Academic Editor: Elena Levantini
Background: Determining predictive biomarkers for immune checkpoint
inhibitors (ICIs) is a current challenge in oncology. Previous studies on
non-small cell lung cancer (NSCLC) have shown how TP53 gene mutations
are correlated with different responses to ICIs. Strong and diffuse
immuno-expression of p53 by immunohistochemistry (IHC) is interpreted as a likely
indicator of a TP53 gene mutation. We aimed to assess the p53 protein
expression via IHC in NSCLC as a predictive biomarker of the response to ICIs.
Methods: This was a retrospective hospital-based study of patients with NSCLC
treated with Nivolumab in the University Hospital of Salamanca. All diagnostic
biopsies were studied via IHC (measuring p53 protein expression, peroxidase
anti-peroxidase immunohistochemistry technique using Leica BOND Polymer
development kits). Survival analysis was performed by subgroups of expression of
p53 and other factors using the Kaplan-Meier estimator and Cox
proportional-hazards model. Results: Seventy-three patients were included (59 men
and 14 women). The median age was 68 (44–84) years. Thirty-six biopsies were
adenocarcinoma, 34 were squamous, and three were undifferentiated. In 41 biopsies
(56.2%), the cellular expression of p53 was
Among malignancies, lung cancer is a with the highest incidence worldwide, with 2.1 million newly diagnosed cases per year [1]. In terms of mortality, a total of 1.8 million people die from lung tumours each year (representing 18.4% of deaths from cancer) [2]. The aetiology is well known; tobacco is the main cause, responsible for 90% of lung cancers [3]. Other known risk factors include asbestos, radon, and polycyclic aromatic hydrocarbons [4]. The most widely used histological classification for the characterisation of lung cancer divides these tumours into four variants: (1) adenocarcinoma, (2) squamous cell carcinoma, (3) large cell carcinoma, and (4) small cell or small cell carcinoma. The first three tumour types are grouped under the term non-small cell lung cancer (NSCLC), which encompasses 85% of lung tumours.
The introduction of immunotherapy treatments [immune checkpoint inhibitors (ICIs)] has revolutionized NSCLC treatment. The inhibition of T cells by tumour cells is performed through two main inhibitory pathways best described for T cells: the cytotoxic T-lymphocyte antigen 4 (CTLA-4) protein receptor and the programmed cell death protein 1 (PD-1) [5, 6]. These are the pathways through which ICIs act by negatively regulating these receptors and consequently stimulating the T lymphocytes. Various factors, such as gene mutations, are known as predictors or modifiers of the response to immunotherapy or, more accurately, to ICIs in solid tumours [7].
The most pertinent somatic mutations in NSCLC are those related to the RAS (Rat Sarcoma Virus), RB (Retinoblastoma Protein), TP53 (Tumour Protein 53), AKT (Protein Kinase B), LKB1 (Liver Kinase B1), and BRAF (Murine Sarcoma Viral Oncogene Homolog B) genes. Alterations in the TP53 gene occur in approximately 50% of NSCLC cases [8]. This percentage is higher in epidermoid tumours and lower in adenocarcinomas because it is associated with tobacco use [9]. The involvement of the TP53 gene in different tumours is widely known because of its function as a suppressor gene. The loss or mutation of the TP53 gene in tumour cells has been shown to influence immune recognition through mechanisms, such as the presentation of major histocompatibility complex 1 or the recruitment of suppressor myeloid cells or Treg lymphocytes [10]. Not only is TP53 key in the direct regulation of the immune system, but it also plays a role in establishing the tumour microenvironment [11]. Therefore, several studies have evaluated the implications of TP53 mutations in predicting the response to ICIs in solid tumours, especially NSCLC [12, 13].
Immunohistochemical (IHC) staining for p53 is mainly correlated with the presence of missense mutations in TP53 [14, 15]. Historically, p53 IHC has been interpreted as negative or positive based on the percentage of stained tumour cell nuclei using variable cut-offs ranging from 5 to 10% [16]. However, the current classification of p53 expression is subdivided into three expression patterns: overexpression, the complete absence of expression, and cytoplasmatic expression. Only 5% of tumours with mutated TP53 show p53 protein expression by the IHC wild type. This three-pattern classification correlates with all possible TP53 mutations, the most common being missense. For all these reasons, the protein expression of p53 by IHC (in all its forms) can be used as a highly sensitive surrogate marker for mutations in TP53 with a failure rate of only 5% [17].
TP53 mutations have traditionally been correlated with resistance to radiation therapy and chemotherapy [18]. Studies on immunotherapy and NSCLC have presented contradictory results regarding the mutational status of TP53. A study by Assoun et al. [19] demonstrated a greater benefit of immunotherapy in patients with NSCLC and mutated TP53. However, a study by Zhao et al. [20] presented contrary results, with lower survival of NSCLC patients treated with immunotherapy treatment in patients with mutated TP53. These results were more marked in patients with low immune expression measured as the tumour mutation burden (TMB), and were aligned with the findigns of Carlisle et al. [21], who reported a trend toward a greater response in tumours with high expression of PD-L1.
In this research, our primary objective was to determine whether the protein expression of p53, as a surrogate marker of the mutational status of TP53, was correlated with the response to immunotherapy treatments in patients with NSCLC. As secondary objectives of the study, we investigated whether this response to ICIs, based on the mutational status of TP53, was influenced by various other factors, such as sex, histology, and the expression of PD-L1.
A retrospective hospital-based study was carried out in selected patients within the Complejo Asistencial Universitario de Salamanca (Salamanca, Spain). The inclusion criteria were a diagnosis of advanced or metastatic NSCLC and second-line immunotherapy treatment with an anti-PD1 drug (Nivolumab independent of PD-L1 expression in the tumour, according to clinical trials CheckMate 017 and CheckMate 057) [22, 23]. The fundamental data collected and studied were age (years), sex, histology, sites of metastases, progression-free survival (PFS) in months, overall survival (OS) in months, the number of doses received, the best response obtained with immunotherapy, and immunotoxicity presented. The study was carried out according to the ethical protocols of the hospital and with the informed consent of the patients for the extraction of the sample. If the information found in this study is favourable for the immunotherapy treatment, it will be included in the NSCLC treatment protocols of the Complejo Asistencial Universitario de Salamanca.
According to the data available in the literature, a p53 protein expression level of 5% is considered to determine a high probability of TP53 mutation (based on the results of a study carried out by Kim et al. [24]. Several studies have shown that the 5% cut-off point has the shortest sensitivity and specificity for assessing TP53 mutations, both in solid tumours (such as adenocarcinoma of the lung or ovary) and in haematological neoplasms [15, 25]. Together with the above, for evaluating the secondary endpoint, the patients were subdivided by positive or negative PD-L1 expression (this subdivision was made because it is the most widely used in clinical trials in NSCLC and second-line immunotherapy, such as the trials CheckMate 017 and CheckMate 057). To carry out this classification of patients, we relied on published clinical trials of second-line immunotherapy in which the main factor for dividing the subgroups was the positive or negative expression of PD-L1 [26, 27].
The expression analysis of the p53 protein was performed with the peroxidase
anti-peroxidase immunohistochemistry technique using the Leica BOND Polymer
development kits. Leica BOND III automatic machines were subsequently used. P53
clon D07 Leica prediluted. The level of expression of p53 was quantified as a
percentage, with 0% indicating no expression and 100% indicating complete
expression of the protein in the cell (Fig. 1). The values of p53 are described
in quantitative terms according to the main studies in the literature on IHC in
p53 [28, 29]. We carried out a semi-quantitative study, analysing ten fields of
20
Assessment of the level of protein expression of p53 by IHC. The figure shows 0% p53 expression (upper left), 30% (upper right), 50% (lower left), and 100% (lower right) staining.
The samples were evaluated by two independent people to avoid bias. Microscopic
analysis was carried out using a Nikon Eclipse Ci microscopy device (Tokyo,
Kanto, Japan). According to the existing literature on IHC as a surrogate marker
for mutations in the TP53 gene (in both solid and haematological
malignancies), it has been considered that a p53 protein expression level of
For statistical analysis, first, PFS was calculated as the months from the
initiation of immunotherapy treatment to clinical or radiological progression. OS
was calculated as the period (in months) from initiating the patient’s
immunotherapy treatment until death. Survival rates were calculated as medians
with 95% confidence intervals (CIs). Survival as a function of p53 expression
was calculated using the Kaplan-Meier method (log-rank and Breslow test) and Cox
regression analysis. To avoid confounding factors, subgroup analyses were
performed for sex, age, and histology. Additional proportional-hazards models
that included the above potential confounders were constructed. The statistical
significance for the analyses in this study was established at p
A total of 73 biopsies were examined, which were taken from patients with NSCLC
who were treated with anti-PD1 as second-line therapy. The median age was 68
(44–84) years; 59 patients were men (80.8%), and 14 were women (19.2%).
Histology corresponded to adenocarcinoma in 36 patients, squamous cell carcinoma
in 34 patients, and undifferentiated carcinoma in three patients. The most
frequent sites of metastatic involvement were the lung with metastatic secondary
nodules (45 patients, 61.6%) and lymph nodes (39 patients, 53.4%), followed by
the bone (14 patients, 19.2%) and liver (ten patients, 13.7%). The mean PD-L1
expression was 2%. PD-L1 expression was negative (0%) in 27 patients and
positive (
Sample | Overall, 73 (100%) | Adenocarcinoma, 36 (49.3%) | Squamous, 34 (46.6%) |
Age (range) | 68 (44–84) | 68 (44–84) | 68 (44–84) |
Sex (M/W) | 59/14 (80.8/19.2%) | 25/11 (69.4/30.6%) | 31/3 (91.2/8.8%) |
P53 expression | |||
• 0% | 35 (47.9%) | 20 (55.5%) | 13 (38.2%) |
• |
41 (56.2%) | 12 (33.3%) | 14 (41.2%) |
• |
32 (43.8%) | 24 (66.7%) | 20 (58.8%) |
• |
26 (35.6%) | 7 (19.4%) | 19 (55.9%) |
• |
22 (30.1%) | 6 (16.6%) | 16 (47.1%) |
• |
14 (19.2%) | 3 (8.3%) | 8 (23.5%) |
PD-L1 | |||
• Negative | 27 (37%) | 18 (50%) | 9 (26.5%) |
• Positive | 46 (63%) | 18 (50%) | 25 (73.5%) |
Survival (months) | |||
• Overall | 13 (95% CI 8.2–17.8) | 16 (95% CI 3.5–28.5) | 12 (95% CI 9–15) |
• Progression-free | 5 (95% CI 3.8–6.2) | 4 (95% CI 2.5–5.5) | 5 (95% CI 2–8) |
Response | |||
• Progression | 40 (54.8%) | 22 (61.1%) | 16 (47.1%) |
• Stabilisation | 16 (21.9%) | 8 (22.2%) | 8 (23.5%) |
• Partial response | 13 (17.8%) | 3 (8.3%) | 9 (26.5%) |
• Complete response | 4 (5.4%) | 3 (8.3%) | 1 (2.9%) |
Toxicity | |||
• Not observed | 47 (64.4%) | 24 (66.7%) | 22 (64.7%) |
• Asthenia | 12 (16.4%) | 5 (13.9%) | 7 (20.6%) |
• Endocrine | 4 (5.5%) | 2 (5.6%) | 2 (5.9%) |
• Dermal | 1 (1.4%) | 1 (2.8%) | 0 (0%) |
• Gastrointestinal | 3 (4.1%) | 1 (2.8%) | 1 (2.9%) |
• Hepatic | 4 (5.5%) | 2 (5.6%) | 2 (5.9%) |
• Renal | 7 (9.6%) | 5 (13.9%) | 2 (5.9%) |
• Cardiac | 1 (1.4%) | 1 (2.8%) | 0 (0%) |
• Pulmonary | 1 (1.4%) | 1 (2.8%) | 0 (0%) |
The table shows the demographic variables of the study population of patients with advanced or metastatic NSCLC under treatment with immunotherapy at the University Hospital of Salamanca. Patients with undifferentiated carcinomas have been excluded from Table 1 due to its lack of representation (three patients). |
The OS of the sample was 13 months (95% CI 8.2–17.8). The PFS was 5 months
(95% CI 3.8–6.2). The OS of patients with adenocarcinoma histology was 16
months (95% CI 3.5–28.5), with a 4-month PFS [95% CI 2.5–5.5]. The OS of
patients with squamous cell carcinoma histology was 12 months (95% CI 9–15)
with a 5-month PFS (95% CI 2–8). The PFS of the PD-L1-negative subgroup was 4
months (95% CI 3–5), whereas it was 6 months (95% CI 3–9) in the
PD-L1-positive subgroup (p = 0.097). The response results were as
follows: (1) four patients (5.4%) had a complete response, (2) 13 patients
(17.8%) had a partial response, (3) stabilisation was observed in 16 patients
(21.9%), and (4) progression was observed in 40 patients (54.8%). The mean
number of doses administered was seven. The observed toxicity was grade
The OS for patients above the 5% p53 cut-off point was 12 months (95% CI 7.5–16.5), and the OS was 20 months (95% CI 3.4–36.6) in patients below the cut-off (HR = 0.590 [95% CI 0.327–1.064]; p = 0.070) (Fig. 2). The PFS was 4 months in patients above the 5% cut-off point (95% CI 3.0–5.0), and it was 7 months (95% CI 3.3–10.7) in patients below the cut-off (HR = 0.630 [95% CI 0.370–1.072]; p = 0.064). No statistical differences were observed with cut-off points of 1% (OS p = 0.317 and PFS p = 0.343) and 10% (OS p = 0.149 and PFS p = 0.181).
Comparison of overall survival (OS) in the entire sample of
patients with protein expression levels of p53 of
In the analysis by histology, statistically significant differences were
observed for adenocarcinoma histology but not for squamous tumours. In
adenocarcinomas, the OS was 8 months (95% CI 0.0–16.0) in patients with p53
expression levels of
Comparison of overall survival (OS) in patients with
adenocarcinoma with p53 protein expression levels of
The only statistically significant differences regarding sex were observed in
PFS in female patients. The PFS was 3 months (95% CI 2.2–3.8) in patients with
p53 levels of
A higher percentage of responses was observed in patients with protein
expression of p53
Responses were obtained as a function of p53 protein expression.
The absolute value is accompanied by the percentage of all patients with the
same p53 expression. All complete responses were in tumours with p53 protein
expression
When the sample was analysed according to the positivity or negativity of PD-L1
expression, statistically significant differences were identified in the subgroup
of patients with negative PD-L1. For this subgroup of patients, OS was 13 months
(95% CI 11.0–15.0) in patients with p53 levels of
The difference in overall survival (OS) of patients in
the sample with negative PD-L1 expression, in which a higher OS was observed in
patients with high p53 protein expression compared with those with p53 expression
levels of
In the evaluation of the subgroup of patients with adenocarcinoma and negative
PD-L1 expression, we observed a 14-month OS (95% CI 5–30) in patients with p53
levels of
The difference in overall survival (OS) between
patients with advanced lung cancer, adenocarcinoma subtype, who were PD-L1
negative as a function of p53 protein expression. A greater tendency for OS was
observed in patients with expression
To validate the chosen cut-off point of p53, a ROC curve has been made to show which cut-off point of p53 protein expression has the most homogeneous sensitivity and specificity. The patients were divided into two subgroups according to whether they obtained a response or not with the immunotherapy treatment, and it was compared with the percentage of p53 expression. Fig. 7 shows the ROC curve where the cut-off point of 5% is the one that presents the most homogeneous sensitivity and specificity of the entire sample.
ROC curve showing the cut-off point of p53 protein expression in 5% as the one that best discriminates between the possibility of mutated and non-mutated TP53.
In this study, we analysed the influence of p53 mutations on the response to immunotherapy in patients with NSCLC. We performed an IHC analysis of an NSCLC sample to measure the protein expression of p53 (section 2.2). This research has a series of limitations, the first of which is due to the retrospective design of the study. Along with this, the number of patients is limited because of the difficulty of processing the samples, given that those performed by fine-needle aspiration were not accepted as valid because of technical difficulties.
Instead of genetic analysis of the tumour, we performed IHC (which can be performed at any type of centre) to determine mutations in TP53 and assess predictive markers of response to immunotherapy [30, 31]. Currently, genetic sequencing technology is not available at any centre [32]; therefore, the IHC technique was chosen as a surrogate marker of mutation in TP53 [33]. The different studies carried out to date do not clearly show the influence of TP53 on the response to immunotherapy treatments in NSCLC. Current studies are contradictory on this matter, and therefore this article aims to make a greater contribution to this field.
The choice of patients with anti-PD1 treatment as second-line therapy was a key
point of our work and is crucial for understanding the analysis and the sample.
Given that until 2 years ago, first-line and second-line immunotherapy treatment
was classified according to PD-L1 expression levels of
The main objective of the analysis was to assess whether p53 mutations (determined through IHC) were correlated with a greater response to immunotherapy treatment and, therefore, may serve as a predictive marker of response [24, 25]. The global data analysis showed a trend toward enhanced survival in both OS and PFS, although the trend was not statistically significant. The absence of statistical significance was possibly due to the small sample size. These differences are consistent with those from other studies in solid tumours, such as those carried out by Assoun et al. [19] in NSCLC and Michel et al. [35] in colorectal cancer. These theoretical data are based on the increased release of antigens produced by mutations in TP53 [36]. This creation of antigens would imply activation of the immune system with the consequent greater response to ICIs [37].
These differences in OS and PFS can be observed in our analysis, which was dependent on the adenocarcinoma subtype and in women (two factors that are likely related in many cases). A study by Sun et al. [38] reported that TP53 mutations could predict response to immunotherapy in the NSCLC adenocarcinoma subtype, although this was independent of the mutational type. According to these findings, p53 expression as a surrogate marker is very likely dependent on and exclusive to the adenocarcinoma subtype. It is possible that in adenocarcinoma subtypes, the influence of p53 is key compared to squamous cells because the TMB in the former is lower. Therefore, in squamous subtypes, high TMB would already be a biomarker of response to ICIs [39]. Together with the above, a higher percentage of responses is observed in patients with high protein expression of p53. There were only complete responses in patients with mutated TP53. In contrast, the percentage of progressions was 14.1% higher in patients without mutations. The results, in this case, were not statistically significant. However, it is possible that the limiting factor of the sample size accounts for this absence of statistical significance.
Another relevant finding is the fact that the high protein expression level of
p53 seems more critical in tumours with negative PD-L1 expression. In these
patients, OS was higher in patients with expression levels of
Taken together, our results favour the determination of p53 before the start of treatment with immunotherapy, mainly in patients with the adenocarcinoma subtype. The prior determination of p53 should be evaluated in those patients whose treatment is based on immunotherapy plus chemotherapy regimens where chemotherapy could be avoided, or on the contrary in those with monotherapy ICIs, where chemotherapy addiction would be interesting.
It can be stated that survival is higher with NSCLC immunotherapy in patients with mutations in TP53, as assessed using a surrogate marker, such as p53 protein expression. This finding seems to be more valid in patients with the adenocarcinoma subtype. The influence of p53 on NSCLC is possibly greater on PD-L1 negative tumours due to the relatively less activation of the immune system in these tumours. Additional studies are required in the future to validate these conclusions. Due to the limitation of the sample size in the research, future studies should include a larger sample size with a greater number of patients with negative PD-L1 and adenocarcinoma subtypes.
Conceptualization—AO-H, EB-M, and JPM-G. Methodology—AO-H, LP-B, and LF-P. Validation—AO-H, EB-M, JPM-G, RG-S, JJC-H, MDL-C, and CP-P. Writing - original draft preparation—AO-H, JM-V, TM-G, RE-C, and RV-T. Investigation—AO-H, LF-P, and LB-H. All authors have read and agreed to the published version of the manuscript.
This study has been approved by the ethics committee of the University Hospital of Salamanca under reference number PI 2021 10 878. It has had the informed consent of all the patients included for the analysis of the samples and has complied with the bioethical protocols current.
For the cancer patients of the University Hospital of Salamanca for their kindness and affection.
This research received no external funding.
The authors declare no conflict of interest.