Cellular senescence at the saphenofemoral junction in patients with healthy,primary varicose and recurrent varicose veins

Abstract

Objectives

Cellular senescence could play a role in the development of venous disease. Superficial venous reflux at the saphenofemoral junction is a common finding in patients with primary varicose veins. Furthermore, reflux in this essential area is associated with higher clinical stages of the disease and recurrent varicose veins. Therefore, this pilot study aimed to investigate cellular senescence in the immediate area of the saphenofemoral junction in patients with healthy veins, primary varicose veins and additionally in patients with recurrent varicose veins due to a left venous stump.

Methods

We analyzed vein specimens of the great saphenous vein immediately at the saphenofemoral junction. Healthy veins were collected from patients who underwent arterial bypass reconstructions. Samples with superficial venous reflux derived from patients who received high ligation and stripping or redo-surgery at the groin, respectively. Sections were stained for p53, p21, and p16 as markers for cellular senescence and Ki67 as a proliferation marker.

Results

A total of 30 samples were examined (10 healthy, 10 primary varicose, and 10 recurrent varicose veins). We detected 2.10% p53⁺ nuclei in the healthy vein group, 3.12% in the primary varicose vein group and 1.53% in the recurrent varicose vein group, respectively. These differences were statistically significant (p = 0.021). In the healthy vein group, we found 0.43% p16⁺ nuclei. In the primary varicose vein group, we found 0.34% p16⁺ nuclei, and in the recurrent varicose vein group, we found 0.74% p16⁺ nuclei. At the p < 0.05 level, the three groups tended to be significant without reaching statistical significance (p = 0.085). There was no difference in respect of p21 and Ki67.

Conclusion

We found significantly higher expression rates of p53 in primary varicose veins at the saphenofemoral junction than in healthy veins. p16 expression tended to be increased in the recurrent varicose vein group. These preliminary findings indicate that cellular senescence may have an impact in the development of varicose veins or recurrence. Further studies addressing this issue are necessary.

Keywords

Varicose veins saphenofemoral junction great saphenous vein cellular senescence

Introduction

Venous reflux at the saphenofemoral junction (SFJ) is a common finding in patients with chronic venous insufficiency.¹ It is associated with higher stages of the disease^1,2 and present in nearly half of the patients with recurrent varicose veins.³ Recurrent varicose veins are a socioeconomic problem as well, as they occur in 13–65% of all patients after varicose vein treatment.⁴ Same-site groin recurrences with a left venous stump have been described as a major cause for recurrent varicose veins.^5,6 Even though the SFJ is an essential area, reasons for the development of superficial venous reflux still remain unclear. The currently leading pathophysiological theory is the so-called venous wall’s weakness hypothesis.⁷ Multiple, presently unknown, pathophysiological mechanisms should lead to a weakness of the venous wall, what secondarily results in an insufficiency of the adjacent venous valves.⁷ Cellular senescence could play a role concerning this aspect, as the serum of varicose veins can induce senescence-related dysfunction of vascular endothelial cells.⁸ In particular, cellular senescence leads to an irreversible growth arrest of cells via activation of p53/p21 and p16 tumor suppressor pathways.^9–11 Levels of these cell-cycle proteins have been described to be higher in sedentary adults when compared to older ones in good condition, who are physically trained.⁹ This might be of clinical relevance as sedentary activities, e.g. prolonged standing or sitting could be associated with a higher prevalence of varicose veins.^12,13 Interestingly, pressure as it occurs in varicose veins can induce cellular senescence too.¹⁴ Furthermore, high pressure should lead to a flow-induced wall remodeling in saphenous veins in rats with higher expression of Ki67, as a marker of cell division.¹⁵ Undoubtedly, age has been described as an independent risk factor for higher stages of the disease.^16,17 Therefore, cellular senescence could be involved in the pathophysiology of chronic venous disease. Due to the fact that reflux is a common finding at the SFJ, ^1,3,18 it seems feasible that cellular senescence causes venous walls weakness in this essential area. This pilot study aimed to investigate cellular senescence in the immediate area of the important SFJ in patients with healthy veins, primary varicose veins, and recurrent varicose veins due to a left venous stump.

Material and methods

Tissue samples

We collected vein specimens of the great saphenous vein (GSV) immediately at the confluence of the GSV into the common femoral vein between the years 2016–2018. We compared these probes in patients with healthy veins and patients with primary varicose or recurrent varicose veins. For better readability, we named the groups “primary varicose veins” and “recurrent varicose veins,” even though patients with higher c classes of the CEAP classification were included too. Main focus was the absence or presence of superficial venous reflux at the SFJ. Therefore, reflux at the SFJ was an essential inclusion criterion in all probes, except for healthy veins. Reflux detected by duplex sonography over 0.5 s was regarded as pathological.¹⁹

A detailed duplex sonographic examination was performed preoperatively. Patients with pathological findings in the deep venous system, e.g. due to deep vein thrombosis, were excluded. The mean diameter of the SFJ was measured in all varicose vein patients in a standing position. In terms of venous disease, the c class of the CEAP classification was noticed. During all surgical procedures, high ligation of the GSV was performed. This means that the great saphenous vein was routinely ligated directly at the opening into the common femoral vein (see Figure 1). Afterwards, a sample of the GSV with a size of about 1 cm was collected directly at the SFJ.

Figure 1.

Redosurgery at the groin to collect recurrent stump specimens. The venous stump is clamped between two Overholt clamps. FA: femoral artery; CFV: common femoral vein; VS: venous stump.

Healthy vein group

Healthy veins derived from patients who underwent femoro-crural or femoro-popliteal bypass reconstructions and solely veins without pathological findings were included. Specifically, the GSV has to be sufficient at the whole length and the diameter of the GSV has to be smaller than 5 mm in order to use the vein as a bypass graft. Moreover, blow-out phenomena, post-thrombotic alterations or other vein degenerations as well as incomplete reflux of the GSV were the exclusion criteria for these samples. In conclusion, these patients did not have superficial venous reflux nor varicose veins or signs of chronic venous insufficiency.

Primary varicose vein group

Specimens in this group derived from patients who received high ligation and stripping of the GSV. Patients were included if superficial venous reflux at the SFJ causes varicose veins at the leg or higher c classes of the CEAP classification. Reflux in this group originates solely from the common femoral vein. Specifically, only samples with an incompetent terminal and preterminal valve, according to a Stuecker class 3,,²⁰ were included. Furthermore, the extent of reflux in the GSV and the proximal and distal point of insufficiency were noticed.

Recurrent varicose vein group

Patients were included if same-site groin recurrence with a residual venous stump of the GSV was the reason for the development of recurrent varicose veins or chronic venous insufficiency. All patients in this group had a previous incomplete high ligation and stripping operation with a left venous stump of the GSV. Indication for redo surgery was a single channel recurrence with a diameter of more than 5 mm and continuing reflux to new-developed varicosities at the tight or calf. As described above, patients with higher c classes of the CEAP classification were included as well. Redo surgery was performed by a lateral approach (see Figure 1). The duplex sonographic detection of a multi-channel recurrence, which could be interpreted as neovascularization, was an exclusion criterion. Additionally, the time interval to primary surgery was noticed.

Immunohistochemistry

All probes were immediately fixed in neutral buffered formalin after the explanation. Samples were embedded in paraffin and serially sliced into 4 µm sections. Each sample was cut to 16 slices with 2 slices per group for immunohistochemistry. Immunostaining was performed, for Ki67, p53, p21, and p16 as cell-cycle markers and cellular senescence. Following antibodies were used Ki67, p21, p53 (DAKO monoclonal mouse), and p16 (ROCHE, CINtec histology cit, monoclonal mouse). Sections were heated and deparaffinizied. Afterwards, permeabilization was performed by heating in a steamer in DAKO Target Retrieval Solution (pH 9.0) (DAKO, Goldstup, Denmark) for 20 min. After blocking with CAS for 30 min, sections were incubated with primary antibodies overnight at 4°C. A Cy3-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, Europe) was used as a secondary antibody for 30 min at room temperature. Nuclei were stained with DAPI (Molecular Probes, Darmstadt, Germany). Sections were counterstained with hematoxylin. For immunostaining with p16, we used a kit (ROCHE, CINtec histology cit, monoclonal mouse) according to the instructions for use. In the absence of a primary antibody, no staining could be observed.

The number of stained cells was manually counted in 10 fields for each vein layer under a light microscope at ×400 magnification. The results were noticed as the number of positively stained cells per 1000 cells.

Statistics and ethics

We documented each sample photographically and utilizing a distinct protocol. Statistical analysis was performed by Microsoft® Excel 2010 (Microsoft Corporation, Redmond, WA, USA) and GNU PSPP Statistical Analysis Software, Release 1.2.0-g07fb-4db (Free Software Foundation, Boston, MA, USA).²⁴ The level of statistical significance was set to p < 0.05. The Ethics Committee of the Medical Faculty of the Ruhr University of Bochum obtained ethical approval – Register Number 4971–14 from 07.06.2014. All participants provided written, informed consent to be involved in the study.

Results

Patient characteristics

The “healthy vein” group included 10 specimens (8 left, 2 right) from 10 different participants (3 females, 7 males). Their average age was 73.6 years (SD = 5.82; 95% CI [69.44, 77.76]). Body height, weight, and corresponding body mass index (BMI) were only available for seven of these participants; their average body height was 170.3 cm (SD = 6.16; 95% CI [164.59, 175.98]), the average body weight 75.3 kg (SD = 13.40; 95% CI [62.89, 87.68]) and the average BMI 26.07 kg/m² (SD = 5.21; 95% CI [21.25, 30.89]). As this was the “healthy” control group, obviously no CEAP classification or a description of reflux was available.

The “primary varicose vein” group comprised 10 specimens (6 left, 4 right) from 8 patients (5 females, 3 males) with an average age of 57.8 years (SD = 13.48; 95% CI [46.60, 69.15]), an average height of 169.9 cm (SD = 6.88; 95% CI [164.13, 175.62]), an average weight of 88.6 kg (SD = 10.41; 95% CI [79.93, 97.32]), and an average BMI of 30.75 kg/m² (SD = 3.72; 95% CI [27.65, 33.86]). Four of the legs presented with CEAP Class C2, another four with C3, and the remaining two with C4. The diameter of the GSV at the SFJ was on average 9.51 mm (SD = 3.12). Patients in this group had reflux in the GSV to the middle of the thigh in three cases and to the middle of the calf in seven cases.

In the group “recurrent varicose veins,” we examined 10 specimens (7 left, 3 right) from a total of 7 female patients. These patients had an average age of 54.0 years (SD = 13.87; 95% CI [41.17, 66.83]), an average height of 169.7 cm (SD = 8.86; 95% CI[161.52, 177.91]), an average body weight of 67.1 kg (SD = 9.55; 95% CI [58.24, 75.90]), and an average BMI of 23.49 kg/m² (SD = 4.61; 95% CI [19.23, 27.76]). Seven of the legs presented with CEAP Class C2, one with C3, and the remaining two with C4. The diameter of the stump of the GSV at the SFJ was in average 7.82 mm (SD = 2.66). The initial surgery was meanly 10.8 years ago (SD = 6.30).

At the p < 0.05 level, the three groups differed significantly by age (F(2,22) = 7.65, p = 0.003) and bodyweight (F(2,19)=7.12, p = 0.005), but not by height or BMI. For further details, see Table 1.

Table 1.

Summary of patients characteristics of healthy, varicose and recurrent varicose specimens of the saphenofemoral junction.

SFJ	Age (years)	Height (cm)	Weight (kg)	Body mass index (kg/m²)	Left	Right	C class	Mean ø SFJ (mm)
Healthy	73.6	170.3	75.3	26.07	8	2	None	<5 mm
Varicose	57.8	169.9	88.6	30.75	6	4	C2: 4, C3: 4, C4: 2	9.5
Recurrent	54.0	169.7	67.1	23.49	7	3	C2: 7, C3: 1, C4: 2	7.8

Clinical class of the CEAP classification. Bold values indicate a significant statistical difference within the groups. SFJ: saphenofemoral junction.

Markers

Ki67

In the “healthy vein” group, we found 1.84% Ki67⁺ nuclei (SD = 1.12; 95% CI [1.04, 2.64]). In the “primary varicose vein” group, we found 1.46% Ki67⁺ nuclei (SD = 0.89; 95% CI [0.83, 2.10]). In the “recurrent varicose vein” group, we found 1.91% Ki67⁺ nuclei (SD = 0.58; 95% CI [1.49, 2.33]). At the p < 0.05 level, the three groups showed no significant differences (p = 0.496).

p53

In the “healthy vein” group, we found 2.10% p53⁺ nuclei (SD = 1.43; 95% CI [1.08, 3.12]). In the “primary varicose vein” group, we found 3.12% p53⁺ nuclei (SD = 0.81; 95% CI [2.54, 3.70]). In the “recurrent varicose vein” group, we found 1.53% p53⁺ nuclei (SD = 1.29; 95% CI [0.61, 2.35]). At the p < 0.05 level, the three groups showed significant differences between the groups (F(2,27)=4.47, p = 0.021). Post hoc comparisons using the Tukey HSD test indicated that the mean p53⁺ percentage for the “recurrent varicose vein” group (M = 1.53, SD = 1.29) was significantly lower than in the “primary varicose vein” group (M = 3.12, SD = 0.81, p = 0.017), and non-significantly lower than in the “healthy vein” group (M = 2.10, SD 1.43, p > 0.05).

p21

In the “healthy vein” group, we found 1.62% p21⁺ nuclei (SD = 1.13; 95% CI [0.81, 2.42]). In the “primary varicose vein” group, we found 1.62% p21⁺ nuclei (SD = 0.60; 95% CI [1.20, 2.05]). In the “recurrent varicose vein” group, we found 1.72% p21⁺ nuclei (SD = 1.40; 95% CI [0.72, 2.72]). At the p < 0.05 level, the three groups showed no significant differences (p = 0.941).

p16

In the “healthy vein” group, we found 0.43% p16⁺ nuclei (SD = 0.31; 95% CI [0.20, 0.65]). In the “primary varicose vein” group, we found 0.34% p16⁺ nuclei (SD = 0.22; 95% CI [0.18, 0.49]). In the “recurrent varicose vein” group, we found 0.74% p16⁺ nuclei (SD = 0.59; 95% CI [0.32, 1.16]). The number of p16 positive nuclei tended to be different in the three groups in ANOVA analysis (p = 0.085). For further details see Figure 2.

Figure 2.

Positively stained cells per 1000 cells of the different markers at the saphenofemoral junction.

Figure 3.

Example of p16 staining in three different probes. A: recurrent varicose vein; B: primary varicose vein; C: healthy vein ×40.

Discussion

Insufficiency of the terminal valve at the SFJ is a major finding in patients with chronic venous disease^1,18 and reflux at the SFJ is present in 47.1% of patients with recurrent varicose veins after surgery (REVAS).³

Insufficiency causes venous hypertension, followed by macroscopic structural changes in the venous wall.²² An increase of collagen and laminin has been described in varicose veins.²³ Overall, a so-called “vascular remodeling” should occur, as varicose veins have been distinguished by damaged endothelial layers, unorganized smooth muscle structures, and increased neoangiogenesis.²⁴ Cellular senescence could play a role in this pathophysiological process as age is a risk factor for chronic venous disesase.^16,17 Endothelial senescence, induced by multiple cellular stressors, is the equivalent of ageing ²⁵ and leads to an irreversible growth arrest of a cell.⁹ The p53 and p21 are key factors in cellular growth arrest and the regulation of apoptosis, as overexpression of p53 can lead to a cell-cycle arrest in the G1 or G2/M phase via the activation of p21.¹⁰ Furthermore, the p16 tumor suppressor pathway can induce cell-cycle arrest⁹ too. Therefore, these markers have been analyzed in this preliminary study.

Ki67

Ki67 is the opponent of p16, p21, and p53 and a well-known marker for cell proliferation.²⁶ There are high Ki67 levels during mitosis as it prevents the chromosome aggregation whereas the expression is reduced in senescent cells or during cell-cycle arrest.²⁷ Filis et al. described a significantly higher expression of Ki67 in varicose veins than in healthy veins limited in the distal parts of the GSV near the ankle probably caused by venous hypertension and vascular remodeling.²⁸ This is supported by the fact that high pressure should lead to an increase of Ki67 in the saphenous vein in a rat model.¹⁵ Nevertheless, Filis et al. did not find differences in respect of Ki67 between proximal and distal veins segments of varicose veins. However, there were no differences in respect of Ki67 positive cells at the SFJ in our data too. Notably, there were no differences between primary varicose veins and recurrent varicose veins, which was confirmed by others as well.²⁶ Additionally, we have to state that El Wajeh et al. analyzed their samples in patients with neovascular recurrent varicose veins at the groin,²⁶ whereas our probes derived from residual venous stumps.

p53

The tumor suppressor p53 has a crucial regulative function during cell cycle.²⁹ p53 expression leads to transient cell-cycle arrest, senescence, or apoptosis of a cell via activation of p21 or other signals.³⁰ Cellular stressors like DNA damage, hypoxia, or oxidative stress can induce p53 expression.³¹ Urbanek et al.¹⁰ reported a higher immunohistochemical expression of p53 in varicose veins when compared with healthy ones. In this study, p53 was significantly higher at the SFJ in patients with primary varicose veins too. Contrary to our expectations, p53 positive cells were significantly lower in the recurrent varicose vein group, when compared to the primary varicose vein group. Chronic venous disease could have a dynamic affection with active and inactive phases. On the other hand, vein wall alterations correlate with reflux and it could be feasible that the different amount of reflux in patients with primary varicose veins and in patients with recurrent varicose veins due to venous stumps may have affected these results. All patients of the recurrent varicose vein group had a scar tissue around venous stump. This could have reduced the arterial perfusion of the vein wall, which could have had an influence in expression of p53 too. Furthermore, it has been stated that the expression of p53 should be associated with early stages of the disease,¹¹ while the expression of p53 should not be relevant in elderly due to increased structural changes of the venous wall.²⁸ This could also explain the small number of stained cells in the recurrent varicose vein group, as these patients had the most extended history of the disease.

Some authors have found a decreased rate of apoptosis in varicose veins in vein segments,^32–34 whereas these authors did not analyze vein segments near the SFJ or did not specify the exact investigated segment of the GSV. Only Filis et al. examined the area of the SFJ and described an increased apoptosis rate in the distal portions of varicose GSVs than the SFJ.²⁸ Other authors did not find a significant difference within distal and proximal segments of varicose veins.³⁴ These authors found higher apoptotic rates in the proximal parts in healthy veins.³⁴ Overall apoptosis seem to be deregulated in patients with varicose veins what can be confirmed by out data.³⁵

p21

p21 is another tumor suppressor which inhibits proliferation of a cell by a stop in the G1 phase. This stop can be induced by p53 as well as from p53 independent mechanisms.³⁶ Especially the p53/p21 pathway has been considered as a cellular senescence mechanism.³⁷ Urbanek et al. reported that p21 is increased in varicose veins.^10,11 These authors found higher mRNA expression levels of p21 in the proximal thigh of the GSV in patients with varicose veins when compared to healthy ones.¹⁰ The levels of p21 expression seems to be higher in proximal parts of the GSV when compared to distal specimens of the vein at the calf.¹¹ They positively correlate with the expression of p53.¹⁰ However, these data result from gene expression assessments, whereas the results of these authors could not be entirely confirmed by immunostaining.¹⁰ We analyze specimens from the SFJ and our data support these findings, as we could not find differences regarding the expression of p21 in patients with healthy, primary varicose, and recurrent varicose veins in this study.

p16

p16 is a widely used marker for endothelial senescence²⁵ associated with the vascular endothelial function.⁹ The p16 pathway functions independently from p53 mechanisms and acts as a backup.³⁷ p16 expression at the SFJ tended to be higher in the recurrent varicose vein group, when compared with primary varicose veins, even though there was no statistical significance. This could be due to the small number of specimens as in this preliminary study only 10 probes were included in each group. Further studies with higher numbers of probes could solve this discrepancy. Permanent reflux in left venous stumps for a long period, due to an incomplete high ligation and stripping, could be an explanation for the expression of p16 in the group of recurrent varicose veins. Obviously, this group of patients had the longest history of varicose veins. p16 expression was higher in the healthy vein group than in the primary varicose vein group. This fact may not be a discrepancy, as patients in the healthy vein group were significantly older than patients in the primary or recurrent varicose vein group. Further examinations of vein specimens of healthy younger patients are necessary to address this aspect in detail. The collection of these probes could be difficult, as young people rarely have an indication for surgery. Moreover, other markers of cellular senescence, e.g. beta-galactosidase, should be analyzed in further investigations. To the best of our knowledge, p16 has not been described in patients with venous disease. See Figure 3 for example of p16 staining in three different probes.

According to the other markers, there exist several histological studies. Most of them deal with vein specimens from the great saphenous vein from the thigh, or lower leg^10,11,32,33 and others did not describe the sampling operation in detail.³⁵ The investigation of patients with primary varicose and recurrent varicose veins due to superficial venous reflux in a clearly defined segment of the SFJ represents a strength of this pilot study.

Limitations

There are several substantial limitations in this study. First, the number of probes in every group is very low, as we designed the study as a pilot project. Second, we have only performed immunostaining with a manageable number of markers and no additional assessments. Therefore, the interpretation of these data is limited. Patients in the healthy group were significantly older as peripheral artery disease is a common disease of elderly and specifically atherosclerosis is associated with endothelial senescence.^38,39 Therefore, these factors could have an influence in our results as well.

Nevertheless, cellular senescence could be involved in the pathophysiology of the chronic venous disease as it is generally associated with age-related disorders.⁴⁰ Inflammation and endothelial dysfunction are relevant too⁴¹ as for example hsCRP (high sensitive c-reactive protein), vWF (von Willebrand factor), or D-Dimer blood values seem to be higher in varicose veins.⁴² Especially endothelial senescence and its relation to vascular endothelial dysfunction could play a key role.⁹ Cell culture experiments demonstrated that the serum of varicose veins patients can induce vascular endothelial senescence and an increase of reactive oxygen species in human umbilical vein endothelial cells.⁸ The expression of p53, p21, and p16 was increased in endothelial cells of antecubital veins in elderly.⁹ Interestingly, young people or older physically trained adults did not show such an increase of these senescence markers.⁹ The expression of these markers should be related with the vascular endothelial function⁹ and the endothelium of patients with chronic venous disease tend to be more damaged with higher c classes of the disease.⁴³ Due to the fact that progression of the disease with recurrent varicose veins affects many patients, this aspect could be also highly relevant for this group of patients.

Conclusion

In conclusion, the impact of cellular senescence in the development of varicose veins is entirely unclear. We did not find any differences in the expression of Ki67 and p21 at the SFJ. We found significantly higher expression rates of p53 at the SFJ in varicose than in healthy veins. The expression of p16 tended to be increased in the recurrent varicose vein group without reaching statistical significance (p = 0.085). This may indicate that initial endothelial and continuing cellular senescence may play a role in the development of varicose veins. The prevention and therapy of cellular senescence has been investigated intensively in the past decades and is still a major target of current research. Thus, inhibitors of autophagy have been identified as promising therapeutic candidate approaches.⁴⁴ This preliminary work indicates that this field of research may be of relevance for chronic venous disease as well. The phlebological community should be aware of the potential relevance of senescence research for their field.

Footnotes

Acknowledgements

This manuscript was created during a habilitation scholarship from the Ruhr University Bochum (supported by Bauerfeind AG). Furthermore, we gratefully thank Dr. Sandra Ueberberg for the technical assistance.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Markus Stücker received fees for consulting of Bauerfeind AG.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Ethical approval

The Ethics Committee of the Medical Faculty of the Ruhr University of Bochum obtained ethical approval – Register Number 4971–14 from 07.06.2014. All participants provided written informed consents to be involved in the study.

Guarantor

DM.

Contributorship

DM and TH conceived the study and developed the protocol. DM, TH, TF and AM collected the probes. AZ was responsible for immunohistochemical analysis. DM, EB and MS were responsible for data analysis. DM and TH wrote the first draft of the manuscript. All authors analyzed and interpreted the data, critically revised the first draft for intellectual content and approved the final version of the manuscript.

ORCID iDs

Dominic Mühlberger

Erich Brenner

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Cellular senescence at the saphenofemoral junction in patients with healthy,primary varicose and recurrent varicose veins – A pilot study

Abstract

Objectives

Methods

Results

Conclusion

Keywords

Introduction

Material and methods

Tissue samples

Healthy vein group

Primary varicose vein group

Recurrent varicose vein group

Immunohistochemistry

Statistics and ethics

Results

Patient characteristics

Markers

Ki67

p53

p21

p16

Discussion

Ki67

p53

p21

p16

Limitations

Conclusion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

Ethical approval

Guarantor

Contributorship

ORCID iDs

References