[Retrospective study of 20 years of post-mortem examination of young adults]. / De meerwaarde van postmortaal onderzoek.

OBJECTIVE: This study is designed to gain insight into the cause of death in deceased young adults, by analyzing autopsies and other post-mortem examinations including their contribution into finding the cause of death DESIGN: Retrospective cohort study. METHOD: Included were adults aged between 18-45 years old who underwent a clinical autopsy at the Isala Klinieken in Zwolle between January 2000 and October 2022. Included patients had a natural cause of deaths and were divided into two categories: expected and unexpected deaths. For each patient the post-mortem examination and their contribution to diagnose the cause of death were determined, among other things. Collected data was processed in a database and analyzed. RESULTS: Between January 2000 and October 2022, 212 autopsies were performed in the 18-45 age group. Of these 212 patients, 54 (25,5%) were expected deaths and 158 (74,5%) unexpected deaths. 116 patients had an unknown cause of death (7 expected vs. 109 unexpected). After post-mortem examination, this number has decreased to 15 deaths (expected 0 vs. unexpected 15). This is a reduction form 54,7% to 7,1%. Of the 96 presumed diagnoses/causes of death for autopsy, 16 (16,7%) cases were reclassified as Goldman score 1 discrepancies. CONCLUSION: Performing post-mortem examinations contributes to reducing the number of unknown causes of death. Post-mortem examinations also provide knowledge about illnesses, the clinical course of syndromes and the actual cause of death in (young) adults, even when mortality is expected.

Differential tissue expression of extracellular vesicle-derived proteins in prostate cancer.

BACKGROUND: Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value. METHODS: Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome. RESULTS: XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009). CONCLUSION: High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker.

Tissue proteomics outlines AGR2 AND LOX5 as markers for biochemical recurrence of prostate cancer.

Although many patients are cured from prostate cancer (PCa) by surgery only, there are still patients who will experience rising prostate-specific antigen (PSA) levels after surgery, a condition known as biochemical recurrence (BCR). Novel protein prognostic markers in PCa tissue might enable finding better treatment for those patients experiencing BCR with a high chance of metastasis. In this study, we aimed to identify altered proteins in prostate cancer tissue, and to evaluate their potential role as prognostic markers. We used two proteomics strategies to analyse 34 prostate tumours (PCa) and 33 normal adjacent prostate (NAP) tissues. An independent cohort of 481 samples was used to evaluate the expression of three proteins: AGR2, FASN and LOX5 as prognostic markers of the disease. Tissue microarray immunohistochemical staining indicated that a low percentage of positive tumour cells for AGR2 (HR (95% CI) = 0.61 (0.43-0.93)), and a low percentage of positive tumour cells for LOX5 expression (HR (95% CI) = 2.53 (1.23-5.22)) are predictors of BCR after RP. In contrast, FASN expression had no prognostic value for PCa.

Gene-expression analysis of gleason grade 3 tumor glands embedded in low- and high-risk prostate cancer.

The Gleason score (GS) of prostate cancer on diagnostic biopsies is an important parameter for therapeutic decision-making. Biopsy GS under-estimates the actual GS at radical prostatectomy in a significant number of patients due to samplingartifact. The aim of this study was to identify markers that are differentially expressed in Gleason grade 3 (GG3) tumor glands embedded in GS 4 + 3 = 7 and GS 3 + 3 = 6 prostate cancer using laser capture microdissection and RNA sequencing.GG3 tumor glands embedded in nine GS 3 + 3 = 6 and nine GS 4 + 3 = 7 prostate cancers were isolated by laser capture microdissection of frozen radical prostatectomy specimens. After RNA amplification and RNA sequencing, differentially expressed genes in both GG3 components were identified by a 2log fold change > 1.0 and p-value < 0.05. We applied immunohistochemistry on a tissue micro-array representing 481 radical prostatectomy samples for further validation on protein level.A total of 501 genes were up-regulated and 421 down-regulated in GG3 glands embedded in GS 4 + 3 = 7 as compared to GS 3 + 3 = 6 prostate cancer. We selected HELLS, ZIC2 and ZIC5 genes for further validation. ZIC5 mRNA was up-regulated 17 fold (p = 8.4E-07), ZIC2 8 fold (p = 1.3E-05) and HELLS 2 fold (p = 0.006) in GG3 glands derived from GS 4 + 3 = 7. HELLS expression of ≥ 1% occurred in 10% GS < 7, 17% GS 7 and 43% GS >7 prostate cancer (p < 0.001). Using a cut-off of ≥ 1%, protein expression of ZIC5 was present in 28% GS < 7, 43% GS 7 and 57% GS > 7 cancer (p < 0.001). ZIC2 was neither associated with GS nor outcome in our validation set. HELLS was independently predictive for biochemical-recurrence after radical prostatectomy (HR 2.3; CI 1.5-3.6; p < 0.01).In conclusion, HELLS and ZIC5 might be promising candidate markers for selection of biopsy GS 6 prostate cancer being at risk for up-grading at prostatectomy.

MET expression during prostate cancer progression.

Tyrosine-kinase inhibitors of the hepatocyte growth factor receptor MET are under investigation for the treatment of hormone-refractory prostate cancer (HRPC) metastasis. Analysis of MET protein expression and genetic alterations might contribute to therapeutic stratification of prostate cancer patients. Our objective was to investigate MET on protein, DNA and RNA level in clinical prostate cancer at various stages of progression. Expression of MET was analyzed in hormone-naive primary prostate cancers (N=481), lymph node (N=40) and bone (N=8) metastases, as well as HRPC (N=54) and bone metastases (N=15). MET protein expression was analyzed by immunohistochemistry (D1C2 C-terminal antibody). MET mRNA levels and MET DNA copy numbers were determined by in situ hybridization. None of the hormone-naive primary prostate cancer or lymph node metastases demonstrated MET protein or mRNA expression. In contrast, MET protein was expressed in 12/52 (23%) evaluable HRPC resections. RNA in situ demonstrated cytoplasmic signals in 14/54 (26%) of the HRPC patients, and was associated with MET protein expression (p=0.025, χ2), in absence of MET amplification or polysomy. MET protein expression was present in 7/8 (88%) hormone-naive and 10/15 (67%) HRPC bone metastases, without association of HRPC (p=0.37; χ2), with MET polysomy in 8/13 (61%) evaluable cases. In conclusion, MET was almost exclusively expressed in HRPC and prostate cancer bone metastasis, but was not related to MET amplification or polysomy. Evaluation of MET status could be relevant for therapeutic stratification of late stage prostate cancer.

Low-Molecular-Weight Protein Tyrosine Phosphatase Predicts Prostate Cancer Outcome by Increasing the Metastatic Potential.

BACKGROUND: Low-risk patients suffering from prostate cancer (PCa) are currently placed under active surveillance rather than undergoing radical prostatectomy. However, clear parameters for selecting the right patient for each strategy are not available, and new biomarkers and treatment modalities are needed. Low-molecular-weight protein tyrosine phosphatase (LMWPTP) could present such a target. OBJECTIVE: To correlate expression levels of LMWPTP in primary PCa to clinical outcome, and determine the role of LMWPTP in prostate tumor cell biology. DESIGN, SETTING, AND PARTICIPANTS: Acid phosphatase 1, soluble (ACP1) expression was analyzed on microarray data sets, which were subsequently used in Ingenuity Pathway Analysis. Immunohistochemistry was performed on a tissue microarray containing material of 481 PCa patients whose clinicopathologic data were recorded. PCa cell line models were used to investigate the role of LMWPTP in cell proliferation, migration, adhesion, and anoikis resistance. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association between LMWPTP expression and clinical and pathologic outcomes was calculated using chi-square correlations and multivariable Cox regression analysis. Functional consequences of LMWPTP overexpression or downregulation were determined using migration and adhesion assays, confocal microscopy, Western blotting, and proliferation assays. RESULTS AND LIMITATIONS: LMWPTP expression was significantly increased in human PCa and correlated with earlier recurrence of disease (hazard ratio [HR]:1.99; p<0.001) and reduced patient survival (HR: 1.53; p=0.04). Unbiased Ingenuity analysis comparing cancer and normal prostate suggests migratory propensities in PCa. Indeed, overexpression of LMWPTP increases PCa cell migration, anoikis resistance, and reduces activation of focal adhesion kinase/paxillin, corresponding to decreased adherence. CONCLUSIONS: Overexpression of LMWPTP in PCa confers a malignant phenotype with worse clinical outcome. Prospective follow-up should determine the clinical potential of LMWPTP overexpression. PATIENT SUMMARY: These findings implicate low-molecular-weight protein tyrosine phosphatase as a novel oncogene in prostate cancer and could offer the possibility of using this protein as biomarker or target for treatment of this disease.

The role of HOXC6 in prostate cancer development.

BACKGROUND: Homeobox (HOX) genes, which are involved in organ development and homeostasis, have been shown to be involved in normal prostate- and PCa development. In this study, we investigate the expression levels of the HOX A-D genes in PCa. The functional relevance and potential of HOX gene as biomarkers are explored. METHODS: We evaluated HOX gene expression in prostate tissues of different grade and stage and related the outcome to clinical parameters. We analyzed AR regulation and function of HOXC6 in PCa cell lines. We developed a urine-based HOXC6 mRNA assay for diagnostic purposes. RESULTS: HOXC6 was one of the most upregulated HOX genes in all primary, metastasized, and castration-resistant PCa. HOXC6 upregulation was specific to the epithelial component of PCa, and HOXC6 was shown to be involved in epithelial cell proliferation. HOXC6 expression was not influenced by androgens nor by treatments targeting the AR signaling pathway. HOXC6 expression was not related to a prognosis after radical prostatectomy, that is, biochemical or local recurrence. We successfully developed an assay for HOXC6 mRNA detection in urine and confirmed that HOXC6 levels are higher in PCa patients. CONCLUSIONS: HOXC6 has a role in all PCa stages, particularly in PCa cell proliferation. Due to its stable expression, HOXC6 is a novel candidate biomarker for PCa not only in early detection but also for monitoring of progression or response to therapy.

Novel long non-coding RNAs are specific diagnostic and prognostic markers for prostate cancer.

Current prostate cancer (PCa) biomarkers such as PSA are not optimal in distinguishing cancer from benign prostate diseases and predicting disease outcome. To discover additional biomarkers, we investigated PCa-specific expression of novel unannotated transcripts. Using the unique probe design of Affymetrix Human Exon Arrays, we identified 334 candidates (EPCATs), of which 15 were validated by RT-PCR. Combined into a diagnostic panel, 11 EPCATs classified 80% of PCa samples correctly, while maintaining 100% specificity. High specificity was confirmed by in situ hybridization for EPCAT4R966 and EPCAT2F176 (SChLAP1) on extensive tissue microarrays. Besides being diagnostic, EPCAT2F176 and EPCAT4R966 showed significant association with pT-stage and were present in PIN lesions. We also found EPCAT2F176 and EPCAT2R709 to be associated with development of metastases and PCa-related death, and EPCAT2F176 to be enriched in lymph node metastases. Functional significance of expression of 9 EPCATs was investigated by siRNA transfection, revealing that knockdown of 5 different EPCATs impaired growth of LNCaP and 22RV1 PCa cells. Only the minority of EPCATs appear to be controlled by androgen receptor or ERG. Although the underlying transcriptional regulation is not fully understood, the novel PCa-associated transcripts are new diagnostic and prognostic markers with functional relevance to prostate cancer growth.

SYK is a candidate kinase target for the treatment of advanced prostate cancer.

Improved targeted therapies are needed to combat metastatic prostate cancer. Here, we report the identification of the spleen kinase SYK as a mediator of metastatic dissemination in zebrafish and mouse xenograft models of human prostate cancer. Although SYK has not been implicated previously in this disease, we found that its expression is upregulated in human prostate cancers and associated with malignant progression. RNAi-mediated silencing prevented invasive outgrowth in vitro and bone colonization in vivo, effects that were reversed by wild-type but not kinase-dead SYK expression. In the absence of SYK expression, cell surface levels of the progression-associated adhesion receptors integrin α2ß1 and CD44 were diminished. RNAi-mediated silencing of α2ß1 phenocopied SYK depletion in vitro and in vivo, suggesting an effector role for α2ß1 in this setting. Notably, pharmacologic inhibitors of SYK kinase currently in phase I-II trials for other indications interfered similarly with the invasive growth and dissemination of prostate cancer cells. Our findings offer a mechanistic rationale to reposition SYK kinase inhibitors for evaluation in patients with metastatic prostate cancer.

Prognostic histopathological and molecular markers on prostate cancer needle-biopsies: a review.

Prostate cancer is diverse in clinical presentation, histopathological tumor growth patterns, and survival. Therefore, individual assessment of a tumor's aggressive potential is crucial for clinical decision-making in men with prostate cancer. To date a large number of prognostic markers for prostate cancer have been described, most of them based on radical prostatectomy specimens. However, in order to affect clinical decision-making, validation of respective markers in pretreatment diagnostic needle-biopsies is essential. Here, we discuss established and promising histopathological and molecular parameters in diagnostic needle-biopsies.

Validation of stem cell markers in clinical prostate cancer: α6-integrin is predictive for non-aggressive disease.

BACKGROUND: Stem cells are postulated to mediate prostate cancer progression, and represent a small fraction of the entire tumor. Various proteins (α2-integrin, α6-integrin, CD117, CD133, EZH2, OCT3/4) are associated with a prostate cancer stem cell phenotype in cell lines and xenografts. Our objective was to investigate expression of stem cell markers in clinical prostate cancer in relation to outcome. METHODS: We validated immunohistochemical expression of stem cell markers in 481 prostate cancer patients and correlated expression with clinicopathologic parameters. RESULTS: Sporadic expression of α2-integrin was present in a fraction of tumor cells (<5%) in 94.7% of tumors and associated with PSA > 10 ng/ml (P = 0.04). α6-Integrin expression (<5%) occurred in 28.4% patients, while ≥5% α6-integrin expression was associated with PSA≤10 ng/ml (P = 0.01), Gleason score <7 (P < 0.01) and pT2-disease (P = 0.02). α6-integrin was predictive for biochemical recurrence (P < 0.01), local recurrence (P = 0.03) and disease specific death (P = 0.03). EZH2 expression was generally low with 2.6% of tumors showing ≥1% positive cells. EZH2 was associated with Gleason score ≥7 (P = 0.01) and biochemical recurrence (P = 0.01). We did not identify expression of CD117, CD133, and OCT3/4 in prostate cancer samples. CONCLUSIONS: Expression of α2-integrin and EZH2 in a small fraction of prostate cancer cells is supportive for their role as stem cell marker. Although α6-integrin was not a unique stem cell marker, it was predictive for prostate cancer biochemical and local recurrence, and disease specific death. The validity of CD117, CD133, and OCT3/4 as prostate cancer stem cell marker is questionable since these proteins were not expressed in clinical prostate cancer.

Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer.

BACKGROUND: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ∼100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery. MATERIALS AND METHODS: Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry. RESULTS: Proteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. CONCLUSIONS: Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer.

ERG immunohistochemistry is not predictive for PSA recurrence, local recurrence or overall survival after radical prostatectomy for prostate cancer.

In prostate cancer genomic rearrangements involving genes encoding ETS transcription factors are commonly present, with androgen-regulated transmembrane protease, serine 2 (TMPRSS2)-v-ets erythroblastosis virus E26 oncogen homologue (ERG) gene fusion occurring in 40-70%. Studies on the predictive value of ERG rearrangement as detected by in-situ hybridization or polymerase chain reaction have resulted in varying outcomes. The objective of this study was to correlate immunohistochemical ERG protein expression with clinico-pathological parameters at radical prostatectomy specimens, and to determine its predictive value for postoperative disease recurrence and progression in a prostate cancer screening cohort. Since androgen receptor is downregulated by ERG in cell lines, we also compared the expression of respective proteins. We selected 481 participants from the European Randomized Study of Screening for Prostate Cancer treated by radical prostatectomy for prostate adenocarcinoma. A tissue microarray was constructed containing representative cores of all prostate cancer specimens as well as 22 xenografts and seven cell lines. Immunohistochemical expression of ERG and androgen receptor was correlated with prostate-specific antigen (PSA), Gleason sum, pT-stage, surgical margins, biochemical recurrence, local recurrence, overall death and disease-specific death. ERG expression was detected in 284 patients (65%). Expression occurred significantly more frequent in patients with PSA ≤10 ng/ml (P=0.024). There was no significant association between ERG and Gleason sum, pT-stage or surgical margin status. PSA (P=0.011), Gleason sum (P=0.003), pT-stage (P=0.001) and surgical margin status (P<0.001) all had independent value for postoperative biochemical recurrence, while positive surgical margin (P=0.021) was the only independent predictor for local recurrence. ERG protein expression did not have prognostic value for the clinical end points in uni- and multivariate analyses. A positive correlation existed between ERG and androgen receptor expression in single tissue cores (P<0.001). In conclusion, immunohistochemical ERG expression has no predictive value for prostate cancer recurrence or progression after radical prostatectomy. Increasing ERG levels are associated with the upregulation of androgen receptor expression in clinical specimens.

Antibody EPR3864 is specific for ERG genomic fusions in prostate cancer: implications for pathological practice.

Genomic rearrangements involving genes encoding erythroblast transformation-specific transcription factors are commonly present in prostate cancer. The TMPRSS2-ERG gene fusion that leads to ERG overexpression occurs in ~70% of prostate cancers. Implementation of fusion gene detection in pathological practice, however, has been hampered by the lack of reliable ERG antibodies. The objective of this study was first to compare ERG immunohistochemistry using the recently described antibody EPR3864 with ERG mRNA by quantitative PCR and, second, to investigate ERG immunohistochemistry in diagnostic prostate cancer needle biopsies. We analyzed 41 primary prostate adenocarcinomas obtained by radical prostatectomy and 83 consecutive prostate cancer needle biopsies. In the prostatectomy specimens, immunohistochemical ERG expression was highly concordant with the ERG mRNA overexpression (sensitivity 100% and specificity 85%). ERG overexpression was due to TMPRSS2-ERG gene fusion in all cases. ERG protein expression was identified in 51/83 adenocarcinomas (61%) on needle biopsies. ERG expression was more frequent in tumors infiltrating ≥2 needle biopsies (P<0.001) or occupying ≥50% of a single biopsy (P=0.018). Expression of ERG also occurred in 11/21 (52%) high-grade prostate intraepithelial neoplasia lesions. In 5/87 (6%) needle biopsies containing benign secretory glands, weak ERG staining was focally observed. In all of these cases, respective glands were adjacent to adenocarcinomas. In conclusion, immunohistochemistry for ERG strongly correlated with ERG mRNA overexpression and was specific for prostate cancer on needle biopsies. Therefore, ERG immunohistochemistry is an important adjunctive tool for pathophysiological studies on ERG gene fusions, and might support the pathological diagnosis of adenocarcinoma in a subset of prostate needle biopsies.