Neurofilament Light Chain as a Biomarker of Global Cognition in Individuals With Possible Vascular Mild Cognitive Impairment.

BACKGROUND: Neurofilament Light Chain (NfL) is a biomarker of axonal injury elevated in mild cognitive impairment (MCI) and Alzheimer's disease dementia. Blood NfL also inversely correlates with cognitive performance in those conditions. However, few studies have assessed NfL as a biomarker of global cognition in individuals demonstrating mild cognitive deficits who are at risk for vascular-related cognitive decline. OBJECTIVE: To assess the relationship between blood NfL and global cognition in individuals with possible vascular MCI (vMCI) throughout cardiac rehabilitation (CR). Additionally, NfL levels were compared to age/sex-matched cognitively unimpaired (CU) controls. METHOD: Participants with coronary artery disease (vMCI or CU) were recruited at entry to a 24-week CR program. Global cognition was measured using the Montreal Cognitive Assessment (MoCA) and plasma NfL level (pg/ml) was quantified using a highly sensitive enzyme-linked immunosorbent assay. RESULTS: Higher plasma NfL was correlated with worse MoCA scores at baseline (ß = -.352, P = .029) in 43 individuals with vMCI after adjusting for age, sex, and education. An increase in NfL was associated with worse global cognition (b[SE] = -4.81[2.06], P = .023) over time, however baseline NfL did not predict a decline in global cognition. NfL levels did not differ between the vMCI (n = 39) and CU (n = 39) groups (F(1, 76) = 1.37, P = .245). CONCLUSION: Plasma NfL correlates with global cognition at baseline in individuals with vMCI, and is associated with decline in global cognition during CR. Our findings increase understanding of NfL and neurobiological mechanisms associated with cognitive decline in vMCI.

Host Expression Profiling From Diagnostic Coronavirus Disease 2019 Swabs Associates Upper Respiratory Tract Immune Responses With Radiologic Lung Pathology and Clinical Severity.

Background: COVID-19 presents with a breadth of symptomatology including a spectrum of clinical severity requiring intensive care unit (ICU) admission. We investigated the mucosal host gene response at the time of gold standard COVID-19 diagnosis using clinical surplus RNA from upper respiratory tract swabs. Methods: Host response was evaluated by RNA-sequencing, and transcriptomic profiles of 44 unvaccinated patients including outpatients and in-patients with varying levels of oxygen supplementation were included. Additionally, chest X-rays were reviewed and scored for patients in each group. Results: Host transcriptomics revealed significant changes in the immune and inflammatory response. Patients destined for the ICU were distinguished by the significant upregulation of immune response pathways and inflammatory chemokines, including cxcl2 which has been linked to monocyte subsets associated with COVID-19 related lung damage. In order to temporally associate gene expression profiles in the upper respiratory tract at diagnosis of COVID-19 with lower respiratory tract sequalae, we correlated our findings with chest radiography scoring, showing nasopharygeal or mid-turbinate sampling can be a relevant surrogate for downstream COVID-19 pneumonia/ICU severity. Conclusions: This study demonstrates the potential and relevance for ongoing study of the mucosal site of infection of SARS-CoV-2 using a single sampling that remains standard of care in hospital settings. We highlight also the archival value of high quality clinical surplus specimens, especially with rapidly evolving COVID-19 variants and changing public health/vaccination measures.

Genomic profiling of dedifferentiated endometrial carcinomas arising in the background of high-grade carcinoma: a targeted next-generation sequencing study.

AIMS: Our understanding of dedifferentiated endometrial carcinoma (DEC), a rare and aggressive malignancy, mainly reflects undifferentiated carcinomas (UC) arising in the setting of low-grade endometrial cancer (DEC-LG). However, cases of UC arising in the setting of high-grade EC (DEC-HG) have been noted in the literature. Our knowledge of the genomics of DEC-HG is limited. To characterise the molecular landscape of DEC-HC, targeted genomic sequencing and immunohistochemical analysis was carried out on seven DEC-HG and four DEC-LG. METHODS AND RESULTS: DEC-HG and DEC-LG, including undifferentiated and differentiated components, both showed a similar frequency and spectrum of mutations. ARID1A mutations were identified in 6/7 (86%) DEC-HG and 4/4 (100%) DEC-LG, while SMARCA4 mutations were present in 4/7 (57%) DEC-HG and in 1/4 (25%) DEC-LG. Concurrent SMARCA4/BRG1 protein loss by immunohistochemistry was observed in 3/4 and 1/1 SMARCA4 mutated DEC-HG and DEC-LG, respectively. Neither genomic alterations nor protein loss in SMARCB1/INI1 were observed in any of our cases. TP53 mutations were detected in 4/7 (57%) DEC-HG and in 2/4 (50%) DEC-LG, while mutation-pattern p53 immunohistochemistry expression was observed in 2/7 (29%) DEC-HG and none of the DEC-LG. MLH1 mutations were observed in 1/7 (14%) DEC-HG and 1/4 (25%) DEC-LG. MSH2 and MSH6 mutations were each detected in 1/7 (14%) DEC-HG, but neither was associated with corresponding loss of protein expression. CONCLUSION: The findings support expanding the definition of DEC to include DEC-HG, a previously under-recognised phenomenon with genomic similarities to DEC-LG.

Mesonephric-like adenocarcinoma of the female genital tract: novel observations and detailed molecular characterisation of mixed tumours and mesonephric-like carcinosarcomas.

AIMS: To report novel observations in five mesonephric-like adenocarcinomas (MLAs) of the female genital tract. METHODS AND RESULTS: We report two endometrial MLAs in association with endometrioid carcinoma and atypical hyperplasia and three (one endometrial, two ovarian) cases with a sarcomatoid component (mesonephric-like carcinosarcoma). Pathogenic KRAS mutations, which are characteristic of MLA, were identified in all cases although interestingly, in one of the mixed carcinomas, this was confined to the endometrioid component. The concurrent MLA, endometrioid carcinoma and atypical hyperplasia components in one case harboured identical EGFR, PTEN and CCNE1 mutations, suggesting that the atypical hyperplasia gave rise to a Müllerian carcinoma with both endometrioid and mesonephric-like components. The carcinosarcomas all contained a component of MLA and a sarcomatous component with chondroid elements. In the ovarian carcinosarcomas, the coexisting epithelial and sarcomatous components shared some mutations including KRAS and CREBBP, suggesting that they are clonally related. Furthermore, in one case CREBBP and KRAS mutations detected in the MLA and sarcomatous components were also detected in an associated undifferentiated carcinoma component, suggesting that it was clonally related to the MLA and sarcomatous components. CONCLUSIONS: Our observations provide additional evidence that MLAs have a Müllerian origin and characterise mesonephric-like carcinosarcomas in which chondroid elements appear to be characteristic. In reporting these findings, we provide recommendations for distinction between a mesonephric-like carcinosarcoma and a MLA with a spindle cell component.

PAXgene Fixation for Pancreatic Cancer: Implications for Molecular and Surgical Pathology.

Genomic profiling of pancreatic cancer using small core biopsies has taken an increasingly prominent role in precision medicine. However, if not appropriately preserved, nucleic acids (NA) from pancreatic tissues are known to be susceptible to degradation due to high intrinsic levels of nucleases. PAXgene fixation (PreAnalytix, Switzerland) represents a novel formalin-free tissue preservation method. We sought to compare the NA and histomorphological preservation of pancreatic cancer tissues preserved with PAXgene-fixed paraffin-embedding (PFPE) and formalin-fixed paraffin-embedding (FFPE). Tissues from 19 patients were obtained prospectively from pancreaticoduodenectomy specimens and evaluated by four gastrointestinal pathologists. The extracted NA were quantified by Nanodrop and Qubit and assessed for quality by qPCR, targeted next-generation sequencing (NGS) assay, and RNA-sequencing. Our results demonstrated that, when assessed blindly for morphological quality, the four pathologists deemed the PFPE slides adequate for diagnostic purposes. PFPE tissues enable greater yields of less fragmented and more amplifiable DNA. PFPE tissues demonstrated significantly improved quality control (QC) metrics in a targeted NGS assay including Median Absolute Pair-wise Difference (MAPD) scores. Our results support the use of PAXgene fixative for the processing of specimens from pancreatic cancers with the potential benefits of improved yields for more amplifiable DNA in low-yield biopsy specimens and its ideal use for amplicon-based NGS assays.

Multisite verification of the accuracy of a multi-gene next generation sequencing panel for detection of mutations and copy number alterations in solid tumours.

Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for information derived from RNA was lower than observed for DNA, due largely to decreased quality metrics associated with the RNA components of the assay, suggesting that the RNA portions of comprehensive NGS assays may be more vulnerable to variations in approach and workflow. Overall the results of this study support the use of the OFA for targeted sequencing for testing of clinical samples and suggest specific internal quality metrics that can be reliable indicators of assay failure. While we believe this evidence can be interpreted to support deep targeted sequencing in general, additional studies should be performed to confirm this.

Delayed MRI Enhancement of Colorectal Cancer Liver Metastases Is Associated With Metastatic Mutational Profile.

BACKGROUND/AIM: Individual tumor genomics plays a key role in determining patient prognosis, response to chemotherapy and in guiding therapy. In prior studies, it was shown that the degree of late enhancement of colorectal liver metastases (CRCLM) target tumor enhancement (TTE) as seen on magnetic resonance imaging (MRI) was associated with overall survival. In order to better understand the relationship between MRI enhancement and survival, the aim of this study was to characterize genomic profiles of tumors clustered by MRI TTE, and investigate the association between TTE and genetic mutations. MATERIALS AND METHODS: Matched tumor and normal tissue samples from patients with weak TTE and strong TTE were analyzed by Next-generation sequencing (NGS) technology using a custom colorectal cancer panel. RESULTS: We discovered a total of 42 non-synonymous somatic mutations from 10 patients with weak TTE and 26 with 10 patients with strong TTE. Adenomatosis Polyposis Coli (APC) was the most commonly altered gene, 18 of those APC mutations were found in the weak TTE and 9 in the strong TTE group. CONCLUSION: An association exists between TTE and mutational status of CRCLM, which may offer some explanation as to why TTE is associated with overall survival in patients with CRCLM.

MiR-139 Induces an Interferon-ß Response in Prostate Cancer Cells by Binding to RIG-1.

BACKGROUND: We previously identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Expression of one of the down-regulated miRNAs, miR-139-5p, was significantly associated with a lower incidence of biochemical recurrence and metastasis. Transcriptome profiling of miR-139-expressing prostate cancer cells revealed up-regulation of genes involved in interferon (IFN) stimulation. The association between miR-139 and IFN-ß was further explored in this study. MATERIALS AND METHODS: We examined miR-139 transfected PC3, Du145 and LNCaP cells and the associated IFN response by transcriptome sequencing, immunoblotting and pulldown assays. RESULTS: Treatment of prostate cancer cells by miR-139 resulted in the up-regulation of IFN-related genes. Specifically, miR-139 induced expression of the IFN-ß protein. The ability of miR-139 to induce IFN-ß was due to its binding to RIG-1 and the induction of IFN-related genes was found to be dependent on RIG-1 expression. CONCLUSION: miR-139 acts as an immune agonist of RIG-1 to enhance IFN-ß response in prostate cancer cells.

MR-guided focused ultrasound liquid biopsy enriches circulating biomarkers in patients with brain tumors.

BACKGROUND: Liquid biopsy is promising for early detection, monitoring of response, and recurrence of cancer. The blood-brain barrier (BBB) limits the shedding of biomarker, such as cell-free DNA (cfDNA), into the blood from brain tumors, and their detection by conventional assays. Transcranial MR-guided focused ultrasound (MRgFUS) can safely and transiently open the BBB, providing an opportunity for less-invasive access to brain pathology. We hypothesized that MRgFUS can enrich the signal of circulating brain-derived biomarkers to aid in liquid biopsy. METHODS: Nine patients were treated in a prospective single-arm, open-label trial to investigate serial MRgFUS and adjuvant temozolomide combination in patients with glioblastoma (NCT03616860). Blood samples were collected as an exploratory measure within the hours before and after sonication, with control samples from non-brain tumor patients undergoing BBB opening (BBBO) alone (NCT03739905). RESULTS: Brain regions averaging 7.8 ± 6.0 cm3 (range 0.8-23.1 cm3) were successfully treated within 111 ± 39 minutes without any serious adverse events. We found MRgFUS acutely enhanced plasma cfDNA (2.6 ± 1.2-fold, P < .01, Wilcoxon signed-rank test), neuron-derived extracellular vesicles (3.2 ± 1.9-fold, P < .01), and brain-specific protein S100b (1.4 ± 0.2-fold, P < .01). Further comparison of the cfDNA methylation profiles suggests a signature that is disease- and post-BBBO-specific, in keeping with our hypothesis. We also found cfDNA-mutant copies of isocitrate dehydrogenase 1 (IDH1) increased, although this was in only one patient known to harbor the tumor mutation. CONCLUSIONS: This first-in-human proof-of-concept study shows MRgFUS enriches the signal of circulating brain-derived biomarkers, demonstrating the potential of the technology to support liquid biopsy for the brain.

CTNNB1 Mutations and Aberrant ß-Catenin Expression in Ovarian Endometrioid Carcinoma: Correlation With Patient Outcome.

CTNNB1 mutations and aberrant ß-catenin expression have adverse prognosis in endometrial endometrioid carcinoma, and recent evidence suggests a prognostic role of ß-catenin in ovarian endometrioid carcinoma. Thus, we aimed to determine the prognostic value of the CTNNB1 mutational status, and its correlation with ß-catenin expression, in a well-annotated cohort of 51 ovarian endometrioid carcinomas. We performed immunohistochemistry for ß-catenin and developed an 11-gene next-generation sequencing panel that included whole exome sequencing of CTNNB1 and TP53. Results were correlated with clinicopathologic variables including disease-free and disease-specific survival. Tumor recurrence was documented in 14 patients (27%), and cancer-related death in 8 patients (16%). CTNNB1 mutations were found in 22 cases (43%), and nuclear ß-catenin in 26 cases (51%). CTNNB1 mutation highly correlated with nuclear ß-catenin (P<0.05). Mutated CTNNB1 status was statistically associated with better disease-free survival (P=0.04, log-rank test) and approached significance for better disease-specific survival (P=0.07). It also correlated with earlier International Federation of Gynecology and Obstetrics stage (P<0.05). Nuclear ß-catenin, TP53 mutations, age, ProMisE group, surface involvement, tumor grade and stage also correlated with disease-free survival. There was no association between membranous ß-catenin expression and disease-free or disease-specific survival. CTNNB1 mutations and nuclear ß-catenin expression are associated with better progression-free survival in patients with OEC. This relationship may be in part due to a trend of CTNNB1-mutated tumors to present at early stage. ß-catenin immunohistochemistry may serve as a prognostic biomarker and a surrogate for CTNN1B mutations in the evaluation of patients with ovarian endometrioid neoplasia, particularly those in reproductive-age or found incidentally without upfront staging surgery.

Mir-139 Regulates Autophagy in Prostate Cancer Cells Through Beclin-1 and mTOR Signaling Proteins.

BACKGROUND/AIM: We previously identified a panel of five miRNAs (including miR-139) associated with biochemical recurrence and metastasis in prostate cancer patients. MATERIALS AND METHODS: We examined miR-139 transfected PC3, DU145 and LNCaP cells by morphology as well as by cell-based assays, confocal microscopy and immunoblotting. RESULTS: We found that treatment of prostate cancer cells with miR-139 resulted in phenotypic changes characteristic of autophagic cells. MiR-139 increased the autophagy-related conversion of the microtubule-associated protein light chain 3 (LC3-I to LC3-II) that was specifically inhibited by the miR-139 antagomir. The upregulation of LC3 II was further confirmed by confocal microscopy. miR-139 regulated activation of both mTOR and Beclin1 the two important autophagy-related molecules. We found that upon miR-139 treatment, the cargo adaptor protein p62 which is degraded during autophagy, accumulates. CONCLUSION: These results suggest that miR-139 is inducing autophagic flux blockade leading to apoptosis in prostate cancer cells through the mTOR and Beclin-1 proteins.

Differentiated exophytic vulvar intraepithelial lesion: Clinicopathologic and molecular analysis documenting its relationship with verrucous carcinoma of the vulva.

Verruciform proliferations of the vulva unrelated to HPV infection are rare. The term differentiated exophytic vulvar intraepithelial lesion (DEVIL) was recently proposed for these lesions, which harbor recurrent PIK3CA mutations. It is still unclear whether DEVIL is related to verrucous carcinoma, a neoplasm characterized by persistence and local recurrence but nil risk of distant spread. Specimens identified using the words "verruciform" and "verrucous" were reviewed. Diagnosis of DEVIL required verruciform acanthosis, hyper and/or parakeratosis, hypogranulosis, cytoplasmic pallor, and bland nuclei. Verrucous carcinoma required, in addition, discontinuous, bulbous, puzzle-like nests in the stroma. A targeted next-generation sequencing using a custom 11-gene panel was performed. Eighteen specimens corresponding to ten patients with DEVIL and/or verrucous carcinoma were included. Median age at presentation was 66 years for DEVIL and 70 years for verrucous carcinoma. A similar spectrum of prevalent mutations was found in both lesions involving HRAS, PIK3CA, and BRAF. DEVIL preceded verrucous carcinoma and/or was diagnosed concurrently or in subsequent follow-up in five patients. In four of these, the same mutation was identified in DEVIL and synchronous or metachronous carcinoma. All cases showed wild-type 53 staining and lacked pathogenic TP53 mutations. DEVIL is a rare form of squamous proliferation characterized by prevalent PIK3CA and HRAS mutations. Its temporal relationship with verrucous carcinoma and their shared mutational profile in some patients suggest that DEVIL is a precursor of verrucous carcinoma. Moreover, given their morphologic and molecular overlap and the nil risk of verrucous carcinoma for distant spread, it is conceivable that DEVIL and verrucous carcinoma represent a spectrum of the same entity.

Pilot Study on the Utility of Circulating HER2/Neu Levels in the Serum of Breast Cancer Patients.

BACKGROUND/AIM: Accurate and timely assessment of the human epidermal growth factor receptor 2 (HER2/neu) overexpression is pivotal for the identification of breast cancer (BC) patients that could benefit from HER2-targeted therapy. Currently approved tissue-based HER2 assays (tHER2) are limited to testing HER2 status on tumor samples obtained at a few points in time during the course of the disease. Herein, we assessed serum HER2 (sHER2) status longitudinally in 81 serial samples prospectively collected from 43 consenting patients pre- and post-therapy to revisit the idea of serum testing in the follow-up of BC patients. PATIENTS AND METHODS: The cohort included 11 patients with early BC (EBC), 17 with locally advanced BC (LABC), and 15 with metastatic BC (MBC). sHER2 concentrations were measured using a quantitative ELISA-based technique, using 15 ng/ml as the cut-off for positivity. RESULTS: At baseline, sHER2 was negative in all EBC patients while positive in 1 LABC and 5 MBC patients. Sixteen BC patients (10 LABC, 1 EBC, and 5 MBC) were tHER2 positive. sHER2 and tHER2 results were discordant in 14 patients. Among the 16 tHER2 positive patients, 9 LABC, 1 EBC and 2 MBC patients were sHER2 negative. Conversely, 2 MBC patients were sHER2 positive, despite being tHER2 negative. A rise or drop of sHER2 by >20% correlated with disease progression or pathological response to therapy, respectively. CONCLUSION: The study demonstrated the technical validity and feasibility of the sHER2 assay. Findings suggest that post initial tissue diagnosis (tHER2), sHER2 assay may supplement subsequent tissue tests to monitor disease status and response to therapy. Further studies to assess the role of HER2 targeted therapies in sHER-positive/tHER2-negative cases upon disease progression are warranted.

MicroRNA-139 is a predictor of prostate cancer recurrence and inhibits growth and migration of prostate cancer cells through cell cycle arrest and targeting IGF1R and AXL.

BACKGROUND: We previously identified a panel of five microRNAs (miRNAs) associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using next-generation sequencing-based whole miRNome sequencing and quantitative polymerase chain reaction-based validation analysis. In this study, we examined the mechanism of action of miR-139-5p, one of the downregulated miRNAs identified in the panel. METHODS: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan-Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that overexpressed miR-139 to confirm its targets as well as examine pathways through which miR-139 may function using cell-based assays. RESULTS: Low miR-139 expression was significantly associated with a variety of prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity, and lymph node status. MiR-139 expression was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis were significantly lower among patients with high miR-139 expression (P = .0004 and .038, respectively). Validation in the TCGA data set showed a significant association between dichotomized miR-139 expression and biochemical recurrence (odds ratio, 0.52; 95% confidence interval, 0.33-0.82). Overexpression of miR-139 in prostate cancer cells led to a significant reduction in cell proliferation and migration compared with control cells, with cells arrested in G2 of cell cycle. IGF1R and AXL were identified as potential targets of miR-139 based on multiple miRNA-binding sites in 3'-untranslated regions of both the genes and their association with prostate cancer growth pathways. Luciferase assays verified AXL and IGF1R as direct targets of miR-139. Furthermore, immunoblotting of prostate cancer cells demonstrated IGF1R and AXL protein expression were inhibited by miR-139 treatment, which was reversed by the addition of miR-139 antagomir. Examination of the molecular mechanism of growth inhibition by miR-139 revealed the downregulation of activated AKT and cyclin D1, with upregulation of the CDK inhibitor p21. CONCLUSIONS: miR-139 is associated with improved prognosis in patients with localized prostate cancer, which may be mediated through downregulation of IGF1R and/or AXL and associated signaling pathway components.

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern, North African, and South European countries.

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population-specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.

Identification of a Novel MicroRNA Panel Associated with Metastasis Following Radical Prostatectomy for Prostate Cancer.

BACKGROUND/AIM: This is a case control study designed to identify one or more novel microRNA sequences associated with metastasis following radical prostatectomy for clinically localized prostate cancer. MATERIALS AND METHODS: Samples were obtained from patients with clinical evidence of metastatic disease following surgery (cases) and patients who showed no evidence of metastasis or biochemical recurrence at least 5 years following surgery (controls) as identified from a single-center, institutional database. Cases and controls were matched for tumor grade and duration of follow-up. RESULTS: Whole miRNome analysis identified 2,792 expressed miRNAs in 19 patient pairs. The 497 miRNA sequences with reads per million over 10, were used for analysis, bootstrapping with backward selection identified a panel of 5-miRNA (miR-17-3p, miR-27a-3p, miR-200a-3p, miR-375, and miR-376b-3p) with a risk score strongly associated with metastasis (AUC=89.5%, 95%CI=79.5-99.5%). Methodologically, most studies use the magnitude of differential expression with or without clinical judgement for selection of predictors for inclusion in panels. In order to strengthen the predictive model, a selection strategy was employed, bootstrapping with automated backwards selection, which relied on the strength of association for inclusion. CONCLUSION: A genome-wide analysis of microRNA expression identified a panel of 5 miRNAs strongly associated with prostate cancer metastasis following radical prostatectomy.

MicroRNA-652 induces NED in LNCaP and EMT in PC3 prostate cancer cells.

MicroRNAs (miRNAs) are small noncoding RNA molecules that post-transcriptionally regulate gene expression. Dysregulation of miRNAs is frequently associated with disease and, in particular, is involved in prostate cancer progression. Next generation miRNA sequencing identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. High expression of one of these five miRNAs, miR-652, correlated significantly with an increased rate of prostate cancer biochemical recurrence. Overexpression of miR-652 in prostate cancer cells, PC3 and LNCaP, resulted in increased growth, migration and invasion. Prostate cancer cell xenografts overexpressing miR-652 showed increased tumorigenicity and metastases. We found that miR-652 directly targets the B" regulatory subunit, PPP2R3A, of the tumor suppressor PP2A, inducing epithelial-mesenchymal transition (EMT) in PC3 cells and neuroendocrine-like differentiation (NED) in LNCaP cells. The mesenchymal marker N-cadherin increased and epithelial marker E-cadherin decreased in PC3 cells overexpressing miR-652. In LNCaP cells and xenografted tumors, overexpression of miR-652 increased markers of NED, including chromogranin A, neuron specific enolase, and synaptophysin. MiR-652 may contribute to prostate tumor progression by promoting NED through decreased PP2A function. MiR-652 expression could serve as a biomarker for aggressive prostate cancer, as well as provide an opportunity for novel therapy in prostate cancer.

Null association between androgen-deprivation therapy and nonprostate cancer mortality among older men with nonmetastatic prostate cancer.

BACKGROUND: Androgen-deprivation therapy (ADT) has been associated with cardiovascular risk factors and the development of cardiovascular disease in men with metastatic prostate cancer. We sought to examine the effect of ADT on nonprostate cancer mortality among patients with nonmetastatic prostate cancer. METHODS: We performed a population-based, retrospective cohort study of men aged 66 years and older treated with surgery or radiotherapy for nonmetastatic prostate cancer in Ontario, Canada from 2002 to 2009 using administrative datasets (including the Ontario Cancer Registry, Ontario Drug Benefit, and Ontario Health Insurance Plan). Analysis was performed between September 2016 and April 2017. ADT exposure was operationalized as a time-varying binary and cumulative dose exposure. Primary and secondary outcomes were nonprostate cancer mortality and cardiovascular mortality, respectively. The Fine and Gray subdistribution method with generalized estimating equations was used to calculate subdistribution hazard ratios (sdHR), while accounting for competing risks. RESULTS: We examined 20,651 men treated for nonmetastatic prostate cancer. Median follow-up was 7.4 years and median ADT exposure was 6.4 months. ADT was not significantly associated with nonprostate cancer mortality (sdHR = 0.75, 95% CI: 0.37-1.50) or cardiovascular mortality (sdHR = 1.16, 95% CI: 0.37-3.63) when operationalized as a binary time-varying exposure. Similar results were obtained when we examined ADT cumulative dose exposure. CONCLUSIONS: ADT is not associated with nonprostate cancer mortality or cardiovascular mortality in a large, population-based cohort of older men with localized prostate cancer treated by surgery or radiation therapy.

Association Between Primary Local Treatment and Non-prostate Cancer Mortality in Men With Nonmetastatic Prostate Cancer.

OBJECTIVE: To assess the association between local treatment modality, surgery or radiotherapy, and non-prostate cancer and cardiovascular mortality in patients treated for nonmetastatic prostate cancer, given the high competing risk of mortality in this population. METHODS: We performed a population-based, retrospective cohort study of men treated for nonmetastatic prostate cancer in Ontario, Canada, from 2002 to 2009. Patients treated with surgery and radiotherapy were matched on demographics, comorbidity, and cardiovascular risk factors. The primary outcome was non-prostate cancer mortality. Outcomes were compared using the Fine and Gray subdistribution method with generalized estimating equations. We used a previously published technique to quantify the prevalence and strength of residual confounding necessary to account for observed results. RESULTS: We examined 5393 pairs of matched men. The 10-year cumulative incidence of non-prostate cancer mortality was higher among patients who underwent radiotherapy (12%) than surgery (8%; adjusted subdistribution hazard ratio [HR] 1.57, 95% confidence interval 1.35-1.83). Patients treated with radiotherapy also had an increased risk of cardiovascular mortality (adjusted HR 1.74, 95% confidence interval 1.27-2.37). Hypothetical residual confounders would have to be both strongly associated with non-prostate cancer mortality (HRs > 2.5) and have highly differential prevalence to nullify the observed effect. CONCLUSION: Among patients carefully matched on cardiovascular risk factors, those treated with radiotherapy had an increased risk of non-prostate cancer mortality and cardiovascular disease. Because of the observational nature of the data, the potential for confounding remains. The magnitude and prevalence of potential residual confounders required to account for differences in treatment effects for prostate cancer was quantified.

Overview of transcriptomic analysis of all human proteases, non-proteolytic homologs and inhibitors: Organ, tissue and ovarian cancer cell line expression profiling of the human protease degradome by the CLIP-CHIP™ DNA microarray.

The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript. Using the CLIP-CHIP™ we mapped the expression profile of proteases and their inhibitors in 23 different human tissues and 6 ovarian cancer cell lines in 104 sample datasets. Hierarchical cluster analysis showed that expression profiles clustered according to their anatomic locations, cellular composition, physiologic functions, and the germ layer from which they are derived. The human ovarian cancer cell lines cluster according to malignant grade. 110 proteases and 42 inhibitors were tissue specific (1 to 3 tissues). Of these 110 proteases 69% (74) are mainly extracellular, 30% (34) intracellular and 1% intramembrane. Notably, 35% (197/565) of human proteases and 30% (47/156) of inhibitors were ubiquitously expressed in all 23 tissues; 27% (155) of proteases and 21% (32) of inhibitors were broadly expressed in 4-20 tissues. Our datasets provide a valuable resource for the community of baseline protease and inhibitor relative expression in normal human tissues and can be used for comparison with diseased tissue, e.g. ovarian cancer, to decipher pathogenesis, and to aid drug development. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.