Treatment-Emergent Neuroendocrine Prostate Cancer: A Clinicopathological and Immunohistochemical Analysis of 94 Cases

Purpose This study aimed to evaluate the pathological characteristics, immunophenotype, and prognosis of treatment-emergent neuroendocrine prostate cancer (T-NEPC). Materials and Methods We collected 231 repeated biopsy specimens of castration-resistant prostate cancer (CRPC) cases between 2008 and 2019. We used histopathological and immunohistochemical evaluations of Synaptophysin (SYN), ChromograninA (CgA), CD56, androgen receptor (AR), and prostate-specific antigen (PSA) to screen out T-NEPC cases. Multivariate analyses were performed to identify factors in the prognosis of T-NEPC. Further, the results were verified in the Surveillance, Epidemiology, and End Results (SEER) program. Results Among the 231 CRPC cases, 94 (40.7%) cases were T-NEPC. T-NEPC were more likely to present with negative immunohistochemistry for AR (30.9%) and PSA (47.9%) than that of CRPC (8.8% and 17.5%, respectively). Kaplan-Meier analysis revealed that patients with T-NEPC (median overall survival [OS]: 17.6 months, 95% CI: 15.3–19.9 months) had significantly worse survival compared with usual CRPC patients (median OS: 23.6 months, 95% CI: 21.3-25.9 months, log-rank P = 0.001), especially in metastasis cases (median OS: 15.7 months, 95% CI: 13.3-18.0 months) and patients with small cell carcinoma component (median OS: 9.7 months, 95% CI: 8.2–11.2 months). Prostate adenocarcinoma with diffuse NE differentiation (median OS: 18.8 months, 95% CI: 15.3–22.3 months) had poor outcome than those with usual CRPC (P = 0.027), while there was no significant change in the focal NE differentiation (median OS: 22.9 months, 95% CI: 18.1–27.7 months, P = 0.136). In the unadjusted model, an excess risk of overall death was observed in T-NEPC with PSA negative (HR = 2.86, 95% CI = 1.39–6.73). Among 476 NEPC cases in the SEER database from 2004 to 2017, we observed a higher hazard of overall death in patients aged 65 years and older (HR = 1.35, 95% CI = 1.08–1.69), patients with PSA ≤ 2.5 ng/ml (HR = 1.90, 95%CI = 1.44–2.52), patients with PSA 2.6–4.0 ng/ml (HR = 2.03, 95%CI = 1.38–2.99), stage IV tumor (HR = 2.13, 95%CI = 1.47–3.08) and other races (HR = 1.85, 95%CI = 1.17–2.94) in total NEPC, adjusting for confounders. Similar hazard ratios were observed in pure NEPC, while there was no significant results among prostate adenocarcinoma with NE differentiation tumors. Conclusion T-NEPC was associated with an unfavorable prognosis, negative immunohistochemistry for PSA in T-NEPC and serum PSA level ≤ 4 ng/ml had a worse prognosis. Urologists and pathologists should recognize the importance of the second biopsy in CRPC to avoid unnecessary diagnosis and treatment delays.


INTRODUCTION
Treatment-emergent neuroendocrine prostate cancer (T-NEPC) mainly occurs in the advanced castration-resistant prostate cancer (CRPC), which is caused by the transformation of ordinary prostate adenocarcinoma after androgen-deprivation therapy (ADT) (1). The main clinical manifestations of T-NEPC include low PSA level, high tumor metastasis load, and rapid resistance to ADT (2). T-NEPC is an aggressive variant of CRPC, with a poor prognosis. Most T-NEPC patients die within 1 to 2 years after diagnosis, accounting for approximately 25% of CRPC deaths (3). The clinical acquaintance of conversion to T-NEPC after resistance to endocrine therapy in prostate adenocarcinoma is insufficient, and secondary biopsies are not routinely performed, cause to the missed diagnosis of T-NEPC. At present, there is still no pathological consensus definition of T-NEPC. The morphological characteristics of T-NEPC are similar to poorly differentiated prostate adenocarcinoma, so the incidence of T-NEPC is greatly underestimated (4). In recent years, with the widespread use of novel, highly potent androgen receptor-targeted therapies (eg, Arbiton, MDV3100), the incidence of T-NEPC may also rise significantly. T-NEPC is not sensitive to ADT treatment, and the treatment is different from adenocarcinoma. Once T-NEPC is diagnosed, systemic chemotherapy based on etoposide combined with cisplatin (EP) or carboplatin(CE) should be performed as soon as possible, as well as radiotherapy, and other potentially effective treatment includes targeted drugs such as Aurora kinase A (AURKA) kinase inhibitors, anti-EGFR and mTOR inhibitors (5,6) if necessary. Therefore, early diagnosis and treatment are of great significance to improve the patient's survival benefit. But there are very few clinical series studies to date, and the clinical characteristics and prognosis of T-NEPC are not very clear. Therefore, we analyzed the clinicopathologic characteristics and outcomes of T-NEPC, intending to improve the understanding of T-NEPC among clinicians and pathologists.

Immunohistochemistry
All the specimens had been fixed by 10% formalin and paraffinembedded routinely. Four µm thickness tissue sections from blocks were stained with hematoxylin and eosin (H&E) and immunohistochemical staining (Syn, CgA, CD56, AR, and PSA). Immunohistochemistry was performed by the avidin-biotinperoxidase complex method (MaiXin Inc, China, prediluted) according to the manufacturer's instruction.

Immunohistochemical Evaluation
Two independent, blinded investigators, XSQ and QFZ evaluated all the slides. AR immunopositivity was located in the nucleus, CD56 immunopositivity was located in both the cell membrane and cytoplasm, PSA, SYN, and CgA immunopositivity were located in the cytoplasm. At least 10% of tumor cells showed at least one NE marker positive was considered as NE differentiation. According to the staining intensity, the expression level of immunohistochemical staining was scored as: 0 point (negative), 1 point (weak expression), 2 points (moderate expression-strong expression); staining cell percentage evaluation score: 0 point (0%-10%), 1 point (10%-50%), 2 points (51%-100%). The scores of staining intensity and the percentage of staining cells were multiplied, and the final score was >1 point was positive.

SEER Cohort
We used the SEER database (released in April of 2020) to identify eligible NEPC cases. This study included men diagnosed with malignant prostate adenocarcinomas between 2004 and 2017 (n = 780,379) because the SEER database did not collect PSA information until 2004. We excluded non-microscopically confirmed cases, patients with a prior cancer history, unknown survival time, men without PSA level. The final analytic sample included 476 NEPC cases ( Figure 1). Due to the use of deidentified data, this study was determined as exempt by the Institutional Review Board of the First Hospital of China Medical University.

Statistical Analysis
Chi-squared tests were applied to analyze the clinicopathological characteristics of subgroups. Survival curves were plotted according to the Kaplan-Meier method and compared using the log-rank test in a univariate analysis. Multivariate analysis was performed using the Cox proportional hazards model. The estimated risks for overall survival (OS) were calculated as hazard ratios (HRs) with 95% confidence intervals (CIs). All statistical analyses were performed using SAS (version 9.4; SAS Institute, Cary, NC) or SPSS 22.0. Statistical significance was assessed as two-sided P<0.05.

Histological Findings
The cases were divided into five groups according to morphological and the expression extent of neuroendocrine markers: 1. Usual CRPC, there were 137 cases, of which 125 cases were AR positive and 113 cases were PSA positive. 2. Prostate adenocarcinoma with focal increased neuroendocrine differentiation (Figures 2A-F), confirmed by immunohistochemistry with neuroendocrine differentiation, but did not meet the criteria of group 3, of which 23 cases were matched, all 23 cases were AR positive, and 17 cases were PSA positive. And there were 16 cases with Gleason score >7, 7 cases with Gleason score ≤7. 3. Prostate adenocarcinoma with diffuse neuroendocrine differentiation (typical prostate acinar adenocarcinoma or prostate ductal adenocarcinoma with at least 1 neuroendocrine marker >50% positive) ( Figures 2G-R), and of which 34 cases were matched, 27 cases were AR positive, and 23 cases were PSA positive. In addition, there were 30 cases with Gleason score > 7, 4 cases with Gleason score ≤ 7. 4. Highly  The cell morphological characteristics of small-cell carcinoma was similar to that of lung small-cell carcinoma, including delicate nuclear chromatin, little cytoplasm, lack of obvious nucleoli, and occasional necrosis, expression of neuroendocrine markers or not. In addition, there were some tumors whose histological morphology was intermediate cell type between small-cell carcinoma and prostate acinar adenocarcinoma, with relatively more cytoplasm and occasionally visible small nucleoli, and showed neuroendocrine differentiation, these cases were also included in this group. We did not include cases of Paneth cell-like differentiation.

Clinicopathological Features of Usual CRPC and T-NEPC
T-NEPC showed a significant association with AR and PSA negative expression (P< 0.001) than Usual CRPC, there was no significant difference in Age, higher Gleason score, metastasis and TNM stage between two groups (Table 1). In T-NEPC patients, poorly differentiated neuroendocrine carcinoma are more prone to AR and PSA negative expression than prostate adenocarcinoma with neuroendocrine differentiation(P<0.001), there was no significant difference in clinicopathological features between prostate adenocarcinoma with diffuse neuroendocrine differentiation and prostate adenocarcinoma with focal neuroendocrine differentiation except AR positive(P = 0.034) ( Table 2).

The Clinical Outcome of Usual CRPC and T-NEPC
A total of 113 cases of usual CRPC and 82 cases of T-NEPC were followed up. There was a significant difference in OS between     (Table 5).

DISCUSSION
The mechanism of T-NEPC was not clear. One explanation was that neuroendocrine tumor cells were resistant to hormonal therapies that target AR signaling. Therefore, ADT could inhibit the proliferation of ordinary prostate adenocarcinoma cells and relieved clinical symptoms of patients, while preserving and enriching neuroendocrine tumor cells. The use of potent AR signaling inhibitors could promote the transformation of adenocarcinoma in the CRPC state to T-NEPC in a clonal manner (7,8). Some other research found that molecular events of T-NEPC such as TMPRSS2-ERG gene rearrangement and fusion genes were similar to prostate adenocarcinoma, sugg est ed that T -NEPC orig in ate d f r om prost a te adenocarcinoma and ADT enabled adenocarcinoma cells to develop into T-NEPC cells through the mechanism of adaptive response/tumor resistance (9,10).  treatment between them, and there were some overlaps in morphology in some cases. In ad dition, prostate adenocarcinoma with diffuse neuroendocrine differentiation Gleason score 5 + 5 = 10 (grade group 5) showed overlapping features of large-cell carcinoma due to its morphological crossing (12). The prognosis of adenocarcinoma with Paneth cell-like neuroendocrine differentiation was completely different from that of T-NEPC, which may be just a morphological change  and should not be classified as T-NEPC. Therefore, we recommended classification T-NEPC as adenocarcinoma with neuroendocrine differentiation, well-differentiated neuroendocrine carcinoma and poor-differentiated neuroendocrine carcinoma. The 2019 WHO classification of digestive system tumors divided the neuroendocrine neoplasm (NEN) into two categories according to mitotic count and the Ki-67 index of hotspots: highly differentiated neuroendocrine tumor (NET) and poorly differentiated neuroendocrine caicinoma (NEC). NET includes NET G1, G2, and G3, which still retained high differential morphological features of NEN, and NEC included large-cell and small-cell carcinoma (13). However, compared with the tumors of the digestive system, the mitotic count and ki-67 index of prostate cancer were generally low, so it could not be classified according to the classification standard of gastrointestinal neuroendocrine tumors. Some study has found that Ki-67 was an independent prognostic predictor of prostate cancer. However, due to a low number of cases, the cut-off value to define a proliferation index of different grades of prostate neuroendocrine carcinoma was not established, and further studies were needed.
When prostate cancer patients were clinically resistant to hormonal therapy, presented with the clinical features such as carcinoid syndrome, lower urinary tract symptoms, osteolytic bone metastases, visceral metastases as progressive metastases on imaging (14). In addition, an increase of serum neuroendocrine markers CGA and NSE, or the low level or significantly elevated level of PSA was disproportionate to tumor progression, the possibility of T-NEPC should be considered (15). And it was recommended to re-biopsy the rapidly progressing lesion to exclude the diagnosis of T-NEPC. Urologists and pathologists should strengthen strategic communication, and pathologists should be reminded to perform immunohistochemical analysis of neuroendocrine markers. Due to the classic prostate adenocarcinoma can also showed different degrees of neuroendocrine differentiation, and scattered or clustered neoplastic neuroendocrine cells could appear in prostate cancer. So immunohistochemistry for neuroendocrine markers were not recommended for general use on prostate acinar adenocarcinoma. Therefore, the diagnostic rate of T-NEPC was also hovering at a low level. We suggested that for CRPC patients with repeated biopsy and metastatic Gleason score 5 + 5 = 10 after the failure of endocrine therapy, routine neuroendocrine markers (Syn, CgA, and CD56) examination should be performed to exclude the diagnosis of T-NEPC. Our results showed that f o c a l n e u r o e n d o c r i n e d i ff e r e n t i a t i o n o f p r o s t a t e adenocarcinoma was not a prognostic factor. Due to the possibility of non-specific staining, in the absence of the typical histomorphological manifestations of T-NEPC, we suggested that T-NEPC pathological diagnosis should follow strict definition and combine with the use of multiple neuroendocrine markers and morphological features: at least 1 neuroendocrine marker with a diffuse (>50%) positive could be diagnosed. However, if the morphological features of typical small-cell carcinoma, we believed that it should be diagnosed as T-NEPC even if all the neuroendocrine indicators were negative. T-NEPC, especially small-cell carcinoma, tended to have AR and PSA negative, however, in adenocarcinoma with neuroendocrine differentiation, AR was often positive and PSA expression was weakened or negative. Recent studies on T-NEPC had shown that AR often positively expressed in tumor cells, but despite the AR protein expression in the nucleus, the transcriptional activity of AR was very low, which indicated that epigenetic mechanisms may inhibit AR function (16). Our study found that the negative PSA expression was an independent prognostic factor, these results were consistent with previous reports (17,18). As T-NEPC and primary neuroendocrine prostate cancer showed similar molecular pathological characteristics (1), we validated our results using the SEER NEPC database. Our results showed that in pure NEPC, the prognosis of patients with low serum PSA level (≤4 ng/ml) were significantly worse than other PSA (>4 ng/ ml) groups.
In summary, T-NEPC had highly invasive, rapid progress, and poor prognosis, and was related to resistance to ADT. Although the effect of chemotherapy was not very satisfactory, the progress of molecular diagnostic testing was expected to create favorable conditions for the development of precision medicine (19). Pathologists and clinicians should pay attention to the timely diagnosis and treatment of T-NEPC to improve the survival time of patients.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
This study was approved by the Ethics Committee of China Medical University. Written informed consent was obtained from all participants.

AUTHOR CONTRIBUTIONS
QZ contributed to the study design, data analysis, interpretation, and manuscript preparation. YH, YZ, DL, JM, and BH contributed to data interpretation and manuscript revision. XQ contributed study design and immunohistochemical evaluation, manuscript revision. All authors contributed to the article and approved the submitted version.