Prostate-specific membrane antigen (PSMA) glycoforms in prostate cancer patients seminal plasma.

INTRODUCTION: Prostate-specific membrane antigen (PSMA) is a US Food and Drug Administration-approved theranostic target for prostate cancer (PCa). Although PSMA is known to be glycosylated, the composition and functional roles of its N-linked glycoforms have not been fully characterized. METHODS: PSMA was isolated from pooled seminal plasma from low-risk grade Groups 1 and 2 PCa patients. Intact glycopeptides were analyzed by mass spectrometry to identify site-specific glycoforms. RESULTS: We observed a rich distribution of PSMA glycoforms in seminal plasma from low and low-intermediate-risk PCa patients. Some interesting generalities can be drawn based on the predicted topology of PSMA on the plasma membrane. The glycoforms at ASN-459, ASN-476, and ASN-638 residues that are located at the basal domain facing the plasma membrane in cells, are predominantly high mannose glycans. ASN-76 which is located in the interdomain region adjacent to the apical domain of the protein shows a mixture of high mannose glycans and complex glycans, whereas ASN-121, ASN-195 and ASN-336 that are located and are exposed at the apical domain of the protein predominantly possess complex sialylated and fucosylated N-linked glycans. These highly accessible glycosites display the greatest diversity in isoforms across the patient samples. CONCLUSIONS: Our study provides novel qualitative insights into PSMA glycoforms that are present in the seminal fluid of PCa patients. The presence of a rich diversity of glycoforms in seminal plasma provides untapped potential for glycoprotein biomarker discovery and as a clinical sample for noninvasive diagnostics of male urological disorders and diseases including PCa. Specifically, our glycomics approach will be critical in uncovering PSMA glycoforms with utility in staging and risk stratification of PCa.

Increased α2,3-sialyl N-glycosylated prostate-specific membrane antigen (PSMA) in post-DRE urine is associated with high grade group prostate cancer.

INTRODUCTION: Aberrant glycosylation of proteins is an important hallmark in multiple cancers. Prostate-specific membrane antigen (PSMA), a highly glycosylated protein with 10 N-linked glycosylation sites, is an Food and Drug Administration approved theranostic for prostate cancer. However, glycosylation changes in PSMA that are associated with prostate cancer disease progression have not been fully characterized. METHODS: We investigated whether urinary PSMA sialylation correlate with high-grade prostate cancer. Urine samples were collected from men after digital rectal examination (DRE) before prostate biopsy. Lectin-antibody enzyme-linked immunoassay was used to quantify α2,3-sialyl PSMA in post-DRE urine samples from subjects with benign prostate tumors, Grade Group 1 prostate cancer and those with Grade Group ≥2 disease. RESULTS: There are significant increases in α2,3-sialylated PSMA in patients with Grade Group ≥2 disease compared to benign (p = 0.0009) and those with Grade Group 1 disease (p = 0.0063). There were no significant differences in α2,3-sialyl PSMA levels between Grade Group 1 and benign prostate tumors (p = 0.7947). CONCLUSIONS: Our study shows that there are significant differences in the abundance of α2,3-sialylated PSMA in post-DRE urines from disease stratified prostate cancer patients, and the increase is correlated with progression and disease severity. The detection of increased PSMA sialyation in post-DRE urines from patients with higher Grade Group ≥2 disease states provides novel untapped potential for the development of prognostic biomarkers for prostate cancer. Specifically, quantitation of α2,3-sialylated PSMA shows potential for discriminating between benign to intermediate grade disease, which is a significant clinical challenge in staging and risk stratification of prostate cancer.

ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression.

Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Assessment of the Banff Working Group classification of definitive BK polyomavirus nephropathy.

Polyomavirus associated nephropathy (PyVAN) continues to be a burden in renal transplantation leading to allograft insufficiency or graft failure. A presumptive diagnosis of PyVAN is made based on the presence of BK polyomavirus in patients' plasma; however, kidney biopsy remains the gold standard to establish a definitive diagnosis. The Banff Working Group on PyVAN proposed a novel classification of definitive PyVAN based on polyomavirus replication/load level and the extent of interstitial fibrosis. The aim of our study was to test the newly defined classes of PyVAN using independent cohorts of 124 kidney transplant patients with PyVAN with respect to the initial presentation and outcome, and to compare our analysis to that previously reported. Detailed analysis of our cohort revealed that the proposed classification of PyVAN did not stratify or identify patients at increased risk of allograft failure. Specifically, while class 3 was associated with the worst prognosis, there was no significant difference between the outcomes in classes 1 and 2. We also found that the timing post-transplantation and inflammation in areas of interstitial fibrosis and tubular atrophy might be additional factors contributing to an unfavorable allograft outcome in patients with PyVAN.

Complex-I Alteration and Enhanced Mitochondrial Fusion Are Associated With Prostate Cancer Progression.

Mitochondria (mt) encoded respiratory complex-I (RCI) mutations and their pathogenicity remain largely unknown in prostate cancer (PCa). Little is known about the role of mtDNA loss on mt integrity in PCa. We determined mtDNA mutation in human and mice PCa and assessed the impact of mtDNA depletion on mt integrity. We also examined whether the circulating exosomes from PCa patients are transported to mt and carry mtDNA or mt proteins. We have employed next generation sequencing of the whole mt genome in human and Hi-myc PCa. The impact of mtDNA depletion on mt integrity, presence of mtDNA, and protein in sera exosomes was determined. A co-culture of human PCa cells and the circulating exosomes followed by confocal imaging determined co-localization of exosomes and mt. We observed frequent RCI mutations in human and Hi-myc PCa which disrupted corresponding complex protein expression. Depletion of mtDNA in PCa cells influenced mt integrity, increased expression of MFN1, MFN2, PINK1, and decreased expression of MT-TFA. Increased mt fusion and expression of PINK1 and DNM1L were also evident in the Hi-myc tumors. RCI-mtDNA, MFN2, and IMMT proteins were detected in the circulating exosomes of men with benign prostate hyperplasia (BPH) and progressive PCa. Circulating exosomes and mt co-localized in PCa cells. Our study identified new pathogenic RCI mutations in PCa and defined the impact of mtDNA loss on mt integrity. Presence of mtDNA and mt proteins in the circulating exosomes implicated their usefulness for biomarker development.

Loss of Expression of AZGP1 Is Associated With Worse Clinical Outcomes in a Multi-Institutional Radical Prostatectomy Cohort.

BACKGROUND: Given the uncertainties inherent in clinical measures of prostate cancer aggressiveness, clinically validated tissue biomarkers are needed. We tested whether Alpha-2-Glycoprotein 1, Zinc-Binding (AZGP1) protein levels, measured by immunohistochemistry, and RNA expression, by RNA in situ hybridization (RISH), predict recurrence after radical prostatectomy independent of clinical and pathological parameters. METHODS: AZGP1 IHC and RISH were performed on a large multi-institutional tissue microarray resource including 1,275 men with 5 year median follow-up. The relationship between IHC and RISH expression levels was assessed using the Kappa analysis. Associations with clinical and pathological parameters were tested by the Chi-square test and the Wilcoxon rank sum test. Relationships with outcome were assessed with univariable and multivariable Cox proportional hazards models and the Log-rank test. RESULTS: Absent or weak expression of AZGP1 protein was associated with worse recurrence free survival (RFS), disease specific survival, and overall survival after radical prostatectomy in univariable analysis. AZGP1 protein expression, along with pre-operative serum PSA levels, surgical margin status, seminal vesicle invasion, extracapsular extension, and Gleason score predicted RFS on multivariable analysis. Similarly, absent or low AZGP1 RNA expression by RISH predicted worse RFS after prostatectomy in univariable and multivariable analysis. CONCLUSIONS: In our large, rigorously designed validation cohort, loss of AZGP1 expression predicts RFS after radical prostatectomy independent of clinical and pathological variables. Prostate 76:1409-1419, 2016. © 2016 Wiley Periodicals, Inc.

Outcomes of Active Surveillance for Clinically Localized Prostate Cancer in the Prospective, Multi-Institutional Canary PASS Cohort.

PURPOSE: Active surveillance represents a strategy to address the overtreatment of prostate cancer, yet uncertainty regarding individual patient outcomes remains a concern. We evaluated outcomes in a prospective multicenter study of active surveillance. MATERIALS AND METHODS: We studied 905 men in the prospective Canary PASS enrolled between 2008 and 2013. We collected clinical data at study entry and at prespecified intervals, and determined associations with adverse reclassification, defined as increased Gleason grade or greater cancer volume on followup biopsy. We also evaluated the relationships of clinical parameters with pathology findings in participants who underwent surgery after a period of active surveillance. RESULTS: At a median followup of 28 months 24% of participants experienced adverse reclassification, of whom 53% underwent treatment while 31% continued on active surveillance. Overall 19% of participants received treatment, 68% with adverse reclassification, while 32% opted for treatment without disease reclassification. In multivariate Cox proportional hazards modeling the percent of biopsy cores with cancer, body mass index and prostate specific antigen density were associated with adverse reclassification (p=0.01, 0.04, 0.04, respectively). Of 103 participants subsequently treated with radical prostatectomy 34% had adverse pathology, defined as primary pattern 4-5 or nonorgan confined disease, including 2 with positive lymph nodes, with no significant relationship between risk category at diagnosis and findings at surgery (p=0.76). CONCLUSIONS: Most men remain on active surveillance at 5 years without adverse reclassification or adverse pathology at surgery. However, clinical factors had only a modest association with disease reclassification, supporting the need for approaches that improve the prediction of this outcome.

A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer.

BACKGROUND: Loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision-making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study. METHODS: We used recently developed probes optimized for sensitivity and specificity in a four-color FISH deletion assay to study the Canary Retrospective multicenter Prostate Cancer Tissue Microarray (TMA). This TMA was constructed specifically for biomarker validation from radical prostatectomy specimens, and is accompanied by detailed clinical information with long-term follow-up. RESULTS: In 612 prostate cancers, the overall rate of PTEN deletion was 112 (18.3%). Hemizygous PTEN losses were present in 55/612 (9.0%) of cancers, whereas homozygous PTEN deletion was observed in 57/612 (9.3%) of tumors. Significant associations were found between PTEN status and pathologic stage (P < 0.0001), seminal vesicle invasion (P = 0.0008), extracapsular extension (P < 0.0001), and Gleason score (P = 0.0002). In logistic regression analysis of clinical and pathological variables, PTEN deletion was significantly associated with extracapsular extension, seminal vesicle involvement, and higher Gleason score. In the 406 patients in which clinical information was available, PTEN homozygous (P = 0.009) deletion was associated with worse post-operative recurrence-free survival (number of events = 189), pre-operative prostate specific antigen (PSA) (P < 0.001), and pathologic stage (P = 0.03). CONCLUSION: PTEN status assessed by FISH is an independent predictor for recurrence-free survival in multivariate models, as were seminal vesicle invasion, extracapsular extension, and Gleason score, and preoperative PSA. Furthermore, these data demonstrate that the assay can be readily introduced at first diagnosis in a cost effective manner analogous to the use of FISH for analysis of HER2/neu status in breast cancer. Combined with published research beginning 17 years ago, both the data and tools now exist to implement a PTEN assay in the clinic.

MALDI imaging mass spectrometry profiling of proteins and lipids in clear cell renal cell carcinoma.

Reducing the incidence and mortality rates for clear cell renal cell carcinoma (ccRCC) remains a significant clinical challenge with poor 5-year survival rates. A unique tissue cohort was assembled of matched ccRCC and distal nontumor tissues (n = 20) associated with moderate risk of disease progression, half of these from individuals who progressed to metastatic disease and the other half who remained disease free. These tissues were used for MALDI imaging MS profiling of proteins in the 2-20 kDa range, resulting in a panel of 108 proteins that had potential disease-specific expression patterns. Protein lysates from the same tissues were analyzed by MS/MS, resulting in identification of 56 proteins of less than 20 kDa molecular weight. The same tissues were also used for global lipid profiling analysis by MALDI-FT-ICR MS. From the cumulative protein and lipid expression profile data, a refined panel of 26 proteins and 39 lipid species was identified that could either distinguish tumor from nontumor tissues, or tissues from recurrent disease progressors from nonrecurrent disease individuals. This approach has the potential to not only improve prognostic assessment and enhance postoperative surveillance, but also to inform on the underlying biology of ccRCC progression.

Clinical validation of an epigenetic assay to predict negative histopathological results in repeat prostate biopsies.

PURPOSE: The DOCUMENT multicenter trial in the United States validated the performance of an epigenetic test as an independent predictor of prostate cancer risk to guide decision making for repeat biopsy. Confirming an increased negative predictive value could help avoid unnecessary repeat biopsies. MATERIALS AND METHODS: We evaluated the archived, cancer negative prostate biopsy core tissue samples of 350 subjects from a total of 5 urological centers in the United States. All subjects underwent repeat biopsy within 24 months with a negative (controls) or positive (cases) histopathological result. Centralized blinded pathology evaluation of the 2 biopsy series was performed in all available subjects from each site. Biopsies were epigenetically profiled for GSTP1, APC and RASSF1 relative to the ACTB reference gene using quantitative methylation specific polymerase chain reaction. Predetermined analytical marker cutoffs were used to determine assay performance. Multivariate logistic regression was used to evaluate all risk factors. RESULTS: The epigenetic assay resulted in a negative predictive value of 88% (95% CI 85-91). In multivariate models correcting for age, prostate specific antigen, digital rectal examination, first biopsy histopathological characteristics and race the test proved to be the most significant independent predictor of patient outcome (OR 2.69, 95% CI 1.60-4.51). CONCLUSIONS: The DOCUMENT study validated that the epigenetic assay was a significant, independent predictor of prostate cancer detection in a repeat biopsy collected an average of 13 months after an initial negative result. Due to its 88% negative predictive value adding this epigenetic assay to other known risk factors may help decrease unnecessary repeat prostate biopsies.

Identification of differentially expressed proteins in direct expressed prostatic secretions of men with organ-confined versus extracapsular prostate cancer.

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.

High expression of Trop2 is associated with aggressive localized prostate cancer and is a candidate urinary biomarker.

Distinguishing indolent from clinically significant localized prostate cancer is a major clinical challenge and influences clinical decision-making between treatment and active surveillance. The development of novel predictive biomarkers will help with risk stratification, and clinical decision-making, leading to a decrease in over or under-treatment of patients with prostate cancer. Here, we report that Trop2 is a prognostic tissue biomarker for clinically significant prostate cancer by utilizing the Canary Prostate Cancer Tissue Microarray (CPCTA) cohort composed of over 1100 patients from a multi-institutional study. We demonstrate that elevated Trop2 expression is correlated with worse clinical features including Gleason score, age, and pre-operative PSA levels. More importantly, we demonstrate that elevated Trop2 expression at radical prostatectomy predicts worse overall survival in men undergoing radical prostatectomy. Additionally, we detect shed Trop2 in urine from men with clinically significant prostate cancer. Our study identifies Trop2 as a novel tissue prognostic biomarker and a candidate non-invasive marker for prostate cancer.

In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine.

Expressed prostatic secretions (EPS) are proximal fluids of the prostate that are increasingly being utilized as a clinical source for diagnostic and prognostic assays for prostate cancer (PCa). These fluids contain an abundant amount of microvesicles reflecting the secretory function of the prostate gland, and their protein composition remains poorly defined in relation to PCa. Using expressed prostatic secretions in urine (EPS-urine), exosome preparations were characterized by a shotgun proteomics procedure. In pooled EPS-urine exosome samples, ~900 proteins were detected. Many of these have not been previously observed in the soluble proteome of EPS generated by our labs or other related exosome proteomes. We performed systematic comparisons of our data against previously published, prostate-related proteomes, and global annotation analyses to highlight functional processes within the proteome of EPS-urine derived exosomes. The acquired proteomic data have been deposited to the Tranche repository and will lay the foundation for more extensive investigations of PCa derived exosomes in the context of biomarker discovery and cancer biology.

Differential Activation of NRF2 Signaling Pathway in Renal-Cell Carcinoma Caki Cell Lines.

Renal-cell carcinoma (RCC) is a heterogeneous disease consisting of several subtypes based on specific genomic profiles and histological and clinical characteristics. The subtype with the highest prevalence is clear-cell RCC (ccRCC), next is papillary RCC (pRCC), and then chromophobe RCC (chRCC). The ccRCC cell lines are further subdivided into prognostic expression-based subtypes ccA or ccB. This heterogeneity necessitates the development, availability, and utilization of cell line models with the correct disease phenotypic characteristics for RCC research. In this study, we focused on characterizing proteomic differences between the Caki-1 and Caki-2 cell lines that are commonly used in ccRCC research. Both cells are primarily defined as human ccRCC cell lines. Caki-1 cell lines are metastatic, harboring wild-type VHL, whereas Caki-2 are considered as the primary ccRCC cell lines expressing wild-type von Hippel-Lindau protein (pVHL). Here, we performed a comprehensive comparative proteomic analysis of Caki-1 and Caki-2 cells using tandem mass-tag reagents together with liquid chromatography mass spectrometry (LC/MS) for the identification and quantitation of proteins in the two cell lines. Differential regulation of a subset of the proteins identified was validated using orthogonal methods including western blot, q-PCR, and immunofluorescence assays. Integrative bioinformatic analysis identifies the activation/inhibition of specific molecular pathways, upstream regulators, and causal networks that are uniquely regulated and associated with the two cell lines and RCC subtypes, and potentially the disease stage. Altogether, we have identified multiple molecular pathways, including NRF2 signaling, which is the most significantly activated pathway in Caki-2 versus Caki-1 cells. Some of the differentially regulated molecules and signaling pathways could serve as potential diagnostic and prognostic biomarkers and therapeutic targets amongst ccRCC subtypes.

Identification of prostate-enriched proteins by in-depth proteomic analyses of expressed prostatic secretions in urine.

Urinary expressed prostatic secretion or "EPS-urine" is proximal tissue fluid that is collected after a digital rectal exam (DRE). EPS-urine is a rich source of prostate-derived proteins that can be used for biomarker discovery for prostate cancer (PCa) and other prostatic diseases. We previously conducted a comprehensive proteome analysis of direct expressed prostatic secretions (EPS). In the current study, we defined the proteome of EPS-urine employing Multidimensional Protein Identification Technology (MudPIT) and providing a comprehensive catalogue of this body fluid for future biomarker studies. We identified 1022 unique proteins in a heterogeneous cohort of 11 EPS-urines derived from biopsy negative noncancer diagnoses with some benign prostatic diseases (BPH) and low-grade PCa, representative of secreted prostate and immune system-derived proteins in a urine background. We further applied MudPIT-based proteomics to generate and compare the differential proteome from a subset of pooled urines (pre-DRE) and EPS-urines (post-DRE) from noncancer and PCa patients. The direct proteomic comparison of these highly controlled patient sample pools enabled us to define a list of prostate-enriched proteins detectable in EPS-urine and distinguishable from a complex urine protein background. A combinatorial analysis of both proteomics data sets and systematic integration with publicly available proteomics data of related body fluids, human tissue transcriptomic data, and immunohistochemistry images from the Human Protein Atlas database allowed us to demarcate a robust panel of 49 prostate-derived proteins in EPS-urine. Finally, we validated the expression of seven of these proteins using Western blotting, supporting the likelihood that they originate from the prostate. The definition of these prostatic proteins in EPS-urine samples provides a reference for future investigations for prostatic-disease biomarker studies.

Site-Specific Intact N-Linked Glycopeptide Characterization of Prostate-Specific Membrane Antigen from Metastatic Prostate Cancer Cells.

The composition of N-linked glycans that are conjugated to the prostate-specific membrane antigen (PSMA) and their functional significance in prostate cancer progression have not been fully characterized. PSMA was isolated from two metastatic prostate cancer cell lines, LNCaP and MDAPCa2b, which have different tissue tropism and localization. Isolated PSMA was trypsin-digested, and intact glycopeptides were subjected to LC-HCD-EThcD-MS/MS analysis on a Tribrid Orbitrap Fusion Lumos mass spectrometer. Differential qualitative and quantitative analysis of site-specific N-glycopeptides was performed using Byonic and Byologic software. Comparative quantitative analysis demonstrates that multiple glycopeptides at asparagine residues 51, 76, 121, 195, 336, 459, 476, and 638 were in significantly different abundance in the two cell lines (p < 0.05). Biochemical analysis using endoglycosidase treatment and lectin capture confirm the MS and site occupancy data. The data demonstrate the effectiveness of the strategy for comprehensive analysis of PSMA glycopeptides. This approach will form the basis of ongoing experiments to identify site-specific glycan changes in PSMA isolated from disease-stratified clinical samples to uncover targets that may be associated with disease progression and metastatic phenotypes.

Prospective comparison of restriction spectrum imaging and non-invasive biomarkers to predict upgrading on active surveillance prostate biopsy.

BACKGROUND: Protocol-based active surveillance (AS) biopsies have led to poor compliance. To move to risk-based protocols, more accurate imaging biomarkers are needed to predict upgrading on AS prostate biopsy. We compared restriction spectrum imaging (RSI-MRI) generated signal maps as a biomarker to other available non-invasive biomarkers to predict upgrading or reclassification on an AS biopsy. METHODS: We prospectively enrolled men on prostate cancer AS undergoing repeat biopsy from January 2016 to June 2019 to obtain an MRI and biomarkers to predict upgrading. Subjects underwent a prostate multiparametric MRI and a short duration, diffusion-weighted enhanced MRI called RSI to generate a restricted signal map along with evaluation of 30 biomarkers (14 clinico-epidemiologic features, 9 molecular biomarkers, and 7 radiologic-associated features). Our primary outcome was upgrading or reclassification on subsequent AS prostate biopsy. Statistical analysis included operating characteristic improvement using AUROC and AUPRC. RESULTS: The individual biomarker with the highest area under the receiver operator characteristic curve (AUC) was RSI-MRI (AUC = 0.84; 95% CI: 0.71-0.96). The best non-imaging biomarker was prostate volume-corrected Prostate Health Index density (PHI, AUC = 0.68; 95% CI: 0.53-0.82). Non-imaging biomarkers had a negligible effect on predicting upgrading at the next biopsy but did improve predictions of overall time to progression in AS. CONCLUSIONS: RSI-MRI, PIRADS, and PHI could improve the predictive ability to detect upgrading in AS. The strongest predictor of clinically significant prostate cancer on AS biopsy was RSI-MRI signal output.

Development and validation of a quantitative reactive stroma biomarker (qRS) for prostate cancer prognosis.

To develop and validate a new tissue-based biomarker that improves prediction of outcomes in localized prostate cancer by quantifying the host response to tumor. We use digital image analysis and machine learning to develop a biomarker of the prostate stroma called quantitative reactive stroma (qRS). qRS is a measure of percentage tumor area with a distinct, reactive stromal architecture. Kaplan Meier analysis was used to determine survival in a large retrospective cohort of radical prostatectomy samples. qRS was validated in two additional, distinct cohorts that include international cases and tissue from both radical prostatectomy and biopsy specimens. In the developmental cohort (Baylor College of Medicine, n = 482), patients whose tumor had qRS > 34% had increased risk of prostate cancer-specific death (HR 2.94; p = 0.039). This result was replicated in two validation cohorts, where patients with qRS > 34% had increased risk of prostate cancer-specific death (MEDVAMC; n = 332; HR 2.64; p = 0.02) and also biochemical recurrence (Canary; n = 988; HR 1.51; p = 0.001). By multivariate analysis, these associations were shown to hold independent predictive value when compared to currently used clinicopathologic factors including Gleason score and PSA. qRS is a new, validated biomarker that predicts prostate cancer death and biochemical recurrence across three distinct cohorts. It measures host-response rather than tumor-based characteristics, and provides information not represented by standard prognostic measurements.

MALDI tissue imaging: from biomarker discovery to clinical applications.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for the generation of multidimensional spatial expression maps of biomolecules directly from a tissue section. From a clinical proteomics perspective, this method correlates molecular detail to histopathological changes found in patient-derived tissues, enhancing the ability to identify candidates for disease biomarkers. The unbiased analysis and spatial mapping of a variety of molecules directly from clinical tissue sections can be achieved through this method. Conversely, targeted IMS, by the incorporation of laser-reactive molecular tags onto antibodies, aptamers, and other affinity molecules, enables analysis of specific molecules or a class of molecules. In addition to exploring tissue during biomarker discovery, the integration of MALDI-IMS methods into existing clinical pathology laboratory practices could prove beneficial to diagnostics. Querying tissue for the expression of specific biomarkers in a biopsy is a critical component in clinical decision-making and such markers are a major goal of translational research. An important challenge in cancer diagnostics will be to assay multiple parameters in a single slide when tissue quantities are limited. The development of multiplexed assays that maximize the yield of information from a small biopsy will help meet a critical challenge to current biomarker research. This review focuses on the use of MALDI-IMS in biomarker discovery and its potential as a clinical diagnostic tool with specific reference to our application of this technology to prostate cancer.

Molecular preservation by extraction and fixation, mPREF: a method for small molecule biomarker analysis and histology on exactly the same tissue.

BACKGROUND: Histopathology is the standard method for cancer diagnosis and grading to assess aggressiveness in clinical biopsies. Molecular biomarkers have also been described that are associated with cancer aggressiveness, however, the portion of tissue analyzed is often processed in a manner that is destructive to the tissue. We present here a new method for performing analysis of small molecule biomarkers and histology in exactly the same biopsy tissue. METHODS: Prostate needle biopsies were taken from surgical prostatectomy specimens and first fixed, each in a separate vial, in 2.5 ml of 80% methanol:water. The biopsies were fixed for 24 hrs at room temperature and then removed and post-processed using a non-formalin-based fixative (UMFIX), embedded, and analyzed by hematoxylin and eosin (H&E) and by immunohistochemical (IHC) staining. The retained alcohol pre-fixative was analyzed for small molecule biomarkers by mass spectrometry. RESULTS: H&E analysis was successful following the pre-fixation in 80% methanol. The presence or absence of tumor could be readily determined for all 96 biopsies analyzed. A subset of biopsy sections was analyzed by IHC, and cancerous and non-cancerous regions could be readily visualized by PIN4 staining. To demonstrate the suitability for analysis of small molecule biomarkers, 28 of the alcohol extracts were analyzed using a mass spectrometry-based metabolomics platform. All extracts tested yielded successful metabolite profiles. 260 named biochemical compounds were detected in the alcohol extracts. A comparison of the relative levels of compounds in cancer containing vs. non-cancer containing biopsies showed differences for 83 of the compounds. A comparison of the results with prior published reports showed good agreement between the current method and prior reported biomarker discovery methods that involve tissue destructive methods. CONCLUSIONS: The Molecular Preservation by Extraction and Fixation (mPREF) method allows for the analysis of small molecule biomarkers from exactly the same tissue that is processed for histopathology.