Ulrike Walz-Eschenlohr1, Susan Witt1,2 and Thomas Dieterle1,3*
Received: June 19, 2023; Published: June 26, 2023
*Corresponding author: Thomas Dieterle, Professor of Cardiology, Földiklinik GmbH & Co. KG and Department of Clinical Research, University Hospital Basel, Roesslehofweg 2-6, D-79856 Hinterzarten, Germany
Introduction: Growing body of evidence suggests that molecular markers could have important prognostic value in non-small cell lung carcinoma (NSCLC) patients. Using targeted therapy based on these markers leads to improved outcomes in lung adenocarcinoma. However, progress of targeted therapy in squamous lung carcinoma is still modest. p16(ink4) protein acts as tumor suppressor.
Aim: Purpose of this study was to evaluate the difference in p16(ink4) expression between squamous and adenocarcinoma of the lung; to evaluate the relationship of p16(ink4) expression to survival outcomes in NSCLC patients, and the difference of their prognostic values between squamous and adenocarcinoma subtypes.
Material and Methods: 100 NSCLC patients (50 squamous and 50 adenocarcinoma) and 80 healthy individuals were included. p16(ink4) was immunohistochemicaly detected on formalin-fixed tissues. One- and 2-year progression-free survival (PFS) and overall survival (OS) were observed.
Results: p16(ink4) expression was significantly lower in squamous type compared to adenocarcinoma. In both squamous and adenocarcinoma, low p16(ink4) expression correlated with worse 1- and 2-year PFS, as well as OS. In squamous lung carcinoma p16(ink4) expression was an independent negative prognostic marker.
Conclusion: Our study confirms the difference of p16(ink4) protein expression in squamous and adenocarcinoma of the lung. Besides anti-VEGF therapy, the regulation of p16(ink4) expression could have a therapeutic potential in NSCLC, especially in squamous lung carcinoma.
Keywords: Non-Small Lung Carcinoma; p16(ink4); VEGF; Expression; Survival
Abbreviations: OS: Overall Survival; PFS: Progression-Free Survival; NSCLC: Non–Small–Cell Lung Carcinoma; VEGF: Vascular Endothelial Growth Factor; MCLC: Macrocellular Lung Carcinoma; ADC: Adenocarcinoma; SCC: Squamous Cell Carcinoma
Lung carcinoma is usually diagnosed in an advanced stage . Clinically it is divided into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) compromising 80-85% patients. Histopathologically NSCLC is divided into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and macrocellular lung carcinoma (MCLC) . Therapy based on this classification showed modest improvement in survival benefits [1,2]. In the pathogenesis of lung carcinoma essential place have numerous biological processes of which tumor suppression and disorders in angiogenesis seems to be crucial. Growing body of evidence suggests that lung carcinoma is even more heterogenous pathological entity rather than 4 basic types . So far, a large number of molecular markers have been found, but further research is needed to determine their clinical significance. In this content, latest improvement in therapy based on molecular changes (targeted therapy) showed significant clinical benefit in advanced adenocarcinoma of lung . On the other side, there is still no targeted therapy of squamous lung carcinoma , which is implication from poorly understood molecular profile of this disease. p16 protein, which is product of INK4a gene, similar to p53, plays important role in the control of cell cycle by inhibition of cyclin-depended kinase 4 (Cdk4) [6,7]. p16(ink4) inactivation could be found in many solid tumours . At the same time, loss of p16(ink4) suppression could be one of the first initiator in promotion of carcinogenesis [7,8]. Clinical value of p16(ink4) in lung carcinoma is still poorly understood and results from several studies are quite contradictory. So, p16(ink4) methylation did not correlated significantly to overall survival  and expression of p16 protein was not significant prognostic factor in resectabile non-small lung carcinoma . On the other side, low or aberrant p16(ink4) expression was strong and independed poor prognostic factor in NSCLC [11-13].
Vascular endothelial growth factor (VEGF) is a signaling protein that is an essential part of the process of vasculogenesis and angiogenesis . Overexpression of VEGF is important in the growth of the tumor mass, malignant invasion and metastasis, and is caused primarily by molecular mechanisms related to hypoxia [14-17]. Similar to p16(ink4), results of studies regarding prognostic value of VEGF in lung carcinoma are inconclusive. Some authors report that sera VEGF expression does not correlate significantly to histological type or clinical stage , and tissue VEGF expression does not correlate to survival rates . Meanwhile, another authors concluded that combination of VEGF-A and VEGFR-2 is independent prognostic factor in advanced lung carcinoma , and sera VEGF expression could be negative prognostic factor [17,20]. To our knowledge, our study is the first to compare p16(ink4) and VEGF expression between tumour and tumour surroundings, as well as between ADC and SCC. The aim of this study was to evaluate the difference in p16(ink4) and VEGF expression between tumour tissue and tumour surroundings in NSCLC patients; between squamous and adenocarcinoma of the lung; to evaluate the relationship of p16(ink4) and VEGF expression to survival outcomes in NSCLC patients, and the difference of their prognostic values between squamous and adenocarcinoma subtypes. The results of this study could improve the molecular mapping of lung carcinoma and may offer valuable insights into identifying potential anti-angiogenic targeted therapy in lung carcinoma.
This retrospective-prospective, longitudinal and consecutive study was begun after it was approved by the Human Research Ethics Committee of Bosnia and Herzegovina and all patients gave their informed written consent. Collection and subsequent preservation (formalin fixation, paraffin embedding) of tissue samples from the participating patients and controls as well as immunohistochemical evaluation were performed at General Hospital Tesanj (Bosnia and Herzegovina).
100 patients with non–small–cell lung carcinoma (NSCLC), including SCC (n=50) and ADC (n=50) (Table 1), and 80 age-, sex- and smoke-habits-matched individuals with no clinical evidence of a malignant disease (control group) were included (Table 2). For pathological examination and immunohistochemistry, forceps biopsies of tumor tissue and surrounding tissue (2 cm radius around primary tumor tissue without malignant cells) were taken from all patients during routine bronchoscopy examination. The diagnosis of SCC or ADC in all tumor tissue samples was confirmed by two experienced pathologist before enrolment into the study. Tissue samples were immediately formalin fixed and then paraffin embedded using standard procedures.
Tissue Sampling and Immunohistochemistry
Sections of 5 µm were cut from paraffin-embedded tissue blocks. From each tissue specimen, sections stained with haematoxylin-eosin were used for morphological examination. For the purposes of the p16 immunohistochemical analysis, sampled tissue was deparaffinised with xylene solution, then rehydrated, washed with alcohol, and washed twice with water. In order to antigen unmasking, all samples were incubated in 0.01 mol/l of citrated buffer (pH 6.0) for 25 minutes. In the next 15 minutes, the tissue material was alternately cooled and rinsed with distilled water, and treated with 3% H202 deluded in methanol. Thereafter the samples were rinsed with water again, then with phosphate saline buffer, and conjugated with 10% horse serum during 30 minutes. Unmasking of p16 antigen (pre-deluded mouse monoclonal antibody) was performed by heating at a temperature of 95°C in 0.001 M EDTA solution (pH 8.0) during 25 minutes, and then been cooled for 15 minutes and rinse in 0.05M Tris-saline buffer with Tween 20.
For the purposes of immunohistochemical analysis of VEGF, sampled tissue was deparfinizirano and rehydrated routinely. Antigen unmasking was performed in 0.01 mol/L citrate buffer with 10 minutes of heating and 30 minutes of cooling. The activity of endogenous peroxidase has been inhibited after incubation with 3% hydrogen peroxide in methanol during 20 minutes. Nonspecific binding was inhibited with 5% bovine serum in phosphate buffered saline at room temperature. After washing, the samples were left overnight at 4°C with anti-human monoclonal anti-VEGF antibody (Clone JH121, NeoMarkers, Lab Vision Corporation, Fremont, CA, USA, 2011). After incubation, the samples were treated with 0.05 M Tris-buffered saline (pH 7.6) in combination with H202. Lung and breast tissue were used as positive control. p16 and VEGF expression on each sample were determined with semiquantitative value of 0 to 3 (0 - no expression; 1 - low deregulated; 2 - moderate expression; 3 - high expression). Pathohistopathological analysis of immunohistochemically stained samples preparations were performed by two experienced cytopathologists independently. Mean result of both analyses was calculated. p16(ink4) and VEGF expression were compared between SCC, ADC and control, as well as in relation to one- and two-year progression-free survival (PFS) and overall survival (OS).
The distribution of data was determined using the D’Agostino and Pearson omnibus normality test. The strength of association between p16 and VEGF expression levels were analyzed with the Mann-Whitney U-test, unpaired t test or Fisher’s exact test as appropriate. All patients included in this study were observed for 1 and 2 year; after this time survival probabilities (PFS and OS) were calculated from the day of diagnosis to the time of event (progress or death, respectively) or loss to follow-up using the Kaplan-Meier method and the log-rank test was used to compare different categories. Statistical analyses were performed using GraphPad Prism 5 software (San Diego, California, USA) and probability values of p<0.05 were accepted as significant according to Bonferroni correction for 5 tests. Statistically significant differences are presented as: *p<0.05; **p<0.01; ***p<0.001.
p16(ink4) and VEGF Expression in Adenocarcinoma and Squamous Carcinoma of Lung and Corresponded Surroundings
Tumor and tumor surrounding showed similar p16(ink4) and VEGF expression in both SCC and ADC patients (p>0.05 for both groups). In both SCC and ADC, p16(ink4) expression was significantly lower in tumor tissue, as well as in tumor surrounding tissue compared to the control (SCC tumor tissue vs. control: p<0.0001; SCC tumor surrounding tissue vs. control: p<0.0001; ADC tumor tissue vs. control: p<0.05; ADC tumor surrounding tissue vs. control: p<0.05). On the other side, VEGF expression, in SCC as well as in ADC patients, was significantly higher in tumor tissue and also in tumor surrounding tissue compared to the control (SCC tumor tissue vs. control: p<0.01; SCC tumor surrounding tissue vs. control: p<0.05; ADC tumor tissue vs. control: p<0.001; ADC tumor surrounding tissue vs. control: p<0.01) (Figure 1).
Figure 1 (A, B, C and D). p16(ink4) (A and C) and VEGF (B and D) expression in SCC (A and B) and ADC (C and D) and their surroundings.
p16(ink4) and VEGF Expression Between Adenocarcinoma and Squamous Carcinoma of Lung
p16(ink4) expression in SCC patients was significantly lower compared to ADC (p<0.0001). On the contrary, VEGF expression was similar in SCC and ADC (p>0.05) (Figure 2).
Figure 2 (A and B). p16(ink4) (A) and VEGF (B) expression between squamous and adenocarcinoma of lung.
Median PFS for the entire group of patients was 86 days (one year) and 453 (two years), while median OS was 178.5 days (one year) and 542 (two years). No significant differences in PFS or OS for the entire group of patients were observed when patients were stratified according to histological type of lung carcinoma or disease stages (data not shown). In both SCC and ADC, higher p16(ink4) expression was significantly associated with longer one-year PFS, as well as with longer OS (p<0.001 for all measurement) (Figure 3). Also, higher p16(ink4) expression was significantly associated with longer two-year PFS and OS in both SCC and ADC (p<0.0001 for all measurement) (Figure 4). Similarly, higher VEGF expression was significantly associated with longer one-year PFS and OS in SCC and ADC (p<0.001 for all measurements) (Figure 5). However, higher VEGF expression was significantly associated with longer two-year PFS (p<0.001) and OS (p<0.001) in ADC, but not in SCC (p>0.05 for both PFS and OS) (Figure 6).
Figure 3 (A, B, C, D). p16(ink4) expression to one-year progression free survival (A and B) and overall survival (C and D) in squamous carcinoma (A and D) and adenocarcinoma of lung (B and D).
Figure 4 (A, B, C, D). p16(ink4) expression to two-year progression free survival (A and B) and overall survival (C and D) in squamous carcinoma (A and D) and adenocarcinoma of lung (B and D).
Figure 5 (A, B, C, D). VEGF expression to one-year progression free survival (A and B) and overall survival (C and D) in squamous carcinoma (A and D) and adenocarcinoma of lung (B and D).
Figure 6 (A, B, C, D). VEGF expression to two-year progression free survival (A and B) and overall survival (C and D) in squamous carcinoma (A and D) and adenocarcinoma of lung (B and D).
Molecular mechanisms that underlie the complex regulation of tumor initiation and angiogenesis are still poorly understood, as well as molecular processes in tumor surrounding. Recently many molecular markers were recognized in adenocarcinoma of lung, which has resulted in the creation of targeted therapies, often based on the inhibition of angiogenesis [1,2]. However, the molecular profile of squamous cell carcinoma of lung is still largely unknown, and treatment of this disease results in a low rate of survival . We demonstrated significantly decreased p16(ink4) expression and increased VEGF expression in tumor tissue and surrounding tissue, but no differences were observed in expression levels of these two markers between tumor tissue and surrounding tissue from NSCLC patients. Also, we demonstrated significant difference of p16(ink4), but not of VEGF expression between SCC and ADC of lung. Furthermore, we observed significant correlation of p16(ink4) expression to all survival rates in both ADC and SCC of lung, while VEGF expression was limited in SCC. These observations strongly suggest more potent p16(ink4) tumor suppressive role in SCC of lung carcinoma compared to ADC, but also significant prognostic value of p16(ink4) expression in SCC. Moreover, the regulation of p16(ink4) expression could have therapeutic potential in NSCLC patients in regard to suppression of tumor growth. Our study showed significantly lower levels of p16(ink4) expression, and significantly higher level of VEGF expression in tumor tissue compared to the control. These results are consistent to the results of other published studies in regard to p16(ink4) supressive role and overexpression of VEGF in tumor tissue . So, the loss of p16(ink4) expression is described in a number of malignancies of different organs [22-24]. This protein stops the cell cycle after inactivation of Rb gene, which is one of the main controllers [25-27]. Besides low expression of tumor suppressor p16(ink4), tumor tissue is characterized with a high expression of pro-angiogenic factor VEGF. This results in the creation of multiple blood vessels within the tumor tissue [28,29] and the very process of induced tumor angiogenesis is very complex and still insufficiently understood [21,30].
In our study, we observed a great similarity in the level of p16(ink4) and VEGF expression between the tumor and surrounding tissue. Several studies report multifocal and extensive molecular changes in non-malignant respiratory epithelium of lung carcinoma patients (genetic instability, deletions and mutations), which are particularly pronounced in the fields around clinically manifest tumors [31-33]. These abnormalities result in reduced expression of tumor suppressors and increased expression of pro-oncogenic and pro-angiogenic factors [33-35], and also in significantly higher molecular pro-oncogenic potential compared to other cells of healthy mucosa broncho-pulmonary system . Besides this, the presence of inflammation and its effect on the angiogenic processes in these fields must be considered. Inflamed and reactive cells abundantly secrete NF-kβ, numerous matrix metalloproteinases, COX-2 and nitrite oxide synthase, and a large number of chemokines and inflammatory cytokines, whose significance was also proven in tumor invasion and angiogenesis . Moreover, stromal cells that secrete a large number of pro-inflammatory cytokines, chemokines, growth factors and proteases contribute to the process of inflammation . All of these changes promote the growth of the tumor [38,39]. In available and reviewed literature, we found only one comparative study of molecular genetic changes in lung carcinoma and its surrounding . These authors report about similar expression of proangiogenic miRNAs (let-7b and miR-126) in lung carcinoma tissue and its surrounding. However, in the reviewed literature, we could not find any study that describes the p16(ink4) and VEGF expression in lung carcinoma surrounding. Consequently, our study, from the point of p16(ink4) and VEGF, again indicate the great importance of the tumor microenvironment in carcinoma development, progression and its involvement in tumor angiogenesis. We observed singnificantly lower p16(ink4) expression, but similar VEGF expression in SCC patients compared to ADC patients. In the available and reviewed literature, we could find only a few studies describing p16(ink4) and VEGF expression between SCC and ADC patients, but the results were opposite. So, Ann, et al.  report no significant difference in loss of heterozygosity frequencies of p16 between SCC and ADC. On the other side, Huang, et al.  found that p16-negative tumours in SCC were significantly more than those in ADC, whereas Leversha, et al.  observed that SCC had substantially increased p16 loss (SCC 75%, ADC 53%). Also, only several studies report about differences of VEGF expression between SCC and ADC, and their results are opposite too. So, some authors reported significantly higher VEGF expression in ADC than in SCC [44,45], and another authors observed no significant difference between SCC and ADC [46,47]. Clinical data suggest a distinct biologic role of the VEGF pathway in the different histologic subtypes of lung carcinoma . Moreover, cigarette smoking seems to correlate positively to p16[INK4α] gene hypermethylation in NSCLC patients . So, our and other observations suggest that some other factors, like histological subtype, differentiation, grade, clinical stage, age or smoking habits of SCC and ACD patients, could distinct influence on p16(ink4), but particularly on VEGF expression . Nevertheless, larger studies are warranted to determine differences of p16(ink4) and VEGF expression between SCC and ADC, since only a limited number of studies in lung carcinoma have been published so far.
Our study showed significant correlation of p16(ink4) expression to all survival rates in both ADC and SCC, while VEGF expression was limited in two year PFS, as well as OS in SCC. The literature data regarding relationship of p16(ink4) expression and survival rates are mutually contradictory also. Specifically, a number of studies defined p16(ink4) as an important prognostic factor in patients with lung carcinoma [11-13,50,51,53-55], while the authors of other studies observed no significant relationship of p16(ink4) and survival rates [9,10]. Similar, relationship of VEGF expression and survival rates in NSCLC patients is still controversial. According to some authors height of VEGF expression was significantly correlated with longer survival [4,15], [16,18,56], but results from other authors deny this relationship [19,20]. Furthermore, some authors observed significant correlation of p16(ink4) expression and survival rates only in younger NSCLC patients , but also in patients with non-advanced stage of SCC . According our and the results from other authors one may conclude that new studies of p16(ink4) and VEGF expression relationship to survival rates are mandatory regarding subtype, differentation, grade and clinical stage of the diseases as well as patients age and smoking habits. In conclusion, our results indicate that significantly decreased p16(ink4) has an impact on prognosis in SCC patients. These findings could provide us an important step toward understanding the complex pathways necessary for development and progression of SCC. Moreover, besides anti-VEGF therapy, p16(ink4) could be proposed as an attractive target for developing treatment strategies that would achieve better clinical outcomes in NSCLC patients, particularly in SCC. However, the present results provide only for conclusions based on correlative analysis and further validation through mechanistic studies seems mandatory.
We are grateful to all patients that participated in this study and to Faruk Turkic (General Hospital Tesanj) for his skilled assistance and invaluable advice in pathohistological procedures.