Prostate Cancer Screening With PSA, Kallikrein Panel, and MRI: The ProScreen Randomized Trial.

Importance: Prostate-specific antigen (PSA) screening has potential to reduce prostate cancer mortality but frequently detects prostate cancer that is not clinically important. Objective: To describe rates of low-grade (grade group 1) and high-grade (grade groups 2-5) prostate cancer identified among men invited to participate in a prostate cancer screening protocol consisting of a PSA test, a 4-kallikrein panel, and a magnetic resonance imaging (MRI) scan. Design, Setting, and Participants: The ProScreen trial is a clinical trial conducted in Helsinki and Tampere, Finland, that randomized 61 193 men aged 50 through 63 years who were free of prostate cancer in a 1:3 ratio to either be invited or not be invited to undergo screening for prostate cancer between February 2018 and July 2020. Interventions: Participating men randomized to the intervention underwent PSA testing. Those with a PSA level of 3.0 ng/mL or higher underwent additional testing for high-grade prostate cancer with a 4-kallikrein panel risk score. Those with a kallikrein panel score of 7.5% or higher underwent an MRI of the prostate gland, followed by targeted biopsies for those with abnormal prostate gland MRI findings. Final data collection occurred through June 31, 2023. Main Outcomes and Measures: In descriptive exploratory analyses, the cumulative incidence of low-grade and high-grade prostate cancer after the first screening round were compared between the group invited to undergo prostate cancer screening and the control group. Results: Of 60 745 eligible men (mean [SD] age, 57.2 [4.0] years), 15 201 were randomized to be invited and 45 544 were randomized not to be invited to undergo prostate cancer screening. Of 15 201 eligible males invited to undergo screening, 7744 (51%) participated. Among them, 32 low-grade prostate cancers (cumulative incidence, 0.41%) and 128 high-grade prostate cancers (cumulative incidence, 1.65%) were detected, with 1 cancer grade group result missing. Among the 7457 invited men (49%) who refused participation, 7 low-grade prostate cancers (cumulative incidence, 0.1%) and 44 high-grade prostate cancers (cumulative incidence, 0.6%) were detected, with 7 cancer grade groups missing. For the entire invited screening group, 39 low-grade prostate cancers (cumulative incidence, 0.26%) and 172 high-grade prostate cancers (cumulative incidence, 1.13%) were detected. During a median follow-up of 3.2 years, in the group not invited to undergo screening, 65 low-grade prostate cancers (cumulative incidence, 0.14%) and 282 high-grade prostate cancers (cumulative incidence, 0.62%) were detected. The risk difference for the entire group randomized to the screening invitation vs the control group was 0.11% (95% CI, 0.03%-0.20%) for low-grade and 0.51% (95% CI, 0.33%-0.70%) for high-grade cancer. Conclusions and Relevance: In this preliminary descriptive report from an ongoing randomized clinical trial, 1 additional high-grade cancer per 196 men and 1 low-grade cancer per 909 men were detected among those randomized to be invited to undergo a single prostate cancer screening intervention compared with those not invited to undergo screening. These preliminary findings from a single round of screening should be interpreted cautiously, pending results of the study's primary mortality outcome. Trial Registration: ClinicalTrials.gov Identifier: NCT03423303.

Expected impact of MRI-targeted biopsy interreader variability among uropathologists on ProScreen prostate cancer screening trial: a pre-trial validation study.

PURPOSE: Prostate cancer (PCa) histology, particularly the Gleason score, is an independent prognostic predictor in PCa. Little is known about the inter-reader variability in grading of targeted prostate biopsy based on magnetic resonance imaging (MRI). The aim of this study was to assess inter-reader variability in Gleason grading of MRI-targeted biopsy among uropathologists and its potential impact on a population-based randomized PCa screening trial (ProScreen). METHODS: From June 2014 to May 2018, 100 men with clinically suspected PCa were retrospectively selected. All men underwent prostate MRI and 86 underwent targeted prostate of the prostate. Six pathologists individually reviewed the pathology slides of the prostate biopsies. The five-tier ISUP (The International Society of Urological Pathology) grade grouping (GG) system was used. Fleiss' weighted kappa (κ) and Model-based kappa for associations were computed to estimate the combined agreement between individual pathologists. RESULTS: GG reporting of targeted prostate was highly consistent among the trial pathologists. Inter-reader agreement for cancer (GG1-5) vs. benign was excellent (Model-based kappa 0.90, Fleiss' kappa κ = 0.90) and for clinically significant prostate cancer (csPCa) (GG2-5 vs. GG0 vs. GG1), it was good (Model-based kappa 0.70, Fleiss' kappa κ 0.67). CONCLUSIONS: Inter-reader agreement in grading of MRI-targeted biopsy was good to excellent, while it was fair to moderate for MRI in the same cohort, as previously shown. Importantly, there was wide consensus by pathologists in assigning the contemporary GG on MRI-targeted biopsy suggesting high reproducibility of pathology reporting in the ProScreen trial.

Cancer origin tracing and timing in two high-risk prostate cancers using multisample whole genome analysis: prospects for personalized medicine.

BACKGROUND: Prostate cancer (PrCa) genomic heterogeneity causes resistance to therapies such as androgen deprivation. Such heterogeneity can be deciphered in the context of evolutionary principles, but current clinical trials do not include evolution as an essential feature. Whether or not analysis of genomic data in an evolutionary context in primary prostate cancer can provide unique added value in the research and clinical domains remains an open question. METHODS: We used novel processing techniques to obtain whole genome data together with 3D anatomic and histomorphologic analysis in two men (GP5 and GP12) with high-risk PrCa undergoing radical prostatectomy. A total of 22 whole genome-sequenced sites (16 primary cancer foci and 6 lymph node metastatic) were analyzed using evolutionary reconstruction tools and spatio-evolutionary models. Probability models were used to trace spatial and chronological origins of the primary tumor and metastases, chart their genetic drivers, and distinguish metastatic and non-metastatic subclones. RESULTS: In patient GP5, CDK12 inactivation was among the first mutations, leading to a PrCa tandem duplicator phenotype and initiating the cancer around age 50, followed by rapid cancer evolution after age 57, and metastasis around age 59, 5 years prior to prostatectomy. In patient GP12, accelerated cancer progression was detected after age 54, and metastasis occurred around age 56, 3 years prior to prostatectomy. Multiple metastasis-originating events were identified in each patient and tracked anatomically. Metastasis from prostate to lymph nodes occurred strictly ipsilaterally in all 12 detected events. In this pilot, metastatic subclone content analysis appears to substantially enhance the identification of key drivers. Evolutionary analysis' potential impact on therapy selection appears positive in these pilot cases. CONCLUSIONS: PrCa evolutionary analysis allows tracking of anatomic site of origin, timing of cancer origin and spread, and distinction of metastatic-capable from non-metastatic subclones. This enables better identification of actionable targets for therapy. If extended to larger cohorts, it appears likely that similar analyses could add substantial biological insight and clinically relevant value.

Breast density and the likelihood of malignant MRI-detected lesions in women diagnosed with breast cancer.

OBJECTIVES: To assess whether mammographic breast density in women diagnosed with breast cancer correlates with the total number of incidental magnetic resonance imaging (MRI)-detected lesions and the likelihood of the lesions being malignant. METHODS: Patients diagnosed with breast cancer meeting the EUSOBI and EUSOMA criteria for preoperative breast MRI routinely undergo mammography and ultrasound before MRI at our institution. Incidental suspicious breast lesions detected in MRI are biopsied. We included patients diagnosed with invasive breast cancers between 2014 and 2019 who underwent preoperative breast MRI. One reader retrospectively determined breast density categories according to the 5th edition of the BI-RADS lexicon. RESULTS: Of 946 patients with 973 malignant primary breast tumors, 166 (17.5%) had a total of 175 (18.0%) incidental MRI-detected lesions (82 (46.9%) malignant and 93 (53.1%) benign). High breast density according to BI-RADS was associated with higher incidence of all incidental enhancing lesions in preoperative breast MRIs: 2.66 (95% confidence interval: 1.03-6.86) higher for BI-RADS density category B, 2.68 (1.04-6.92) for category C, and 3.67 (1.36-9.93) for category D compared to category A (p < 0.05). However, high breast density did not predict higher incidence of malignant incidental lesions (p = 0.741). Incidental MRI-detected lesions in the contralateral breast were more likely benign (p < 0.001): 18 (27.3%)/48 (72.7%) vs. 64 (58.7%)/45 (41.3%) malignant/benign incidental lesions in contralateral vs. ipsilateral breasts. CONCLUSION: Women diagnosed with breast cancer who have dense breasts have more incidental MRI-detected lesions, but higher breast density does not translate to increased likelihood of malignant incidental lesions. CLINICAL RELEVANCE STATEMENT: Dense breasts should not be considered as an indication for preoperative breast MRI in women diagnosed with breast cancer. KEY POINTS: • The role of preoperative MRI of patients with dense breasts diagnosed with breast cancer is under debate. • Women with denser breasts have a higher incidence of all MRI-detected incidental breast lesions, but the incidence of malignant MRI-detected incidental lesions is not higher than in women with fatty breasts. • High breast density alone should not indicate preoperative breast MRI.

Laser desorption tissue imaging with Differential Mobility Spectrometry.

Pathological gross examination of breast carcinoma samples is sometimes laborious. A tissue pre-mapping method could indicate neoplastic areas to the pathologist and enable focused sampling. Differential Mobility Spectrometry (DMS) is a rapid and affordable technology for complex gas mixture analysis. We present an automated tissue laser analysis system for imaging approaches (iATLAS), which utilizes a computer-controlled laser evaporator unit coupled with a DMS gas analyzer. The system is demonstrated in the classification of porcine tissue samples and three human breast carcinomas. Tissue samples from eighteen landrace pigs were classified with the system based on a pre-designed matrix (spatial resolution 1-3 mm). The smoke samples were analyzed with DMS, and tissue classification was performed with several machine learning approaches. Porcine skeletal muscle (n = 1030), adipose tissue (n = 1329), normal breast tissue (n = 258), bone (n = 680), and liver (n = 264) were identified with 86% cross-validation (CV) accuracy with a convolutional neural network (CNN) model. Further, a panel tissue that comprised all five tissue types was applied as an independent validation dataset. In this test, 82% classification accuracy with CNN was achieved. An analogous procedure was applied to demonstrate the feasibility of iATLAS in breast cancer imaging according to 1) macroscopically and 2) microscopically annotated data with 10-fold CV and SVM (radial kernel). We reached a classification accuracy of 94%, specificity of 94%, and sensitivity of 93% with the macroscopically annotated data from three breast cancer specimens. The microscopic annotation was applicable to two specimens. For the first specimen, the classification accuracy was 84% (specificity 88% and sensitivity 77%). For the second, the classification accuracy was 72% (specificity 88% and sensitivity 24%). This study presents a promising method for automated tissue imaging in an animal model and lays foundation for breast cancer imaging.

Population-based randomized trial of screening for clinically significant prostate cancer ProScreen: a pilot study.

OBJECTIVES: To evaluate the feasibility of a population-based screening trial using prostate-specific antigen (PSA), a kallikrein panel and multiparametric magnetic resonance imaging (MRI) aimed at minimizing overdiagnosis, while retaining mortality benefit. PATIENTS AND METHODS: Feasibility of the screening algorithm was evaluated in terms of participation, screening test results and cancer detection. A random sample of 400 men aged 65 years was identified from the population registry and invited for screening with three stepwise tests (PSA, kallikrein panel and MRI). Men with PSA levels ≥3 ng/mL were further tested with the kallikrein panel, and those with positive findings (risk >7.5%) were referred for prostate MRI. Men with positive MRI (Prostate Imaging Reporting and Data System [PI-RADS] score 3-5) had targeted biopsies only. Men with negative MRI, but PSA density ≥0.15 underwent systematic biopsies. RESULTS: Of the 399 men invited, 158 (40%) participated and 27 had PSA levels ≥3 ng/mL (7% of the invited and 17% of the participants). Of these, 22 had a positive kallikrein panel (6% of the invited and 81% of the PSA-positive men). Finally, 10 men (3% of the invited and 45% of 4Kscore [kallikrein panel]-positive) had a suspicious MRI finding (PI-RADS score ≥3) and five were diagnosed with a clinically significant prostate cancer (Gleason Grade Group [GG] ≥2) at fusion biopsy (3% of the participants), with two GG 1 cases (1%). Additional testing (kallikrein panel and MRI) after PSA reduced biopsies by 56%. CONCLUSION: The findings constitute proof of principle for our screening protocol, as we achieved a substantial detection rate for clinically significant cancer with few clinically insignificant cases. Participation, however, was suboptimal.

Radiological and pathological assessment of response to neoadjuvant CDK4/6 inhibitor and endocrine treatments in a real-life setting-initial results.

BACKGROUND: Neoadjuvant endocrine therapy is an alternative to neoadjuvant chemotherapy in women with inoperable luminal-like breast cancers. Neoadjuvant cyclin-dependent kinase 4/6 inhibitor treatment combined with endocrine treatment (CDK4/6I + E) is interesting given the combination's utility in the treatment of metastatic breast cancer. Currently, the literature on the radiological response evaluation of patients treated with neoadjuvant CDK4/6I + E in a real-life setting is scarce. PURPOSE: To conduct a radiological response evaluation of patients treated with neoadjuvant CDK4/6I + E in a real-life setting. MATERIAL AND METHODS: We retrospectively reviewed clinical, pathological, and radiological findings of six patients with luminal-like breast cancers treated with neoadjuvant CDK4/6I + E treatment. The radiological neoadjuvant CDK4/6I + E response was evaluated with the RECIST 1.1 criteria and the pathological residual disease was assessed using the Residual Cancer Burden (RBC) criteria. RESULTS: None of the patients achieved a complete radiological magnetic resonance imaging (MRI)-determined response or a complete pathological response; three (50%) patients had a partial radiological response; in the three others, the disease remained stable radiologically. All of the tumors were rendered susceptible to surgical treatment. Two out of six (33.3%) patients had a moderate response (RBC-II); four (66.7%) had an extensive residual disease (RBC-III) in the final surgical sample. CONCLUSION: Although none of the patients achieved a pathologically complete response, neoadjuvant CDK4/6I + E treatment rendered all tumors operable. MRI appears to be reliable in the assessment of the neoadjuvant CDK4/6I + E treatment response in a real-life setting. Larger studies are warranted to confirm these results.

Combined Longitudinal Clinical and Autopsy Phenomic Assessment in Lethal Metastatic Prostate Cancer: Recommendations for Advancing Precision Medicine.

BACKGROUND: Systematic identification of data essential for outcome prediction in metastatic prostate cancer (mPC) would accelerate development of precision oncology. OBJECTIVE: To identify novel phenotypes and features associated with mPC outcome, and to identify biomarker and data requirements to be tested in future precision oncology trials. DESIGN SETTING AND PARTICIPANTS: We analyzed deep longitudinal clinical, neuroendocrine expression, and autopsy data of 33 men who died from mPC between 1995 and 2004 (PELICAN33), and related findings to mPC biomarkers reported in the literature. INTERVENTION: Thirty-three men prospectively consented to participate in an integrated clinical-molecular rapid autopsy study of mPC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Data exploration with correction for multiple testing and survival analysis from the time of diagnosis to time to death and time to first occurrence of severe pain as outcomes were carried out. The effect of seven complications on the modeled probability of dying within 2 yr after presenting with the complication was evaluated using logistic regression. RESULTS AND LIMITATIONS: Feature exploration revealed novel phenotypes related to mPC outcome. Four complications (pleural effusion, severe anemia, severe or controlled pain, and bone fracture) predict the likelihood of death within 2 yr. Men with Gleason grade group 5 cancers developed severe pain sooner than those with lower-grade tumors. Surprisingly, neuroendocrine (NE) differentiation was frequently observed in the setting of high serum prostate-specific antigen (PSA) levels (≥30 ng/ml). In 4/33 patients, no controlled (requiring analgesics) or severe pain was detected, and strikingly, 14/15 metastatic sites studied in these men did not express NE markers, suggesting an inverse relationship between NE differentiation and pain in mPC. Intracranial subdural metastasis is common (36%) and is usually clinically undetected. Categorization of "skeletal-related events" complications used in recent studies likely obscures the understanding of spinal cord compression and fracture. Early death from prostate cancer was identified in a subgroup of men with a low longitudinal PSA bandwidth. Cachexia is common (body mass index <0.89 in 24/31 patients) but limited to the last year of life. Biomarker review identified 30 categories of mPC biomarkers in need of winnowing in future trials. All findings require validation in larger cohorts, preferably alongside data from this study. CONCLUSIONS: The study identified novel outcome subgroups for future validation and provides "vision for mPC precision oncology 2020-2050" draft recommendations for future data collection and biomarker studies. PATIENT SUMMARY: To better understand variation in metastatic prostate cancer behavior, we assembled and analyzed longitudinal clinical and autopsy records in 33 men. We identified novel outcomes, phenotypes, and aspects of disease burden to be tested and refined in future trials.

Expression of the miR-200 family in tumor tissue, plasma and urine of epithelial ovarian cancer patients in comparison to benign counterparts.

OBJECTIVE: Plasma, but also urine sample could represent a simple liquid biopsy for ovarian cancer biomarker detection. The miRNA-200 family has been shown to be dysregulated in ovarian cancer. The aim of this study was to isolate three members of miR-200 family from tumor tissue, plasma and urine of high-grade serous ovarian cancer patients in comparison with samples from patients with benign ovarian tumors. This is a methodological pilot study of a prospective ovarian cancer patient cohort investigating the potential of liquid biopsies and the role of miRNAs in ovarian cancer treatment. RESULTS: MiR-200a, miR-200b and miR-200c were isolated from samples of nine ovarian cancer patients and seven patients with benign ovarian tumor. The most significant finding is that all three miRNAs were detectable in all sample types. Tumor tissue and plasma, but not urine analysis was able to discriminate malignant and benign samples. A correlation between the miRNA-200 expression in urine and plasma was observed in malignant samples only. Plasma and urine with respect to miRNA detection show potential according to this study, but larger studies are needed to clarify the usefulness of these liquid biopsies in ovarian cancer. TRIAL REGISTRATION: ClinicalTrials.gov NCT02758652, May 2, 2016.

Histopathology and immunohistochemical analysis of 5-fluorouracil and triamcinolone treated keloids in double-blinded randomized controlled trial.

Keloids are a major complication related to surgical wound healing and very challenging condition to treat. Many treatment options are available, but the efficacy of the treatment is poor in most of cases and some keloids do not respond to the treatment at all. We compared the efficacy of intralesional 5-fluorouracil (5-FU) and triamcinolone (TAC) injections in a double-blind randomized controlled trial (RCT). Forty-three patients with 50 keloid scars were treated with either intralesional TAC or 5-FU-injections over 6 months. We wanted to find out whether biological features (cell density, cell proliferation rate, vascular density, myofibroblast numbers, steroid hormone receptor expression) in keloids could be used to predict the response to therapy and define the biological changes that take place in patients receiving a response. As there was no statistically significant difference in the remission rate between TAC and 5-FU treatments, all patients were combined and analyzed as responders and nonresponders. Although responders have slightly more myofibroblasts than the nonresponders in their keloids in the pretreatment biopsy samples, we could not identify a single predictive factor that could identify those patients that respond to drug injections. The good clinical response to therapy is associated with the simultaneous reduction of myofibroblasts in the keloid. This study demonstrates that myofibroblasts are reduced in number in those keloids that were responsive to therapy, and that both 5-FU and TAC injections are useful for keloid treatment.

Cytokeratin-Supervised Deep Learning for Automatic Recognition of Epithelial Cells in Breast Cancers Stained for ER, PR, and Ki-67.

Immunohistochemistry (IHC) of ER, PR, and Ki-67 are routinely used assays in breast cancer diagnostics. Determination of the proportion of stained cells (labeling index) should be restricted on malignant epithelial cells, carefully avoiding tumor infiltrating stroma and inflammatory cells. Here, we developed a deep learning based digital mask for automated epithelial cell detection using fluoro-chromogenic cytokeratin-Ki-67 double staining and sequential hematoxylin-IHC staining as training material. A partially pre-trained deep convolutional neural network was fine-tuned using image batches from 152 patient samples of invasive breast tumors. Validity of the trained digital epithelial cell masks was studied with 366 images captured from 98 unseen samples, by comparing the epithelial cell masks to cytokeratin images and by visual evaluation of the brightfield images performed by two pathologists. A good discrimination of epithelial cells was achieved (AUC of mean ROC = 0.93; defined as the area under mean receiver operating characteristics), and well in concordance with pathologists' visual assessment (4.01/5 and 4.67/5). The effect of epithelial cell masking on the Ki-67 labeling index was substantial. 52 tumor images initially classified as low proliferation (Ki-67 < 14%) without epithelial cell masking were re-classified as high proliferation (Ki-67 ≥ 14%) after applying the deep learning based epithelial cell mask. The digital epithelial cell masks were found applicable also to IHC of ER and PR. We conclude that deep learning can be applied to detect carcinoma cells in breast cancer samples stained with conventional brightfield IHC.

Identification of breast tumors from diathermy smoke by differential ion mobility spectrometry.

INTRODUCTION: Breast cancer is the most frequent cancer in women worldwide. The primary treatment is breast-conserving surgery or mastectomy with an adequate clearance margin. Diathermy blade is used extensively in breast-conserving surgery. Surgical smoke produced as a side product has cancer-specific molecular features. Differential mobility spectrometry (DMS) is a rapid and affordable technology for analysis of complex gas mixtures. In our study we examined surgical smoke from malignant and benign breast tissue created with a diathermy blade using DMS. MATERIAL AND METHODS: Punch biopsies of 4 mm diameter from breast cancer surgical specimens were taken during gross dissection of fresh surgical specimen and placed in a well plate. The measurement system is a custom-built device called automatic tissue analysis system (ATAS) based on a DMS sensor. Each specimen was incised with a diathermy blade and the surgical smoke was analyzed. RESULTS: We examined 106 carcinoma samples from 21 malignant breast tumors. Benign samples (n = 198) included macroscopically normal mammary gland (n = 82), adipose tissue (n = 88) and vascular tissue (n = 28). The classification accuracy when comparing malignant samples to all benign samples was 87%. The sensitivity was 80% and the specificity was 90%. The classification accuracy of carcinomas to ductal and lobular was 94%, 47%, respectively. CONCLUSIONS: Benign and malignant breast tissue can be identified with ATAS. These results lay foundation for intraoperative margin assessment with DMS from surgical smoke.

Atorvastatin Versus Placebo for Prostate Cancer Before Radical Prostatectomy-A Randomized, Double-blind, Placebo-controlled Clinical Trial.

We tested whether intervention with atorvastatin affects the prostate beneficially compared with placebo in men with prostate cancer in a randomized clinical trial. A total of 160 statin-naïve prostate cancer patients scheduled for radical prostatectomy were randomized to use 80mg atorvastatin or placebo daily from recruitment to surgery for a median of 27 d. Blinding was maintained throughout the trial. In total, 158 men completed the follow-up, with 96% compliance. Overall, atorvastatin did not significantly lower tumor proliferation index Ki-67 or serum prostate-specific antigen (PSA) compared with placebo. In subgroup analyses, after a minimum of 28 d of atorvastatin use, Ki-67 was 14.1% lower compared with placebo (p = 0.056). Among high-grade cases (International Society of Urological Pathology Gleason grade 3 or higher), atorvastatin lowered PSA compared with placebo: median change -0.6 ng/ml; p = 0.024. Intraprostatic inflammation did not differ between the study arms (p = 0.8). Despite a negative overall result showing no effect of statins on Ki67 or PSA overall, in post hoc exploratory analyses, there appeared to be benefit after a minimum duration of 28 d. Further studies are needed to verify this. PATIENT SUMMARY: Cholesterol-lowering atorvastatin does not lower prostate cancer proliferation rate compared with placebo overall, but exploratory analyses suggest a benefit in longer exposure.

Expression of neuroendocrine differentiation markers in lethal metastatic castration-resistant prostate cancer.

Neuroendocrine differentiation (NED) is a common phenomenon in prostate cancer, and it has been associated with poor prognosis in some studies of primary prostate cancer. Incidence and patterns of NED in metastatic prostate cancer sites have not been examined widely. In this study, we studied expression of three commonly used markers of NED (chromogranin A, neuron specific enolase and synaptophysin) in 89 metastases from 31 men that died of castration-resistant prostate cancer and underwent rapid autopsy, and in 89 hormone-naïve primary tumors removed by radical prostatectomy. In addition, we examined NED association with androgen receptor, ERG and Ki-67 expression in metastatic tumor sites. Morphologically, 1 of 31 cases was classified as small cell carcinoma, and the remaining 30 were classified as usual prostate adenocarcinoma using a recently proposed classification of prostate cancers with NED. Metastases showed more expression of neuron specific enolase and synaptophysin compared to prostatectomies (6.3% of cells vs. 1.0%, p < 0.001 and 4.0% vs. 0.4%, p < 0.001, respectively). At least focal expression of one of the markers was seen in 78% of metastases. Strong expression was relatively uncommon, seen in 3/89 (chromogranin A), 8/89 (neuron specific enolase), and 5/89 (synaptophysin) metastases. Expression of chromogranin A and synaptophysin correlated with each other (r = 0.64, p < 0.001), but expression of neuron specific enolase did not correlate with the two other markers. Extent of NED varied significantly between different metastatic sites in individual patients. Absent androgen receptor expression was associated with strong expression of chromogranin A (p = .02) and neuron specific enolase (p = .02), but not with focal expression of any marker. No clear association was found between expression of NE markers and ERG or Ki-67. In conclusion, NED is a common and heterogeneous phenomenon in metastatic, castration-resistant prostate cancer. NED is more often present in castration-resistant prostate cancer compared to hormone-naïve disease, and it is associated with androgen receptor negativity. More research is needed to understand significance of NED in the progression of prostate cancer.

Feasibility of Prostate PAXgene Fixation for Molecular Research and Diagnostic Surgical Pathology: Comparison of Matched Fresh Frozen, FFPE, and PFPE Tissues.

Advances in prostate cancer biology and diagnostics are dependent upon high-fidelity integration of clinical, histomorphologic, and molecular phenotypic findings. In this study, we compared fresh frozen, formalin-fixed paraffin-embedded (FFPE), and PAXgene-fixed paraffin-embedded (PFPE) tissue preparation methods in radical prostatectomy prostate tissue from 36 patients and performed a preliminary test of feasibility of using PFPE tissue in routine prostate surgical pathology diagnostic assessment. In addition to comparing histology, immunohistochemistry, and general measures of DNA and RNA integrity in each fixation method, we performed functional tests of DNA and RNA quality, including targeted Miseq RNA and DNA sequencing, and implemented methods to relate DNA and RNA yield and quality to quantified DNA and RNA picogram nuclear content in each tissue volume studied. Our results suggest that it is feasible to use PFPE tissue for routine robot-assisted laparoscopic prostatectomy surgical pathology diagnostics and immunohistochemistry, with the benefit of significantly improvedDNA and RNA quality and RNA picogram yield per nucleus as compared with FFPE tissue. For fresh frozen, FFPE, and PFPE tissues, respectively, the average Genomic Quality Numbers were 7.9, 3.2, and 6.2, average RNA Quality Numbers were 8.7, 2.6, and 6.3, average DNA picogram yields per nucleus were 0.41, 0.69, and 0.78, and average RNA picogram yields per nucleus were 1.40, 0.94, and 2.24. These findings suggest that where DNA and/or RNA analysis of tissue is required, and when tissue size is small, PFPE may provide important advantages over FFPE. The results also suggest several interesting nuances including potential avenues to improve RNA quality in FFPE tissues and confirm recent suggestions that some DNA sequence artifacts associated with FFPE can be avoided.

Digital image analysis of the tissue surface areas of site-designated and bilaterally pooled prostate biopsies.

Initial reports about the length of bilaterally pooled biopsies showed alarming tissue loss compared to individual biopsies, but the current understanding of "noodle biopsies" and better embedding techniques may have improved their quality. Here, we implemented digital image analysis to study the differences in tissue surface areas between individual and pooled cores. Prostate biopsy reports from 1242 consecutive patients were reviewed. Urologist-dependent bias on the biopsy quality was eliminated by identifying four urologists who submitted equally individual and bilaterally pooled biopsies. Digital image analysis was applied to the tissue surface areas of 936 virtual slides containing 1440 biopsy cores (12 cores per patient x 120 patients) taken by the four urologists. The median (range) surface areas were 73.8 mm² (40.1-102.5) for the site-designated (n=57) and 77.1 mm² (49.5-119.2) for the bilaterally pooled biopsies (n=63) (p=0.19). For three urologists, the median surface areas were 69.5 mm² (60.4-93.2), 75.5 mm² (48.2-98.7) and 78.2 mm² (47.1-92.7) for the site-designated and 79.2 mm² (49.5-116.4), 69.3mm² (49.6-119.2) and 79.2 mm² (55.1-96.7) for the pooled biopsies, respectively (p=0.58-0.75). For one urologist, the median surface area was marginally higher for the pooled biopsies, 68.1 mm² (40.1-102.5) vs. 81.6 mm² (62.7-108.8) (p=0.03). In conclusion, the histological yields of individual and pooled prostate biopsies were practically equal. The results should not be considered as a recommendation to increasingly submit unspecified bilateral cores but to encourage pathology laboratories to embed and cut all received prostate biopsies with special attention, regardless of submission type.

Androgen Receptor Deregulation Drives Bromodomain-Mediated Chromatin Alterations in Prostate Cancer.

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses.

Integrated clinical, whole-genome, and transcriptome analysis of multisampled lethal metastatic prostate cancer.

We report the first combined analysis of whole-genome sequence, detailed clinical history, and transcriptome sequence of multiple prostate cancer metastases in a single patient (A21). Whole-genome and transcriptome sequence was obtained from nine anatomically separate metastases, and targeted DNA sequencing was performed in cancerous and noncancerous foci within the primary tumor specimen removed 5 yr before death. Transcriptome analysis revealed increased expression of androgen receptor (AR)-regulated genes in liver metastases that harbored an AR p.L702H mutation, suggesting a dominant effect by the mutation despite being present in only one of an estimated 16 copies per cell. The metastases harbored several alterations to the PI3K/AKT pathway, including a clonal truncal mutation in PIK3CG and present in all metastatic sites studied. The list of truncal genomic alterations shared by all metastases included homozygous deletion of TP53, hemizygous deletion of RB1 and CHD1, and amplification of FGFR1. If the patient were treated today, given this knowledge, the use of second-generation androgen-directed therapies, cessation of glucocorticoid administration, and therapeutic inhibition of the PI3K/AKT pathway or FGFR1 receptor could provide personalized benefit. Three previously unreported truncal clonal missense mutations (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) were expressed at the RNA level and assessed as druggable. The truncal status of mutations may be critical for effective actionability and merit further study. Our findings suggest that a large set of deeply analyzed cases could serve as a powerful guide to more effective prostate cancer basic science and personalized cancer medicine clinical trials.

Microseminoprotein-Beta Expression in Different Stages of Prostate Cancer.

Microseminoprotein-beta (MSMB, MSMB) is an abundant secretory protein contributed by the prostate, and is implicated as a prostate cancer (PC) biomarker based on observations of its lower expression in cancerous cells compared with benign prostate epithelium. However, as the current literature on MSMB is inconsistent, we assessed the expression of MSMB at the protein and mRNA levels in a comprehensive set of different clinical stages of PC. Immunohistochemistry using monoclonal and polyclonal antibodies against MSMB was used to study protein expression in tissue specimens representing prostatectomies (n = 261) and in diagnostic needle biopsies from patients treated with androgen deprivation therapy (ADT) (n = 100), and in locally recurrent castration-resistant PC (CRPC) (n = 105) and CRPC metastases (n = 113). The transcript levels of MSMB, nuclear receptor co-activator 4 (NCOA4) and MSMB-NCOA4 fusion were examined by qRT-PCR in prostatectomy samples and by RNA-sequencing in benign prostatic hyperplasia, PC, and CRPC samples. We also measured serum MSMB levels and genotyped the single nucleotide polymorphism rs10993994 using DNA from the blood of 369 PC patients and 903 controls. MSMB expression in PC (29% of prostatectomies and 21% of needle biopsies) was more frequent than in CRPC (9% of locally recurrent CRPCs and 9% of CRPC metastases) (p<0.0001). Detection of MSMB protein was inversely correlated with the Gleason score in prostatectomy specimens (p = 0.024). The read-through MSMB-NCOA4 transcript was detected at very low levels in PC. MSMB levels in serum were similar in cases of PC and controls but were significantly associated with PC risk when adjusted for age at diagnosis and levels of free or total PSA (p<0.001). Serum levels of MSMB in both PC patients and controls were significantly associated with the rs10993994 genotype (p<0.0001). In conclusion, decreased expression of MSMB parallels the clinical progression of PC and adjusted serum MSMB levels are associated with PC risk.