Proteomic-based stratification of intermediate-risk prostate cancer patients.

Gleason grading is an important prognostic indicator for prostate adenocarcinoma and is crucial for patient treatment decisions. However, intermediate-risk patients diagnosed in the Gleason grade group (GG) 2 and GG3 can harbour either aggressive or non-aggressive disease, resulting in under- or overtreatment of a significant number of patients. Here, we performed proteomic, differential expression, machine learning, and survival analyses for 1,348 matched tumour and benign sample runs from 278 patients. Three proteins (F5, TMEM126B, and EARS2) were identified as candidate biomarkers in patients with biochemical recurrence. Multivariate Cox regression yielded 18 proteins, from which a risk score was constructed to dichotomize prostate cancer patients into low- and high-risk groups. This 18-protein signature is prognostic for the risk of biochemical recurrence and completely independent of the intermediate GG. Our results suggest that markers generated by computational proteomic profiling have the potential for clinical applications including integration into prostate cancer management.

68Ga-PSMA-11 PET/MRI versus multiparametric MRI in men referred for prostate biopsy: primary tumour localization and interreader agreement.

BACKGROUND: Magnetic resonance imaging (MRI) is recommended by the European Urology Association guidelines as the standard modality for imaging-guided biopsy. Recently positron emission tomography with prostate-specific membrane antigen (PSMA PET) has shown promising results as a tool for this purpose. The aim of this study was to compare the accuracy of positron emission tomography with prostate-specific membrane antigen/magnetic resonance imaging (PET/MRI) using the gallium-labeled prostate-specific membrane antigen (68Ga-PSMA-11) and multiparametric MRI (mpMRI) for pre-biopsy tumour localization and interreader agreement for visual and semiquantitative analysis. Semiquantitative parameters included apparent diffusion coefficient (ADC) and maximum lesion diameter for mpMRI and standardized uptake value (SUVmax) and PSMA-positive volume (PSMAvol) for PSMA PET/MRI. RESULTS: Sensitivity and specificity were 61.4% and 92.9% for mpMRI and 66.7% and 92.9% for PSMA PET/MRI for reader one, respectively. RPE was available in 23 patients and 41 of 47 quadrants with discrepant findings. Based on RPE results, the specificity for both imaging modalities increased to 98% and 99%, and the sensitivity improved to 63.9% and 72.1% for mpMRI and PSMA PET/MRI, respectively. Both modalities yielded a substantial interreader agreement for primary tumour localization (mpMRI kappa = 0.65 (0.52-0.79), PSMA PET/MRI kappa = 0.73 (0.61-0.84)). ICC for SUVmax, PSMAvol and lesion diameter were almost perfect (≥ 0.90) while for ADC it was only moderate (ICC = 0.54 (0.04-0.78)). ADC and lesion diameter did not correlate significantly with Gleason score (ρ = 0.26 and ρ = 0.16) while SUVmax and PSMAvol did (ρ = - 0.474 and ρ = - 0.468). CONCLUSIONS: PSMA PET/MRI has similar accuracy and reliability to mpMRI regarding primary prostate cancer (PCa) localization. In our cohort, semiquantitative parameters from PSMA PET/MRI correlated with tumour grade and were more reliable than the ones from mpMRI.

Single-cell proteomics defines the cellular heterogeneity of localized prostate cancer.

Localized prostate cancer exhibits multiple genomic alterations and heterogeneity at the proteomic level. Single-cell technologies capture important cell-to-cell variability responsible for heterogeneity in biomarker expression that may be overlooked when molecular alterations are based on bulk tissue samples. This study aims to identify prognostic biomarkers and describe the heterogeneity of prostate cancer and the associated microenvironment by simultaneously quantifying 36 proteins using single-cell mass cytometry analysis of over 1.6 million cells from 58 men with localized prostate cancer. We perform this task, using a high-dimensional clustering pipeline named Franken to describe subpopulations of immune, stromal, and prostate cells, including changes occurring in tumor tissues and high-grade disease that provide insights into the coordinated progression of prostate cancer. Our results further indicate that men with localized disease already harbor rare subpopulations that typically occur in castration-resistant and metastatic disease.

Hot needles can confirm accurate lesion sampling intraoperatively using [18F]PSMA-1007 PET/CT-guided biopsy in patients with suspected prostate cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA)-targeted PET is increasingly used for staging prostate cancer (PCa) with high accuracy to detect significant PCa (sigPCa). [68 Ga]PSMA-11 PET/MRI-guided biopsy showed promising results but also persisting limitation of sampling error, due to impaired image fusion. We aimed to assess the possibility of intraoperative quantification of [18F]PSMA-1007 PET/CT uptake in core biopsies as an instant confirmation for accurate lesion sampling. METHODS: In this IRB-approved, prospective, proof-of-concept study, we included five consecutive patients with suspected PCa. All underwent [18F]PSMA-1007 PET/CT scans followed by immediate PET/CT-guided and saturation template biopsy (3.1 ± 0.3 h after PET). The activity in biopsy cores was measured as counts per minute (cpm) in a gamma spectrometer. Pearson's test was used to correlate counts with histopathology (WHO/ISUP), tumor length, and membranous PSMA expression on immunohistochemistry (IHC). RESULTS: In 43 of 113 needles, PCa was present. The mean cpm was overall significantly higher in needles with PCa (263 ± 396 cpm) compared to needles without PCa (73 ± 44 cpm, p < 0.001). In one patient with moderate PSMA uptake (SUVmax 8.7), 13 out of 24 needles had increased counts (100-200 cpm) but only signs of inflammation and PSMA expression in benign glands on IHC. Excluding this case, ROC analysis resulted in an AUC of 0.81, with an optimal cut-off to confirm PCa at 75 cpm (sens/spec of 65.1%/87%). In all 4 patients with PCa, the first or second PSMA PET-guided needle was positive for sigPCa with high counts (156-2079 cpm). CONCLUSIONS: [18F]PSMA-1007 uptake in PCa can be used to confirm accurate lesion sampling of the dominant tumor intraoperatively. This technique could improve confidence in imaging-based biopsy guidance and reduce the need for saturation biopsy. TRIAL REGISTRATION NUMBER: NCT03187990, 15/06/2017.

Diagnostic performance of 68Ga-PSMA-11 PET/MRI-guided biopsy in patients with suspected prostate cancer: a prospective single-center study.

PURPOSE: Ultrasound-guided biopsy (US biopsy) with 10-12 cores has a suboptimal sensitivity for clinically significant prostate cancer (sigPCa). If US biopsy is negative, magnetic resonance imaging (MRI)-guided biopsy is recommended, despite a low specificity for lesions with score 3-5 on Prostate Imaging Reporting and Data System (PIRADS). Screening and biopsy guidance using an imaging modality with high accuracy could reduce the number of unnecessary biopsies, reducing side effects. The aim of this study was to assess the performance of positron emission tomography/MRI with 68Ga-labeled prostate-specific membrane antigen (PSMA-PET/MRI) to detect and localize primary sigPCa (ISUP grade group 3 and/or cancer core length ≥ 6 mm) and guide biopsy. METHODS: Prospective, open-label, single-center, non-randomized, diagnostic accuracy study including patients with suspected PCa by elevation of prostate-specific antigen (PSA) level and a suspicious lesion (PIRADS ≥3) on multiparametric MRI (mpMRI). Forty-two patients underwent PSMA-PET/MRI followed by both PSMA-PET/MRI-guided and section-based saturation template biopsy between May 2017 and February 2019. Primary outcome was the accuracy of PSMA-PET/MRI for biopsy guidance using section-based saturation template biopsy as the reference standard. RESULTS: SigPCa was found in 62% of the patients. Patient-based sensitivity, specificity, negative and positive predictive value, and accuracy for sigPCa were 96%, 81%, 93%, 89%, and 90%, respectively. One patient had PSMA-negative sigPCa. Eight of nine false-positive lesions corresponded to cancer on prostatectomy and one in six false-negative lesions was negative on prostatectomy. CONCLUSION: PSMA-PET/MRI has a high accuracy for detecting sigPCa and is a promising tool to select patients with suspicion of PCa for biopsy. TRIAL REGISTRATION: This trial was retrospectively registered under the name "Positron Emission Tomography/Magnetic Resonance Imaging (PET/MRI) Guided Biopsy in Men with Elevated PSA" (NCT03187990) on 06/15/2017 ( https://clinicaltrials.gov/ct2/show/NCT03187990 ).

Multi-region proteome analysis quantifies spatial heterogeneity of prostate tissue biomarkers.

It remains unclear to what extent tumor heterogeneity impacts on protein biomarker discovery. Here, we quantified proteome intra-tissue heterogeneity (ITH) based on a multi-region analysis of prostate tissues using pressure cycling technology and SWATH mass spectrometry. We quantified 6,873 proteins and analyzed the ITH of 3,700 proteins. The level of ITH varied depending on proteins and tissue types. Benign tissues exhibited more complex ITH patterns than malignant tissues. Spatial variability of ten prostate biomarkers was validated by immunohistochemistry in an independent cohort (n=83) using tissue microarrays. PSA was preferentially variable in benign prostatic hyperplasia, while GDF15 substantially varied in prostate adenocarcinomas. Further, we found that DNA repair pathways exhibited a high degree of variability in tumorous tissues, which may contribute to the genetic heterogeneity of tumors. This study conceptually adds a new perspective to protein biomarker discovery: it suggests that recent technological progress should be exploited to quantify and account for spatial proteome variation to complement biomarker identification and utilization.

Comprehensive immunohistochemical analysis of PD-L1 shows scarce expression in castration-resistant prostate cancer.

BACKGROUND: We aimed to analyze the frequency and distribution of PD-L1 expression in specimens from prostate cancer (PC) patients using two different anti-PD-L1 antibodies (E1L3N, SP263). MATERIALS AND METHODS: PD-L1 immunohistochemistry was performed in a tissue microarray consisting of 82 castration-resistant prostate cancer (CRPC) specimens, 70 benign prostate hyperplasia (BPH) specimens, 96 localized PC cases, and 3 PC cell lines, using two different antibodies (clones E1L3N, and SP263). Staining images for CD4, CD8, PD-L1, and PanCK of a single PD-L1 positive case were compared, using a newly developed dot-wise correlation method for digital images to objectively test for co-expression. RESULTS: Depending on the antibody used, in tumor cells (TC) only five (E1L3N: 6%) and three (SP263: 3.7%) samples were positive. In infiltrating immune cells (IC) 12 (SP263: 14.6%) and 8 (E1L3N: 9.9%) specimens showed PD-L1 expression. Two PC cell lines (PC3, LnCaP) also displayed membranous immunoreactivity. All localized PCs or BPH samples tested were negative. Dot-wise digital correlation of expression patterns revealed a moderate positive correlation between PD-L1 and PanCK expression, whereas both PanCK and PD-L1 showed a weak negative Pearson correlation coefficient between CD4 and CD8. CONCLUSIONS: PD-L1 was not expressed in localized PC or BPH, and was only found in a minority of CRPC tumors and infiltrating immune cells. Protein expression maps and systematic dot-wise comparison could be a useful objective way to describe the relationship between immuno- and tumor-related proteins in the future, without the need to develop multiplex staining methods.

KPNA2 is overexpressed in human and mouse endometrial cancers and promotes cellular proliferation.

Endometrial cancer is the most frequently occurring malignancy of the female genital tract in Western countries. Although in many cases surgically curable, about 30% of the tumours represent an aggressive and untreatable disease. In an attempt to establish a reliable prognostic marker for endometrial carcinomas disregarding their histological diversity, we investigated the expression of KPNA2, a mediator of nucleocytoplasmic transport, and other cell proliferation-associated proteins and their correlation with cancer progression. We analysed patient tissue microarrays (TMAs) assembled from 527 endometrial cancer tissue specimens and uterus samples from a Trp53 knockout mouse model of endometrial cancer. Our data show that KPNA2 expression was significantly up-regulated in human endometrial carcinomas and associated with higher tumour grade (p = 0.026), higher FIGO stage (p = 0.027), p53 overexpression (p < 0.001), activation of the PI3K/AKT pathway, and epithelial-mesenchymal transition. Increased nuclear KPNA2 immunoreactivity was identified as a novel predictor of overall survival, independent of well-established prognostic factors in Cox regression analyses (hazard ratio 1.7, 95% CI 1.13-2.56, p = 0.01). No significant association between KPNA2 expression and endometrial cancer subtype was detected. In the mouse model, KPNA2 showed increased expression levels from precancerous (EmgD, EIC) to far-advanced invasive lesions. We further investigated the cell proliferation capacity after siRNA-mediated KPNA2 knockdown in the human endometrial cancer cell line MFE-296. KPNA2 silencing led to decreased proliferation of the cancer cells, suggesting interplay of the protein with the cell cycle. Taken together, increased expression of KPNA2 is an independent prognostic marker for poor survival. The mechanism of enhanced nucleocytoplasmic transport by KPNA2 overexpression seems a common event in aggressive cancers since we have shown a significant correlation of KPNA2 expression and tumour aggressiveness in a large variety of other solid tumour entities. Introducing KPNA2 immunohistochemistry in routine diagnostics may allow for the identification of patients who need more aggressive treatment regimens.