Molecular Characterization of Prostate Cancer in Middle Eastern Population Highlights Differences with Western Populations with Prognostic Implication

Background Immunohistochemistry for ERG, PTEN and SPINK1 was performed in cohort of localized PCa (n=340). Data correlated to pathological and clinical outcomes and compared to Western populations.Results ERG expression and PTEN loss noted in 123/288 (42.7%) and 91/297 (30.6%) of patients, respectively. SPINK1 expression assessed in subset of cases, noted in 6/150 (4%) of patients. Only ERG expression was associated with grade groups, being more common in lower grade groups (1-3 vs 4-5; p=0.04). In contrast to Western population, PTEN loss foci were more likely to be ERG negative, observed in 81% of tumor foci and patients with PTEN neg/ERG pos were more likely to exhibit biochemical recurrence (OR 2.831; 95% CI: 1.10-726, p=0.03). This association remained significant in multivariate analysis (OR 2.68; 95% CI: 0.98-7.33, p=0.05), after adjusting for GG, path stage and surgical margin.Conclusion This study documents significant differences in key molecular events in PCa in Middle Eastern population compared to Western populations that could explain differences in PCa incidence, progression and prognostication. ERG, PTEN and SPINK1 genomic alteration occur less frequently and the enrichment of ERG for PTEN loss is not observed. Additionally, patients with combined PTEN loss/ERG positive are at highest rate for BCR vs North American Caucasian population where PTEN loss alone seems to be associated with the worst clinical outcome. The data presented here further support differences in clonal evolution between Middle Eastern and Western population in relation to PCa and add further

One previous study performed in Middle East (Jordan) in 2017 with 193 prostatic cancer cases suggested ERG to be present in slightly lower incidence at 33.2% compared to Caucasian American [21].
PTEN is one of the most commonly inactivated tumor suppressor genes in prostate cancer [22] and PTEN genomic deletions or protein loss have been associated with several cancer progression including PCA [23][24][25]. Several studies have documented association between PTEN genomic deletions or protein loss and worse clinical outcome in PCA patients [26,28,29] which has been gaining significant value in clinical practice given its potential endorsement by the Food and Drug Administration (FDA) as clinically valued biomarker in PCA [26]. Several studies have also documented association between ERG gene rearrangements/expression and increased incidence of PTEN deletions/loss of expression (i.e. there is enrichment of in PTEN deletion in tumor foci with ERG gene rearrangements (Bismar, et al. 2010) [23]. We also reported significant association of homozygous PTEN genomic deletions and ERG gene rearrangements with lymph node metastasis, hormone-refractory PCA and cancer-specific death [ Herein, we comprehensively characterized ERG, PTEN and SPINK1 expression using combined IHC in a cohort of men with PCA patients from Middle Eastern population, who were treated by radical prostatectomy as single therapy. We assessed the incidence, overlap and prognostication of individual and combined markers in relation to key clinical and pathological parameter.

Study population:
Our study population consisted of 340 men with PCA from 1998-2012 and who were managed by radical prostatectomies. Clinical information on follow-up was available for 329 patients collected through the AUB clinical Registry in relation to biochemical recurrence post radical prostatectomy. Five tissue microarrays (TMAs) were constructed with average four cores (range 2-6) for each patient using manual tissue microarrayer (Beecher Instruments, Silver Spring, MD). The University of Calgary Cumming School of Medicine institutional review board approved the study.

ERG, PTEN and SPINK1 status by immunohistochemistry (IHC)
We carried out a dual-color ERG/PTEN and ERG/SPINK1 IHC staining protocol as

Pathological analysis
All cases were re-reviewed and Gleason scores/groups were assessed by the study pathologists based on the 2014 WHO/ISUP criteria. The two common patterns were sampled on the TMAs for evaluation. Both ERG and SPINK1 intensities were assessed as (0; negative; 1, positive) based on previous correlation with ERG rearrangement detected by FISH (data not shown). The expression of ERG in endothelial cells, acted as internal control being highly expressed. PTEN intensity was assessed as (0; negative, 1; weak, 2; moderate and 3; high), with high defined as intensity equal to benign adjacent epithelium. Furthermore, PTEN intensity was further analyzed as intact "positive" vs absent "negative" to avoid smaller numbers within subgroups.

Statistical analysis
Descriptive statistics were used to describe the study variables. Mean and standard deviation were reported for continuous variables and frequency and proportions for the categorical variables. Chi-square tests were to compare two categorical variables. Fisher's exact test were reported if the cell frequency were less than 5.
Binary logistic regression analysis was used for the outcome variable PSA relapse (yes vs. no). Odds ratio (OR) and the corresponding 95% confidence intervals were reported. The association of the combined score of PTEN and ERG with PSA relapse were analyzed using univariate and multivariate models. For the multivariate model Gleason score, surgical margin and pathological stage were included in the analysis.
SPSS version 25 was used to conduct all the statistical analysis, a p-value <0.05 was used for statistical significance. Two sided tests were used for all the analysis.
PTEN did not show any significant association within this distribution (p=0.11). CI=0.98-7.33), p=0.05) ( Table 4) No significant association was found between SPINK1 positive vs negative and BCR (data not shown).

Discussion
The epidemiology of PCA in Middle Eastern Arab populations are not well studied in contrast to North America and Western Europe. This is likely due to the absence of established PSA screening, lack of population-based registries, and existence of prospective oncology databases [41].
In this study, we assessed a large and more homogenous cohort of 340 radical prostatectomy and documented ERG expression to be present in 42% of PCA cases in Middle Eastern Arab population with prognostic implication in association to PTEN loss and SPINK1 expression.
Although the rate of ERG expression detected in this study is at lower end of that observed in in North America and Western Europe, which is documented generally around 45%-50% [42], it is still significantly higher than those reported in Asian and African American populations of about 15% and 27% respectively [43, 44].

One earlier study by Aldaoud et al. reported on ERG expression in Middle Eastern
population suggested the incidence to be lower among Arab population at 33.2% compared to North American and European population and the study failed to document any prognostic value or association to Gleason grade grouping [21]. The reason for such discrepancy in incidence between our study and that of Aldaoud et al, is that they reported ERG incidence in mixed cases spanning peripheral zone (assessed through needle biopsy) and transition zone (assess by TURP). This is significant issue, as it is well known and documented by our group and others that ERG expression is significantly higher in peripheral zone tumors compared to those in transition zone [3], hence, combining both zones for reporting ERG incidence will artificially suggest lower incidence of ERG.
Loss of PTEN expression is well known to be associated with disease progression and patient's prognosis in several cancers and has also been implicated as potential targets for PI3K related therapies [21] and PTEN loss, as assessed by a validated and simple IHC protocol, was documented to be associated with a two-fold increase in risk of lethal progression independent of clinical-pathologic parameters [26].
Another study performed by Shan, et al in Arab men with PCA confirmed that PTEN is downregulated and associated with disease progression in PCA [45]. In the current study, the rate of PTEN loss was noted within the same range compared to Caucasian and African American cohorts, however, we did not observe significant association between loss of PTEN expression and increased Gleason grade grouping as suggested in North American and Western populations (p=0.11), Most studies reported that SPINK1 protein is detected in about 9% of PCA cases of Caucasian cohorts vs 23% in African American with some suggestion of prognostic value [44,48]. In our study, SPINK1 protein expression was noted at much lower incidence of only 4% which is significantly lower than those reported in Western population above. However, we did not observe any prognostic association for SPINK1 which may be due to the fact of lower-case number overall.

Previous reports in Western and
Previous studies found SPINK1 protein expression to be inversely associated with PTEN gene deletion and exclusive of loss of PTEN intensity [27] and positively associated with higher PTEN protein expression [49]. In this study we also observed notable inverse relation between PTEN negative intensity and SPINK positivity.
Although some recent studies have reported co-expression of ERG and SPINK1 in up to 4% of prostate cancers[27, 41, 49], the majority of reports confirm ERG and SPINK1 being mutually exclusive [34], which was also noted in our study.
The above results indicate that molecular landscape of PCA in Middle Eastern population differs from other races. This might be explained by genetic differences, environmental, lifestyle and dietary factors, especially since it has been reported that gut microbiome reflective of diet may have a role on gene expression and cancer risk cancers [50].
The clinical value of our study stems from the fact that pathological staging system and Gleason score do not explain racial difference in PCA outcomes. Therefore, such markers may have a role in the future in personalizing prognosis estimates for patients and guide novel targeted treatment.     39. Figure 1 A, Immunohistochemistry for ERG positive (brown) and PTEN negative in Gleason grade group