Immunohistochemical Analysis of Prostein Expression in Archived Prostatic Core Biopsies from Prostate Cancer Patients in Western Kenya

Omondi Swaya Tyrus (  omondityrus18@gmail.com ) Department of Medical laboratory Science, School of Public Health and Biomedical Sciences, Masinde Muliro University of Science and Technology, Kenya Dedan Opondo Division Of Urology, Synergy Clinics, Kisumu David O. Atandi Scanlab Laboratories, Nakuru Benard Guyah Department of Biomedical Science and Technology, School of Public Health and Community Development, Maseno University, Kenya Ng’wena Gideon Magak Department of Medical physiology, School of Medicine, Maseno University, Kenya

. In East Africa, prostate cancer ranks third in both incidence and mortality, and leads to an estimated 9,000 (9% of all male cancers) cases and 7,300 (8.5% of all male cancer) deaths annually (Ferlay et al., 2011). It is signi cant to note that PCa incidences increased by 64.5% between 1990 and 2010 (Lozano et al., 2012). In 2019, the prevalence of Pca in Western Kenya was at 7.0% (Macharia et Schoenborn et al., 2013). This has led to overtreatment and unnecessary biopsies in some cases. PSA also often fails to indicate accurately which patients are responding to a given treatment and is sometimes decreased or increased in higher grade or metastatic tumors. As a biomarker PSA lacks speci city and sensitivity (Epstein et al., 2014& Romero et al., 2014. Due to increased number of specimens with limited number of suspicious glands and minimal atypia (Magi-Galluzzi, 2018), histomorphological ndings in the biopsied tissues sometimes are di cult to report. Distinguishing between aggressive and indolent tumors is a major challenge (Sequeiros et al., 2013). Moreover, differentiating between high-grade urothelial carcinoma (UC) and high-grade Prostate adenocarcinoma (PAC) is frequently a diagnostic and prognostic challenge. According to recent European guidelines, PCa validated biomarkers are urgently needed for guiding the pre-treatment decision processes (Mottet et al., 2017). These scenarios can be improved by using Prostate speci c and sensitive immunohistochemical biomarkers, which would adjunct PSA levels and help the pathologist to make much more accurate differential diagnosis.
Prostein (also known as prostate cancer-associated protein 6 / P501S / SLC45A3) is a protein present in the Golgi apparatus of benign and malignant prostatic glandular epithelium. It is encoded by the Solute carrier family 45, member 3 (SLC45A3) gene, an androgen-regulated gene and a prostate speci c marker expressed in prostatic glandular cells (Xu et al., 2001). It shows perinuclear cytoplasmic localization in immunohistochemical experiments (Xu et al., 2001;Sheridan et al., 2007). Because it is highly speci c for prostate glandular cells, this target is useful for differentiating prostatic metastases from other carcinomas such as urothelial carcinomas or colorectal carcinomas (Xu et  Since prostate cancer is highly heterogeneous and its incidence and mortality vary strikingly among ethnic, racial, and national groups and this is an area worth exploring. native black African patients. There have been no reported studies on manual immunohistochemical diagnostic utility of Prostein in detection of prostate cancer among the African populations. The current study aims to evaluate manual immunohistochemical expression levels of Prostein, P501s, in archived formalin xed para n embedded (FFPE) prostatic core biopsy specimens consecutively collected between January 2018 and May 2021 from prostate cancer patients in Western Kenya.

Materials And Methods
This study involved 106 archived formalin xed para n embedded prostatic core biopsy specimens consecutively collected between January 2018 and May 2021 from prostate cancer patients at Jaramogi Oginga Odinga Teaching and Referral Hospital, Pathology Department and Division of urology at Synergy Clinics, Kisumu. All the clinical prostate tissue samples used in this study were accompanied by demographic information and pathological reports such as Tribe, Age, and type of malignancy. Nonprostatic tissues from the neighboring organs such as Testis, Penile; distant organs such as Liver and Oesophagus were included in the study as control specimen.
Following approval by Jaramogi Oginga Odinga Teaching and Referral Hospital Ethics and review committee (IERC/JOORTH/353/2021); the eligible patients or their families were contacted and consent obtained via recorded telephonic interview and their demographics obtained as well as their histopathology reports retrieved from the hospital laboratory management information system. The tissue blocks, selected using non-probability approach, were retrieved from the laboratory archive, deidenti ed, sectioned and manually stained using prostein immunohistochemical stains. Immunohistochemistry Immunohistochemistry (IHC) was performed manually following manufacturer's instructions. The FFPE tissue specimens were cut into sections of 4 μm using Micros Razor rotary microtome. After preparation of the tissue, the sections were mounted on charged poly-l-lysine FLEX IHC Microscope Slides (Code K8020) at and wrinkle-free. The tissue sections mounted on the slides were dewax at 60 0 C for 45Minutes in a hot oven then subjected to 3-in -1 pretreatment procedure with HIER using 1:50 diluted EnVision FLEX Target Retrieval Solution, High pH (50x) (Code K8004) in Dako PT Link. Depara nization, rehydration and Heat induced epitope retrieval were performed in Dako PT Link at automated Pre-heat temperature: 65 °C; epitope retrieval temperature and time: 97 °C for 20 minutes; cool down to 65 °C. The slide rack was then removed from PT tank and immediately dipped into jar/tank (e.g., PT Link Rinse Station (Code PT109)) containing diluted room temperature EnVision FLEX Wash Buffer (20x) (Code K8007). The slides were left in Wash Buffer for 5 minutes.
The immunolabeling procedures and incubation times were carried out manually according to the manufacturers' instruction using Dako Envision Labelled monomer-HRP anti-mouse (Dako, Glostrup, Denmark). The protocol involved each slide being rinsed with wash Buffer for further cooling to room temperature and pressure. Thereafter, 100µL FLEX Ready to Use Primary Antibody was added to the slides and incubated for 5minutes. This was again rinsed twice with Wash Buffer and then followed by addition of 100µL EnVision™ FLEX /HRP (RTU) and subjected to incubation for 20minutes. The slides were rinsed in Wash buffer and incubated for 5minutes then ooded with 200µL Envision TM FLEX Substrate working solution prepared by mixing 1ml of Substrate with one drop of 3,3′-Diaminobenzidine (DAB) and incubated further for another 20minutes. The slides were further rinsed using wash buffer and nally counterstained using 100µL EnVision™ FLEX Hematoxylin (RTU) incubated for 20minutes and rinsed in two changes of Deionized water and two changes of Distilled water. The incubations were done at room temperature.
After staining the slides were dehydrated in an increasing concentration of alcohol (70%, 90%, 95%, 100% 100%), cleared in two changes of xylene and mounted using aqueous Dako Glycergel™ Mounting Medium, Code C0563 then examined and con rmed by a surgical pathologist and immunohistochemical features recorded.
A negative control, section treated with a tris-buffer solution instead of primary antibody, were run simultaneously using the same protocol as the patient specimens and control biopsies. Caution was taken not to allow tissue sections to dry out during the treatment or during the following immunohistochemical staining procedure.
Interpretation of the immunohistochemical stains Photomicrograph of prostein immunohistochemical staining pattern in PCa Benign, HGPIN and nonprostatic specimens was taken at x4, x10 and x40 objective lens using Euromex Oxion Microscope. The expression proportion and intensity of immunopositivity were scored, using a method similar to Yin et al.; Zhao et al. and Hao et al. (Yin et al., 2007;Zhao, et al., 2003 andHao et al., 2000). The intensity of immunopositivity was scored from 0 to 3 as follows: score 0 = non-stained; score 1 = weak; score 2 = moderate; and score 3 = strong. The percentage of positively stained cells for each staining intensity was then estimated in the respective biopsy section. The nal composite score was determined after multiplying the intensity of positivity and percentage of positivity in the respective lesions (Yin et al., 2007). For example, if 50% of tumor cells are scored 1 (1=Weak), 25% scored 2 (2= Moderate), and the remaining 25% scored 3 (3=Strong), the composite staining score of this case is [50% × 1] + [25% × 2] + [25% × 3] = 1.75. A board-certi ed surgical pathologists con rmed the evaluation of the specimens from this study. The data was presented as mean ± standard errors (SE).

Data Analysis
The frequency and descriptive statistic for age, tribe, tumor type, staining immunolocalization and intensity were analyzed using IBM SPSS version 23.0. For each dataset within groups, the distribution, mean and standard deviation within the 95% con dence limits are shown. Correlation between age and the intensity of prostein expression was evaluated by non-parametric Spearman correlation test.

Results
In the present study, the median age of the subjects was 72. Prostein expression had a cytoplasmic immunolocalization in most of the prostatic core biopsies cases (95.1%) with varying degree of homogeneous staining pattern. Prostein was strongly expressed (55.7%), weakly expressed in 24.5%, moderately expressed in 17.0% and non-stained in 2.8% of the prostatic core biopsies. 95.3% of staining pattern was majorly cytoplasmic with 1.9% cases of Nucleocytoplasmic staining characteristics and 2.8% non-stained.
Most prevalent tumor was acinar adenocarcinoma (81.7%) followed by 5.7% intraductal Carcinoma, 1.9% High grade Prostatic Intraepithelial Neoplasms. 4.7% of the cases were Benign prostatic hyperplasia. The acinar adenocarcinoma was stained as follows; 51% strong, 23% weak, 16% moderate and 3% unstained. Intraductal carcinoma were stained as follows 2% strong, 4% moderate and 3%weak. The most common type of prostate malignancy among the prostatic core biopsies was acinar adenocarcinoma (91.2%) followed by intraductal carcinoma (5.9%). 2.9% of the tissues had High grade Prostatic Intraepithelial Neoplasms (HGPIN) ( gure 3). The sections were stained with Mouse monoclonal antibody 10E3-G4-D3 against Prostein. The intensity of the immunohistochemical analysis shows (Arrows) a strong punctate plasma membrane staining pattern with mouse monoclonal anti-prostein antibody, Clone 10E3. The stained areas are clustered within the cytoplasm in a perinuclear location. The staining characteristics has a completely random polarity from cell to cell but remained polarized within each cell (x400).
The intensity of the immunohistochemical analysis shows (Arrows) a moderate punctate plasma membrane staining pattern with mouse monoclonal anti-prostein antibody, Clone 10E3. Prostein is expressed in the cytoplasm showing circumscribed, cytosolic brown dense granular staining pattern.
The intensity of the immunohistochemical analysis shows (Arrows) a faintly stained punctate plasma membrane staining pattern with mouse monoclonal anti-prostein antibody, Clone 10E3. The granules are relatively faint and punctate showing focal loss of expression. However, the granules were still visible in the apical region of the cells using higher magni cations.
The intensity of the immunohistochemical analysis shows (Arrows) a moderately stained punctate plasma membrane staining pattern with mouse monoclonal anti-prostein antibody, Clone 10E3. The polarity of the staining characteristics appears remained polarized within each cell.
The immunohistochemical ndings indicated that none of the four non-prostatic tissues (Liver Penile, Testis, and Esophagus) that were stained with mouse monoclonal anti-prostein antibody were positive for Prostein. Prostein was exclusively expressed only in the Prostatic epithelium tissues. The expression is speci c for prostate and is not detected in other tumor tissues, including liver, penile, testis, and esophageal tissues (Figure 4e).

Discussion
The ndings of the present study have demonstrated that prostein, P501s, is expressed in both benign and malignant prostate tumor tissues with a brown punctate cytoplasmic staining pattern (Figure 4a-d; Table 1). The immunohistochemical staining showed stained areas clustered within the cytoplasm in a perinuclear location. This con rmed that Prostein has a cytoplasmic immunolocalization.  , 2003). The polarity of the staining in moderately and strongly stained tissue sections were completely random within each cell (Figure 4a-b, d).
However, in weakly stained cases, the granules were relatively faint and punctate, but were still visible in the apical region of the cells using higher magni cations (Figure 4c). The staining characteristics is consistent with the manufacturer's positive controlled pictorial atlas ( Figure 5). The luminal epithelial cells show a moderate to strong granular cytoplasmic staining reaction. The staining characteristics from cell to cell was completely random but remained polarized within each cell.
None of the non-Prostatic specimens in our study including liver, penile, testicular and esophagus tissues showed prostein immunopositivity (Figure 4e). This is because prostein is a prostate speci c biomarker and are only expressed by epithelial tissues of the prostate glands. These ndings indicate that prostein is a potential biomarker for prostate cancer diagnosis. Previous studies demonstrated that prostein appears to be expressed immunohistochemically in an exclusively prostate-speci c pattern and could not be detected even at mRNA expression levels in any of the non-prostatic tissues ( These results therefore con rm prostein as a prostate-speci c marker with potential utility in the diagnosis of prostatic origin of metastatic adenocarcinomas among the African population. There was no statistical correlation between age and the intensity of prostein expression as evaluated by non-parametric spearman's analysis. The age group was between 70 and 79 (mean 72.22±0.96) years and is in contrast with earlier studies (Madani et al., 2011& Rashed et al., 2012. The photomicrographs taken at different objective lenses showed excellent comparison of the cytoplasmic immunolocalization of the marker. However, preanalytical factors such as Ischemic time, amount and duration of xation as well as tumor heterogeneity seems to affect the prostein staining characteristics. This results into nonstaining features of some areas or sections (2.8%). Prescott and colleagues attributed 42.1% of the diagnostic discrepancies in immunohistochemistry to poor antibody selection (Prescott et al., 1995). Nevertheless, according to the ndings from this study, prostein immunohistochemistry can be used even in resource limited setting as adjunct prostate cancer speci c and sensitive biomarker. This will enhance both the accuracy and reduce the false positive and negative diagnostic rates of poorly differentiated prostate cancer. While Prostein immunostaining may be of value for the differential diagnosis of clinically signi cant Prostate cancer in H&E diagnostically challenging cases, a correlation with the Gleason grades should be considered.

Conclusion
Our ndings indicate that Prostein, P501S, is exclusively expressed with an excellent brown punctate cytoplasmic granular staining pattern in prostate tissues of African Men. We propose the utility of Prostein as an additional marker in the diagnosis of poorly differentiated prostatic carcinoma of unknown origin. Further studies can be conducted on the prostein expression and staging pattern of prostate adenocarcinoma and as a target for development of prostein-speci c antibody therapeutic regimen for prostate cancer among the African population.
Declarations Authors 1. Tyrus Omondi Swaya: Concept development, Design of the work; the acquisition, analysis, interpretation of data ( Figure 1-3), and have drafted the work and substantively revised manuscript writing. Also ensured that all listed authors have approved the manuscript before submission.
2. Dedan Opondo: Supervision and Uro-oncologist; Design of work, Data analysis, Sample retrieval, substantively revised Manuscript reviewing and have substantially modi ed version that involves the author's contribution to the study. He has also agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.
3. David O. Atandi: Immunohistochemical staining protocol and analysis of the staining intensities and substantively revised manuscript writing. He has also agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.
4. Benard Guyah: Supervision; Immunological concept development, Proposal writing, Data collection, Data analysis (Figure 1-3), and substantively revised manuscript writing and modi ed version that involves the author's contribution to the study. He has also agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

5.
Ng'wena Gideon Magak: Supervision; Physiological concept development, Proposal writing, Data collection, Data analysis ( Figure 1-3), and substantively revised manuscript writing and modi ed version that involves the author's contribution to the study. He has also agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Financial support and Sponsorship
This research received no external funding.

Con ict of interest
The authors declares that there was no con ict or competing of interest. and consent obtained via recorded telephonic interview. Their demographics were obtained for the retrieval of their tissue blocks as well as their histopathology reports retrieved from the hospital histology laboratory and management information system.

Data Availability
The datasets generated and analyzed during the current study are available as additional supporting les, in spreadsheet format alongside other supporting documents. Part of the data contain information that would compromise research participant privacy thus are available on the manuscript as pictorials.  Tumor type (A) and Immunoreactivity (B).   Immunohistochemical expression pattern of prostein, Clone 10E in normal prostate: Luminal epithelial cells show a moderate to strong granular cytoplasmic staining reaction (www. Dako/Agilent/Prostein).