted in the Department of Pathology at Tertiary Care Teaching Hospital over a period of 1 year. A total of 80 primary lung carcinoma cases included over a period of one year with unequivocal morphological diagnosis irrespective of age, gender and nature of biopsy material (endoscopic biopsy/ needle core biopsy / resected specimen).

Cases diagnosed as small cell carcinoma of lung, as metastatic lung cancers, poorly differentiated non-small cell carcinomas-non-committal diagnosis (NSCLC-NOS) and with inadequate material for IHC study were excluded from present study.

Primary antibodies

Two antibodies were used in this study, mouse monoclonal p63 antibody and mouse monoclonal p40 antibody. Normal skin and laryngeal squamous cell carcinoma tissues were used as the positive controls for p63 and p40, respectively.

Hematoxylin and eosin stained slides were reviewed by pathologists to confirm the diagnosis. Tumours were graded on the basis of WHO classification. 

IHC Analysis were done by using antigen retrieval method: BIO GENEX-EZ Retriever system V.2 (temperature controlled microwaving). Percentage of SqCC and ADC showing positivity for P40 and P63 immunomarker and Sensitivity and specificity was calculated. Immunohistochemistry with p40 antibody was performed at in department. Antigen retrieval was performed with CC1 buffer (Cell Conditioning 1; citrate buffer pH 6.0, Ventana Medical Systems). Immunohistochemistry for p63 (TP63; 4A4, Dako, 1:700 dilution) was performed. P63 (4A4) recognizes an epitope shared by TAp63 and DNp63 isoforms, whereas p40 recognizes an epitope which is unique to DNp63.

Immunohistochemical staining analysis

The staining intensity of both p40 and p63 markers were scored as 0, 1+, 2+, or 3+ and the percentage of immunoreactive cells were recorded. Cases were considered positive if 5% or more of the tumour cells showed brown nuclear staining. Cases with less than 5% staining or no areas of positive staining were regarded as negative.

A total of 80 cases of NSCLC were investigated. More than half of the cases presented with distant metastasis at diagnosis (63.2%). For squamous cell carcinoma, 35 (87.5%) cases were males and Five cases (12.5%) were females. Adenocarcinoma had a more similar sex distribution, with 21 cases (52.5%) in males and 19 cases (47.5%) in females. The clinical information is summarised in Table 1.

Table 1. Distribution of Gender of patients diagnosed with lung squamous cell carcinoma and lung adenocarcinoma

SD, standard deviation.

Table 2: Distribution of Mean Age of patients diagnosed with lung squamous cell carcinoma and lung adenocarcinoma

The mean age at squamous cell carcinoma diagnosis was 60 years, ranging from 36 to 80 years. The mean age for adenocarcinoma was 59 years, ranging from 32 to 76 years in table 2.

Table 3: Distribution of Histologic grade of patients diagnosed with lung squamous cell carcinoma and lung adenocarcinoma

Squamous cell carcinomas comprised 9 well, 14 moderately, and 17 poorly differentiated tumours. Among the adenocarcinomas, there were 10 well, 16 moderately, and 14 poorly differentiated carcinomas in table 3.

Table 4. Expression of p40 and p63 in lung squamous cell carcinoma and lung adenocarcinoma

Values are presented as number (%).

p40 immunostaining

Thirty cases of primary lung squamous cell carcinoma (75%) were positive for p40 (Table 4). Remarkably, 10 cases that were previously diagnosed as primary lung squamous cell carcinoma showed negative staining. On the other hand, 40 cases that were previously diagnosed as Adenocarcinoma showed negative staining.

P63 immunostaining

Thirty three cases of primary lung squamous cell carcinoma (82.5%) were positive for p63. Moreover, 7 cases that were primary lung squamous cell carcinoma showed negative staining. On the other hand, 15 cases of Adenocarcinoma (37.5%) were positive for p63. In addition, 25 cases that were diagnosed as Adenocarcinoma showed negative staining in table 4.

Two cases of lung adenocarcinoma showed weak p40 expression (1+). However, the immunoreactive cells were minimal (in less than 5% of tumour cells) and scattered with no specific pattern. Immunopositivity of p40 and p63 according to tumour differentiation is shown in Table 5.

Table 5. Immunoreactivity for p40 and p63 in lung SCC and lung ADC

Table 6. Immunopositivity of p40 and p63 according to tumour differentiation

Values are presented as number (%).

Table 7.Histologic findings and IHC profile of p40-negative lung squamous cell carcinoma

IHC, immunohistochemistry; CK, cytokeratin; TTF1, thyroid transcription factor 1.

Table 7 summarizes immunohistochemical profile of p40-negative squamous cell carcinoma and histologic grading.

The findings in this study confirm and expand upon several recent reports, suggesting that p40 antibody (detecting ΔN isoform of p63) is markedly superior to the standard p63 4A4 antibody (detecting both ΔN and TA isoforms) in the diagnosis of pulmonary squamous cell carcinoma. The key finding in this study is that in a large series of whole-tissue sections, p40 was equivalent to p63 in sensitivity for squamous cell carcinoma; all squamous carcinomas were positive for both markers, and reactivity for both markers was consistently diffuse.

The extent and intensity of p40 reactivity in squamous cell carcinoma were indistinguishable from that of p63. The second key finding is that p40 was markedly superior to p63 in specificity. Although p63 showed significant reactivity in adenocarcinomas (31% of cases) and large cell lymphomas (54% of cases), only rare adenocarcinomas (3% of cases) had labelling for p40, which was always minimal (≤5% of tumor cells), and absolutely all lymphomas were completely negative for p40.

Overall our findings confirm the initial observations made in several smaller studies in which immunohistochemistry for p40 was evaluated in lung adenocarcinomas and squamous cell carcinomas. The first study to describe immunohistochemistry with p40 antibody was by Hibi et al^[6] in 2000, where p40 was found to be entirely sensitive and specific for squamous cell carcinoma, based on analysis of 23 lung carcinomas. This finding, however, has remained dormant until recently, when Pelosi et al^[7] examined whole-tissue sections of 20 pulmonary adenocarcinomas, and subsequently 46 matched small-biopsy/cytology samples and surgical resections of non-small cell carcinomas, ^[8] and found p40 antibody, unlike p63 4A4 antibody, to be 100% squamous-specific.

In the one case of p40 labeling in adenocarcinoma, the staining was described as weak and focal. Finally, Del Vescovo et al^[9] investigated p40 reactivity in 50 lung resections, and found it to be 95.8% sensitive and 100% specific for squamous cell carcinoma. Although the remarkable specificity of p40 compared with p63 was suggested in these prior investigations, the main contribution of the current study is that here we performed a comprehensive comparison of p63 and p40 in a large series of whole-tissue sections of lung carcinomas (n=318), allowing us to establish definitively the markedly superior specificity and equivalent sensitivity of p40 compared with p63 for squamous cell carcinoma.

Given the exceedingly high specificity of p40 for squamous cell carcinoma, the significance of rare adenocarcinomas with p40 immunoreactive cells (which in all cases was minimal, not exceeding 5% of tumor cells) is unclear. These adenocarcinomas labeled diffusely for TTF-1 and there was no overt morphological evidence of squamous differentiation (data not shown). Additional studies will be needed to investigate the potential significance of p40 reactivity in these rare adenocarcinomas, particularly in tumors with a peculiar basal-like distribution of immunoreactive cells. From a practical standpoint, because of minimal extent of p40 reactivity (compared with consistently diffuse positivity in squamous cell carcinoma) and diffuse expression of TTF-1 (which is not expected for squamous cell carcinoma), this reactivity should not present a difficulty in the distinction from squamous cell carcinoma. Of note, in this series we have not encountered focal but significant p40 reactivity (10–40%) in either adenocarcinoma or squamous cell carcinoma; therefore the interpretation of such reactivity.

In addition to the analysis of p40 versus p63 in non-small cell carcinomas, this study represents the largest review of these markers in various large cell lymphomas. This is relevant because occasionally these tumors can be mistaken for lung cancer, and p63 reactivity in this setting can be misleading. Anecdotally, a case submitted to one of our institutions in consultation as suspected squamous cell carcinoma on the basis of p63 labeling was later proven to be lymphoma on further workup (WDT and NR, unpublished observations). p63 labeling in large cell lymphomas in our study is in the range of what has been previously reported. ^[10-16]

Overall, p63 4A4 is one of the most widely utilized multipurpose antibodies in the diagnostic immunohistochemistry of tumors. Common applications outside of lung include the diagnosis of invasion in prostate and breast cancer, where p63 is used to document the loss of basal and myoepithelial cells, respectively, and the diagnosis of squamous carcinomas of various sites, as well as urothelial and myoepithelial neoplasms. ^[17] Unexpected p63 reactivity in large cell lymphomas and various other tumor types may also present a diagnostic dilemma in these extra-pulmonary settings, and it may therefore be of interest to explore the utility of p40 at other sites.

We find that p40 is equivalent to p63 in sensitivity for pulmonary squamous cell carcinoma, but it has a marked advantage over p63 in that it is also remarkably specific. In rare cases in which p40 labeling is seen in adenocarcinoma, it is very focal, limited to isolated tumor cells, which is readily distinguishable from the diffuse reactivity in squamous cell carcinomas. We suggest that a strong consideration should be given for a routine use of p40 in place of p63 as a marker of pulmonary squamous cell carcinoma.

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