Prostate malignant tumor and benign prostatic hyperplasia microenvironments in black African men: Limited infiltration of CD8+ T lymphocytes, NK‐cells, and high frequency of CD73+ stromal cells

Abstract Background Anti‐cancerous immunology has yet to be investigated in the African black population, despite being the dawn of precision medicine. Aim Here we investigated the tumor microenvironment of prostate cancer and benign prostatic hyperplasia (BPH) in black Africans. Methods Through immunohistochemistry analysis of prostate cancer and BPH patients' biopsies, we investigated the expression and distribution of CD73, CCD8 T‐lymphocytes, and natural killer cells. In addition, we looked at tumor‐infiltrating features CD8 T‐lymphocytes and natural killer cells. Results We show for the first time in black Africans a high expression of CD73 in epithelial‐stromal cells and virtually no infiltration of CD8 T lymphocytes and natural killer cells in the tumoral area. In addition, CD73 was seven (7) times more likely to be expressed in prostate cancer stromal tissues than in benign prostatic hyperplasia tissues (odds ratio = 7.2; χ2 = 21; p < .0001). In addition, PSA concentration was significantly higher in prostate cancer patients than in BPH patients (p < .001). Also, the PSA‐based ROC. analysis showed an area under the curve of 0.87 (p < .0001). Conclusion CD73 expression is more likely expressed in prostate cancer stromal tissues than in benign prostatic hyperplasia tissues. The features of prostate cancer in Black Africans suggest CD73 expression as a possible target for immunotherapy in this population.

With around 1.4 million new cases and 375 000 deaths worldwide, prostate cancer was the most common cancer and the fifth leading cause of death among men over 60 in 2020. 2 In Africa, with an incidence of 29.7 cases per 100 000 people and a mortality rate of 16.3 cases per 100 000, prostate cancer is becoming a public concern. 2 The high mortality rate in this region is mainly attributed to the stage at which the disease is diagnosed. 75% of diagnoses are advanced, and the disease is immediately aggressive, particularly in those under 60 years of age. 3,4 In Gabon, the incidence of prostate cancer was estimated to be between 30 and 42 newly diagnosed cases per 100 000 inhabitants. The mortality was estimated between 14 and 19 cases per 100 000 inhabitants. 4 Benign prostatic hyperplasia (BPH), a pathology for which lower urinary tract symptoms are common manifestations, affects more than half of men above 65 years old. 1 Although Prostate cancer occurs independently of benign prostatic hyperplasia, there might be some overlapping in symptoms, biomarker expression, or secretion. 5 Prostate cancers are most often detected based on elevated blood levels of prostate-specific antigen, a glycoprotein (PSA > 4 ng/mL) generally expressed by prostate tissue, 3 and nonspecific lower urinary tract symptoms. 6 Also, common prostate diseases, including BPH, and prostatitis, have varying degrees of elevated blood PSA levels and nonspecific lower urinary tract symptoms (LUTS). 5,6 Therefore, a tissue biopsy is a gold standard for confirming prostate cancer and defining the tumor microenvironment.
The tumor microenvironment (TME) is a complex heterogeneous milieu built around different cell types (immune cells, tumor cells, and stromal cells) and a network of molecules produced by these cells. This complex microenvironment dictates tumor differentiation, dissemination, immune evasion, and even resistance to therapy. 7 The standard therapeutic strategy against prostate cancer is based on palliative treatment based on androgen deprivation, aimed at reducing testosterone levels. 8 However, castration resistance becomes inevitable after an average period of 18 months, typically requiring the introduction of systemic chemotherapy with docetaxel. 9 Castration-resistant forms now benefit from new hormone therapies (abiraterone acetate and enzalutamide), new systemic chemotherapy (cabazitaxel), and bone-tropic targeted therapies against bone metastases such as denosumab. [10][11][12] These treatments can be combined with complementary radiotherapy and/or chemotherapy, depending on case. 13 Nevertheless, the ability of the tumor to develop resistance to anti-androgenic therapies often compromises the prognosis of patients. Less than a third of them survive 5 years after diagnosis. 14 The development of new therapeutic approaches to circumvent the progression of these tumors represents an increasingly crucial and urgent need for managing this type of cancer. Among the many concepts under study, augmenting the immune response to cancer has been proposed as a valid therapeutic option, offering an alternative approach to improving survival. 15,16 For some years, the idea that immunotherapy could be the solution has become more evident. Evasion of the immune system has recently been recognized as one of the defining properties of a cancer cell. 17,18 The close interactions between tumor epithelial cells and the microenvironment, which are dynamic over time, establish immunosuppression. This is a defining feature of cancer and a crucial step in tumor progression and establishing metastases. 7,19,20 Several immunosuppression mechanisms can be put in place by the tumor microenvironment to decrease the antitumor efficacy of the immune system. 20 Adenosine generated by the membrane enzyme CD73 is one of these factors. 21 The involvement of this enzyme in alterations in the regulation of major pathophysiological processes, including carcinogenesis and immune escape from tumors, including prostate cancer, has been reported. [21][22][23][24] Within this context, we explored the immune microenvironment of prostate cancer in African men. We mainly looked at CD73 expression within the tumor and stromal zone and the tumor-infiltrating capability of CD8 T-lymphocytes and natural killer cells.

| MATERIALS AND METHODS
This is a cross-sectional cytological, histological, and Immunohistochemical (IHC) 25 analysis of biopsies from prostate cancer and BPH patients.

| Patients
The target population was men aged 40 and above who had a prostate biopsy and were diagnosed with prostate cancer or BPH between January 2018 and December 2020 by the Pathological Anatomy and Cytology Laboratory of the Faculty of Medicine in Libreville.
Patients were selected based on their medical records and histopathology analysis results. We included patients with well-defined cancer histopathology analysis results and enough biopsy samples to perform immunohistochemistry. Patients with undefined histopathological results and insufficient biopsies for immunohistochemistry analysis were excluded.
The medical and histopathology records included anthropometric, clinical, bloodwork (including PSA), and social data. Also recorded were the organ removed, the sample type, the sample preanalytical treatment, the number of cores obtained during the biopsy, the biopsy approach, and the final diagnosis.
For each patient, we retrieved information about their age, the concentration of total prostate-specific antigen (ng/mL), the number of cores obtained during the biopsy, and the Gleason score. Patients with less than 12 cores at biopsy, follow-up biopsies after diagnosis, transurethral resection of the prostate, and an unspecified prostatespecific antigen value, were not included.

| Collection and morphological analysis
Stored prostate biopsy samples fixed with 10% buffered formalin and embedded in paraffin were first comparatively analyzed and matched to the Hematoxylin-Eosinstained slide used for the initial diagnosis.
Based on this analysis, samples from 100 out of 134 patients were eligible for an Immunohistochemical study (53 prostate cancers and 47 BPH patients). The 34 remaining patients were excluded due to missing or low-integrity samples.

| Oversee independent analysis of biopsy samples
All the selected blocks of biopsy samples were sent to France at the "France Tissue Bank" laboratory in Fréjus for a second independent analysis and immunohistochemistry-based analysis. Two (2) pathologists from the "France Tissue Bank" laboratory (France) confirmed prostate cancer and benign prostatic hypertrophy diagnosis made in Libreville (Gabon).

| Controls
We used amygdala tissues to control the reading and interpretation of CD8+ T cell labeling. The latter has the characteristic of expressing CD8 T lymphocytes in the normal state. For the reading and interpretation of CD56+ marked cells, we chose the neuroblastoma tissue as a positive control because neuroblastoma strongly expresses CD56. Placental tissue served as a positive control for reading CD73 expression as recommended by the manufacturer.
Negative control slides without the primary antibody were included in all lots.

| Reading and digitalization of stained slides
Two pathologists independently scored positive slides. CD73+, CD56+, and CD8+ were counted in five randomly selected high-power fields at Â40 magnification, and counts were scored positive or negative. Each slide was digitized using a Philips UFS Â40 magnification scanner (Philips) in TIFF format.
Complementary information on the material used can be found in Data S1.

| Ethics and administrative authorization
Ethical approval was obtained from the institutional ethics committee.
All data in this study were de-identified, and the ethics committee

| T CD8+ distribution, cancer versus BPH patients
Data showed that prostate cancer patients are nearly three times more likely to have Lymphocytes T CD8+ in their stromal area than BPH patients (odds ratio = 2.8; χ 2 = 4.3; p = .03) ( Figure 5B (b1-b3)).  Figure 6A). The receiver operating characteristics (ROC) curve analysis showed an area under the curve (AUC) of 0.87 (p < .0001) ( Figure 6A,B). The cutoff analysis is shown in Table 2.

| DISCUSSION
There is mounting evidence that immune surveillance evasion through Nevertheless, the characteristics of prostate cancer in Black Africans presented here suggest the potential clinical use of CD73 expression as a target for immunotherapy in this population. 38 Indeed, there are pieces of evidence supporting that black Africans might be more susceptible to prostate cancer than others. Published data showed that prostate inflammation, which would constitute an important induction factor of neoplastic transformation and malignant progression in the prostate, is more pronounced in black men. 39 Also, it has been laid out that genetics, diet (alteration of the metabolic pathway), and lifestyle converge to drive the molecular pathogenesis of prostate cancer. 39,40 The changing lifestyle in developing countries characterized by increased exposure to sources of pollution (urbanization), exposure, increased usage of tobacco and alcohol, and more consumption of meat, sugar, and processed foods is considered a non-negligible risk factor for the development of cancers in Africa. 41 A recent study in the United States showed that prostate tumors from self-identified Black men or men of African genetic ancestry had increased quantities of plasma cells and increased immune activity. In addition, NK activity was associated with improved outcomes after surgery. 42 Interestingly, CD8+ T-cell content and high activity (as measured by high cytolytic activity) were not associated with metastasis-free survival. 42 Also, it has been shown that a higher cytolytic score correlates with an immunosuppressive tumor microenvironment and reduced survival in brain cancer. 43  involving T-lymphocytes. 44 Also, the CD73 was seven (7)

| CONCLUSION
The tumor area of black sub-Saharan African men with prostate cancer