Abstract
Background/Aim: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death. Patients eligible for surgery have better overall survival rate than patients who are not eligible. We investigated the prognostic value of p16 in patients who underwent surgery for lung cancer, in association with other factors such as PD-L1 and Ki-67. Materials and Methods: Expression of p16 was evaluated along with the presence of Ki-67 and PD-L1 in 256 NSCLC patients treated only surgically. Results: Adenocarcinoma was the prevalent histotype (56%) followed by squamous cell (29%) and differentiation grade of 3 was the most common (60%). p16 was detected in 83 patients (30%): low positivity (<10% cells) was observed in 30 samples (11%) and high positivity (>10 % cells) in 53 patients (20%). Ki-67 was detected in 89 patients (34%) with mild positivity in 46 patients (10-25% cells), moderate positivity (26-75% cells) in 30 patients (11%), and high positivity (>75% cells) in 13 patients (5%). An influence of p16 expression (p<0.05) along with grading and staging on overall survival (OS) was found. The average OS was 36 months, but the OS increased up to 54 months when patients were stratified according to p16 expression levels. The stratification by staging showed a significant prognostic value for p16 at an early stage (p<0.014). Conclusion: p16 significantly influences prognosis, notably at an early stage, along with other variables such as grading and staging.
- p16 prognostic value
- ki-67
- resected lung cancer
- early and late stage survival
Lung cancer is the leading cause of cancer death (1). Adenocarcinoma is the most common histotype, followed by squamous cell carcinoma and large cell carcinoma (2). Smoking habit is the main avoidable cause of both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and at the same time it brings about chronic inflammatory comorbidities (3, 4).
It is usually asymptomatic and therefore diagnosed at an advanced stage. Genetic factors and proteins have been investigated as prognostic markers to better determine lung cancer evolution (5).
Among the proteins and factors involved in carcinogenesis and tumor cell proliferation is p16 (6). It is recognised as a tumor suppressor that inhibits phosphorylation of the retinoblastoma protein (pRB) anti-oncogene by binding to the cyclin-dependent kinase complexes (CDK4-CDK6) leading to the arrest in the G1 phase of the cell cycle.
Conversely, p16 gene inhibition leads to phosphorylation of the retinoblastoma protein, unblocking the cell cycle with a subsequent uncontrolled progression of cell growth and increased proliferation in all cancer types (6, 7). Several genetic alterations can induce inactivation of p16 including homozygous deletions, promoter hypermethylation, point mutations and loss of heterozygosity (LOH), but the most frequent are the first two (7). Homozygous deletions and promoter hypermethylation (on the CpG islands of the transcriptional start site) usually lead to its dysregulation, whereas point mutations and small deletions (in particular miss-sense) alter the structure and activity of p16 (8). The interaction with the pRB pathway represents a mechanism of defence of the organism (9).
By contrast, Ki-67 is a nuclear protein expressed in proliferating tumor cells and associated with a high cell growth index (10). It is able to interact with heterochromatin regulators and proteins involved in nucleolar processes. It is associated with smoking habit and tumor aggressiveness (11).
Programmed cell death protein 1 ligand (PD-L1) is another protein involved in cancer progression. It binds to programmed cell death protein 1 (PD-1), an inhibitory receptor of T lymphocytes that reduces T cell function by suppressing signalling pathways of the TCR receptor (12).
We assumed that p16 has variable expression depending on the stage and biological characteristics of the tumor. Thus, it could be used as a prognostic factor in surgically resected patients useful to identify subgroups with different prognosis. We analyzed the relationship between p16 and other tumor-related factors, such as Ki-67 and PD-L1.
Inclusion criteria included a histology sample coming from lung resection. We ruled out patients previously treated with either chemotherapy or radiotherapy.
Materials and Methods
Study population. Among 350 patients who underwent surgery for lung cancer from 2015 to 2018, we considered only 256 histological samples of NSCLC available for PDL1, p16 and Ki67 detection. All subjects had given their written informed consent and that the study protocol was approved by the Ravenna Hospital Institute's committee on human research. Smoking habit, cancer histotype, staging and survival were evaluated.
PD-L1, p16 and Ki-67 were determined on tissue specimens derived from lung pulmonary resection. Inclusion criteria were, age >18 and diagnosis of lung cancer in patients who underwent surgery. Patients previously treated with chemotherapy or radiotherapy were excluded.
The overall survival was considered as the time from diagnosis to death. The staging was performed by total body CT scan at first and then by histology of surgical samples according to the eight edition of lung cancer staging (13).
The study was approved by the Ethic Committee of the Tumor Institute of Ravenna.
Samples description. The micro-array tissue technique (MTA) was used to detect p16 and Ki-67.
Briefly, two cores of 2 mm were punched out from different areas of formalin-fixed paraffin embedded tumor blocks. Tissue sections of 4 μm were dried and applied on glass slides.
The histology was revised according to IASLC-ATS-ERS classification. The staging was provided according to the 8th edition of TNM for NSCLC (14).
Immunohistochemistry. The p16 INK-4A assessment was performed using a set of two different reagents (CINtec® PLUS Kit Roche, mtm laboratories AG, Heidelberg, Germany): a primary monoclonal rabbit antibody clone 274-11AC3 directed against human Ki-67 protein, and a primary monoclonal mouse antibody clone E6H4 directed to human p16-INK4a protein. A two-step immunohistochemical assay was performed: after microwave antigen retrieval and endogenous peroxidase block, a cocktail of the primary antibodies was added for 30 min at a temperature between 22 and 25°C. Visualization reagents comprising a polymer reagent conjugated to horseradish peroxidase and goat anti-mouse antibody (15 min) and a polymer reagent conjugated to alkaline phosphatase and goat anti-rabbit antibody (15 min) were used. The samples were then developed with two different chromogen reactions based on horseradish peroxidase-mediated conversion of DAB chromogen (10 min), and alkaline phosphatase-mediated conversion of Fast Red chromogen (15 min) to visible reaction products at the respective antigen sites. A squamous cell carcinoma of the cervix with known positive staining for p16INK4A was used as the positive control; an immunoglobulin class-matched non-immune antibody was substituted for the primary antibody in the negative control. After counterstaining, a two-step mounting protocol was applied: in the first step an aqueous mounting medium was added, followed by the addition of permanent mounting medium in the second step. The results were evaluated by light microscopy. The immunohistochemical reaction produced two different colours: a red staining of nuclei which indicates Ki-67 expression and a brown precipitate (cytoplasm and/or nuclei) indicating p16-INK4a.
Immunohistochemical scoring was determined according to previous histological studies (15).
The p16 was considered as a positive test result when a diffuse staining was observed; a focal staining pattern of <10% isolated cells or small cells clusters was considered a low positivity whereas a positivity in >10% of cells was considered high positivity.
Scoring for Ki-67 included nuclear staining only, and was scored as 0 (no staining), low 1 (<25%), moderate 2 (26-75%), high 3 (>76%).
Statistical analysis. The values were expressed as mean±SD or average and interquartile range as appropriate. The level of statistical significance was set at p<0.05. The OS was determined in the whole population and according to the p16 expression using the Kaplan-Meier method.
Comparison between p16 positive and p16 negative patients was performed by the log -rank test assuming that the ratio of hazard functions (deaths per time) was the same at all time points.
Multivariate and univariate analysis were performed using overall survival as dependent variable. A linear regression analysis was performed to correlate Ki-67 with p16 expression.
A cox proportional-hazard regression analysis was also performed to detect the influences of variables on survival. A contingency table was applied to estimate the difference of p16 expression between two histotypes. SPSS version 24.0 was used for analysis.
Results
The case study included 256 samples among NSCLC patients who underwent surgery and had tissue available for immunohistochemistry. The population was mainly consisted of males, Table I.
The mean age was 67.3 years. Adenocarcinoma was the prevalent histotype (roughly 56% of patients), whereas squamous cell represented about 29% of patients. Grade 3 was the most common grade of differentiation (60% of cases).
Patients were mainly former smokers (70%) and EGFR was mutated in about 10% of patients, consistently with the literature.
Stage IIB was more frequent than the others stages (23%) and lobectomy was the surgical technique mostly used (66%). The median time to progression (TTP) was 60 months (5 years) and the overall survival (OS) was 36 months. p16 was positive in 30% of samples, of which two-thirds were high positive. The Ki-67 was positive in roughly 34% of samples, with mild positivity in 46 patients (10-25% cells), moderate positivity (26-75% cells) in 30 patients (11%), and high positivity (>75% cells) in 13 patients (5%). The PD-L1 was positive in 39% of patients of which 20% had high positive expression, higher than 50%.
Table II shows that some considered covariates differentially affected survival in the univariate model.
Gender (p<0.25), PD-L1 expression (p<0.9), and Ki-67 (p<0.5) did not affect significantly OS, whereas age (p<0.007), grading (p<0.002), and staging (p<0.01) affected significantly OS.
Regarding p16, it seems to significantly influence prognosis (p<0.05).
Table III includes the variables that were significant in the univariate model. Age and staging also significantly affected OS in the multivariate model.
Table IV displays the association of p16 expression with each considered stage.
It was found that a high level of expression of p16 is notably associated with early stage such as IA1 and IA2.
Table V indicates the difference of p16 expression in adenocarcinoma vs. squamous cell; the difference was not found to be significant.
The Kaplan-Meyer survival curve for the whole population is depicted in Figure 1. The average survival was 36 months. The OS in the subgroup of patients who expressed p16 in less than 10% of cells was 54 months.
Figure 2 shows that the OS was not significantly affected by the p16 expression level (p=0.25).
Figure 3 shows that OS was not significantly different (p=0.10) between patients with less and higher than 10% positive cells.
Stratification of OS by staging, indicated that in early stage there is a significant difference between patients with low expression and patients with higher expression (Figures 4 and 5). The difference in OS for stage IA2 was found statistically significant with a better OS with mild expression of p16 (p=0.014).
Figure 6 displays the relationship between Ki-67 and p16 showing that Ki-67 is mostly negative in p16 positive samples. Furthermore, immunohistochemical (IHC) analysis indicated that negative Ki-67 is associated with low p16 expression (Figure 7).
Discussion
The present study concerns 256 patients who underwent surgery for lung cancer. This is a significant number, because the current resection rate of patients diagnosed with lung cancer is very low (14). The patients were mainly former smokers and it is known that cigarette smoking adversely affects several genetic and translational pathways including p16 and p53 dysregulation, leading to an alteration in G/S phase transition (16, 17).
Several transcriptional factors could be counterbalanced by tumor suppressor genes (16). While p16 is clearly a tumor suppressor who limits proliferation, aberrant overexpression of p16-ink4a is observed in different cancers (18); its overexpression has been demonstrated to be associated with human papilloma virus infection (HPV) (19, 20). The p16 has mainly been studied in squamous cells tumors, whereas in our cohort we found altered expression in both adenocarcinoma and squamous cell lung cancer. Moreover, different authors have reported that an increased expression of p16 is significantly associated with reduced OS in human cancers such as breast cancer, and it is also related to senescence and high-grade cancer (21, 22).
In the current study, we found that high expression of p16 is associated with positive Ki-67 indicating that p16 is present but is probably dysregulated by deletion or phosphorylation and is associated mainly with the RB loss of function (22). Thus, our results indicate that p16 could be a potential marker of lung cancer evolution and aggressiveness. Our results also indicated that PD-L1 and Ki-67 do not affect significantly OS. By contrast, staging, tumor grading, patients' age and p16 expression seem to affect separately the prognosis.
There is evidence that, in addition to its function in cell cycle regulation, p16 is also implicated in other processes, such as apoptosis, cell invasion and angiogenesis, and it is associated with high grading (23).
The p16 inactivation is a critical step involved in tumor progression and high malignancy development probably combined with an altered immune system response (24, 25). The compromise of p16 function by gene missense mutations can be balanced by the increased p16 levels observed in some tumours (26). This was probably found to be associated with the quantitative high levels of p16, that in turn could foster the relapse of a malignant disease (26).
Our data support this hypothesis, since a high expression was associated with a poor prognosis.
In our study the significant prognostic value of p16, notably in early stage resected lung cancer, was irrespective of the histotype, whereas in the literature an association with squamous cell histotype has been reported (27). The microtissue array method that was applied in the present study has proven to be a quick and effective method consistently with the literature. It is very useful when a large population is studied, because only small tissue samples are needed for analysis (28).
Conclusion
The p16 protein confirms its prognostic value in malignant lung cancer notably in early stage disease, and is not associated with a specific histotype. It is involved in tumour growth along with other factors, such as Ki-67 and PD-L1. Age, grading and staging are also factors affecting prognosis.
Acknowledgements
The Authors thank Dr. Navarini and Prof. Ciccozzi for the support in the statistical analyses.
Footnotes
Authors' Contributions
Dr. Pezzuto envisioned the work, Dr. Carico performed the immunohistochemistry test, Dr. D'Arcangelo, Dr Cappuzzo, Dr Guerrini, Prof Ricci, Dr D'Ascanio contributed to the results.
Funding
Resources were derived from a master's degree in the cytopathology of Sapienza University in Rome, Italy.
Conflicts of Interest
There are no conflicts of interest to declare regarding this study.
- Received December 31, 2019.
- Revision received January 11, 2020.
- Accepted January 15, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved