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BACKGROUND: Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. METHODS: A robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). RESULTS: This new assay (telomere chromogenic in situ hybridization ["Telo-CISH"]) produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. CONCLUSIONS: In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.
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Lesões Pré-Cancerosas , Próstata , Masculino , Humanos , Hibridização in Situ Fluorescente/métodos , Hibridização In Situ , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/genética , TelômeroRESUMO
Microscopic examination of prostate cancer has failed to reveal a reproducible association between molecular and morphologic features. However, deep-learning algorithms trained on hematoxylin and eosin (H&E)-stained whole slide images (WSI) may outperform the human eye and help to screen for clinically-relevant genomic alterations. We created deep-learning algorithms to identify prostate tumors with underlying ETS-related gene (ERG) fusions or PTEN deletions using the following 4 stages: (1) automated tumor identification, (2) feature representation learning, (3) classification, and (4) explainability map generation. A novel transformer-based hierarchical architecture was trained on a single representative WSI of the dominant tumor nodule from a radical prostatectomy (RP) cohort with known ERG/PTEN status (n = 224 and n = 205, respectively). Two distinct vision transformer-based networks were used for feature extraction, and a distinct transformer-based model was used for classification. The ERG algorithm performance was validated across 3 RP cohorts, including 64 WSI from the pretraining cohort (AUC, 0.91) and 248 and 375 WSI from 2 independent RP cohorts (AUC, 0.86 and 0.89, respectively). In addition, we tested the ERG algorithm performance in 2 needle biopsy cohorts comprised of 179 and 148 WSI (AUC, 0.78 and 0.80, respectively). Focusing on cases with homogeneous (clonal) PTEN status, PTEN algorithm performance was assessed using 50 WSI reserved from the pretraining cohort (AUC, 0.81), 201 and 337 WSI from 2 independent RP cohorts (AUC, 0.72 and 0.80, respectively), and 151 WSI from a needle biopsy cohort (AUC, 0.75). For explainability, the PTEN algorithm was also applied to 19 WSI with heterogeneous (subclonal) PTEN loss, where the percentage tumor area with predicted PTEN loss correlated with that based on immunohistochemistry (r = 0.58, P = .0097). These deep-learning algorithms to predict ERG/PTEN status prove that H&E images can be used to screen for underlying genomic alterations in prostate cancer.
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BACKGROUND: Most prostate cancers are "immune cold" and poorly responsive to immune checkpoint inhibitors. However, the mechanisms responsible for the lack of a robust antitumor adaptive immune response in the prostate are poorly understood, which hinders the development of novel immunotherapeutic approaches. AIMS: Most inflammatory infiltrates in the prostate are centered around benign glands and stroma, which can confound the molecular characterization of the antitumor immune response. We sought to analytically validate a chromogenic-based multiplex immunohistochemistry (IHC) approach applicable to whole slide digital image analysis to quantify T cell subsets from the tumor microenvironment of primary prostatic adenocarcinomas. As an initial application, we tested the hypothesis that PTEN loss leads to an altered antitumor immune response by comparing matched regions of tumors within the same individual with and without PTEN loss. MATERIALS & METHODS: Using the HALO Image Analysis Platform (Indica Labs), we trained a classifier to quantify the densities of eight T cell phenotypes separately in the tumor epithelial and stromal subcompartments. RESULTS: The iterative chromogenic approach using 7 different antibodies on the same slide provides highly similar findings to results using individually stained slides with single antibodies. Our main findings in carcinomas (benign removed) include the following: i) CD4+ T cells are present at higher density than CD8+ T cells; ii) all T cell subsets are present at higher densities in the stromal compartment compared to the epithelial tumor compartment; iii) most CD4+ and CD8+ T cells are PD1+; iv) cancer foci with PTEN loss harbored increased numbers of T cells compared to regions without PTEN loss, in both stromal and epithelial compartments; and v) the increases in T cells in PTEN loss regions were associated with ERG gene fusion status. DISCUSSION: This modular approach can apply to any IHC-validated antibody combination and sets the groundwork for more detailed spatial analyses. CONCLUSION: Iterative chromogenic IHC can be used for whole slide analysis of prostate tissue samples and can complement transcriptomic results including those using single cell and spatial genomic approaches.
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Neoplasias da Próstata , Microambiente Tumoral , Humanos , Imuno-Histoquímica , Contagem de Linfócitos , Masculino , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/patologiaRESUMO
Mitochondria regulate ATP production, metabolism, and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Because most measurements of mtDNA use homogenates of complex tissues, little is known about cell-type-specific mtDNA copy number heterogeneity in normal physiology, aging, and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, an in situ hybridization approach to generate a single-cell-resolution atlas of mtDNA content in mammalian tissues was validated. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared with differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared with cells within glomeruli and collecting duct epithelial cells. In mice, decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. This study provides a novel approach for quantifying species- and cell-type-specific mtDNA copy number and dynamics in any normal or diseased tissue that can be used for monitoring the effects of interventions in animal and human studies.
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Proliferação de Células , DNA Mitocondrial/análise , Células-Tronco , Envelhecimento/fisiologia , Animais , Atlas como Assunto , Variações do Número de Cópias de DNA , Feminino , Humanos , Hibridização In Situ/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Ocular adnexal (OA) sebaceous carcinomas generally demonstrate more aggressive clinical and histopathological phenotypes than extraocular cases, but the molecular drivers implicated in their oncogenesis remain poorly defined. A retrospective review of surgical and ocular pathology archives identified eleven primary resection specimens of OA sebaceous carcinomas with adequate tissue for molecular analysis; two extraocular cases were also examined. Next-generation sequencing was used to evaluate mutations and copy number changes in a large panel of cancer-associated genes. Fluorescence in situ hybridization (FISH) confirmed MYC copy number gain in select cases, and immunohistochemistry to evaluate MYC protein expression. The commonest mutations occurred in TP53 (10/13) and RB1 (7/13). Additional mutations in clinically actionable genes, or mutations with a frequency of at least 25%, included the NF1 (3/12), PMS2 (4/12), ROS1 (3/12), KMT2C (4/12), MNX1 (6/12), NOTCH1 (4/12), PCLO (3/12), and PTPRT (3/12) loci. Low level copy number gain suggestive of amplification of the MYC locus was seen in two cases, and confirmed using FISH. MYC protein expression, as assessed by immunohistochemistry, was present in almost all sebaceous carcinoma cases. Our findings support the concept that alterations in TP53 and RB1 are the commonest alterations in sebaceous carcinoma, and suggest that MYC may contribute to the oncogenesis of these tumors.
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Neoplasias Oculares/genética , Neoplasias de Anexos e de Apêndices Cutâneos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas de Ligação a Retinoblastoma/genética , Neoplasias das Glândulas Sebáceas/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Dosagem de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
Intraductal carcinoma of the prostate (IDC-P) most often appears associated with high-grade invasive prostate carcinoma (PCa), where it is believed to represent retrograde spread. However, IDC-P rarely occurs as an isolated finding at radical prostatectomy or with concurrent low-grade (Grade Group 1) invasive carcinoma. We hypothesized that isolated IDC-P (iIDC-P) in these unusual cases may represent a distinct in situ lesion and molecularly profiled 15 cases. iIDC-P was characterized by copy number alteration (CNA) profiling and targeted next generation sequencing in cases with sufficient tissue (n = 7). Immunohistochemistry for PTEN and ERG was performed on the total cohort (n = 15), where areas of iIDC-P and associated invasive disease were evaluated separately (n = 9). By copy number profiling, iIDC-P alterations were similar to those previously described in high-grade invasive PCa (PTEN, RB1, and CHD1 loss; MYC gain). However, in four cases, targeted sequencing revealed a striking number of activating oncogenic driver mutations in MAPK and PI3K pathway genes, which are extraordinarily rare in conventional PCa. In addition, pathogenic mutations in DNA repair genes were found in two cases of iIDC-P (BRCA2, CHEK2, CDK12) and other known PCa-associated mutations (FOXA1, SPOP) in two cases. Overall, ERG was expressed in 7% (1/15) of the iIDC-P lesions and PTEN was lost in 53% (8/15). Discordance for ERG or PTEN status between IDC-P and the low-grade PCa was observed in five of nine cases, with intact PTEN in the invasive tumor and PTEN loss in IDC-P in four. Despite a CNA profile similar to conventional PCa, iIDC-P is enriched with potentially targetable oncogenic driver mutations in MAPK/PI3K genes. Based on PTEN and ERG status, iIDC-P is not likely a precursor to the associated low-grade invasive PCa, but represents a molecularly unique in situ tumor of unclear clinical significance. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Biomarcadores Tumorais/genética , Carcinoma in Situ/genética , Carcinoma Ductal/genética , Mutação , Oncogenes , Neoplasias da Próstata/genética , Idoso , Carcinoma in Situ/classificação , Carcinoma in Situ/patologia , Carcinoma Ductal/classificação , Carcinoma Ductal/patologia , Variações do Número de Cópias de DNA , Dosagem de Genes , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Gradação de Tumores , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/genética , Fenótipo , Fosfatidilinositol 3-Quinase/genética , Neoplasias da Próstata/classificação , Neoplasias da Próstata/patologia , Regulador Transcricional ERG/genética , TranscriptomaRESUMO
BACKGROUND: Urogenital infection with Schistosoma haematobium is a risk factor for the development of squamous cell carcinoma of the urinary bladder. The pathophysiology is thought to be mediated in part by inflammation, cellular damage, and bladder regeneration induced by the parasitic infection. Herein, we report an unusual case of schistosomiasis of the prostate that was found concurrent with prostate adenocarcinoma in a radical prostatectomy specimen from a man in the United States. METHODS: The infecting Schistosoma species was characterized via histomorphology and acid-fast stain. The concurrent Gleason score 6 prostate cancer was assessed for ETS transcription factor ERG (ERG), phosphatase and tensin homolog (PTEN), p27, and p53 status using immunohistochemistry (IHC). Cellular proliferation and the presence of intermediate cells in prostatic atrophy were assessed via immunostaining for Ki67 and CK903, respectively. RESULTS: Histomorphology and acid-fast stain of the infecting species were consistent with S. haematobium. We classified the Gleason score 6 prostate adenocarcinoma via IHC as ERG positive, PTEN intact, p27 intact, and without p53 nuclear accumulation. The prostatic epithelium immediately adjacent to the schistosomiasis-related granulomatous inflammation was atrophic and accompanied by increased cellular proliferation and the presence of intermediate cells. Upon literature review, we determined that prostate schistosomiasis is associated with a young age of prostate cancer diagnosis and highly aggressive prostate cancer. CONCLUSIONS: This is a rare case of prostate schistosomiasis in the United States; however, prostate schistosomiasis occurs frequently in endemic areas. The patient had traveled to a Schistosoma-endemic region, which was the likely location of exposure to the parasite. To our knowledge, this is the first report of the association of proliferative inflammatory atrophy and intermediate cells with schistosomiasis of the prostate. We propose that prostate schistosomiasis may be considered as a risk factor for the development of prostate cancer in geographic regions where Schistosoma species are endemic.
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Adenocarcinoma/parasitologia , Carcinogênese/patologia , Próstata/parasitologia , Neoplasias da Próstata/parasitologia , Esquistossomose/patologia , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Humanos , Inflamação/parasitologia , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Esquistossomose/complicaçõesRESUMO
BACKGROUND: Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer. METHOD: We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively. RESULTS: Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection. DISCUSSION: These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.
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Adenocarcinoma/patologia , Linhagem Celular Tumoral , PTEN Fosfo-Hidrolase/genética , Próstata/patologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Adenocarcinoma/genética , Alelos , Animais , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos , Neoplasias da Próstata/genéticaRESUMO
BACKGROUND: Current and recent smoking have been associated with a greater risk of prostate cancer recurrence and mortality, though the underlying mechanism is unknown. METHODS: To determine if telomere shortening, which has been associated with poor outcomes, may be a potential underlying mechanism, we prospectively evaluated the association between smoking status and telomere length in 567 participants in the Health Professionals Follow-up Study, who were surgically treated for prostate cancer. Using tissue microarrays (TMA), we measured telomere length in cancer and benign tissue, specifically stromal cells in the same TMA spot using a telomere-specific fluorescence in situ hybridization assay. Smoking status was collected via questionnaire 2-years before diagnosis. Adjusting for age, pathologic stage and grade, the median and standard deviation of the per-cell telomere signals were determined for each man for stromal cells and cancer cells by smoking categories. In sub-analyses, we restricted to men without major co-morbidities diagnosed before prostate cancer. RESULTS: Overall, there were no associations between smoking status and telomere length or variability in stromal cells or cancer cells. However, among men without comorbidities, current smokers and former smokers who quit <10 years ago had the most variable telomere length in stromal cells (29.3% more variable than never smokers; P-trend = 0.0005) and in cancer cells (27.7% more variable than never smokers; P-trend = 0.05). Among men without comorbidities, mean telomere length did not differ by smoking status in stromal cells or cancer cells. CONCLUSION: Telomere variability in prostate cells may be one mechanism through which smoking influences poor prostate cancer outcomes.
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Próstata/patologia , Neoplasias da Próstata/patologia , Fumar , Células Estromais/patologia , Homeostase do Telômero/fisiologia , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Encurtamento do Telômero/fisiologiaRESUMO
BACKGROUND: Recurrent ERG gene fusions, the most common genetic alterations in prostate cancer, drive overexpression of the nuclear transcription factor ERG, and are early clonal events in prostate cancer progression. The nuclear transcription factor MYC is also frequently overexpressed in prostate cancer and may play a role in tumor initiation and/or progression. The relationship between nuclear ERG and MYC protein overexpression in prostate cancer, as well as the clinicopathologic characteristics and prognosis of ERG-positive/MYC high tumors, is not well understood. METHODS: Immunohistochemistry (IHC) for ERG and MYC was performed on formalin-fixed, paraffin-embedded tissue from prostate cancer tissue microarrays (TMAs), and nuclear staining was scored semi-quantitatively (IHC product score range = 0-300). Correlation between nuclear ERG and MYC protein expression and association with clinicopathologic parameters and biochemical recurrence after radical prostatectomy was assessed. RESULTS: 29.1% of all tumor nodules showed concurrent nuclear ERG and MYC protein overexpression (i.e., ERG-positive/MYC high), including 35.0% of secondary nodules. Overall, there was weak positive correlation between ERG and MYC expression across all tumor nodules (rpb = 0.149, P = 0.045), although this correlation was strongest in secondary nodules (rpb = 0.520, P = 0.019). In radical prostatectomy specimens, ERG-positive/MYC high tumors were positively associated with the presence of extraprostatic extension (EPE), relative to all other ERG/MYC expression subgroups, however, there was no significant association between concurrent nuclear ERG and MYC protein overexpression and time to biochemical recurrence. CONCLUSIONS: Concurrent nuclear ERG and MYC protein overexpression is common in prostate cancer and defines a subset of locally advanced tumors. Recent data indicates that BET bromodomain proteins regulate ERG gene fusion and MYC gene expression in prostate cancer, suggesting possible synergistic targeted therapeutics in ERG-positive/MYC high tumors. Prostate 76:845-853, 2016. © 2016 Wiley Periodicals, Inc.
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Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/genética , Humanos , Imuno-Histoquímica , Masculino , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Fusão Oncogênica , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Análise Serial de Tecidos , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismoRESUMO
When distinguishing between indolent and potentially harmful prostate cancers, the Gleason score is the most important variable, but may be inaccurate in biopsies due to tumor under-sampling. This study investigated whether a molecular feature, PTEN protein loss, could help identify which Gleason score 6 tumors on biopsy are likely to be upgraded at radical prostatectomy. Seventy one patients with Gleason score 6 tumors on biopsy upgraded to Gleason score 7 or higher at prostatectomy (cases) were compared with 103 patients with Gleason score 6 on both biopsy and prostatectomy (controls). A validated immunohistochemical assay for PTEN was performed, followed by fluorescence in situ hybridization (FISH) to detect PTEN gene deletion in a subset. PTEN protein loss and clinical-pathologic variables were assessed by logistic regression. Upgraded patients were older than controls (61.8 vs 59.3 years), had higher pre-operative PSA levels (6.5 vs 5.3 ng/ml) and a higher fraction of involved cores (0.42 vs 0.36). PTEN loss by immunohistochemistry was found in 18% (13/71) of upgraded cases compared with 7% (7/103) of controls (P=0.02). Comparison between PTEN immunohistochemistry and PTEN FISH showed the assays were highly concordant, with 97% (65/67) of evaluated biopsies with intact PTEN protein lacking PTEN gene deletion, and 81% (13/16) of the biopsies with PTEN protein loss showing homozygous PTEN gene deletion. Tumors with PTEN protein loss were more likely to be upgraded at radical prostatectomy than those without loss, even after adjusting for age, preoperative PSA, clinical stage and race (odds ratio=3.04 (1.08-8.55; P=0.035)). PTEN loss in Gleason score 6 biopsies identifies a subset of prostate tumors at increased risk of upgrading at radical prostatectomy. These data provide evidence that a genetic event can improve Gleason score accuracy and highlight a path toward the clinical use of molecular markers to augment pathologic grading.
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PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Idoso , Biópsia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prostatectomia , Neoplasias da Próstata/cirurgiaRESUMO
We have described a rare group of prostate adenocarcinomas that show aberrant expression of p63, a protein strongly expressed in prostatic basal cells and absent from usual-type acinar prostate cancers. The partial basal-like immunophenotype of these tumors is intriguing in light of the persistent debate surrounding the cell-of-origin for prostate cancer; however, their molecular phenotype is unknown. We collected 37 of these tumors on radical prostatectomy and biopsy and assessed subsets for a diverse panel of molecular markers. The majority of p63-expressing tumors were positive for the ΔNp63 isoform (6/7) by immunofluorescence and p63 mRNA (7/8) by chromogenic in situ hybridization. Despite p63 positivity, these tumors uniformly expressed luminal-type cytokeratin proteins such as CK18 (13/13), CK8 (8/8), and markers of androgen axis signaling commonly seen in luminal cells, including androgen receptor (10/11), NKX3.1 (8/8), and prostein (12/13). Conversely, basal cytokeratins such as CK14 and CK15 were negative in all cases (0/8) and CK5/6 was weakly and focally positive in 36% (4/11) of cases. Pluripotency markers including ß-catenin, Oct4, and c-kit were negative in p63-expressing tumors (0/11). Despite nearly universal expression of androgen receptor and downstream androgen signaling targets, p63-expressing tumors lacked ERG rearrangements by fluorescence in situ hybridization (0/14) and ERG protein expression (0/37). No tumors expressed SPINK1 or showed PTEN protein loss (0/19). Surprisingly, 74% (14/19) of p63-expressing tumors expressed GSTP1 protein at least focally, and 33% (2/6) entirely lacked GSTP1 CpG island hypermethylation by bisulfite sequencing. In contrast to usual prostatic adenocarcinomas, prostate tumors with p63 expression show a mixed luminal/basal immunophenotype, uniformly lack ERG gene rearrangement, and frequently express GSTP1. These data strongly suggest that p63-expressing prostate tumors represent a molecularly distinct subclass and further study of this rare tumor type may yield important insights into the role of p63 in prostatic biology and the prostate cancer cell-of-origin.
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Adenocarcinoma/metabolismo , Proteínas de Membrana/biossíntese , Neoplasias da Próstata/metabolismo , Adenocarcinoma/patologia , Imunofluorescência , Humanos , Hibridização In Situ , Masculino , Reação em Cadeia da Polimerase , Neoplasias da Próstata/patologia , Isoformas de Proteínas/biossínteseRESUMO
Fibroblast activation protein (FAP) is a serine protease upregulated at sites of tissue remodeling and cancer that represents a promising therapeutic and molecular imaging target. In prostate cancer, studies of FAP expression using tissue microarrays are conflicting, such that its clinical potential is unclear. Furthermore, little is known regarding FAP expression in benign prostatic tissues. Here we demonstrated, using a novel iterative multiplex IHC assay in standard tissue sections, that FAP was nearly absent in normal regions, but was increased consistently in regions of proliferative inflammatory atrophy (PIA). In carcinoma, FAP was expressed in all cases, but was highly heterogeneous. High FAP levels were associated with increased pathological stage and cribriform morphology. We verified that FAP levels in cancer correlated with CD163+ M2 macrophage density. In this first report to quantify FAP protein in benign prostate and primary tumors, using standard large tissue sections, we clarify that FAP is present in all primary prostatic carcinomas, supporting its potential clinical relevance. The finding of high levels of FAP within PIA supports the injury/regeneration model for its pathogenesis and suggests that it harbors a protumorigenic stroma. Yet, high levels of FAP in benign regions could lead to false positive FAP-based molecular imaging results in clinically localized prostate cancer.
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Gleason grade group (GG) is the most powerful prognostic variable in localized prostate cancer; however, interobserver variability remains a challenge. Artificial intelligence algorithms applied to histopathologic images standardize grading, but most have been tested only for agreement with pathologist GG, without assessment of performance with respect to oncologic outcomes. We compared deep learning-based and pathologist-based GGs for an association with metastatic outcome in three surgical cohorts comprising 777 unique patients. A digitized whole slide image of the representative hematoxylin and eosin-stained slide of the dominant tumor nodule was assigned a GG by an artificial intelligence-based grading algorithm and was compared with the GG assigned by a contemporary pathologist or the original pathologist-assigned GG for the entire prostatectomy. Harrell's C-indices based on Cox models for time to metastasis were compared. In a combined analysis of all cohorts, the C-index for the artificial intelligence-assigned GG was 0.77 (95% confidence interval [CI]: 0.73-0.81), compared with 0.77 (95% CI: 0.73-0.81) for the pathologist-assigned GG. By comparison, the original pathologist-assigned GG for the entire case had a C-index of 0.78 (95% CI: 0.73-0.82). PATIENT SUMMARY: Artificial intelligence-enabled prostate cancer grading on a single slide was comparable with pathologist grading for predicting metastatic outcome in men treated by radical prostatectomy, enabling equal access to expert grading in lower resource settings.
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Intraductal carcinoma of the prostate is a marker of aggressive disease. However, intraductal carcinoma exists on a morphologic continuum with high-grade prostatic intraepithelial neoplasia (PIN) and distinguishing intraductal carcinoma from PIN is a common diagnostic dilemma with significant clinical implications. We evaluated whether immunostains for PTEN and ERG can sensitively identify intraductal carcinoma and accurately distinguish it from high-grade PIN. A combined immunostain for PTEN, ERG, p63 and CK903 was developed and validated. Radical prostatectomy specimens with lesions meeting criteria for intraductal carcinoma (n=45), intraductal cribriform proliferations falling short of intraductal carcinoma (n=15), and PIN lesions (n=39) were retrospectively identified and assessed for PTEN and ERG. Cytoplasmic PTEN loss was identified in 84% (38/45) of the intraductal carcinoma and 100% (15/15) of intraductal cribriform proliferation cases. In contrast, cytoplasmic PTEN loss was never observed in PIN (0/39; P<0.0001). Of the 53 cases of intraductal carcinoma or intraductal cribriform proliferation with cytoplasmic PTEN loss, it was homogeneously lost in 42 cases (79%). Weak, focal nuclear positivity for PTEN was retained in 31 of these 42 cases (74%). ERG expression was identified in 58% (26/45) of intraductal carcinoma and 67% (10/15) of intraductal cribriform proliferations compared with 13% (5/39) of PIN. Concordance between the PTEN/ERG status of the intraductal carcinoma lesions and the concurrent invasive carcinoma was high (>95% and P<0.0001 for each), and substantially less for PIN and the concurrent invasive tumor (83% for PTEN and 67% for ERG; P=NS for each). Cytoplasmic PTEN loss occurs in the majority of intraductal carcinoma and intraductal cribriform proliferation cases. Cytoplasmic PTEN loss was never observed in PIN (100% specificity). Our study identifies PTEN loss as a potentially useful marker to distinguish intraductal carcinoma from PIN and provides a plausible molecular explanation for why intraductal carcinoma is associated with poor prognosis.
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Biomarcadores Tumorais/análise , Carcinoma Ductal/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismo , Carcinoma Ductal/diagnóstico , Citoplasma/química , Citoplasma/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , PTEN Fosfo-Hidrolase/análise , Neoplasia Prostática Intraepitelial/diagnóstico , Neoplasias da Próstata/diagnósticoRESUMO
PURPOSE: To evaluate the impact of an inpatient pharmacy consult on discharge medications following bariatric surgery. METHODS: A pharmacy consult for discharge medication review for bariatric surgery patients was instituted at an academic medical center. The intervention included conducting a medication history, reviewing home medications for updates post-bariatric surgery, creating and documenting a discharge medication plan, and providing patient education. The impact of the intervention was evaluated by comparing medication classes, doses, and formulations prescribed during the intervention relative to a historical control group. RESULTS: The study included 85 patients who received pharmacist intervention and 167 patients who did not receive pharmacist intervention following bariatric surgery. The prescription of an extended-release medication at discharge in the intervention group was reduced by 19.3% (28.7% vs. 9.4%, p = 0.0005). For patients on hypertension medications, 94.0% had their regimen reduced in the intervention group compared with 37.5% of patients in the control group (p < 0.001). Of patients on insulin at baseline, 87.5% of patients in the intervention group had dose reductions at discharge vs. 66.7% of patients in the control group (p = 0.37). No patients in the intervention group were discharged with oral antihyperglycemic medications or non-insulin injectable medications vs. 33.3% (p = 0.12) and 20.0% (p = 0.47), respectively, in the control group. Readmission rates at 30 days were insignificantly lower in the intervention group (3.5% vs. 4.2%, p = 1). CONCLUSIONS: Clinical pharmacist involvement in the discharge medication reconciliation process for bariatric surgery patients reduced prescribing of unadjusted medication classes, doses, and drug formulations.
Assuntos
Serviço de Farmácia Hospitalar , Farmácia , Humanos , Alta do Paciente , Readmissão do Paciente , Pacientes Internados , Reconciliação de Medicamentos , FarmacêuticosRESUMO
Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. Here, a robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). This new assay ("Telo-CISH") produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.
RESUMO
Glutathione S-transferase pi 1 (GSTP1) is lowly expressed in normal prostate luminal cells and becomes induced in most proliferative inflammatory atrophy (PIA) lesions. GSTP1 becomes silenced in prostatic intraepithelial neoplasia (PIN) and prostate adenocarcinoma (CaP) via cytosine-phospho-guanine (CpG) island promoter hypermethylation. However, GSTP1 methylation patterns in PIA and PIN, and their relationship to patterns in CaP are poorly understood. We used bisulfite genomic sequencing to examine patterns of GSTP1 promoter CpG island methylation in laser capture microdissected benign, PIA, PIN, and CaP regions from 32 subjects that underwent radical prostatectomy. We analyzed 908 sequence clones across 24 normal epithelium, 37 PIA, 18 PIN, and 23 CaP regions, allowing assessment of 34,863 CpG sites with allelic phasing. Normal and PIA lesions were mostly unmethylated with 0.52 and 1.3% of total CpG sites methylated, respectively. PIN and CaP lesions had greater methylation with 24% and 51% of total CpG sites methylated, respectively. The degree of GSTP1 methylation showed progression from PIA << PIN < CaP. PIN lesions showed more partial methylation compared with CaP lesions. Partially methylated lesions were enriched for methylation changes at AP1 and SP1 transcription factor binding sites. These results demonstrate that methylation density in the GSTP1 CpG island in PIN was intermediate relative to that in normal prostate epithelium/PIA and CaP lesions. These results are consistent with gradual spreading of DNA methylation centered at the SP1/AP1 transcription factor binding sites in precursor lesions, with subsequent spreading of methylation across the entire CpG island in transition to CaP. PREVENTION RELEVANCE: DNA hypermethylation at the GSTP1 promoter progressively spreads from being unmethylated in normal prostate to intermediate levels in precursor lesions to extensive methylation in cancer. This molecular progression of GSTP1 promoter methylation patterns in early prostate carcinogenesis could be useful for identification and interception of prostate cancer precursors.
Assuntos
Neoplasia Prostática Intraepitelial , Neoplasias da Próstata , Masculino , Humanos , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Metilação de DNA , Ilhas de CpG/genética , Glutationa Transferase/genética , Neoplasias da Próstata/patologia , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/patologiaRESUMO
Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by two orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.