Esther I. Verhoef

Disease-specific survival of patients with invasive cribriform and intraductal prostate cancer at diagnostic biopsy.

Invasive cribriform and intraductal carcinoma in radical prostatectomy specimens have been associated with an adverse clinical outcome. Our objective was to determine the prognostic value of invasive cribriform and intraductal carcinoma in pre-treatment biopsies on time to disease-specific death. We pathologically revised the diagnostic biopsies of 1031 patients from the first screening round of the European Randomized Study of Screening for Prostate Cancer (1993–2000). Ninety percent of all patients (n=923) had received active treatment, whereas 10% (n=108) had been followed by watchful waiting. The median follow-up was 13 years. Patients who either had invasive cribriform growth pattern or intraductal carcinoma were categorized as CR/IDC+. The outcome was disease-specific survival. Relationships with outcome were analyzed using multivariable Cox regression and log-rank analysis. In total, 486 patients had Gleason score 6 (47%) and 545 had ≥7 (53%). The 15-year disease-specific-survival probabilities were 99% in Gleason score 6 (n=486), 94% in CR/IDC− Gleason score ≥7 (n=356) and 67% in CR/IDC+ Gleason score ≥7 (n=189). CR/IDC− Gleason score 3+4=7 patients did not have statistically different survival probabilities from those with Gleason score 6 (P=0.30), while CR/IDC+ Gleason score 3+4=7 patients did (P<0.001). In multivariable analysis, CR/IDC+ status was independently associated with a poorer disease-specific survival (HR 2.6, 95% CI 1.4–4.8, P=0.002). We conclude that CR/IDC+ status in prostate cancer biopsies is associated with a worse disease-specific survival. Our findings indicate that men with biopsy CR/IDC− Gleason score 3+4=7 prostate cancer could be candidates for active surveillance, as these patients have similar survival probabilities to those with Gleason score 6.

Three-dimensional microscopic analysis of clinical prostate specimens

Microscopic evaluation of prostate specimens for both clinical and research purposes is generally performed on 5‐μm‐thick tissue sections. Because cross‐sections give a two‐dimensional (2D) representation, little is known about the actual underlying three‐dimensional (3D) architectural features of benign prostate tissue and prostate cancer (PCa). The aim of this study was to show that a combination of tissue‐clearing protocols and confocal microscopy can successfully be applied to investigate the 3D architecture of human prostate tissue.

CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDHhigh sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDHlow. Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDHhigh sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDHhigh sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Human PDE4D isoform composition is deregulated in primary prostate cancer and indicative for disease progression and development of distant metastases

Phosphodiesterase 4D7 was recently shown to be specifically over-expressed in localized prostate cancer, raising the question as to which regulatory mechanisms are involved and whether other isoforms of this gene family (PDE4D) are affected under the same conditions. We investigated PDE4D isoform composition in prostatic tissues using a total of seven independent expression datasets and also included data on DNA methylation, copy number and AR and ERG binding in PDE4D promoters to gain insight into their effect on PDE4D transcription. We show that expression of PDE4D isoforms is consistently altered in primary human prostate cancer compared to benign tissue, with PDE4D7 being up-regulated while PDE4D5 and PDE4D9 are down-regulated. Disease progression is marked by an overall down-regulation of long PDE4D isoforms, while short isoforms (PDE4D1/2) appear to be relatively unaffected. While these alterations seem to be independent of copy number alterations in the PDE4D locus and driven by AR and ERG binding, we also observed increased DNA methylation in the promoter region of PDE4D5, indicating a long lasting alteration of the isoform composition in prostate cancer tissues. We propose two independent metrics that may serve as diagnostic and prognostic markers for prostate disease: (PDE4D7 - PDE4D5) provides an effective means for distinguishing PCa from normal adjacent prostate, whereas PDE4D1/2 - (PDE4D5 + PDE4D7 + PDE4D9) offers strong prognostic potential to detect aggressive forms of PCa and is associated with metastasis free survival. Overall, our findings highlight the relevance of PDE4D as prostate cancer biomarker and potential drug target.

Characteristics and outcome of Prostate cancer patients with overall biopsy Gleason score 3+4=7 and highest Gleason score 3+4=7 or > 3+4=7

Prostate cancer heterogeneity and multifocality might result in different Gleason scores (GS) at individual biopsy cores. According to World Health Organisation/International Society of Urologic Pathology (WHO/ISUP) guidelines, the GS in each biopsy core should be recorded with optional reporting of overall GS for the entire case. We aimed to compare the clinicopathological characteristics and outcome of men with overall biopsy GS 3 + 4 = 7 with highest GS 3 + 4 = 7 (HI = OV) to those with highest GS > 3 + 4 = 7 (HI > OV).

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.

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 sampling artifact. 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.

Epithelial-mesenchymal transition in human prostate cancer demonstrates enhanced immune evasion marked by IDO1 expression

Cancer invasion and metastasis are driven by epithelial-mesenchymal transition (EMT), yet the exact mechanisms that account for EMT in clinical prostate cancer are not fully understood. Expression of N-cadherin is considered a hallmark of EMT in clinical prostate cancer. In this study, we determined the molecular mechanisms associated with N-cadherin expression in patients with prostate cancer. We performed laser capture microdissection of matched N-cadherin-positive and -negative prostate cancer areas from patient samples (n = 8), followed by RNA sequencing. N-cadherin expression was significantly associated with an immune-regulatory signature including profound upregulation of indoleamine 2,3-dioxygenase (IDO1; log2-fold change = 5.1; P = 2.98E-04). Fluorescent immunostainings of patient samples confirmed expression of IDO1 protein and also its metabolite kynurenine in primarily N-cadherin-positive areas. N-cadherin-positive areas also exhibited a local decrease of intraepithelial cytotoxic (CD8+) T cells and an increase of immunosuppressive regulatory T cells (CD4+/FOXP3+). In conclusion, EMT in clinical prostate cancer is accompanied by upregulated expression of IDO1 and an increased number of regulatory T cells. These data indicate that EMT, which is an important step in tumor progression, can be protected from effective immune control in patients with prostate cancer.Significance: These findings demonstrate EMT is linked to an immunosuppressive environment in clinical prostate cancer, suggesting that patients with prostate cancer can potentially benefit from combinatorial drug therapy. Cancer Res; 78(16); 4671-9. ©2018 AACR.

Absent and abundant MET immunoreactivity is associated with poor prognosis of patients with oral and oropharyngeal squamous cell carcinoma

Although the receptor tyrosine kinase (RTK) MET is widely expressed in head and neck squamous cell carcinoma (HNSCC), its prognostic value remains unclear. This might be due to the use of a variety of antibodies and scoring systems. Here, the reliability of five commercial C-terminal MET antibodies (D1C2, CVD13, SP44, C-12 and C-28) was evaluated before examining the prognostic value of MET immunoreactivity in HNSCC. Using cancer cell lines, it was shown that D1C2 and CVD13 specifically detect MET under reducing, native and formalin-fixed paraffin-embedded (FFPE) conditions. Immunohistochemical staining of routinely FFPE oral SCC with D1C2 and CVD13 demonstrated that D1C2 is most sensitive in the detection of membranous MET. Examination of membranous D1C2 immunoreactivity with 179 FFPE oral and oropharyngeal SCC – represented in a tissue microarray – illustrated that staining is either uniform (negative or positive) across tumors or differs between a tumors center and periphery. Ultimately, statistical analysis revealed that D1C2 uniform staining is significantly associated with poor 5-year overall and disease free survival of patients lacking vasoinvasive growth (HR = 3.019, p < 0.001; HR = 2.559, p < 0.001). These findings might contribute to reliable stratification of patients eligible for treatment with biologicals directed against MET.

Large cribriform growth pattern identifies ISUP grade 2 prostate cancer at high risk for recurrence and metastasis

Invasive cribriform and intraductal carcinoma are associated with adverse clinical outcome in patients with Gleason score 7 prostate cancer. It is yet unclear whether invasive cribriform and intraductal carcinoma of the prostate both have independent prognostic value, or whether field size of invasive cribriform carcinoma has impact on disease outcome. Our objective was to determine the prognostic impact of intraductal and invasive cribriform prostate cancer histological subtypes in radical prostatectomies. We reviewed 420 prostatectomy specimens with ISUP grade 2 prostate cancer, assessed the percentages of Gleason grade 4 and tertiary 5, and performed immunohistochemistry for basal cells to discriminate intraductal from invasive cribriform growth. Small and large invasive cribriform fields were distinguished based on a diameter of at least twice the size of adjacent pre-existent normal glands. Clinicopathological parameters and biochemical recurrence-free survival were used as endpoints. Cribriform architecture was observed in 228 (54.3%) men, 103 (24.5%) of whom had intraductal, 194 (46.2%) small invasive, and 34 (8.1%) large invasive cribriform growth. Large invasive cribriform architecture was associated with older age (P < 0.001), higher percentage Gleason grade 4 (P = 0.001), extraprostatic expansion (P < 0.001), and more frequent lymph node metastases (P = 0.002), when compared with small invasive cribriform and/or intraductal carcinoma. Univariate analysis identified PSA, pT-stage, surgical margin status, and intraductal and invasive cribriform growth as significant predictors for biochemical recurrence-free survival. In multivariable Cox regression analysis, pT-stage (hazard ratio = 1.64, 95% CI: 1.02–2.63, P = 0.04), positive surgical margins (hazard ratio = 3.28, 95% CI: 2.06–5.23, P < 0.001), and large cribriform growth (hazard ratio = 4.36, 95% CI: 2.08–9.17, P < 0.001) were independent predictors for biochemical recurrence-free survival, while intraductal carcinoma, small cribriform growth, and percentage of Gleason grade 4 were not. In conclusion, large cribriform fields represent an aggressive subpattern of invasive cribriform prostate cancer and are an independent predictive factor for biochemical recurrence-free survival in ISUP grade 2 prostate cancer patients.