Abstract
Aim:
p16 and p53 are frequently altered intracellular pathways in cancers. We investigated the aberrant expression of p16 and its relationship with p53 and HPV status in primary non-small-cell lung carcinoma.
Patients & methods:
Lung tumor tissue microarray (n = 163), immunohistochemical study of p16 and p53, and HPV in-situ hybridization were analyzed.
Results:
p16 and p53 were detected in 50.7 and 57.3% of adenocarcinoma (ADCs; n = 75), and 35.2 and 63.6% of squamous cell carcinoma (n = 88). HPV was detected in 16 and 10.2% of ADC and squamous cell carcinoma. In ADCs, p16 positive tumors demonstrated a favorable median overall survival time of 60.9 months, compared with p16 negative tumors of 46.9 months (p < 0.05). Furthermore, we did not find significant relationships between p16 expression and HPV status, nor with p53 expression.
Conclusion:
p16 play an unique role in lung cancer survival. The mechanism of p16 needs to be further studied.
Keywords: : adenocarcinomas, biomarker, clinical survival, human papilloma virus, HPV, immunohistochemistry, in situ hybridization, non-small-cell lung carcinoma, p16, p53, squamous cell carcinomas
Lung cancer is a heterogeneous group of tumors. Its incidence continues to increase worldwide [1,2]. Recent large scale comprehensive molecular profiling, such as the Cancer Genome Atlas and the International Cancer Genome Consortium (ICGC) studies and ours, has identified multiple driver gene mutations in non-small-cell lung cancers (NSCLC), including both adenocarcinomas (ADC) and squamous cell carcinoma (SQCC) [3–8]. These studies have greatly contributed in updating the molecular knowledge of lung cancers, and facilitated the development of targeted therapies [3,4,9]. However, despite these advancements, lung cancer is still among the leading causes of the cancer-related death in the USA and worldwide [1,2]. Thus, understanding the molecular mechanisms of lung cancer development, progression and treatment susceptibility is still a critical step to further improve patient survival.
In addition of EGFR, KRAS and ALK alterations, the loss of tumor suppressor p16INK4a (best known as p16) has also been detected in NSCLC [10–14]. p16, together with the key tumor suppressors P14ARF and P15INK4b, is encoded by the INK4/ARF locus, which is one of the mostly affected genomic regions in human cancers [10–14]. Several mechanisms have also been related to the aberrant expression of p16 in NSCLC, such as alternate reading frame of the CDKN2A locus, epigenetic silencing of the entire INK4/ARF, deletions and mutation of the CDKN2A and/or promoter hypermethylation of the CDKN2A [11–14]. These mechanisms, however, only account for two-thirds of NSCLC cases that have aberrant expression of p16. The molecular explanation for the remaining cases still needs to be further studied. It has been reported that the p53 intracellular signaling pathway is also involved in the p16 expression [13,14].
Furthermore, the expression of p16 is highly correlated with the detection of high-risk human papilloma virus (hrHPV), and has been used as a surrogate marker for predicting the HPV status in the head and neck squamous cell carcinoma (HNSQCC) [15,16]. Clinically, it is difficult to discriminate the origin of p16 positive lung tumor as a primary or metastasis carcinoma. Recent studies have also indicated that hrHPV may play a certain role in lung cancers via the dysregulation of intracellular p16INK4a and p53 signaling pathways [17–20].
Taken together, the potential role of p16 and its relationship with the HPV status and p53 in different subtypes of NSCLCs are controversial and not fully understood. In this study, we investigated the expression of p16 and p53 by immunohistochemistry (IHC), together with the detection of HPV in surgically resected primary lung tumor tissues.
Patients and methods
Case collection
In our study, only untreated tumors were included. Surgical resected primary NSCLCs were obtained from archive of the department of pathology at the John Hopkins Medical Institutions. The WHO classification of lung cancer is used for subclassification of NSCLC [2]; and the American Joint Cancer Committee eighth edition is used for the pathological staging (pT) of the primary tumor [21]. A total of 163 primary NSCLCs (including 75 cases of ADCs and 88 cases of SQCCs) were included in the study. Clinical information was correlated with study findings. Patients’ identity and clinical information were protected under the ethical criteria of the John Hopkins Medical Institutions. Our study was approved by the Johns Hopkins institutional review board.
Construction of primary NSCLC tumor tissue microarrays (TMAs)
The lung tumor tissue tissue microarrays (TMAs) were constructed using surgical resected tumor tissue. All tumor tissues were fixed in 10% formaldehyde and embedded in paraffin blocks prior to TMA construction [22]. The area containing cancer were highlighted on the hematoxylin and eosin stained slide by two pathologists (E Gabrielson and QK Li). Both tumor and tumor-matched normal lung tissue were included and cored with a diameter of 0.6 mm for TMA construction. For the TMA of lung ADC, four cores of tumor and two cores of normal lung per case was included. For the TMA of lung SQCC, three cores of tumor and two cores of normal lung per case was included.
IHC study
Mouse monoclonal antibodies against human p16 (clone INK4a/E6H4, 1:200 dilution, Ventana, AZ, USA) and mouse monoclonal antibodies against human p53 (Bp53-11, 1:200 dilution, Ventana) were used according to manufacturer’s suggestion. In our study, the mouse monoclonal antibody of p53 recognizes both the mutant and wild-type of the p53 nuclear phosphoproteins.
The lung cancer TMAs were cut into a thickness of four microns. The deparaffinization of TMA sections was performed in xylene, and followed by rehydration with different graded alcohol solution prior to incubation with primary antibodies. The antigen retrieval procedure was performed at 70°C for 40 min [22,23]. All IHC stains were achieved using the XT autostainer (Ventana) in the clinical IHC laboratory at the Johns Hopkins Hospital. Primary antibodies were diluted according to manufacturers’ protocols.
A semiquantitatively four-tiered scoring system was used for evaluation of the expression of p16 and p53 based on the intensity and percentage of nuclear staining patterns. 0 = entirely negative staining (0%); 1 = weak staining (<20% of tumor nuclei); 2 = moderate staining (20–50% of tumor nuclei); 3 = strong staining (>50% of tumor nuclei). The staining pattern of TMAs slides were evaluated double-blind by two pathologists (E Gabrielson and QK Li). Appropriate positive and negative controls were included in the IHC assay.
In-situ hybridization assay for HPV RNA
In-situ hybridization (ISH) assay for HPV mRNA was performed using the XT autostainer (Ventana) and the RNAscope HPV kit according to the manufacturer’s instructions (Advanced Cell Diagnostics, CA, USA). In brief, the primary lung ADC and SQCC TMAs were cut into 4-μm section. The sections were pretreated with heat and protease, and then hybridized with probes directed against a subset of HPV genotypes (including HPV 6/11 and hrHPV16, 18 and 31/33), followed by incubation with a horseradish peroxidase-based signal amplification system to the target probes using a color development system of 3,3′-diaminobenzidine. Positive (the housekeeping gene ubiquitin C) and negative (the bacterial gene DapB) control probes were also included in the assay [24]. The positive staining was identified as brown, punctate dots in nucleus and cytoplasm of tumor cells. Staining patterns were semiquantitatively scored as: negative: <10 dots/cell in less than 10% cells (visible at 40× magnification); positive: >10 dots/cell in more than 10% cells (visible at 40× magnification). Appropriate positive control (formalin fixed paraffin embedded cervical SQCC tissues) and negative control (phosphate-buffered saline without primary antibody) were included in the ISH assay for HPV mRNA.
Statistical analysis
Characteristics of clinicopathological information were analyzed using the SPSS statistical software (version 24.0, SPSS Inc., IL, USA), and χ2 and Fisher’s exact tests were used to calculate the p-value. The relationship among HPV detection, p16 expression and p53 expression were correlated with pathological characteristics of NSCLS patients using Spearman rank correlation analysis. Kaplan–Meyer analysis and the log-rank test were also used for survival analysis. All tests were performed using bilateral 95% CIs. If the p-value was less than 0.05, it was considered statistically significant (p < 0.05).
Results
Clinical information
In our study, a total of 163 cases of primary NSCLCs were included. In 75 cases of primary lung ADCs, the median age of patients was 63 years, ranging from 45 to 86 years; and the male and female ratio was: 1:0.97. The average tumor size was 3.09 cm, ranging from 0.5 to 9.0 cm. Among tumors, 38 cases were pT1, 31 cases were pT2, six cases were pT3/pT4. In 88 cases of primary lung SQCCs, the median age of patients was 64.5 years, ranging from 40 to 81 years; and the male and female ratio was: 1:0.52. The average tumor size was 4.35 cm, ranging from 1.0 to 11.0 cm. Among tumors, 24 cases were pT1, 28 cases were pT2, 25 cases were pT3 and 11 cases were pT4 tumors. For lung ADCs, there were nine cases (12.0%) with an EGFR or KRAS mutation. The clinicopathological characteristics of patients were summarized in Table 1. The representative morphological features of tumors were shown in Supplementary Figure 1.
Table 1. . Clinico-pathological features of lung cancer patients.
| Characteristics | ADCs, n = 75 (%) | SQCCs, n = 88 (%) |
|---|---|---|
| Gender | ||
| Male | 38 (50.7%) | 58 (65.9%) |
| Female | 37 (49.3%) | 30 (34.1%) |
| Age (years) | ||
| Median | 63 | 64.5 |
| Range | 45–86 | 40–81 |
| Tumor size (cm) | ||
| Average | 3.09 | 4.35 |
| Range | 0.5–9.0 | 1.0–11.0 |
| Differentiation grade | ||
| Well | 9 (12.0%) | 4 (4.5%) |
| Moderate | 51 (68.0%) | 60 (68.2%) |
| Poor | 15 (20.0%) | 24 (27.3%) |
| Smoking status | ||
| Non-smokers | 15 (20.0%) | 11 (12.5%) |
| Smokers† | 60 (80.0%) | 77 (87.5%) |
| Pathological stage | ||
| pT1 | 38 (50.7%) | 24 (27.3%) |
| pT2 | 31 (41.3%) | 28 (31.8%) |
| pT3 | 5 (6.7%) | 25 (28.4%) |
| pT4 | 1 (1.3%) | 11 (12.5%) |
| EGFR/KRAS status | ||
| Non-mutation | 66 (88.0%) | – |
| Mutation | 9 (12.0%) | – |
| Survival time (months) | ||
| Median | 54 | 46 |
| Range | 0–140 | 0–208 |
Includes both current and former smokers.
ADC: Adenocarcinoma; SQCC: Squamous cell carcinoma.
Detection of p16 & p53
In 75 ADCs (Figure 1A & B), the expression of p16 was detected in 50.7% (38 of 75) of cases, including weakly positive in 30.7% (23 of 75), moderate positive in 16.0% (12 of 75) and strong positive in 4.0% (3 of 75) case. The expression was not detected in 49.3% (37 of 75) of ADCs. The expression of p53 was detected in 57.3% (43 of 75) of cases, including weakly positive in 26.7% (20 of 75), moderate positive in 12.0% (9 of 75) and strong positive in 18.6% (14 of 75) cases. The expression was not detected in 42.7% (43 of 75) of ADCs.
Figure 1. . p16 and p53 staining patterns in primary lung tissue microarrays.
(A) Immunostaining pattern of p16 in ADC, (B) immunostaining pattern of p53 in ADC, (C) immunostaining pattern of p16 in SQCC and (D) immunostaining pattern of p53 in SQCC. Staining of p16 and p53 were scored based on the intensity and percentage of nuclear staining patterns using a semiquantitatively four-tiered system: negative = no staining (0%); 1 = weak staining (<20% of tumor nuclei); 2 = moderate staining (20–50% of tumor nuclei); 3 = strong staining (>50% of tumor nuclei).
ADC: Adenocarcinoma; SQCC: Squamous cell carcinoma.
In 88 SQCCs (Figure 1C & D), the expression of p16 was detected in 35.2% (31 of 88) of cases, including weakly positive in 12.5% (11 of 88), moderate positive in 9.1% (8 of 88) and strong positive in 13.6% (12 of 88) cases. The expression of p16 was not detected in 64.8% (57 of 88) of cases. The expression of p53 was detected in 63.6% (56 of 88) of cases, including weakly positive in 11.4% (10 of 88), moderate positive in 27.2% (24 of 88) and strong positive in 25.0% (22 of 88) cases. The expression of p53 was not detected in 36.4% (32 of 88) of cases.
The overall expression rate of p16 was much higher in ADCs (50.7%, 38/75 cases) than that of SQCCs (35.2%, 31/88 cases; p = 0.024; Table 2). However, the stronger expression (score 3+) of p16 was significantly higher in SQCCs (13.6%, 12/88 cases) compared with the ADCs (4.0%, 3/75 cases; p = 0.027). We further examined the potential relationship of p16 and p53 in ADCs and SQCCs, and did not find a significantly correlation between p16 and p53 (r = 0.119 and 0.085; p = 0.308 and 0.429; Table 3).
Table 2. . Immunostaining scores and correlation of p16 and p53 in ADCs and SQCCs.
| Markers | IHC score | Positive rate | |||
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | ||
| p16 | |||||
| ADC (n = 75) | 37 (49.3%) | 23 (30.7%) | 12 (16.0%) | 3 (4.0%) | 50.7% |
| SQCC (n = 88) | 57 (64.8%) | 11 (12.5%) | 8 (9.1%) | 12 (13.6%) | 35.2% |
| p53 | |||||
| ADC (n = 75) | 32 (42.7%) | 20 (26.7%) | 9 (12.0%) | 14 (18.6%) | 57.3% |
| SQCC (n = 88) | 32 (36.4%) | 10 (11.4%) | 24 (27.2%) | 22 (25.0%) | 63.6% |
ADC: Adenocarcinoma; IHC: Immunohistochemistry; SQCC: Squamous cell carcinoma.
Table 3. . Immunostaining scores and correlation of p16 and p53 in ADCs and SQCCs.
| p16 | p53 | r | p-value | ||
|---|---|---|---|---|---|
| Positive | Negative | ||||
| ADC | Positive | 24 | 14 | 0.119 | 0.308 |
| Negative | 19 | 18 | |||
| SQCC | Positive | 18 | 13 | 0.085 | 0.429 |
| Negative | 38 | 19 | |||
ADC: Adenocarcinoma; IHC: Immunohistochemistry; SQCC: Squamous cell carcinoma.
Correlation of p16 & p53 expression with clinico-pathological findings
To further understand any association between p16 and p53, we correlated the p16 and p53 expression with the age, gender tumor pathological stage, differential grade, smoking status, EGFR/KRAS mutational status and median overall survival time (mOS) of the patients (Table 4). In the multi-variant analysis, we did not find any correlation between p16 and p53 with patients’ age, gender, tumor pathological stage, differential grade and EGFR/KRAS mutational status. However, a strong correlation between p53 and smoking status was observed in SQCC patients (p = 0.007).
Table 4. . Correlation of p16 and p53 expression with clinico-pathological features of patients.
| Characteristics | p16 | p53 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ADCs (n = 75) | SQCCs (n = 88) | ADCs (n = 75) | SQCCs (n = 88) | |||||||||
| (+) | (−) | p-value | (+) | (−) | p-value | (+) | (−) | p-value | (+) | (−) | p-value | |
| Age (years) | 0.414 | 0.106 | 0.409 | 0.966 | ||||||||
| <60 | 11 | 14 | 14 | 16 | 16 | 9 | 19 | 11 | ||||
| ≥60 | 27 | 23 | 17 | 41 | 27 | 23 | 37 | 21 | ||||
| Gender | 0.907 | 0.227 | 0.713 | 0.61 | ||||||||
| Male | 19 | 19 | 23 | 35 | 21 | 17 | 18 | 12 | ||||
| Female | 19 | 18 | 8 | 22 | 22 | 15 | 38 | 20 | ||||
| Pathological stage | 0.14 | 0.526 | 0.165 | 0.853 | ||||||||
| pT1 | 23 | 14 | 10 | 14 | 21 | 16 | 17 | 7 | ||||
| pT2 | 14 | 18 | 7 | 21 | 21 | 11 | 17 | 11 | ||||
| pT3 | 1 | 4 | 9 | 16 | 1 | 4 | 15 | 10 | ||||
| pT4 | 0 | 1 | 5 | 6 | 0 | 1 | 7 | 4 | ||||
| Differentiation grade | 0.108 | 0.291 | 0.139 | 0.619 | ||||||||
| Well | 7 | 2 | 0 | 4 | 3 | 6 | 2 | 2 | ||||
| Moderate | 26 | 25 | 23 | 37 | 33 | 18 | 37 | 23 | ||||
| Poor | 5 | 10 | 8 | 16 | 7 | 8 | 17 | 7 | ||||
| Smoking status | 0.166 | 0.933 | 0.726 | 0.007‡ | ||||||||
| Nonsmoker | 10 | 5 | 4 | 7 | 8 | 7 | 11 | 0 | ||||
| Smoker† | 28 | 32 | 27 | 50 | 35 | 25 | 45 | 32 | ||||
| EGFR/KRAS status | 0.736 | – | 0.286 | – | ||||||||
| Nonmutation | 34 | 32 | – | – | 36 | 30 | – | – | ||||
| Mutation | 4 | 5 | – | – | 7 | 2 | – | – | ||||
| mOS (months) | 60.9 | 46.9 | 0.048‡ | 40.7 | 49.5 | 0.19 | 54.1 | 54.0 | 0.846 | 48.4 | 42.9 | 0.701 |
Include both current and former smokers.
Indicates p < 0.05, considered statistically significant.
ADC: Adenocarcinoma; mOS: Median overall survival time; SQCC: Squamous cell carcinoma.
In ADCs, the expression of p16 showed a favorable prognosis with mOS of 60.9 months, compared with p16 negative tumors of 46.9 months (p = 0.048; Figure 2A). In SQCCs, the expression of p16 showed a slightly worse mOS of 40.7 months, compared with 49.5 months of p16 negative tumors, this difference, however, did not reach statistical significance (p = 0.19; Figure 2B). In contrast to p16 expression, we did not find any survival benefit of p53 expression in NSCLC. In ADC, the mOS was 54.1 months in p53 positive tumors and 54.0 months in negative tumors, respectively. Similarly, in SQCCs, the mOS was 48.4 months in p53 positive tumors and 42.9 months in negative tumors, respectively. We did not find any relationship of p53 expression with the mOS (Figure 2C & D).
Figure 2. . Kaplan-Meier analysis of non-small cell lung carcinomas patients with differential p16 and p53expression and high-risk human papilloma virus status.
(A) Overall survival of p16 positive and negative tumors in ADC patients (p = 0.048); (B) overall survival of p16 positive and negative tumors in SQCC patients (p = 0.190); (C) overall survival of p53 positive and negative tumors in ADC patients (p = 0.846); (D) overall survival of p53 positive and negative tumors in SQCC patients (p = 0.701); (E) overall survival of p16/HPV positive and negative tumors in ADC patients (p = 0.013); and (F) overall survival of p16/HPV positive and negative tumors in SQCC patients (p = 0.343).
ADC: Adenocarcinoma; HPV: Human papillomavirus; SQCC: Squamous cell carcinoma.
Correlation HPV status with p16 & p53
Several HPV probes, including subtypes of 6/11, 16, 18, 31/33, were used in our study. The representative scoring system is shown in the Supplementary Figure 2A. In 75 ADCs, the HPV RNA was detected in 16% (12 of 75) cases; and the hrHPV16/18 was the dominant subtype detected in 66.7% of cases (8 of 12 cases). Similarly, in 88 SQCCs, the HPV was detected in 10.2% (9 of 88) cases with the dominant subtype of hrHPV16/18 detected in 55.6% of cases (five of nine cases). Data is summarized in the Supplementary Figure 2B.
We further analyzed the relationship of HPV status with p16 and p53 expression in both ADCs and SQCCs using Spearmen rank correlation analysis. We did not find any significant correlations between HPV status and the expression of p16 and p53 (Supplementary Table 1). In the clinic-pathological correlation analysis, there were no statistically significant correlations of HPV status with gender, pathological stage, tumor grade, smoking status and mOS (Supplementary Table 2).
We then analyzed the data with p16 plus HPV as one category. In ADCs, the expression of p16/HPV positive tumors showed a favorable prognosis with mOS of 78 months, compared with p16/HPV negative tumors of 45 months (p = 0.013, Figure 2E). In SQCCs, the expression of p16/HPV positive tumors showed a worse mOS of 63 months, compared with 76 months of p16/HPV negative tumors, this difference, however, did not reach statistical significance (p = 0.343; Figure 2F).
Discussion
p16 signaling pathway plays a critical role in cell-cycle regulation, involving in cell proliferation and tumorigenesis [12,16–18,24–26]. The expression of p16 is regulated by the INK4/ARF locus, which is one of the mostly affected genomic regions in human cancers [10–14]. The expression of p16 has been detected in a variety of tumors, particularly in cervical and HNSQCC [15–17,24]. In our study, the expression of p16 was detected in both primary lung ADCs and SQCCs. In ADCs, the expression of p16 was 50.7% (38 of 75), including weakly positive in 30.7% (23 of 75), moderate positive in 16.0% (12 of 75) and strong positive in 4.0% (3 of 75) cases. In 88 SQCCs, the expression of p16 was detected in 35.2% (31 of 88) of cases, including weakly positive in 12.5% (11 of 88), moderate positive in 9.1% (8 of 88) and strong positive in 13.6% (12 of 88) cases. The overall expression pattern of p16 was much higher in ADCs (50.7%, 38/75 cases) than that of SQCCs (35.2%, 31/88 cases; p = 0.024), however, tumors with higher intensity IHC staining for p16 (score 3+) was observed in SQCCs (13.6%, 12/88 cases) compared with ADCs (4.0%, 3/75 cases; p = 0.027). This differential expression pattern of p16 in NSCLC is interesting, and may suggest that the molecular role of p16 expression might be different in lung cancers than that of HNSQCCs.
The expression of p16 has been considered to be a favorable factor in HNSQCCs [15,16]. We found that p16-positive ADCs had a better mOS of 60.9 months compared with 46.9 months in p16-negative tumors. However, the survival benefit of p16 expression was not found in SQCCs. In SQCCs, the expression of p16 showed a slightly lower mOS of 40.7 months, compared with 49.5 months in p16 negative tumors, this difference did not reach statistical significance (p = 0.19). The similar prognostic influence was also found in the analysis of p16 plus HPV as one category, which further suggests the critical role of HPV in the occurrence and development of lung cancer. Alteration in the p53 status has been reported to be a marker for poor prognosis in patients with pulmonary ADCs [27], we therefore evaluated the association between the p53 expression status and the survival benefits among patients with ADCs and SQCCs, however, in our study we found no correlated between the p53 expression and the overall survival in both ADCs and SQCCs patients. Our finding of differential clinical outcomes with aberrant expression of p16 in different subtypes of NSCLC might suggest a different biological role of p16 in different subtype of lung cancers, in contrast to that of HPV-related HNSQCCs [15–17,28].
In some cases, the presence of HPV oncoprotein E6 and E7 could explain the overexpression of p16. While, the p16 overexpression may also be involved in activation of proto-oncogenes, inactivation of tumor suppressor genes and related to the alterations of p16-Rb signaling pathway. Several studies have demonstrated that the dysregulation of Rb results in an increased p16 expression in tumor cells and cancer tissue due to a feedback mechanism [29,30]. The loss of Rb is frequently occurred in numerous malignant tumors and is associated with uncontrollable cell proliferation [31]. Thus, the relationship between p16 and Rb may possibly explain the overexpression of p16 protein in malignant tumors. In addition, the expression of p16 is also involved in the regulation of multiple target genes and proteins including the p53 intracellular signaling pathway (p14ARF/mdm2/p53) [17–20]. The tumor suppressor gene p53 is an important regulator of the cell cycle and plays a key role in many cellular pathways controlling cell proliferation, survival and genomic integrity. The dysregulation of p53 pathway leads to uncontrolled proliferation of tumor cells as well [32–34]. The expression of the tumor suppressor protein p53 is one of the common abnormalities in a variety of cancers, including lung cancer [33,34]. To further study the potential regulatory mechanism of p16 expression in NSCLC, we studied p53 expression in tumor tissues. We found that the aberrant expression of p53 was detected in 57.3% (43/75) of ADCs and 63.6% (56/88) of SQCCs. However, we did not find that the p53 expression was correlated with p16 expression, nor the clinic-pathological features, including patients’ age, gender, pathological stage, tumor grade and EGFR/KRAS status. Our data only demonstrated that p53 expression was correlated with the smoking status in SQCCs patients. Our findings were similar to Shinohara et al. study, where they reported smoking history was correlated with p53 expression in HNSQCC [28].
Over the past decades, many studies have investigated the potential role of HPV in lung cancer [20,26,35–37]. The clinical significance of HPV in lung cancer is still controversial [36]. It has been reported that the detection rate of hrHPV in lung cancers is approximately 26% worldwide, ranging from 0% in certain area of China [35,38–40] to more than 40% in Europe [39,40]. In our study, the overall detection rate of HPV was around 16.0 and 10.2% in ADC and SQCC, respectively. We also correlated the HPV status with the expression of p16 in NSCLC. In contrast to HNSQCC, our data did not show any relationship of p16 expression with the detection of HPV in both ADCs and SQCC. Many factors have also been linked to the heterogeneous detection rate of HPV status in lung cancers, such as geographic location of study population, gender, smoking status, histological type of the tumor and detection methods [26,35]. A recent meta-analysis of more than 31,000 cases from 17 countries has also demonstrated controversial findings [35]. Taken together, further studies are necessary to address the question whether HPV has any permissive role in the development of lung cancers.
To date, it has been reported that about 5% of tumors are caused by HPVs worldwide, especially the hrHPV infections [41]. The need for understanding HPV-associated immune response, including the development of cancer vaccines, prompted the intense research activities. Most of HPV vaccines are developed to induce an adaptive immune response against the E6 and E7 oncoproteins of HPV16 and/or HPV18. In HPV infection, the viral E6 and E7 proteins may form a unique fusion protein, or function individually to enhance antigen presentation by antigen presenting cells in order to improve the immunogenicity in the host [42]. Furthermore, the therapeutic HPV vaccines undergoing clinical evaluations are DNA-based vaccines and administered in combination with conventional chemotherapy and radiotherapy, immune stimulatory molecules such as cytokines and adjuvants or some monoclonal antibodies blocking immune checkpoints blockade and/or angiogenesis [43]. Several vaccines are in Phase I/II clinical trials for the treatment of cervical cancers and HNSQCCs. We look forward to their potential utility in treatment of lung cancers.
Previous studies have demonstrated that p16 signaling pathway plays a critical role in the treatment and prognosis of certain tumors. For example, HNSQCCs with p16 expression are sensitive to radiotherapy, and have a better survival time than those of p16 negative tumors [15,16]. In addition, several clinical studies of CDK4/6 inhibitors (such as Palbociclib and Abemaciclib) targeting the p16 signaling pathway, are under investigation for the treatment of subset of tumors, including lung cancers [44–46]. These clinical trials demonstrate promising outcomes and could potentially improve patients’ survival. Taken together, recent clinical trials and studies suggest that p16 and its signaling pathway may be therapeutic targets in subtype of tumors.
Finally, it is well known that the phenotype of a tumor could be potentially affected by chemo- and targeted-therapy [2–4,9]. For example, our studies have shown that expression of EGFR, KRAS and epithelial markers in NSCLC are affected by prior chemo- and targeted therapy [6,8,22]. p16 signaling pathway is regulated and involved in many other intracellular signaling pathways [11–15]. Whether chemo- and other anti-cancer therapies affect the expression of p16 is still not well understood. Therefore, only untreated tumors were included in our study. The mechanism of p16 status and tumor’s response to the treatment needs to be further studied.
Conclusion
In summary, our data demonstrated that the aberrant expression of p16 and p53 was detected in 50.7 and 57.3% of ADCs (n = 75), and in 35.2 and 63.6% of SQCCs (n = 88). In ADC, p16 positive tumors had a favorable mOS of 60.9 months, in the comparison with p16 negative tumors of 46.9 months (p < 0.05). The expression of p16 appeared to be correlated with better prognosis in ADCs. In contrast to HNSQCC, the expression of p16 was not correlated with p53, nor with the status of HPV. Although hrHPV has been detected in a subset of lung cancers, the potential role of HPV still needs to be further investigated. While our study is based on a small patient cohort, the unique role of p16 and associated pathways also need to be further investigated in lung cancers.
Summary points.
Previous studies have shown that the aberrant expression of p16 is regulated by the INK4/ARF locus, which is one of the mostly affected genomic regions in human cancers.
The expression of p16 is highly correlated with the detection of high-risk human papilloma virus; and it has been used as a surrogate marker for prediction of HPV status in cervical and head and neck squamous cell carcinoma.
Detection of p16 & p53
In primary lung adenocarcinomas (ADCs, n = 75), p16 and p53 expression were detected in 50.7 and 57.3%, respectively.
In primary lung squamous cell carcinomas (SQCCs, n = 88), p16 and p53 expression were detected in 35.2 and 63.6%, respectively.
Correlation of p16 & p53 expression with clinico-pathological findings
In ADC, p16 positive tumors had a favorable median overall survival time of 60.9 months, than that of p16 negative tumors of 46.9 months (p < 0.05).
A strong correlation between p53 and smoking status was observed in SQCC patients (p = 0.007, p < 0.05).
Correlation HPV status with p16 & p53
We further analyzed the relationship of HPV status with p16 and p53 expression in both ADCs and SQCCs using Spearmen rank correlation analysis, and did not find any significant correlations between HPV status and the expression of p16 and p53.
In contrast to head and neck squamous cell carcinoma, the expression of p16 was not correlated with p53, nor with the status of HPV.
Discussion
p16-associated pathways may play more complex and differential biological roles in lung cancers.
Our study suggested that p16 may be a potential biomarker in primary lung ADCs.
Supplementary Material
Acknowledgments
Authors thank Xiao-Jun Ma and YuLing Lou from Advanced Cell Diagnostics, (CA, USA) for their helping with hrHPV RNA ISH staining.
Footnotes
Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/10.2217/bmm-2018-0441
Financial & competing interests disclosure
This work is partially supported by Dr Ji and Li Family Cancer Research Foundation (QK Li). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The study was approved by the Johns Hopkins Institutional Review Board. Patients’ identity and clinical information were protected under the ethical criteria of the John Hopkins Medical Institutions.
References
Papers of special note have been highlighted as: • of interest; •• of considerable interest
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