Cytologic-Histologic Correlation Practices for Nongynecologic Cytology Specimens: A Survey by the College of American Pathologists Cytopathology Committee.

CONTEXT.­: Cytologic-histologic correlation (CHC) is a Clinical Laboratory Improvement Amendments-mandated requirement for gynecologic cytology, but no similar requirement exists for nongynecologic cytology. This study presents the findings from a College of American Pathologists' survey of nongynecologic cytology practice patterns. OBJECTIVE.­: To survey the current CHC practices for nongynecologic cytology. DESIGN.­: Data were analyzed from a survey developed by the committee and distributed to participants in the Nongynecologic Cytopathology Education Program mailing. RESULTS.­: Adoption of CHC for nongynecologic cytology cases is worldwide, with 88.5% of institutions performing CHC on these specimens, a substantial increase from previous years. Performance of CHC varied by institution type, with clinic or regional/local independent laboratories and national/corporate laboratories performing CHC significantly less frequently than hospitals, university hospitals/academic medical centers, and Veterans Administration/Department of Defense hospital institutions. Most CHC was performed concurrently in real time, when the corresponding surgical specimen was reviewed. Selection for real-time concurrent CHC was by the interpreting pathologist, the pathologist diagnosing the surgical biopsy sample or cytopathology case, or both. Sampling was by far the most common reason for discordance. A 2-step difference was the most frequent threshold for discordance between cytology and surgical specimens, but this criterion varied among institutions, with no majority definition. The positive predictive value of a positive cytology finding was calculated rarely in North American institutions but was calculated more frequently in international institutions. CONCLUSIONS.­: CHC practices for nongynecologic cytopathology mirror those found for CHC of gynecologic cytopathology.

Utility and performance of cell blocks in urine cytology: Experience at three teaching hospitals.

BACKGROUND: The use of cell block (CB) preparation is underused in urine cytology (UC) and varies among hospitals. In addition to confirming a diagnosis, CBs can be useful in cases of metastatic disease, diagnoses requiring immunohistochemical (IHC) staining, and for ancillary studies. The role of this study is to examine the performance of CBs for UC at three affiliated teaching hospitals. MATERIALS AND METHODS: A retrospective review of UC cases with a CB was conducted at a county hospital, Veterans Affairs hospital, and tertiary university-based hospital. For each specimen, patient demographics, specimen type, volume, original diagnosis, and IHC stains were recorded. Each case was reviewed for diagnosis based on ThinPrep alone, diagnosis based on ThinPrep and CB, utility of CB for diagnosis, and CB cellularity. RESULTS: A total of 250 UC specimens with CB from 186 patients was identified. Bladder washes were the most common (72.1%). IHC stains were performed on 17.2% of cases. On blinded review, CB preparation was deemed useful in 61.2% of cases, with the highest rate for suspicious for high-grade urothelial carcinoma (SHGUC) cases (87.0%). The diagnosis based on ThinPrep review changed with incorporation of CB in 13.2% of cases, with the highest rate for SHGUC cases (43.5%). CONCLUSIONS: The results demonstrate that use of CB in UC confirms the final diagnosis in more than one-half of cases and changes the diagnosis in a subset of cases. Use of CB was most helpful in the SHGUC category. Further evaluation of the types of cases in which CB are prepared is warranted.

Current State of Cytologic-Histologic Correlation Implementation for North American and International Laboratories: Results of the College of American Pathologists Cytopathology Committee Laboratory Practices in Gynecologic Cytology Survey.

CONTEXT.­: The College of American Pathologists (CAP) updated the Laboratory Accreditation Program Cytopathology Checklist to assist laboratories in meeting and exceeding the Clinical Laboratory Improvement Amendments standards for gynecologic cytologic-histologic correlation (CHC). OBJECTIVE.­: To survey the current CHC practices. DESIGN.­: Data were analyzed from a survey developed by the committee and distributed to participants in the CAP Gynecologic Cytopathology PAP Education Program mailing. RESULTS.­: Worldwide, CHC practice is nearly universally adopted, with an overall rate of 87.0% (568 of 653). CHC material was highly accessible. CHC was commonly performed real time/concurrently at the time the corresponding surgical pathology was reviewed. Investigation of CHC discordances varied with North American laboratories usually having a single pathologist review all discrepant histology and cytology slides to determine the reason for discordance, while international laboratories have a second pathologist review histology slides to determine the reason for discordance. The cause of CHC discordance was primarily sampling issues. The more common statistical metrics for CHC monitoring were the total percentage of cases that correlated with subsequent biopsies, screening error rate by cytotechnologist, and interpretative error rate by cytotechnologist. CONCLUSIONS.­: Many laboratories have adopted and implemented the CHC guidelines with identifiable differences in practices between North American and international laboratories. We identify the commonalities and differences between North American and international institutional practices including where CHC is performed, how CHC cases are identified and their accessibility, when CHC is performed, who investigates discordances, what discordances are identified, and how the findings affect quality improvement.

Cytologic features of micropapillary variant urothelial carcinoma in urinary tract cytology: Case series and review of literature.

BACKGROUND: The micropapillary variant of urothelial carcinoma (MPVUC) is rare and aggressive. Surgical specimens often show atypical micro-clusters (AMCs) of cells with hyperchromatic, pyknotic, peripheral, irregular nuclei with variable nuclear to cytoplasmic ratios. We reviewed urinary tract cytology (UTC) from patients with MPVUC and hypothesized that AMCs would be present similar to those in surgical specimens. METHODS: The archives were searched from 2000 to 2020 for patients with surgical cases with either MPVUC or conventional high-grade urothelial carcinoma (HGUC) and with prior abnormal UTC. Two pathologists reviewed UTC cases and controls in a blinded manner for AMCs, with quantitation of none, low, moderate, and high. Interrater reliability was compared by quadratic weighted Cohen's Kappa test. The association between numerical average score and MPVUC status was determined by logistic regression. RESULTS: Five patients with invasive MPVUC, one patient with a noninvasive micropapillary component, and 15 control patients with conventional HGUC were included. All patients had prior or concurrent abnormal UTC samples. Increasing category of quantities of AMCs on cytology was associated with micropapillary status (OR 7.9, 95% CI 2.7-118, p = .045), with moderate agreement between raters (Cohen's Kappa 0.54, 95% CI 0.19-0.89, p = .004). CONCLUSIONS: In patients with MPVUC on surgical specimen, AMCs were frequently observed on cytology. Similar atypical clusters were observed in patients with nonmicropapillary HGUC, albeit at lower frequency. However, given the WHO recommendation to diagnose micropapillary only if an invasive micropapillary component is present, a specific diagnosis of MPVUC on UTC cannot be based solely on the presence of AMCs.

Performance of specific morphologic features in distinguishing low-grade squamous intraepithelial lesions from high-grade squamous intraepithelial lesions in borderline cases: a College of American Pathologists Cytopathology Committee multiobserver study.

INTRODUCTION: Distinguishing between low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL) can be difficult on certain Papanicolaou (Pap) tests, hindering interobserver concordance. We investigated the variables influencing the interpretation of LSIL versus HSIL in Pap test slides rejected from the College of American Pathologists PAP education program. MATERIALS AND METHODS: Eleven cytologists, who were unaware of the reference interpretation, examined 21 Pap slides (11 submitted as LSIL and 10 as HSIL) rejected from the PAP education program and recorded the number of LSIL cells, HSIL cells, keratinized dysplastic cells, LSIL clusters with mixed HSIL cells, atypical squamous metaplasia, atypical glandular cells, the presence of inflammation or infectious organisms, and the overall interpretation (LSIL or HSIL). We evaluated the significance of these 11 variables using a nonlinear mixed model analysis. RESULTS: LSIL had greater concordance (92 of 121 responses; 76.0% concordance) than HSIL (68 of 110 responses; 61.8% concordance; P < 0.001). The only predictors of misclassified cases were the number of atypical squamous metaplastic cells and the number of HSIL cells (P < 0.001). The more of these cells identified, the more likely the reviewers were to classify the slide as HSIL. The reproducibility of the diagnosis was fair (Gwet's agreement coefficient, 0.33). CONCLUSIONS: Interobserver reproducibility is a challenge for a subset of cases with features intermediate between LSIL and HSIL. Atypical squamous metaplasia and dysplastic nuclei with a nuclear/cytoplasmic ratio greater than one half of the cell volume (HSIL) present on a Pap test influenced the likelihood that a reviewer would interpret the case as HSIL rather than LSIL.

Suppressor effect of catechol-O-methyltransferase gene in prostate cancer.

Catechol-estrogens can cause genetic mutations and to counteract their oncogenicity, the catechol-O-methyltransferase (COMT) gene is capable of neutralizing these reactive compounds. In this study, we determined the functional effects and regulation of COMT in prostate cancer. Both the Cancer Genome Atlas (TCGA) and immunohistochemical analysis of clinical specimens demonstrated a reduction of COMT expression in prostate cancer. Also, western analyses of prostate cancer cell lines show COMT levels to be minimal in DuPro and DU145 and thus, these cells were used for further analyses. Re-expression of COMT led to suppressed migration ability (wound healing assay) and enhanced apoptosis (flow cytometric analyses), and when challenged with 4-hydroxyestradiol, a marked reduction of cell proliferation (MTT assay) was observed. Xenograft growth in athymic mice also resulted in inhibition due to COMT. As a mechanism, western analyses show cleaved CASP3 and BID were increased whereas XIAP and cIAP2 were reduced due to COMT. As COMT expression is low in prostate cancer, its regulation was determined. Databases identified several miRNAs capable of binding COMT and of these, miR-195 was observed to be increased in prostate cancer according to TCGA. Real-time PCR validated upregulation of miR-195 in clinical prostate cancer specimens as well as DuPro and DU145 and interestingly, luciferase reporter showed miR-195 capable of binding COMT and overexpressing miR-195 could reduce COMT in cells. These results demonstrate COMT to play a protective role by activating the apoptosis pathway and for miR-195 to regulate its expression. COMT may thus be a potential biomarker and gene of interest for therapeutic development for prostate cancer.

A lncRNA TCL6-miR-155 Interaction Regulates the Src-Akt-EMT Network to Mediate Kidney Cancer Progression and Metastasis.

Metastasis is the leading cause of mortality from kidney cancer, and understanding the underlying mechanism of this event will provide better strategies for its management. Here we investigated the biological, functional, and clinical significance of lncTCL6 and its interacting miR-155 in clear cell renal cell carcinoma (ccRCC). We employed a comprehensive approach to investigate the lncTCL6-miR-155-Src/Akt-mediated epithelial-to-mesenchymal transition (EMT) pathway as a novel regulatory mechanism in ccRCC progression. Expression analyses revealed that lncTCL6 is downregulated in ccRCC compared with normal tissues. Overexpression of lncTCL6 in ccRCC cell lines impaired their oncogenic functions, such as cell proliferation and migration/invasion, and induced cell-cycle arrest and apoptosis; conversely, depletion of lncTCL6 rescued these phenotypic effects. Furthermore, lncTCL6 directly interacted with miR-155. Unlike lncTCL6, miR-155 was overexpressed in ccRCC. Stable knockdown of miR-155 phenocopied the effects of lncTCL6 overexpression. Conversely, reconstitution of miR-155 and suppression of lncTCL6 in noncancerous renal cell HK2 induced tumorigenic characteristics. Patients with higher expression of lncTCL6 and lower expression of miR-155 had better survival probability. When overexpressed, lncTCL6 recruited STAU1 and mediated decay of Src mRNA, followed by a marked downregulation of an integrated network of Src target genes involved in migration, invasion, and EMT. However, the interaction between miR-155 and lncTCL6 attenuated the regulatory role of lncTCL6 on Src-mediated EMT. In conclusion, this study is the first report documenting the lncTCL6-miR155-Src/Akt/EMT network as a novel regulatory mechanism in aggressive ccRCC and a promising therapeutic target to inhibit renal cancer. SIGNIFICANCE: This study's investigation of noncoding RNA interactions in renal cell carcinoma identify miRNA-155-lncRNA TCL6-mediated regulation of the Src-Akt-EMT network as a novel mechanism of disease progression and metastasis.

MicroRNA determinants of neuroendocrine differentiation in metastatic castration-resistant prostate cancer.

Therapy-induced neuroendocrine prostate cancer (NEPC), an extremely aggressive variant of castration-resistant prostate cancer (CRPC), is increasing in incidence with the widespread use of highly potent androgen receptor (AR)-pathway inhibitors (APIs) such as Enzalutamide (ENZ) and Abiraterone and arises via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED). The molecular basis of NED is not completely understood leading to a lack of effective molecular markers for its diagnosis. Here, we demonstrate for the first time, that lineage switching to NE states is accompanied by key miRNA alterations including downregulation of miR-106a~363 cluster and upregulation of miR-301a and miR-375. To systematically investigate the key miRNAs alterations driving therapy-induced NED, we performed small RNA-NGS in a retrospective cohort of human metastatic CRPC clinical samples + PDX models with adenocarcinoma features (CRPC-adeno) vs those with neuroendocrine features (CRPC-NE). Further, with the application of machine learning algorithms to sequencing data, we trained a 'miRNA classifier' that could robustly classify 'CRPC-NE' from 'CRPC-Adeno' cases. The performance of classifier was validated in an additional cohort of mCRPC patients and publicly available PCa cohorts. Importantly, we demonstrate that miR-106a~363 cluster pleiotropically regulate cardinal nodal proteins instrumental in driving NEPC including Aurora Kinase A, N-Myc, E2F1 and STAT3. Our study has important clinical implications and transformative potential as our 'miRNA classifier' can be used as a molecular tool to stratify mCRPC patients into those with/without NED and guide treatment decisions. Further, we identify novel miRNA NED drivers that can be exploited for NEPC therapeutic targeting.

Potential dual functional roles of the Y-linked RBMY in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a highly heterogeneous liver cancer with significant male biases in incidence, disease progression, and outcomes. Previous studies have suggested that genes on the Y chromosome could be expressed and exert various male-specific functions in the oncogenic processes. In particular, the RNA-binding motif on the Y chromosome (RBMY) gene is frequently activated in HCC and postulated to promote hepatic oncogenesis in patients and animal models. In the present study, immunohistochemical analyses of HCC specimens and data mining of The Cancer Genome Atlas (TCGA) database revealed that high-level RBMY expression is associated with poor prognosis and survival of the patients, suggesting that RBMY could possess oncogenic properties in HCC. To examine the immediate effect(s) of the RBMY overexpression in liver cancer cells, cell proliferation was analyzed on HuH-7 and HepG2 cells. The results unexpectedly showed that RBMY overexpression inhibited cell proliferation in both cell lines as its immediate effect, which led to vast cell death in HuH-7 cells. Transcriptome analysis showed that genes involved in various cell proliferative pathways, such as the RAS/RAF/MAP and PIP3/AKT signaling pathways, were downregulated by RBMY overexpression in HuH-7 cells. Furthermore, in vivo analyses in a mouse liver cancer model using hydrodynamic tail vein injection of constitutively active AKT and RAS oncogenes showed that RBMY abolished HCC development. These findings support the notion that Y-linked RBMY could serve dual tumor-suppressing and tumor-promoting functions, depending on the spatiotemporal and magnitude of its expression during oncogenic processes, thereby contributing to sexual dimorphisms in liver cancer.

Elevated Tumor Lactate and Efflux in High-grade Prostate Cancer demonstrated by Hyperpolarized 13C Magnetic Resonance Spectroscopy of Prostate Tissue Slice Cultures.

Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) 13C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-13C]pyruvate to [1-13C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP 13C MR could distinguish low-risk (Gleason score ≤3 + 4) from high-risk (Gleason score ≥4 + 3) PCa. To test this and the ability of HP 13C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. 31P spectra demonstrated good viability and dynamic HP 13C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased LDHA expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-13C]pyruvate MR.

Practice Patterns in Urinary Cytopathology Prior to the Paris System for Reporting Urinary Cytology.

CONTEXT.­: The Paris System for Reporting Urinary Cytology has been disseminated since its inception in 2013; however, the daily practice patterns of urinary tract cytopathology are not well known. OBJECTIVE.­: To assess urinary tract cytopathology practice patterns across a variety of pathology laboratories to aid in the implementation and future update of the Paris System for Reporting Urinary Cytology. DESIGN.­: A questionnaire was designed to gather information about urinary tract cytopathology practices and mailed in July 2014 to 2116 laboratories participating in the College of American Pathologists interlaboratory comparison program. The participating laboratories' answers were summarized. RESULTS.­: Of the 879 of 2116 laboratories (41%) that participated, 745 (84.8%) reported processing urinary tract specimens in house. The laboratories reported processing various specimen types: voided urine, 735 of 738 (99.6%); bladder washing/barbotage, 639 of 738 (86.6%); and catheterized urine specimens, 653 of 738 (88.5%). Some laboratories used multiple preparation methods, but the most commonly used preparation techniques for urinary tract specimens were ThinPrep (57.4%) and Cytospin (45.5%). Eighty-eight of 197 laboratories (44.7%) reported preparing a cell block, but with a low frequency. Adequacy criteria were used by 295 of 707 laboratories (41.7%) for voided urine, and 244 of 707 (34.5%) assessed adequacy for bladder washing/barbotage. More than 95% of the laboratories reported the use of general categories: negative, atypical, suspicious, and positive. Polyomavirus was classified as negative in 408 of 642 laboratories (63.6%) and atypical in 189 of 642 (29.4%). One hundred twenty-eight of 708 laboratories (18.1%) performed ancillary testing, and of these, 102 of 122 (83.6%) reported performing UroVysion. CONCLUSIONS.­: Most laboratories use the ThinPrep method followed by the Cytospin technique; therefore, the criteria published in The Paris System for Reporting Urinary Cytology, based mostly on ThinPrep and SurePath, should be validated for Cytospin, and relevant information should be included in the revised edition of The Paris System for Reporting Urinary Cytology.

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

BRN4 Is a Novel Driver of Neuroendocrine Differentiation in Castration-Resistant Prostate Cancer and Is Selectively Released in Extracellular Vesicles with BRN2.

PURPOSE: Neuroendocrine prostate cancer (NEPC), an aggressive variant of castration-resistant prostate cancer (CRPC), often emerges after androgen receptor-targeted therapies such as enzalutamide or de novo, via trans-differentiation process of neuroendocrine differentiation. The mechanistic basis of neuroendocrine differentiation is poorly understood, contributing to lack of effective predictive biomarkers and late disease recognition. The purpose of this study was to examine the role of novel proneural Pit-Oct-Unc-domain transcription factors (TF) in NEPC and examine their potential as noninvasive predictive biomarkers.Experimental Design: Prostate cancer patient-derived xenograft models, clinical samples, and cellular neuroendocrine differentiation models were employed to determine the expression of TFs BRN1 and BRN4. BRN4 levels were modulated in prostate cancer cell lines followed by functional assays. Furthermore, extracellular vesicles (EV) were isolated from patient samples and cell culture models, characterized by nanoparticle tracking analyses, Western blotting, and real-time PCR. RESULTS: We identify for the first time that: (i) BRN4 is amplified and overexpressed in NEPC clinical samples and that BRN4 overexpression drives neuroendocrine differentiation via its interplay with BRN2, a TF that was previously implicated in NEPC; (ii) BRN4 and BRN2 mRNA are actively released in prostate cancer EVs upon neuroendocrine differentiation induction; and (iii) enzalutamide treatment augments release of BRN4 and BRN2 in prostate cancer EVs, promoting neuroendocrine differentiation induction. CONCLUSIONS: Our study identifies a novel TF that drives NEPC and suggests that as adaptive mechanism to enzalutamide treatment, prostate cancer cells express and secrete BRN4 and BRN2 in EVs that drive oncogenic reprogramming of prostate cancer cells to NEPC. Importantly, EV-associated BRN4 and BRN2 are potential novel noninvasive biomarkers to predict neuroendocrine differentiation in CRPC.

Upregulation of miR-130b Contributes to Risk of Poor Prognosis and Racial Disparity in African-American Prostate Cancer.

Prostate cancer incidence and mortality rates are higher in African-American (AA) than in European-American (EA) men. The main objective of this study was to elucidate the role of miR-130b as a contributor to prostate cancer health disparity in AA patients. We also determined whether miR-130b is a prognostic biomarker and a new therapeutic candidate for AA prostate cancer. A comprehensive approach of using cell lines, tissue samples, and the TCGA database was employed. We performed a series of functional assays such as cell proliferation, migration, invasion, RT2-PCR array, qRT-PCR, cell cycle, luciferase reporter, immunoblot, and IHC. Various statistical approaches such as Kaplan-Meier, uni-, and multivariate analyses were utilized to determine the clinical significance of miR-130b. Our results showed that elevated levels of miR-130b correlated with race disparity and PSA levels/failure and acted as an independent prognostic biomarker for AA patients. Two tumor suppressor genes, CDKN1B and FHIT, were validated as direct functional targets of miR-130b. We also found race-specific cell-cycle pathway activation in AA patients with prostate cancer. Functionally, miR-130b inhibition reduced cell proliferation, colony formation, migration/invasion, and induced cell-cycle arrest. Inhibition of miR-130b modulated critical prostate cancer-related biological pathways in AA compared with EA prostate cancer patients. In conclusion, attenuation of miR-130b expression has tumor suppressor effects in AA prostate cancer. miR-130b is a significant contributor to prostate cancer racial disparity as its overexpression is a risk factor for poor prognosis in AA patients with prostate cancer. Thus, regulation of miR-130b may provide a novel therapeutic approach for the management of prostate cancer in AA patients.

Role of a novel race-related tumor suppressor microRNA located in frequently deleted chromosomal locus 8p21 in prostate cancer progression.

The prostate cancer (PCa) genome is characterized by deletions of chromosome 8p21-22 region that increase significantly with tumor grade and are associated with poor prognosis. We proposed and validated a novel, paradigm-shifting hypothesis that this region is associated with a set of microRNA genes-miR-3622, miR-3622b, miR-383-that are lost in PCa and play important mechanistic roles in PCa progression and metastasis. Extending our hypothesis, in this study, we evaluated the role of a microRNA gene located in chromosome 8p-miR-4288-by employing clinical samples and cell lines. Our data suggests that (i) miR-4288 is widely downregulated in primary prostate tumors and cell lines; (ii) miR-4288 expression is lost in metastatic castration-resistant PCa; (ii) miR-4288 downregulation is race-related PCa alteration that is prevalent in Caucasian patients and not in African Americans; (iii) in Caucasians, miR-4288 was found to be associated with increasing tumor grade and high serum prostate-specific antigen, suggesting that miR-4288 downregulation/loss may be associated with tumor progression specifically in Caucasians; (iv) miR-4288 possess significant potential as a molecular biomarker to predict aggressiveness/metastasis; and (v) miR-4288 is anti-proliferative, is anti-invasive and inhibits epithelial-to-mesenchymal transition; and (vi) miR-4288 directly represses expression of metastasis/invasion-associated genes MMP16 and ROCK1. Thus, the present study demonstrates a tumor suppressor role for a novel miRNA located with a frequently lost region in PCa, strengthening our hypothesis that this locus is causally related to PCa disease progression via loss of microRNA genes. Our study suggests that miR-4288 may be a novel biomarker and therapeutic target, particularly in Caucasians.

NMR quantification of lactate production and efflux and glutamate fractional enrichment in living human prostate biopsies cultured with [1,6-13 C2 ]glucose.

PURPOSE: Image-guided prostate biopsies are routinely acquired in the diagnosis and treatment monitoring of prostate cancer, yielding useful tissue for identifying metabolic biomarkers and therapeutic targets. We developed an optimized biopsy tissue culture protocol in combination with [1,6-13 C2 ]glucose labeling and quantitative high-resolution NMR to measure glycolysis and tricarboxcylic acid (TCA) cycle activity in freshly acquired living human prostate biopsies. METHODS: We acquired 34 MRI-ultrasound fusion-guided prostate biopsies in vials on ice from 22 previously untreated patients. Within 15 min, biopsies were transferred to rotary tissue culture in 37°C prostate medium containing [1,6-13 C2 ]glucose. Following 24 h of culture, tissue lactate and glutamate pool sizes and fractional enrichments were quantified using quantitative 1 H high resolution magic angle spinning Carr-Purcell-Meiboom-Gill (CPMG) spectroscopy at 1°C with and without 13 C decoupling. Lactate effluxed from the biopsy tissue was quantified in the culture medium using quantitative solution-state high-resolution NMR. RESULTS: Lactate concentration in low-grade cancer (1.15 ± 0.78 nmol/mg) and benign (0.74 ± 0.15 nmol/mg) biopsies agreed with prior published measurements of snap-frozen biopsies. There was substantial fractional enrichment of [3-13 C]lactate (≈70%) and [4-13 C]glutamate (≈24%) in both low-grade cancer and benign biopsies. Although a significant difference in tissue [3-13 C]lactate fractional enrichment was not observed, lactate efflux was significantly higher (P < 0.05) in low-grade cancer biopsies (0.55 ± 0.14 nmol/min/mg) versus benign biopsies (0.31 ± 0.04 nmol/min/mg). CONCLUSION: A protocol was developed for quantification of lactate production-efflux and TCA cycle activity in single living human prostate biopsies, allowing metabolic labeling on a wide spectrum of human tissues (e.g., metastatic, post-non-surgical therapy) from patients not receiving surgery.

Low-Grade Squamous Intraepithelial Lesion or High-Grade Squamous Intraepithelial Lesion? Concordance Between the Interpretation of Low-Grade Squamous Intraepithelial Lesion and High-Grade Squamous Intraepithelial Lesion in Papanicolaou Tests: Results From the College of American Pathologists PAP Education Program.

CONTEXT.­: Obtaining diagnostic concordance for squamous intraepithelial lesions in cytology can be challenging. OBJECTIVE.­: To determine diagnostic concordance for biopsy-proven low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) Papanicolaou test slides in the College of American Pathologists PAP Education program. DESIGN.­: We analyzed 121 059 responses from 4251 LSIL and HSIL slides for the interval 2004 to 2013 using a nonlinear mixed-model fit for reference diagnosis, preparation type, and participant type. We evaluated interactions between the reference diagnosis and the other 2 factors in addition to a repeated-measures component to adjust for slide-specific performance. RESULTS.­: There was a statistically significant difference between misclassification of LSIL (2.4%; 1384 of 57 664) and HSIL (4.4%; 2762 of 63 395). There was no performance difference between pathologists and cytotechnologists for LSIL, but cytotechnologists had a significantly higher HSIL misclassification rate than pathologists (5.5%; 1437 of 27 534 versus 4.0%; 1032 of 25 630; P = .01), and both were more likely to misrepresent HSIL as LSIL ( P < .001) than the reverse. ThinPrep LSIL slides were more likely to be misclassified as HSIL (2.4%; 920 of 38 582) than SurePath LSIL slides (1.5%; 198 of 13 196), but conventional slides were the most likely to be misclassified in both categories (4.5%; 266 of 5886 for LSIL, and 6.5%; 573 of 8825 for HSIL). CONCLUSIONS.­: More participants undercalled HSIL as LSIL (false-negative) than overcalled LSIL as HSIL (false-positive) in the PAP Education program, with conventional slides more likely to be misclassified than ThinPrep or SurePath slides. Pathologists and cytotechnologists classify LSIL equally well, but cytotechnologists are significantly more likely to undercall HSIL as LSIL than are pathologists.

Correction: Cytochrome P450 1B1 inhibition suppresses tumorigenicity of prostate cancer via caspase-1 activation.

[This corrects the article DOI: 10.18632/oncotarget.16598.].

Improved diagnostic precision of urine cytology by implementation of The Paris System and the use of cell blocks.

BACKGROUND: The Paris System for Reporting Urinary Cytology (TPS) published in 2016 provides a standardized approach to evaluating urine cytology. In the authors' practice, a TPS-like approach was adopted in 2012 using similarly defined cytologic criteria and correlating cystoscopic findings, and they also began incorporating the use of cell block (CB) material. The objective of the current study was to assess whether this TPS-like approach with the use of CB, as well as direct implementation of TPS, improved the diagnostic value of urine cytology. METHODS: In total, 188 consecutive urine cytology specimens from 2010 through 2016 that had concurrent or subsequent histologic specimens available were retrospectively analyzed for diagnostic correlation. Urine cytology performance was compared between the periods 2010 to 2012 (pre-TPS-like), 2013 to 2016 (TPS-like), and after TPS reclassification, including blind review by a cytopathologist. The contribution of available CB material to final diagnoses also was assessed. RESULTS: Both the TPS-like approach and TPS reclassification resulted in significantly lowering the rate of atypical urothelial cells (AUC) diagnosis from 48%, to 21%, to 9% (from pre-TPS, to TPS-like, to after TPS reclassification, respectively; P < 0.01) while increasing the positive predictive value of an AUC diagnosis for high-grade urothelial carcinoma from 21%, to 43%, to 83%, respectively. The use of CBs contributed to a definitive final diagnosis in 24 of 36 cases (66.7%). CONCLUSIONS: In support of the new TPS recommendations, the application of stringent "TPS-like" and TPS criteria improves the value of an AUC diagnosis for clinicians and patients by lowering the AUC rate and increasing the positive predictive value of AUC for high-grade urothelial carcinoma. CBs can be used to help resolve problematic cases for more definitive diagnostic categorization.