Formalin-fixed Paraffin-embedded Prostate Research Articles

Multinational, Multicenter Evaluation of Prostate Cancer Tissue in Sub-Saharan Africa: Challenges and Opportunities.

Prostate cancer disproportionately affects men of African descent, yet their representation in tissue-based studies is limited. This multinational, multicenter pilot study aims to establish the groundwork for collaborative research on prostate cancer in sub-Saharan Africa. The Men of African Descent and Carcinoma of the Prostate network formed a pathologist working group representing eight institutions in five African countries. Formalin-fixed paraffin-embedded prostate tissue specimens were collected from Senegal, Nigeria, and Ghana. Histology slides were produced and digitally scanned. A central genitourinary pathologist (P.L.) and eight African general pathologists reviewed anonymized digital whole-slide images for International Society of Urological Pathology grade groups and other pathologic parameters. Discrepancies were re-evaluated, and consensus grading was assigned. A virtual training seminar on prostate cancer grading was followed by a second assessment on a subcohort of the same tissue set. Of 134 tissue blocks, 133 had evaluable tissue; 13 lacked cancer evidence, and four were of insufficient quality. Post-training, interobserver agreement for grade groups improved to 56%, with a median Cohen's quadratic weighted kappa of 0.83 (mean, 0.74), compared with an initial 46% agreement and a quadratic weighted kappa of 0.77. Interobserver agreement between African pathologist groups was 40%, with a quadratic weighted kappa of 0.66 (95% CI, 0.51 to 0.76). African pathologists tended to overgrade (36%) more frequently than undergrade (18%) compared with the reference genitourinary pathologist. Interobserver variability tended to worsen with a decrease in tissue quality. Tissue-based studies on prostate cancer in men of African descent are essential for a better understanding of this common disease. Standardized tissue handling protocols are crucial to ensure good tissue quality and data. The use of digital slide imaging can enhance collaboration among pathologists in multinational, multicenter studies.

Molecular analysis of archival diagnostic prostate cancer biopsies identifies genomic similarities in cases with progression post-radiotherapy, and those with de novo metastaticdisease.

It is important to identify molecular features that improve prostate cancer (PCa) risk stratification before radical treatment with curative intent. Molecular analysis of historical diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies from cohorts with post-radiotherapy (RT) long-term clinical follow-up has been limited. Utilizing parallel sequencing modalities, we performed a proof-of-principle sequencing analysis of historical diagnostic FFPE prostate biopsies. We compared patients with (i) stable PCa (sPCa)postprimary or salvage RT,(ii) progressing PCa (pPCa) post-RT,and (iii) de novo metastatic PCa(mPCa). A cohort of 19 patients with diagnostic prostate biopsies (n = 6 sPCa, n = 5 pPCa, n = 8 mPCa) and mean 4 years 10 months follow-up (diagnosed 2009-2016) underwent nucleic acid extraction from demarcated malignancy. Samples underwent 3'RNA sequencing (3'RNAseq) (n = 19), nanoString analysis (n = 12), and Illumina 850k methylation (n = 8) sequencing. Bioinformatic analysis was performed to coherently identify differentially expressed genes and methylated genomic regions (MGRs). Eighteen of 19 samples provided useable 3'RNAseq data. Principal component analysis (PCA) demonstrated similar expression profiles between pPCa and mPCa cases, versus sPCa. Coherently differentially methylated probes between these groups identified ~600 differentially MGRs. The top 50 genes with increased expression in pPCa patients were associated with reduced progression-free survival post-RT (p < 0.0001) in an external cohort. 3'RNAseq, nanoString and 850k-methylation analyses are each achievable from historical FFPE diagnostic pretreatment prostate biopsies, unlocking the potential to utilize large cohorts of historic clinical samples. Profiling similarities between individuals with pPCa and mPCa suggests biological similarities and historical radiological staging limitations, which warrant further investigation.

Abstract 7028: Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers

Abstract Prostate cancer is the second leading cause of cancer related deaths among men in the United States. Health disparities in men diagnosed with prostate cancer are observed between patients of African and those of European ancestry. To elucidate molecular mechanisms behind prostate tumorigenesis and racial disparities, researchers have identified genetic alterations such as gene fusions, deletions, and mutations that occur at varying frequencies among different ethnic groups. However, results are inconsistent across studies, suggesting that racial disparities may be multifactorial. Our approach to further understand the mechanisms underlying racial disparities is to compare DNA methylation profiles between cancers from different ethnic groups using formalin-fixed paraffin-embedded (FFPE) prostate cancer tissue samples. For this study, FFPE prostate cancer specimens from 32 Black men and 30 age-matched White men were obtained in the form of tissue sections on glass slides. To profile DNA methylation in these samples, tumor and normal tissue regions (in a subset of cases) were outlined on a hematoxylin &amp; eosin (H&amp;E) reference slide and the respective regions were macro-dissected off the corresponding unstained slides from the same block. The areas of macro-dissected regions were measured using ImageJ, and the number of slides were taken to normalize DNA yield. Next, we compared magnetic bead and column based DNA extraction methods. We evaluated them on their relative DNA yield per tissue size, and identified that the bead-based extraction method had both higher and more consistent DNA yield. In total, we used 95 unique tissue regions to profile global DNA methylation levels using Illumina Epic array. We found that a subset of samples harbored an aberrant DNA methylation pattern. Therefore, we extracted DNA from additional FFPE tissue slides using the bead-based method and repeated the assay with a larger amount of DNA, successfully rescuing the majority of the aberrant samples. Next, we will evaluate associations between DNA methylation levels in different ethnic groups, clinicopathological features, and follow-up data to help identify potential DNA methylation biomarkers and therapeutic targets linked to prostate cancer from different ethnicities. Citation Format: Colton Duran Stensrud, Leonardo Gonzalez-Smith, Claire Stevens, Huan Cao, Jenaye Mack, Olumide Arigbede, Daniel Weisenberger, Sarah G. Buxbaum, Sara M. Falzarano, Suhn Rhie. Optimization of DNA extraction methods and DNA methylation array quality from FFPE prostate tumor tissues to identify DNA methylation biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7028.

Association between copy number alterations estimated using low-pass whole genome sequencing of formalin-fixed paraffin-embedded prostate tumor tissue and cancer-specific clinical parameters

Copy number alterations (CNAs) are frequently observed in early-stage prostate cancer and are associated with disease recurrence and tumor aggressiveness. Cost-effective assessment of CNAs could enhance clinical utility of CNAs. Here, we combined the cost-effectiveness of low-pass (low coverage) whole genome sequencing (LPWGS) and the routine availability of formalin-fixed paraffin-embedded (FFPE) tumor tissue for assessing CNAs in a cohort of 187 men with early-stage localised prostate cancer. We detected well known CNAs in 8p, 8q, 13q and 16q and recurrent gains of the oncogene MYC and losses of the tumor suppressor genes NKX3-1, PTEN and RB1, indicating assay reliability. The estimated burden of CNAs was significantly associated with Gleason score, pathological T stage, surgical margin status and biochemical recurrence. Further, genomic losses or gains in specific chromosomal arms were significantly associated with worse BCR-free survival. Copy number signatures extracted from the LPWGS data showed potential for risk stratifying patients, where signatures S1 and S2 showed significant association to worse BCR-free survival compared to S3. Our study provides clinical validation of the associations between CNAs and tumor aggressiveness in an independent and representative RP cohort, while demonstrating the feasibility of performing LPWGS of FFPE tumor tissue for cost-effective assessment of CNAs.

Bioorthogonal Chemical Labeling Probes Targeting Sialic Acid Isomers for N-Glycan MALDI Imaging Mass Spectrometry of Tissues, Cells, and Biofluids.

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.

Abstract 2091: Open-source single-cell spatial image analysis pipeline for ten-plex immunofluorescence of histological prostate cancer tissue sections

Abstract Introduction: Multiplexed fluorescence immunohistochemistry (mfIHC) allows the detection of multiple protein markers on the same histological tissue section, generating valuable information on specific cell subsets and cellular states in their spatial tissue context. Here, we demonstrate a proof-of-concept study for profiling different tumor microenvironment cell subsets in formalin-fixed paraffin-embedded prostate cancer tissue microarrays. Materials and methods: We applied automated mfIHC simultaneous staining of 31 slides with 3.5-µm sections using an Autostainer (Epredia, Kalamazoo, USA). The protocol included four rounds of cyclic staining and scanning of the slides with AxioScanZ.1 (Zeiss, Germany). Each cycle included DAPI for nuclear staining. Here, we applied this pipeline to detect the following targets: D2-40 (lymphatics), CD3 (T lymphocytes), CD56 (NK cells/neural cells/neuroendocrine cells), CD11b+ myeloid cells, PDGFRB (fibroblasts/pericytes), CD20 (B lymphocytes), CD31 (endothelium), CD45 (leukocytes), HLADR+ antigene-presenting cells, as well as a cocktail with PanCK and ECADH to detect epithelial cells. Three different prostate cancer TMA cohorts with n=907 patient samples were analyzed here using this approach.The scanned images were exported as Big TIFF individual channel whole-slide images. Individual TMA spot images were marked with Fiji ROI1 1-Click Tool macro (ImageJ 1.53t), and separate rounds were registered with Python (version 3.11.0, Python Software Foundation) using DAPI channel as reference. Nuclear segmentation was created with nucleAIzer trained modified Mask R-CNN model (https://github.com/lopaavol/nuclei_segmentation). All TMA spot images were run through image curation, where artefacts are excluded by machine learning using ilastik software (version 1.3.3post3). Then, CellProfiler (version 4.2.1) was applied to combine image curated tissue masks and segmented nuclei for cell-based channel intensity measurements. Finally, Jupyter Notebook was applied to classify cell subsets into specific phenotypes using intensity-based thresholding for each channel. Results and Conclusions: We present a detailed pipeline for 10-plex mfIHC with open-source single-cell image analysis tools. The approach can be applied to histological laboratories when combined with a standard five fluorescent channel scanner or imager and with basic computing skills. As the system applies secondary antibodies for the detection, it is flexible for optimizing new antibody panels, and the user is not limited to pre-labelled antibody panels often connected with commercial staining and imaging systems. The approach is especially suited for large-scale high-throughput tissue profiling studies when high n-values are needed for linking associated patient clinical data with spatial phenotype data. Citation Format: Jenni Säilä, Annabrita Schoonenberg, Katja Välimäki, Timo-Pekka Lehto, Carolin Stürenberg, Andrew Erickson, Antti Rannikko, Olli Kallioniemi, Lassi Paavolainen, Tuomas Mirtti, Teijo Pellinen. Open-source single-cell spatial image analysis pipeline for ten-plex immunofluorescence of histological prostate cancer tissue sections [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2091.

Abstract 1961: Discovery and validation of prostate cancer biomarkers of biochemical recurrence in low-risk prostate cancer patients

Abstract Introduction: Prostate cancer (CaP) is predominantly indolent disease with a small fraction of patients undergoing metastatic progression. Therefore, it is critical to identify prognostic markers for the early detection of an aggressive subtype. We hypothesized that tissue specimens from early-stage, low risk CaP may harbor predictive prognostic signatures for disease progression after radical prostatectomy. Previously, we had systematically evaluated and optimized the NanoString platform for CaP gene expression analysis in formalin fixed paraffin embedded (FFPE) whole mounted prostate specimens. The goal of the study is to validate the CPDR findings on the differential mRNA expression of candidate markers from patients with BCR (biochemical recurrence) and non-BCR in an independent cohort. Methods: This is a retrospective case cohort study based on 78 tumor-normal paired (N=156) archived whole mounted FFPE prostate samples received from biobank at UTHSCSA. We performed an optimized NanoString analysis of 203-CaP probe set to evaluate the association of markers with BCR outcome in a racially diverse patient population comprising of 21.6% AA and 78.38% CA men. The cohort comprised of 28 patients with BCR, and 50 patients with no BCR (based on a minimum of 5 years of follow-up). ERG gene expression was validated by immunohistochemistry (IHC) assay using CPDR ERG-MAb (9FY). Normalized nCounter gene expression data was analyzed using the NanoString nSolver (v 4.0) platform. Fisher exact and Wilcoxon-Mann-Whitney tests were used for categorical and continuous variables respectively. Results: We found 96.1% (73/76) concordance between ERG IHC and NanoString datasets, which provides strong QC and validation for NanoString data. ERG positive tumors had strong expression of both TMPRSS2-ERG fusion and several other ERG splice forms. Additionally, the prostate epithelial cell markers PSA, MSMB and PAP had the highest signals in all samples reflecting the prostate epithelial origin of the specimens. Differential gene expression analysis showed that a total of 37 genes were found to be significantly (P adj &amp;lt;0.05) different across tumors and normal. Established CaP genes like AMACR, PCA3, OR51E2, ERG, HOXC6, DLX1, CACNA1D and PSGR were most highly over-expressed in tumors compared to matched normal while GSTP1, CAV1, PAP and MSMB were significantly downregulated in tumors compared to adjacent normal prostate epithelia. Race stratified analysis in this randomly selected patient cohort showed that AA men were found to be significantly younger (median age: 56 years AA vs. 63 years CA; p 0.01) and had higher frequency of BCR events (p 0.004) than CA men. Data analysis for disease outcome across AA and CA men is ongoing. Conclusions: We confirmed that NanoString approach is useful for evaluation of prognostic biomarker candidates in prostate cancer using FFPE specimens. Citation Format: Indu Kohaar, Kaitlyn Bejar, Denise Young, Yingjie Song, Jiji Jiang, Jacob Kagan, Sudhir Srivastava, Javier Hernandez, Gregory Chesnut, Isabell A. Sesterhenn, Robin J. Leach, Gyorgy Petrovics. Discovery and validation of prostate cancer biomarkers of biochemical recurrence in low-risk prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1961.

Abstract 2247: Prostate cancer-related mortality risk assessment in a cohort of southern Nigerian men

Abstract Background: Overexpression of breast cancer susceptibility gene 2 (BRCA2) in patients with prostate cancer is associated with a high Gleason score at diagnosis, poorly differentiated tumors, poor prognosis, and disease recurrence. Such aggressive tumors possess high iron storage which promotes inflammation, angiogenesis, and cell proliferation. Expression of iron progressively increases with cancer grade and stage. Purpose: This study evaluated the expression of BRCA2 protein and Iron expression in Gleason’s graded prostate cancer tissues, in a bid to determine the disease aggressiveness and mortality risk among Southern Nigerian men. Methods: In this retrospective study, 86 formalin-fixed paraffin-embedded prostate cancer tissues (diagnosed in 2018 and 2019) were reviewed using Gleason’s grading system. Tissues stained for iron and BRCA2 proteins using histochemical and immunohistochemical staining techniques, respectively. Staining intensity was scored using a scale of 0, +1, +2, and +3. For BRCA2 and Iron expressions, score 0 and +1 were considered negative while score +2 and +3 were considered positive. Pearson’s chi-square test was used to determine the association between Gleason’s grade and expression of BRCA2 and iron. Results: The prevalence of prostate cancer in the age groups of 50-59, 60-69, 70-79, 80-89, and 90-99 years was 10.8%, 32.4%, 40.5%, 13.5% and 2.7%, respectively. Using the Gleason’s grading pattern, the frequency of Grade (mean age in years) I, II, III, IV and V was 9.3% (70.25 ± 6.95), 16.3% (77.29 ± 6.23), 16.3% (75.57 ± 12.59), 25.6% (72.75 ± 7.80) and 32.6% (67.42 ± 9.76), respectively. Higher frequency of BRCA2 and Iron expression were observed in ≥G4 (37.5% and 72.7%) than in ≤G3 (versus 16.7% and 11.8%) at p= 0.191 and p&amp;lt; 0.001, respectively. The prevalence of BRCA2 negative and positive tissues was 65.1% and 34.9%, respectively whereas the prevalence of iron negative and positive tissues was 48.7% and 51.3%, respectively. There was an inverse relationship between tumour grade and age (p= 0.108), and direct relationships between tumour grade and BRCA2 (p= 0.059), tumour grade and iron (p= 0.000), BRCA2 and Iron expressions (p= 0.027), BRCA2 expression and age (p= 0.019), and Iron and age (p= 0.281). Patients with prostate cancer who are within the age group of 50-59 years and those with ≥G4 require close monitoring for improved survival. Conclusion: This study revealed a high prevalence of aggressive prostate cancer and suggests that there is a high mortality risk among Southern Nigerian men diagnosed with the disease. Citation Format: Jude O. Okoye, Vivian I. Ogbonnaya, Chiemeka M. Emeka, Okechi Obioma. Prostate cancer-related mortality risk assessment in a cohort of southern Nigerian men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2247.

First results of the PROMPT trial: Precision oncology allocation in patients with early castration-resistant prostate cancer following routine molecular profiling.

40 Background: Castration-resistant prostate cancer (CRPC) comprises distinct molecular actionable subtypes. Radboudumc, a tertiary referral center for PCa, hosts a Molecular Tumor Board (MTB), and frequently sees CRPC patients (pts) for referral for genetic testing. In the period 2017-2020, almost 50% of heavily pre-treated CRPC pts received a recommendation for a genetically matched therapy (GMT). In 2020 we initiated the PROMPT trial (NCT04746300) to prospectively study the impact of routine early molecular characterization in CRPC. Increased GMT utilization of targeted- and immunotherapy may improve outcome and quality of life of pts with metastatic CRPC, and lead to lower medical resource utilization (MRU). Here we report on first results from our observational trial. Methods: Within the PROMPT trial, prior to receiving first- or second line standard of care CRPC therapy, up to 300 pts were offered molecular tumor characterization with the TruSightOncology 500 panel (TSO500). Formalin-fixed paraffin-embedded prostate or metastatic tissue biopsies were used, preferably newly obtained. To assess both mutations and copy number alterations, the TSO500 required a minimal tumor cell percentage (TC%) of 30%. Results were discussed within the Radboudumc MTB for GMT recommendation. Actionable targets were defined per Precision Medicine Working group criteria and, when druggable, within the Drug Rediscovery Protocol trial (NCT02925234). All pts with tumor mutations carrying with a risk for cancer predisposition were referred for genetic counselling. Follow-up with patient-reported outcomes (EORTC QLQ-C30, EQ5D, BPI &amp; EPIC-26 questionnaires) and MRU was conducted every 3 months until withdrawal or death. Results: From February 2020 until October 2021 we included 284 consecutive CRPC pts with a median age of 70 years (range 46-86) with a median follow-up of 6.8 months. Newly obtained biopsies and archival material was used in 131 and 126 pts, respectively. Median TC% was 60% (range: 20-90%). TSO500 results could be reported in 254 (89%) cases, with at least one putative clinically relevant aberration in 188 evaluable pts (74%). In 100 pts (39,4%) ≥ 1 druggable target was found. Most common actionable alterations were in PTEN (19%), BRCA2 (9%) or in mismatch-repair genes or resulting in high tumor mutational burden (5%). Out of the 100 druggable pts, 31 pts (31%) initiated a form of GMT, 4 pts (4%; 2 BRCA2, 2 PTEN) died prior to receiving GMT, in 65 pts (65%) GMT is pending as they receive standard of care. Conclusions: Routine molecular profiling early in the CRPC setting is feasible in a tertiary referral center with a MTB and high volume of CRPC pts. Almost 40% CRPC pts harbored an actionable target with 31% of these pts already allocated to a GMT. Failure rates of NGS were low at 11%.

Abstract 2347: Metabolomics profiling of formalin-fixed paraffin-embedded prostate tissues

Abstract The metabolic landscape of the prostate changes with malignant transformation, and metabolomics is a means of identifying diagnostic and prognostic biomarkers for prostate cancer. Epidemiology and registry studies containing archival formalin-fixed paraffin-embedded (FFPE) specimens are a rich resource for this work but this method of tissue preservation presents a challenge due to loss of metabolites. We optimized global metabolic profiling in FFPE tissue and identified metabolites distinguishing tumor from normal prostate tissue, and aggressive from non-aggressive prostate cancer. For optimization of metabolomics in FFPE tissues, we obtained matched frozen and FFPE tumor tissue from 62 prostate cancer cases who underwent radical prostatectomy (RP) at the University of North Carolina (UNC) hospital, of which 24 also had adjacent normal prostate tissue. To identify metabolites associated with tumor aggressiveness, we obtained matched tumor and normal FFPE tissue from 60 prostate cancer patients (Gleason ≥4+3, n=30; Gleason ≤3+4, n=30) who underwent RP participating in the UNC Health Registry/Cancer Survivorship Cohort. Following log transformation and imputation of missing values with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between groups. Random forest analysis evaluated predictive accuracy of metabolites for distinguishing tumor from normal prostate tissue. We identified 768 compounds of known identity in frozen and 119 in matched FFPE tissues. Therefore, only 15% of the metabolites detected in frozen samples were recovered from corresponding FFPE tissue, with 80% of these common to both FFPE and matched frozen tissues. We found a predictive accuracy of 78% and 67% for frozen and FFPE tissue, respectively, for distinguishing tumor from normal. Of 39 metabolites significantly altered between matched tumor and normal FFPE tissue, 47% belonged to the lipid class, and despite a greater number of significantly altered metabolites in frozen tissue (246), the proportion belonging to the lipid class was similar (50%). Citrate, integral to normal prostate metabolism, and citrulline, involved in nitric oxide synthesis, had significantly lower levels in aggressive versus non-aggressive tumors. Cell membrane phospholipids were found at significantly higher levels in aggressive tumors. Common patterns of altered metabolites in tumor vs. normal prostate were observed irrespective of tissue preservation method, despite loss of a large proportion of metabolites during the process of FFPE. As such, FFPE studies are feasible particularly when lipid metabolism is of interest as this class appears one of the best preserved in FFPE. This work will pave the way for incorporating metabolomics profiling of FFPE specimens into epidemiology and registry-based studies of prostate cancer. Citation Format: Sophia Rachael Halliday, Linnea T. Olsson, Alina Hamilton, Sivapriya Ramamoorthy, Jason Kitchen, Erin Kirk, Laura Farnan, Adrian Gerstel, Melissa A. Troester, Jannette T. Bensen, Sara E. Wobker, Emma Allott. Metabolomics profiling of formalin-fixed paraffin-embedded prostate tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2347.

Molecular detection of urogenital mollicutes in patients with invasive malignant prostate tumor

BackgroundThe etiology of prostate cancer (PCa) is multiple and complex. Among the causes recently cited are chronic infections engendered by microorganisms that often go unnoticed. A typical illustration of such a case is infection due to mollicutes bacteria. Generally known by their lurking nature, urogenital mollicutes are the most incriminated in PCa. This study was thus carried out in an attempt to establish the presence of these mollicutes by PCR in biopsies of confirmed PCa patients and to evaluate their prevalence.MethodsA total of 105 Formalin-Fixed Paraffin-Embedded prostate tissues collected from 50 patients suffering from PCa and 55 with benign prostate hyperplasia were subjected to PCR amplification targeting species-specific genes of 5 urogenital mollicutes species, Mycoplasma genitalium, M. hominis, M. fermentans, Ureaplasma parvum, and U. urealyticum. PCR products were then sequenced to confirm species identification. Results significance was statistically assessed using Chi-square and Odds ratio tests.ResultsPCR amplification showed no positive results for M. genitalium, M. hominis, and M. fermentans in all tested patients. Strikingly, Ureaplasma spp. were detected among 30% (15/50) of PCa patients. Nucleotide sequencing further confirmed the identified ureaplasma species, which were distributed as follows: 7 individuals with only U. parvum, 5 with only U. urealyticum, and 3 co-infection cases. Association of the two ureaplasma species with PCa cases proved statistically significant (P < 0.05), and found to represent a risk factor. Of note, Ureaplasma spp. were mostly identified in patients aged 60 and above with prostatic specific antigen (PSA) level > 4 ng/ml and an invasive malignant prostate tumor (Gleason score 8–10).ConclusionsThis study uncovered a significant association of Ureaplasma spp. with PCa arguing in favour of their potential involvement in this condition. Yet, this finding, though statistically supported, warrants a thorough investigation at a much larger scale.

Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues

The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64–69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SignificanceHere we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE tissue analysis.

Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation–Mass Spectrometry Assays

TMPRSS2-ERG gene fusion, a molecular alteration found in nearly half of primary prostate cancer cases, has been intensively characterized at the transcript level. However limited studies have explored the molecular identity and function of the endogenous fusion at the protein level. Here, we developed immunoprecipitation–mass spectrometry assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms, and its interactome in VCaP prostate cancer cells. Our assays quantified total ERG (∼27,000 copies/cell) and its four unique isoforms and revealed that the T1E4-ERG isoform accounted for 52 ± 3% of the total ERG protein in VCaP cells, and 50 ± 11% in formalin-fixed paraffin-embedded prostate cancer tissues. For the first time, the N-terminal peptide (methionine-truncated and N-acetylated TASSSSDYGQTSK) unique for the T1/E4 fusion was identified. ERG interactome profiling with the C-terminal, but not the N-terminal, antibodies identified 29 proteins, including mutually exclusive BRG1- and BRM-associated canonical SWI/SNF chromatin remodeling complexes. Our sensitive and selective IP-SRM assays present alternative tools to quantify ERG and its isoforms in clinical samples, thus paving the way for development of more accurate diagnostics of prostate cancer.

Abstract PO-094: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N- glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexanda E. Stanback, Derek B Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-094.

Abstract PO-085: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N-glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexandra E. Stanback, Derek B. Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-085.

Development and Validation of a Deep Learning Algorithm for Gleason Grading of Prostate Cancer From Biopsy Specimens

For prostate cancer, Gleason grading of the biopsy specimen plays a pivotal role in determining case management. However, Gleason grading is associated with substantial interobserver variability, resulting in a need for decision support tools to improve the reproducibility of Gleason grading in routine clinical practice. To evaluate the ability of a deep learning system (DLS) to grade diagnostic prostate biopsy specimens. The DLS was evaluated using 752 deidentified digitized images of formalin-fixed paraffin-embedded prostate needle core biopsy specimens obtained from 3 institutions in the United States, including 1 institution not used for DLS development. To obtain the Gleason grade group (GG), each specimen was first reviewed by 2 expert urologic subspecialists from a multi-institutional panel of 6 individuals (years of experience: mean, 25 years; range, 18-34 years). A third subspecialist reviewed discordant cases to arrive at a majority opinion. To reduce diagnostic uncertainty, all subspecialists had access to an immunohistochemical-stained section and 3 histologic sections for every biopsied specimen. Their review was conducted from December 2018 to June 2019. The frequency of the exact agreement of the DLS with the majority opinion of the subspecialists in categorizing each tumor-containing specimen as 1 of 5 categories: nontumor, GG1, GG2, GG3, or GG4-5. For comparison, the rate of agreement of 19 general pathologists' opinions with the subspecialists' majority opinions was also evaluated. For grading tumor-containing biopsy specimens in the validation set (n = 498), the rate of agreement with subspecialists was significantly higher for the DLS (71.7%; 95% CI, 67.9%-75.3%) than for general pathologists (58.0%; 95% CI, 54.5%-61.4%) (P < .001). In subanalyses of biopsy specimens from an external validation set (n = 322), the Gleason grading performance of the DLS remained similar. For distinguishing nontumor from tumor-containing biopsy specimens (n = 752), the rate of agreement with subspecialists was 94.3% (95% CI, 92.4%-95.9%) for the DLS and similar at 94.7% (95% CI, 92.8%-96.3%) for general pathologists (P = .58). In this study, the DLS showed higher proficiency than general pathologists at Gleason grading prostate needle core biopsy specimens and generalized to an independent institution. Future research is necessary to evaluate the potential utility of using the DLS as a decision support tool in clinical workflows and to improve the quality of prostate cancer grading for therapy decisions.

Abstract A33: Untapped biospecimens and novel mass spectrometry scanning techniques for DNA adductomics

Abstract A major impediment in the biomonitoring of DNA adducts is the lack of fresh-frozen biopsy samples available for biomarker research. However, archived formalin-fixed, paraffin-embedded (FFPE) tissues with a clinical diagnosis of disease are often accessible. We have established a method to fully unravel DNA crosslinks in FFPE specimens under mild conditions that preserve the structural integrity of DNA adducts. Our targeted, quantitative mass spectrometry measurements employing ion trap or high-resolution Orbitrap mass spectrometry require less than ten micrograms of DNA with limits of quantification at three adducts per 109 nucleotides. We have successfully screened FFPE tissues of rodents exposed to tobacco and dietary carcinogens and reported adduct levels comparable to those of matching fresh-frozen tissues. Our technology has been employed to identify a DNA adduct of aristolochic acid, a potent urothelial carcinogen present in Chinese herbal medicines, in human FFPE kidney blocks stored at ambient temperature for up to nine years. The method also detected DNA adducts of the bladder carcinogen 4-aminobiphenyl in human FFPE bladder, and a DNA adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a heterocyclic aromatic amine formed in cooked meats and a potential prostate carcinogen, in FFPE prostate specimens of prostate cancer patients. Thus, the technology is versatile and can be employed to screen for DNA adducts formed with a wide range of environmental and dietary carcinogens. The ability to retrospectively analyze FFPE tissues for DNA adducts for which there is a clinical diagnosis of disease opens a previously untapped source of biospecimens for molecular epidemiology studies seeking the causal role of environmental chemicals in cancer etiology. With the recent improvements in the sensitivity and scanning rates of high-resolution MS instruments, such as quadrupole time-of-flight and Orbitrap MS detectors, it is now possible to screen for a wide array of DNA damage in the human genome using DNA adductomics approaches. We have adapted data-dependent and data-independent scanning techniques originated from proteomics and metabolomics to screen for DNA adducts of the genome. DNA adductomics is a new and developing technology for human exposure assessment. As the analytic technology matures and bioinformatics tools become available for analysis of the mass spectral data, DNA adductomics can advance our understanding of the role chemical exposures play in DNA damage and disease risk. Citation Format: Byeong Hwa Yun, Jingshu Guo, Scott Walmsley, Paari Murugan, Christopher J. Weight, Peter W. Villalta, Robert J. Turesky. Untapped biospecimens and novel mass spectrometry scanning techniques for DNA adductomics [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A33.

Abstract A014: Raman microscopy to assess biochemical recurrence risk after radical prostatectomy

Abstract Background: There is an urgent need for pathologists to better define patients with high-risk prostate cancer. One of the promising tools is Raman micro-spectroscopy, also known as Raman microscopy, a nondestructive and label-free imaging technique based on light scattered after reflection. Our group has recently developed a rapid standardized protocol for the preparation of formalin-fixed, paraffin-embedded (FFPE) diagnostic tissues suitable for Raman microscopy. The objective of this study was to evaluate the potential of Raman microscopy to assess the prognosis of prostate cancer patients with FFPE tissues from radical prostatectomy. Methods: Patients treated by first-line radical prostatectomy between 1994 and 2004 at Centre hospitalier de l’Université de Montréal (CHUM) were included in this study. FFPE prostate cancer tissues from surgery were used for the construction of tissue microarrays (TMAs). To enable Raman microscopy, TMA sections of 4 µm were placed on low-cost aluminum slides. The rapid dewaxing protocol of the hospital was used (8 minutes), followed by 20 minutes of vacuum drying. All Raman spectra were acquired with the Renishaw inVia confocal Raman microscope equipped with a 785-nm line focus laser. After removing background contributions (e.g., autofluorescence) for each spectrum with Wire 4.4 software, a custom toolbox in MATLAB was used to predict biochemical recurrence. Chemometric methods and calculated ratios were used for the analysis of Raman microscopy images. Results: A total of 320 Raman spectra from 80 patients were analyzed from prostate cancer TMAs, representing 25 patients with biochemical recurrence within 18 months after radical prostatectomy and 55 without. Using a Support Vector Machine (SVM) technique and correlation feature selection for classification, our results with Raman microscopy identified biochemical recurrence with an accuracy of 83.7%, a sensitivity of 84.0% and a specificity of 83.6%. Raman peak assignment of features was used to investigate the molecular differences between these two patient groups. We found that the molecular constituents of RNA and phosphorylated proteins were more important in prostate cancer with biochemical recurrence. In contrast, Raman peaks of the phospholipid head of cell membranes, DNA, and collagen were more intense in prostate cancer without biochemical recurrence. For the visualization of these different molecular constituents of prostate cancer, we developed two methods of Raman microscopy imaging. The first method involved analysis of chemometric data (i.e., extraction of chemical information) to identify the whole tissue (phenylalanine), nuclei (DNA), and red blood cells (hemoglobin), followed by background removal. The images of our chemometric analysis created a virtual staining of hematoxylin and eosin (H&amp;E). The second method involved testing several ratios of Raman peaks associated with proteins, lipids, DNA and RNA. Calculated ratios distinguished specific structures of the prostate tissue, such as the cancerous and normal glands, by different colors. Conclusions: This is the first study demonstrating the potential of Raman microscopy for the prediction of biochemical recurrence within 18 months following radical prostatectomy for prostate cancer. Raman microscopy imaging of tissues is a promising method for the recognition of specific structures, which could help pathologists in the accuracy of diagnosis. The accessibility of this technology to clinicians could be useful for patient follow-up and treatment strategies. Citation Format: Andrée-Anne Grosset, Catherine St-Pierre, Karl St-Arnaud, Kelly Aubertin, Michael Jermyn, Frédéric Leblond, Dominique Trudel. Raman microscopy to assess biochemical recurrence risk after radical prostatectomy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A014.

Differential Expression of Cannabinoid Receptors, CB1R and CB2R, in Prostate Tumor Samples with Perineural Invasion

Perineural invasion (PNI) affects over 20% of patients with prostate cancer who often recur with metastasis primarily to the bones. Despite its association with the patient's poor prognosis and dissemination of various cancers, knowledge of the regulatory mechanisms that drive invasion of prostate cancer cells through and around nerves is limited. Cannabinoid receptors (CBRs) are highly enriched in prostate cancer cells and exogenous application of CBR ligands attenuates the cell's survival, migration and invasion ability. Our objective is to define whether activation of the endocannabinoid system (ECS) associates with PNI and to identify the endogenous regulatory mechanisms through which the ECS promotes prostate cancer cell invasion through nerves. We used immunohistochemistry technique to determine the expression levels of the CB1R and the CB2R in formalin‐fixed paraffin embedded (FFPE) prostate tumor samples with PNI. Preliminary data from our laboratory show that CB1R is highly enriched in FFPE prostate tumor samples with PNI when compared to CB2R. Given the increase of the CB1R in PNI FFPE samples, we used total internal fluorescence reflection microscopy (TIRFm) to elucidate in vivo the trafficking kinetics of CB1Rs in highly invasive versus less invasive prostate cancer cells, PC3 and 22RV1, respectively. We found that PC3 cells display constitutive CB1R trafficking. However, this was not the case for the less invasive prostate cancer cells, 22RV1. Our results suggest that the elevated CB1R levels detected in the FFPE PNI positive samples is not coincidental, but rather an indication of an association of the ECS and PNI. Ongoing studies aim to determine how nerve‐PC tumor interactions encourage PNI by using an in vitro model in which we can modulate the ECS.Support or Funding InformationGrants: RCMI‐NIMHD 8G12MD007600 to JFO.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores.

Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores. A major challenge is low quantity and quality of extracted nucleic acids, which can limit our ability to perform genetic analyses, and have a significant influence on overall study design. This study was aimed at identifying the most reliable and reproducible method of obtaining sufficient high-quality nucleic acids from FFPE tissues. We compared the yield and quality of nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extraction protocols, using 14 commercially available kits. Nucleic acid yield was determined by fluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleic acids in quantities that are compatible with downstream molecular applications. However, the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and five DNA extraction protocols, including protocols from two kits for dual-extraction of RNA and DNA from the same tissue source, were prioritized for further quality assessment based on the yield and purity of their products. Specifically, their compatibility with downstream reactions was assessed using both NanoString nCounter gene expression assays and reverse-transcriptase real-time PCR for RNA, and methylation-specific PCR assays for DNA. The kit deemed most suitable for FFPE tissue was the AllPrep kit by Qiagen because of its yield, quality, and ability to purify both RNA and DNA from the same sample, which would be advantageous in biomarker studies.