Can we use Ki67 expression to predict prostate cancer aggressiveness?

ABSTRACT Introduction: specialists have an urge for biomarkers that can discriminate indolent prostate cancer from aggressive tumors. Ki67 is a proliferation marker, and its expression is associated with the aggressiveness of several cancers. Objective: analyze the expression of Ki67 in prostate cancer samples correlating with the aggressiveness of the disease. Methods: Ki67 mRNA levels were determined utilizing data from a TCGA cohort (Tumor(n)=492 and control(n)=52). The protein expression was determined on 94 biopsies from patients by immunohistochemical assay. Results: in mRNA, the Ki67 upregulation is associated with cancer tissue (p<0.0001) and worst disease-free survival (p=0.035). The protein upregulation is associated with increase of the ISUP score (p<0.0001), cancer stage (p=0.05), biochemical recurrence (p=0.0006) and metastasis (p<0.0001). We also show a positive correlation between Ki67 expression and ISUP score (r=0.5112, p<0.0001) and disease risk stratification (r=0.3388, p=0.0009). Ki67 expression is a factor independently associated with biochemical recurrence (p=0.002) and metastasis (p<0.0001). Finally, the patients with high Ki67expression shows better survival regarding biochemical recurrence (p=0.008) and metastasis (p=0.056). Patients with high Ki67 expression are 2.62 times more likely to develop biochemical recurrence (p=0.036). Conclusion: Ki67 upregulation is associated with prostate cancer aggressiveness.


INTRODUCTION
P rostate cancer (PC) is one of the most prevalent cancers globally and the cause of death of thousands of men every year 1 .Although radical prostatectomy (the most common treatment for PC) has good results in general, it has some side effects, such as erectile dysfunction and urinary incontinence 2 .
Since overtreatment is a challenge in PC, it is critical to predict which patients need invasive treatment since they harbor aggressive cancer and which would have indolent disease.Due to the significant heterogeneity of prostate cancer, classical prognostic factors, such as serum PSA, pathological staging, and Gleason score, are not sufficiently accurate to separate indolent from aggressive cancers in a reliable way 3 .In this sense, molecular prognostic biomarkers can be essential tools in the clinical management of PC.
The Ki67 protein, widely used as a proliferation marker, is expressed in all cell cycle phases, except G0 and G1 4 .Because of this, the potential as a prognostic biomarker of this protein is evaluated in several types of cancer, such as breast, lung, bladder, gastric, and prostate cancer [5][6][7][8][9] .In addition, the use of Ki67 is potentially applicable, as it is technically accessible and easy to interpret 10 .
Whereas there is a need for new molecular biomarkers to classify PC, in this manuscript, we evaluated whether the expression of Ki67 can predict the aggressiveness of the disease.

TCGA cohort
We use RNA-seq data from The Cancer Genome Atlas (TCGA) PC datasets.The cohort consisted of 492 samples of prostate adenocarcinoma and 52 samples of paired normal samples.
All analyses were made using the online Gene Expression Profiling Interactive Analysis (GEPIA) database 11 .The unit of gene expression is Transcripts per Million.All images are original from GEPIA, with minor styles adjustments.

Patients
A total of 94 biopsies from patients with prostate cancer, treated surgically in 1998, 1999, 2006,   and 2007, at Hospital das Clínicas of the Medical School of University of São Paulo, were selected.To obtain clinical and etiological data, electronic medical records were evaluated.The characteristics of the patients are summarized in Table 1.First, the deparaffinization of the Tissue Microarray slides was carried out in an oven at 60-65ºC for 1 hour and washed with Xylol and decreasing dilutions of alcohol followed by washing with distilled water.Then, the antigen was recovered by heat in a universal Diva antigen recovery buffer (1:100) and heated for 10 minutes at 110ºC in an electric pressure cooker (Decloacker).
The slides were then dehydrated, mounted with coverslips, and observed in an optical microscope by an expert pathologist.

Analysis of results
The graphs and the statistical analysis were performed using GraphPad Prism 8 SPSS software (23.0).
We used Student's t-test for two groups and one-way ANOVA with Bonferroni's correction for three or more groups for the hypothesis test.
Correlation analysis used Pearson's r test for the hypothesis test.Logistic regression is applied to evaluate if Ki67 expression is independently associated with prognostic factors.For all statistical analyses, we set a level of significance of 5% (p≤0.05).

Ki67 mRNA upregulation is associated with the onset and progression of Prostate Cancer
To analyze the transcription of Ki67 in prostate cancer, we utilized TCGA PC datasets.When we compared the expression of Ki67 mRNA between cancer and normal tissue, we observed a significant upregulation (p<0.01) of the transcript in the disease (Figure 1A).
Next, we separated the tumors according to mRNA expression and observed patients with the worst disease-free survival overexpressed Ki67 (HR=1.6,p=0.035) (Figure 1B).

Upregulation of Ki67 protein expression is associated with prostate cancer aggressiveness
Then, by immunohistochemical assay, we checked whether Ki67 protein levels were associated with the aggressiveness of primary PC in clinical samples of the disease.The progressive upregulation of Ki67 is associated with the increase of the International Society of Urological Pathology score (ISUP) (p<0.0001)(Figure 2A).In particular, ISUP 4 and ISUP 5 tumors shows the highest expression, with significant differences between ISUP 1 and ISUP 4 (p=0.0009)and between ISUP 5 and ISUP 1, 2 and 3 (p<0.0001,p=0.0046 and p= 0.0027, respectively).
The upregulation of Ki67 was also associated with the non-confined disease (Figure 2B) and the presence of biochemical recurrence (Figure 2C) and metastasis (Figure 2D) (p=0.05,p=0.0006, and p<0.0001, respectively).No association between PSA levels and Ki67 expression was found (data not showed).

Quantitative analysis showed that the upregulation of Ki67 correlates with a worse prognosis for prostate cancer
For a possible application of Ki67 expression in the clinical practice of PC, we need to evaluate its expression quantitatively.For this, we categorize our patients taking into account the ISUP score and the risk stratification for prostate cancer (AUA/ASTRO/SUO Guideline 3,12 ), and performed a correlation analysis.
The increase in Ki67 correlates positively with the worst prognosis of the disease, considering both the ISUP score (r=0.5112,p<0.0001) and risk stratification (r=0.3388,p=0.0009) (Table 2).
In addition, we performed a logistic regression to identify whether Ki67 expression is independently associated with prognostic factors.In this result, we found that Ki67 expression was a factor independently associated with biochemical recurrence (p=0.002) and metastasis (p<0.0001).

DISCUSSION
Usually, early detection of cancer is associated with better results, but for patients with PC, this can also result in overdiagnosis and overtreatment, stressing the need for new biomarkers that could predict the aggressiveness of the disease 13 .In this sense, using molecular markers can improve personalized medicine, improving the clinical management of patients with PC 14 .In this work, we analyzed whether the expression of Ki67, a classic cancer proliferation marker, can be used to predict PC aggressiveness. of the disease, there is currently insufficient evidence to support its clinical use 16 .
Considering mRNA levels, we analyzed TCGA datasets and showed that the upregulation of Ki67 is associated with the malignant tissue and the worst

Maia
Can we use Ki67 expression to predict prostate cancer aggressiveness?
disease-free survival.This suggests that Ki67 has a role in the onsets of carcinogenesis and the progression of cancer.
Although TCGA has already provided critical data on the biogenesis of PC, in the routine, immunohistochemical assays are more accessible because they show the protein expression of the biomarker and are already used by most pathologists 17 .Using this technique, we found that the upregulation of Ki67 is associated with a higher ISUP score, extraprostatic disease, biochemical recurrence, and metastasis.Curiously, we did not find any association between PSA levels and Ki67 expression, and some authors discuss that although PSA is important for disease screening, the prognostic value of PSA levels is low and have been associated with a high rate of overdiagnosis/overtreatment in clinical trials [18][19][20] .The use of Ki67 expression can help to discriminate when increased PSA is clinically significant.
In a more quantitative approach, we demonstrate a positive correlation between Ki67 expression and ISUP score and disease risk stratification.
This is important because both classifications are recent and consider disease-specific survival curves 3,21 .There is a consensus among urologists that biochemical recurrence is associated with a poor prognosis and metastasis with PC worst (and potentially fatal) state.Here we show that positive regulation of Ki67 is an independent predictor of these two factors 22 .Patients with high Ki67 expression show the worst survival curves regarding biochemical recurrence and metastasis.Besides that, in our cohort, the patients with high Ki67 expression are 2.62 times more likely to develop biochemical recurrence, suggesting that this protein may be a predictor of more aggressive cancers.
Ki67 expression shows no association with PC prognosis in a relatively old study 23 .Despite this, more recent studies generally agree that upregulation of Ki67 is associated with the worsening of the disease.A paper showed that Ki67 expression predicts biochemical recurrence and death from PC 24 .Additionally, a multicenter study confirms that Ki67 is an independent predictor of biochemical recurrence after radical prostatectomy 25 .Finally, an article with more than 1000 surgical specimens of PC concludes that high Ki67 expression was strongly associated with a higher Gleason score, cancer stage, seminal vesicle invasion, extracapsular extension, and the greater probability of disease recurrence 26 .
In summary, by mRNA and protein levels, we showed that the upregulation is associated with the aggressiveness of PC.Our results corroborate similar observations in the literature, and with that, we propose that the Ki67 immunohistochemical assay should be incorporated into the prognostic evaluation of PC 9,[27][28][29][30] .

R E S U M O R E S U M O
slides containing the PC specimens simultaneously, allowing homogeneity in the evaluations.

Figure 2 .
Figure 2. Association between Ki67 protein expression and prostate cancer aggressiveness.In A, we have the progressive Ki67 upregulation with the increase of the ISUP score.In B, we have the upregulation of Ki67 associated with the extraprostatic disease.In C and D, we have the upregulation of Ki67 associated with the presence of biochemical recurrence and metastasis, respectively.**p<0.001,*p<0.01.
Figure 1.Ki67 mRNA levels in prostate cancer.In A Ki67, transcription is upregulated in cancer (Log2Fold Change Cutoff=0.9).In B, the upregulation of Ki67 is associated with worse disease-free survival.*p<0.01;HR= Hazard Ratio.

Table 2 -
Correlation between Ki67 expression and prognostic factors.