Bio-Clinical Parameters with a Predictive Role in Advanced Prostate Cancer

Background: Advanced forms of prostate cancer include a heterogeneous group of patients with an uneven prognosis. The lack of consensus on a precise denition of this group of patients prevents clinical decision-making and adequate patient counseling. Methods:In this retrospective study, we investigated a series of preoperative bio-clinical parameters (age, patient comorbidities, tumor clinical stage, serum PSA, preoperative Gleason score, the neutrophil / lymphocyte ratio value and hormonal prole of patients), in relation to some postoperative parameters (histopathological type of tumor, tumor pathological stage, postoperative Gleason score, extra-prostatic extension, positive margins, seminal vesicle invasion, perineural invasion, positive lymphadenopathy), in a group of 96 patients with radical prostatectomy (RP) between 2016 and 2020, for a better understanding of the behavior of the disease and adequate counseling of patients proposed for RP. Results: Of all the clinical and biological parameters analyzed, only the clinical T stage ≥ cT3a was statistically associated with an unfavorable pathological prognosis (p = 0.03). In the multivariate logistic regression model, the presence of a clinical tumor stage at clinical examination ≥ cT3a was associated with a 4.73-fold increase in the risk of unfavorable pathological prognosis. Moreover, the presence of a clinical T stage ≥ cT3a identies with a sensitivity of 68.74% and a specicity of 65.71% patients with an unfavorable pathological prognosis. At the same time, the neutrophil / lymphocyte ratio (NLR) value was statistically signicantly correlated only with the presence of secondary lymph node determinations (p = 0.006), and the serum level of dehydroepiandrostenedione was statistically signicantly correlated with the presence of extra-prostatic extension (p = 0.05). Conclusions: The results obtained can have a signicant impact in improving the knowledge of the pathogenesis of advanced forms of prostate cancer and in making clinical decisions, with immediate positive clinical effect (survival, quality of life) and socio-economic.


Introduction
Despite advances in modern medicine, prostate cancer (PCa) remains a major public health problem affecting the male population, with a growing prevalence. It ranks rst in incidence and ranks 2nd in terms of cancer mortality in men (1). According to Globocan Romania, in 2020, PCa represents 14.9% of new cases of cancer in men. Prostate cancer is di cult to treat due to molecular, cellular and clinical heterogeneity (2). Currently, there are not enough means to be able to characterize the aggressive behavior of PCa and there is no unanimously accepted consensus on precise diagnostic methods or optimal therapeutic management for these patients. The factors that determine the risk of developing PCa are not well known, although age and genetic factors have a signi cant impact on the risk and progression of the clinical disease. Current prognostic factors for newly diagnosed patients with PCa used in current clinical practice are serum levels of preoperative PSA, histological grade Gleason, and / or clinical stage of the tumor assessed by a digital rectal examination (DRE) (3). In addition, several biomarkers have been extensively studied and could be used to identify patients with aggressive disease. The neutrophil / lymphocyte ratio (NLR) is a systemic in ammatory marker, which has been shown to be associated with a poor prognosis in several types of neoplasms, with implications for carcinogenesis and metastasis (4). It has been observed that an in ammatory environment is an essential component of all types of neoplasms, regardless of whether their occurrence is determined by the presence of chronic in ammation or not (5). Moreover, the in ammatory mechanisms in cancer are complex, as the presence of a neoplasm also causes an in ammatory response of the host, which leads to the development of a pro-neoplastic environment. Therefore, the in ammatory tumor environment contains innate cells, such as macrophages, neutrophils and mast cells, but also adaptive immune cells (T and B lymphocytes). Depending on the intercellular communication through cytokines and the expression of different immune mediators, the balance between the in ammatory environments that allows tumor growth is maintained, respectively the one that ghts against it. In advanced tumors, the balance tends much towards protumor in ammation, responsible for angiogenesis, invasion and metastasis. The importance of in ammatory markers has also been con rmed in PCa. Regarding castration-resistant prostate cancer (CPRC), under treatment with enzalutamide, Conteduca et al. observed that patients with NLR over 3 before the start of treatment had a 4.2month lower progression-free survival than patients with NLR below 3 (6). Overall, the overall survival of NLR patients over 3 was signi cantly reduced in compared with those with NLR below 3 (10.4 months vs 16.9 months, p < 0.0001). Therefore, not only the increased value of pre-treatment NLR, but also its persistence during therapy has prognostic value for patients with castration-resistant PCa. Despite advances in surgical technique or patient selection, up to 40% of patients diagnosed with PCa progress to biochemical relapse, often rapid post-surgical and a subsequent unfavorable prognosis marked by metastatic progression and even death by PCa. Thus, a personalized diagnostic and treatment approach for these patients will have a positive impact on mortality and morbidity caused by this disease.

Methodology
The current prognostic factors used in clinical practice are insu cient to characterize the aggressive behavior of PCa. Thus, our study aimed to identify predictive factors that improve the diagnosis of prostate cancer and adapt the therapeutic attitude according to the prognosis of each patient. The secondary outcome was to evaluate the predictive role of NLR in terms of oncological outcomes in the group of patients with localized and locallyadvanced prostate cancer in whom radical prostatectomy (RP) was performed.
The study included 96 patients diagnosed with localized and locally advanced PCa and who were hospitalized for radical prostatectomy with pelvic lymphadenectomy at the Constanța County Emergency Clinical Hospital from January 2016 until December 2020.
The major criterion for inclusion in the study group was the histopathological diagnosis of prostate adenocarcinoma, and the exclusion criteria from the study were the administration of neo-adjuvant hormone therapy and the presence of urinary tract infection at the time of intervention. Histopathological examination of prostatectomy pieces included the following parameters: histological type, Gleason score, pathological stage of the disease, the presence of the intraductal component, lympho-vascular invasion, respectively perineural in ltration, resection margin status and excised lymph node status. Patients were evaluated according to the Charlson Comorbidity Index (CCI) (7). Based on the distribution of the CCI score in our cohort, patients were classi ed into two categories of CCI score: ≤ 3 or > 4. Reporting histological grading was done according to ISUP 2014 and the guide for reporting pathological elements with prognostic value according to Association guidelines European Institute of Urology in force (3). All cases were recorded in a database that included a preoperative clinical parameter (age, patient comorbidities, tumor clinical stage, serum PSA, preoperative Gleason score), but also some postoperative parameters (histopathological type of tumor, tumor pathological stage, postoperative Gleason score, extra-prostatic extension, positive margins, seminal vesicle invasion, perineural invasion, positive lymphadenopathy). In addition, the neutrophil / lymphocyte ratio value and hormonal pro le of patients was analyzed by serum determination of free testosterone, DHT, DHEA, DHEAS, androstenedione. Data obtained were analyzed and graphs were constructed using SPSS version 20.0 software (SPSS, Chicago, IL) and MedCalc version 19.0.3 software (MedCalc, Ostend, Belgium). The effectiveness of selected bio-clinically parameters to function as prognostic biomarkers were evaluated by using Receiver operating characteristics (ROC). Sensitivity and speci city were then de ned by the optimal cut-off point, which refers to the maximized value of the area under the ROC (Youden index). The univariate prognostic analysis revealed the parameters which affected the prognosis of PCa patients, as hormones expression levels and clinicopathological characteristics. The multivariate logistic regression model was used to identify the clinical and biological parameters associated with an unfavorable pathological prognosis.

Results
The clinical and biological preoperative characteristics of the patients included in the study are presented in Table   1. The mean age of the patients included in the study was 63 years (± 5.2 years). The median pre-treatment PSA values were 18 ng / ml (95% CI: 9-28 ng / ml), and the median number of positive biopsies was 5 (95% CI: 4-6).
The preoperative Gleason score had a mean value of 7.6 ± 0.8, and 46 cases (47.9%) had a score value of ≥ 8. The extra-prostatic tumor extent assessed by DRE (clinical tumor stage ≥ cT3a) was reported in more than half of cases (56.3%; 54 patients). In our group, 15 patients (15.6%) were > 60 years old, the preoperative PSA value ≥ 20 and the preoperative Gleason score ≥ 8. PSA -prostate speci c antigen; CCI -Charlson comorbidity index; In Table 2, we present the relationship between the preoperative clinical-biological parameters. In the study group, the age of the patients correlated poorly with the preoperative Gleason score (r = 0.429, p = 0.002) and with the Charlson comorbidity index (r = 0.349, p = 0.010), and the preoperative PSA value showed a statistically signi cant correlation but inversely proportional to the pre-operator Gleason score (r = − 0.363, p = 0.014). The postoperative clinical and biological characteristics of the patients are presented in Table 3. The postoperative Gleason Score had an average value of 7.6 ± 0.8, and 37.5% had a pathological Gleason Score higher than 8. At the same time, 63 patients (65.6%) had concordance between pre-and postoperative Gleason score; 15 patients (15.6%) had a higher postoperative Gleason score compared to the preoperative Gleason score (upgrading), and 19 patients (19.8%) had a lower postoperative Gleason score than the reported preoperative (downstaging). In the studied group, the most common histological type encountered was acinar adenocarcinoma (88.5%), followed by mixed form (9.4%) and only 2.1% had the ductal type at the pathological examination. Of these, 89 patients   Prognostic value of neutrophil / lymphocyte ratio in prostate cancer In the studied group, the median NLR was 1.8, 95% CI: 1.7-1.9. We observed that the NLR value was statistically signi cantly correlated only with the presence of secondary lymph node determinations (p = 0.006). There was no statistically signi cant correlation between NLR and prostate cancer characteristics (Table 5). We noticed that an NLR value above 1.99 has a moderate predictive value for the presence of positive lymph nodes, with an AUC of 0.75, sensitivity 77.7%, speci city 65.3%, p = 0.001 (Fig. 1). Relationship between the value of preoperative and histopathological parameters (Table 6) The Evaluation of clinical-biological parameters associated with an unfavorable pathological prognosis

Univariate analysis
Regarding the analysis of clinical-biological parameters associated with an unfavorable pathological prognosis, the data are summarized in Table 7, which includes the main variables analyzed. Of all the clinical and biological parameters analyzed, only the clinical stage T ≥ cT3a was statistically associated with an unfavorable pathological prognosis (67.7% vs. 35.3%, p = 0.03).

Multivariate analysis (logistic regression)
The multivariate logistic regression model included the following parameters: preoperative PSA value, clinical stage T ≥ cT3a, the association between age > 60 years + preoperative PSA ≥ 20 ng / ml + preoperative Gleason score ≥ 8 and serum testosterone level. The results of this analysis are presented in Table 8. As in the case of the univariate analysis, only the presence of a clinical stage T ≥ cT3a was the parameter retained in the nal model being independently associated with an unfavorable histological prognosis. The presence of a clinical stage T ≥ cT3a was associated with a 4.73-fold increase in the risk of an unfavorable pathological prognosis (Table 8). The prognostic utility for independent determinants of the identi ed unfavorable pathology To evaluate the prognostic utility of the clinical T stage ≥ cT3a for identi cation patients with an unfavorable pathological prognosis, the analysis of areas under the receiver operator curves (ROC) was used. The presence of a clinical T stage ≥ cT3a identi es with a sensitivity of 68.74% and a speci city of 65.71% patients with an unfavorable pathological prognosis (Table 9, Fig. 3).   (8, 9). Moreover, the PSA level was inversely correlated with the Gleason score. And in another study, the serum level of preoperative PSA > 20 ng/ml was statistically signi cantly correlated with lymph node invasion and vesicle invasion (10). Due to recent studies, radical prostatectomy for patients with preoperative PSA greater than 20 ng/ml is considered a viable option, demonstrating encouraging results in this group of patients. In our cohort of PCa patients, 44.8% of patients had preoperative PSA levels > 20 ng/ml. Pre-operative PSA values correlated positively with the presence of positive lymphadenopathy (r = 0.28, p = 0.04) and the presence of lymph node invasion in more than 2 lymph nodes observed at the anatomopathological examination (r = 0.34, p = 0.01), but it was not statistically signi cantly correlated with the other elements of unfavorable pathology. Also, the Gleason score compared to prostate biopsy is considered a signi cant predictor of pathological outcomes after radical prostatectomy, and in addition, may predict unfavorable postoperative evolution. In our study, 37.5% of patients had a preoperative Gleason score higher than 8. The preoperative Gleason score correlated positively with therapy of immediate or delayed androgenic deprivation). They found that for a Charlson score between 0 and 1, the overall survival rates at 15, 20, and 25 years were 26%, 15%, and 8%, respectively; at a Charlson score of > 1, overall survival rates at 15, 20, and 25 years were 11%, 6%, and 3%, respectively (16). Obviously, in this study, a higher Charlson score was correlated with a more unfavorable result (16). Although the Charlson score is probably the most commonly used comorbidity assessment index in the context of RP, in our study, CCI was not associated with unfavorable pathological aspects for patients with advanced PCa. However, we consider that this parameter, alone or in combination with the other parameters, will be useful in assessing the speci c survival for the patients in our study.
The relationship between preoperative levels of endogenous sex hormones and RP outcomes is controversial in the literature. Some studies have reported a higher incidence of poorly differentiated tumors in patients with PCa and low serum testosterone levels (17). Three other retrospective studies have shown that low total testosterone is associated with unfavorable pathological features in RP specimens, but found no association with biochemical recurrence (18,19). In addition, a clinical study from 2006, suggested that age-adjusted free testosterone correlates with poorly differentiated prostate tumors (20). Many circulating androgenic compounds, including testosterone, androstenedione (A), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) can be converted to the metabolically active dihydrotestosterone (DHT). DHT, which is the most potent intraprostatic androgen, appears to play a signi cant role in initiating PCa. The inevitable occurrence of androgenrefractory PCa after hormone treatment suggests that androgens play a rather "permissive" role in tumor progression (20). Morote J et al. (2009), in a study of subjects with nonmetastatic PCa and independent androgens, pointed out that the lower the testosterone levels, the longer period of disease progression (21). In our study, the serum levels of testosterone, dihydrotestosterone, DHEA-S and androstenedione were not statistically signi cantly correlated with unfavorable prognosis at the histopathological examination of the biopsy specimen. In contrast, the serum level of dehydroepiandrostenedione was statistically signi cantly correlated with the presence of pathological extra-prostatic extension (r = 0.29, p = 0.05). The researchers tried to improve the characterization of high-risk patients and created different predictive models by combining preoperative variables to predict postoperative outcomes, such as prediction of extracapsular extension, seminal vesicle invasion, lymph node invasion (22 In last years, markers of systemic in ammation and especially the neutrophils / lymphocytes rate have been studied in relation to the occurrence and prognosis of several types of neoplasms. Although the mechanisms of the immune response and its in uence on neoplastic development are not fully known at present, many studies con rm a signi cant link between them (4). In a review published by Tang L. et al. (2016) it was shown that an increased NLR associated an increased risk of recurrence for localized and locally-advanced PCa, respectively a lower survival for patients with locally advanced PCa compared to those who had a low ratio before treatment (27). Moreover, a meta-analysis of 14 studies that included a total of 16,266 patients con rmed the prognostic role of NLR for a shorter survival and a shorter non-recurrence interval even for metastatic CRPC (mCRPC) (28). The results of our study con rm what has been previously published and support the premise that in ammation has a role in promoting metastasis, but does not in uence the intrinsic characteristics of the tumor (Gleason score, tumor stage). NLR has a prognostic value for the presence of secondary lymph node determinations (p = 0.006). It is estimated that for these patients, overexpressed lymphadenectomy has the potential to improve oncological results.

Conclusions
Simultaneous analysis of a panel of biomarkers to predict the pathological stage is a strong point of the present study, but nevertheless, when analyzing the research results, we must consider the characteristics of the group, the small number of patients and the possibility of combining preoperative parameters or the inclusion of other risk factors in order to be able to predict with great accuracy the nal pathological stage. In order to widely introduce such models into clinical practice, it is necessary to further improve the models currently available and to develop more reliable, exible, simple and easily accessible tools by incorporating conventional clinicopathological factors as well as molecular biomarkers. We consider that the results obtained can be useful for current clinical practice allowing us to analyze the predictive power of preoperative parameters for the subsequent oncological evolution of patients with advanced PCa treated by RP, including the risk of metastatic progression or death by PCa.

Declarations
Ethics approval and consent to participate The study was conducted according to good laboratory practice and in accordance with the national and institutional standards and informed consent was obtained from all patients. The study was approved by the Local Ethics Commission for the Approval of Clinical and Research Developmental Studies.

Consent for publication
Authors con rmed that this work can be published. The content of this manuscript is original and it has not yet been accepted or published elsewhere.

Availability of data and material
Research Data are not shared.

Con icts of interest
There are no con icts to declare. Correlation between the presence of extraprostatic extension and the serum level of dehydroepiandrostenedione (r = 0.29, p = 0.05).

Figure 3
Usefulness of clinical T stage in identifying patients with unfavorable pathological prognosis (area under the curve 0.662; 95% CI 0.499 -0.826).