This study included cases biopsied after January 2017 due to the shift from systemic prostate needle biopsies to the addition of extra biopsies based on pre-biopsy magnetic resonance imaging findings at our institute. As per the previous methodology of our institution, Primary pathological findings were first recorded by YO and reviewed by a second pathologist (YM) according to the 2014 International Society of Urological Pathology system (14-16).
In the event of disagreement on diagnostic findings, another trained pathologist was recruited for discussion until a final consensus was reached. From January 2017, clinical information, including age, body mass index, and pre-biopsy serum prostate-specific antigen (PSA) levels, and pathological findings, such as the presence of tumor lesions, histological type (including suspected NE tumors), number of positive scores, highest Gleason grade (only for adenocarcinoma cases), lesion length, and immunohistochemical confirmation of NE marker expression, were recorded by YO for database generation. Cases were extracted from the database if NE tumors were suspected based on morphological findings and immunohistochemical confirmation of NE markers, such as synaptophysin and chromogranin A. Subsequently, cases were re-reviewed using actual specimens. Serum neuron-specific enolase (NSE) and pro-gastrin-releasing peptide (ProGRP) levels were confirmed in the most recent values at the time of biopsy. Metastasis to other organs and prognosis were confirmed using electronic medical records. The diagnostic criteria for small cell neuroendocrine carcinoma (SCNC) of the prostate were based on the World Health Organization classification (2), with emphasis on morphology and immunoreactivity of NE markers. Given that prostate cancer can exhibit partial NE differentiation, adenocarcinoma with NE in this study was defined as cases with diffuse immunoreactivity for NE markers (prostatic carcinoma with diffuse NE differentiation), based on a previous report (5). The definition of "diffuse" is controversial, but in this study, we included cases in which at least 50% were positive. In each of the cases in this study, only a prostate biopsy was conducted, and no additional surgically resected specimens were obtained.
Immunohistochemical examinations were performed on available paraffin blocks using primary antibodies against the following markers: androgen receptor (AR; Nichirei, clone AR441; 1:1 dilution), chromogranin A (clone 1E2; 1:5 dilution), Ki-67 (Dako, clone MIB-1; 1:50 dilution), prostein (Dako, clone 10E3; 1:100 dilution), PSA (Dako, clone ER-PR8; 1:50 dilution), p53 (Nichirei, clone DO-7; 1:1 dilution), and synaptophysin (Roche, clone 4B5; 1:1 dilution). CD56 was not assessed in this study due to its low specificity (17). The H-score was used for immunohistochemical evaluation and was calculated by multiplying the staining intensity (0, negative; 1, weakly positive; 2, moderately positive; and 3, strongly positive) by the percentage of cells exhibiting that staining intensity (0–100), resulting in a score in the range of 0–300 (18). For the Ki-67 labeling index, hotspots were assessed (19-21). Areas containing a high number of Ki-67-positive cells (at least 500 cells) were selected, and the percentage of positive cells was calculated. For cases of mixed SCNC-adenocarcinoma, immunostaining for adenocarcinoma and small cell carcinoma areas was performed.
Examination of hotspot driver mutations using a gene panel
To explore the possibility of novel therapeutic strategies, we examined hotspot driver mutations using the Ion AmpliSeqTM Cancer Hotspot Panel v2 (CHPv2, Thermo Fisher Scientific, Inc.). Next-generation sequencing (NGS) was performed as described previously (22-24). Tumor tissues were obtained from thin sections (4 µm thickness per slice) prepared from formalin-fixed and paraffin-embedded (FFPE) tissue blocks by manually dissecting the tumor area. DNA was extracted from FFPE tissues using the GeneRead FFPE Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and was quantified using Qubit 2 (Thermo Fisher Scientific, Inc., Waltham, MA). DNA (10 ng) was applied to library construction with the CHPv2 based on Ion Torrent AmpliSeqTM technology (Thermo Fisher Scientific, Inc.) and subjected to sequencing with the Ion PGM next-generation sequencer (Thermo Fisher Scientific, Inc.). The CHPv2 for NGS is a gene panel for analyzing well-characterized mutation hotspots of the following 50 cancer-related genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. The sequenced data were processed and analyzed using Torrent Suite 5.12.2 and Ion Reporter version 5.16 (Thermo Fisher Scientific Inc.). Quality control reports were acquired from Torrent Suite. To identify somatic variants, single nucleotide variations and insertion or deletion mutations with a coverage of 20 base pairs and encoding amino acid sequence substitutions comparable to the UCSC hg19 reference genome sequence were first selected. Next, single-nucleotide polymorphisms (SNPs) were excluded using these sequences as queries against COS-MIC (http://cancer.sanger.ac.uk/cosmic), dbSNPs (NCBI, NIH; https://www.ncbi.nlm.nih.gov/projects/SNP/), and other publicly accessible databases. Finally, mutations with >5% allele frequency were defined as somatic nonsynonymous mutations and further evaluated. The biological significance of the identified somatic mutations was assessed using COSMIC, ClinVar (NCBI; https://www.ncbi.nlm.nih.gov/clinvar/).
Statistical analyses were performed using International Business Machines (IBM) Statistical Package for the Social Sciences Statistics version 25 (IBM Corp., Armonk, NY, USA). Serum NSE, ProGRP, and PSA levels and the H-score and Ki-67 labeling index were subjected to statistical analysis. Student’s t-test was used to compare two groups. One-way analysis of variance (one-way ANOVA) and the Bonferroni post hoc test were used to compare three groups. A p-value <0.05 was considered statistically significant.