SiNVICT: ultra-sensitive detection of single nucleotide variants and indels in circulating tumour DNA.

MOTIVATION: Successful development and application of precision oncology approaches require robust elucidation of the genomic landscape of a patient's cancer and, ideally, the ability to monitor therapy-induced genomic changes in the tumour in an inexpensive and minimally invasive manner. Thanks to recent advances in sequencing technologies, 'liquid biopsy', the sampling of patient's bodily fluids such as blood and urine, is considered as one of the most promising approaches to achieve this goal. In many cancer patients, and especially those with advanced metastatic disease, deep sequencing of circulating cell free DNA (cfDNA) obtained from patient's blood yields a mixture of reads originating from the normal DNA and from multiple tumour subclones-called circulating tumour DNA or ctDNA. The ctDNA/cfDNA ratio as well as the proportion of ctDNA originating from specific tumour subclones depend on multiple factors, making comprehensive detection of mutations difficult, especially at early stages of cancer. Furthermore, sensitive and accurate detection of single nucleotide variants (SNVs) and indels from cfDNA is constrained by several factors such as the sequencing errors and PCR artifacts, and mapping errors related to repeat regions within the genome. In this article, we introduce SiNVICT, a computational method that increases the sensitivity and specificity of SNV and indel detection at very low variant allele frequencies. SiNVICT has the capability to handle multiple sequencing platforms with different error properties; it minimizes false positives resulting from mapping errors and other technology specific artifacts including strand bias and low base quality at read ends. SiNVICT also has the capability to perform time-series analysis, where samples from a patient sequenced at multiple time points are jointly examined to report locations of interest where there is a possibility that certain clones were wiped out by some treatment while some subclones gained selective advantage. RESULTS: We tested SiNVICT on simulated data as well as prostate cancer cell lines and cfDNA obtained from castration-resistant prostate cancer patients. On both simulated and biological data, SiNVICT was able to detect SNVs and indels with variant allele percentages as low as 0.5%. The lowest amounts of total DNA used for the biological data where SNVs and indels could be detected with very high sensitivity were 2.5 ng on the Ion Torrent platform and 10 ng on Illumina. With increased sequencing and mapping accuracy, SiNVICT might be utilized in clinical settings, making it possible to track the progress of point mutations and indels that are associated with resistance to cancer therapies and provide patients personalized treatment. We also compared SiNVICT with other popular SNV callers such as MuTect, VarScan2 and Freebayes. Our results show that SiNVICT performs better than these tools in most cases and allows further data exploration such as time-series analysis on cfDNA sequencing data. AVAILABILITY AND IMPLEMENTATION: SiNVICT is available at: https://sfu-compbio.github.io/sinvictSupplementary information: Supplementary data are available at Bioinformatics online. CONTACT: cenk@sfu.ca.

Enhanced Rotator-Cuff Repair Using Platelet-Rich Plasma Adsorbed on Branched Poly(ester urea)s.

Platelet-rich plasma (PRP) is a clinically relevant source of growth factors used commonly by surgeons. The clinical efficacy of PRP use as reported in the literature is widely variable which is likely attributed to poorly defined retention time of PRP at the repair site. To overcome this limitation, branched poly(ester urea) (PEU) nanofibers were used to adsorb and retain PRP at the implant site in an acute rotator-cuff tear model in rats. The adsorption of PRP to the branched-PEU 8% material was characterized using quartz crystal microbalance (QCM) and immuno-protein assay. After adsorption of PRP to the nanofiber sheet, the platelets actively released proteins. The adhesion of platelets to the nanofiber material was confirmed by immunofluorescence using a p-selectin antibody. In vivo testing using a rat rotator-cuff repair model compared five groups; no repair (control), suture repair only, repair with disc implant (Disc), repair with PRP-soaked disc (Disc PRP), and a PRP injection (PRP). Mechanical testing at 84 d for the four surgical repair groups resulted in a higher stiffness (11.8 ± 3.8 N/mm, 13.5 ± 3.8 N/mm, 16.8 ± 5.8 N/mm, 12.2 ± 2.6 N/mm, respectively) for the Disc PRP group. Histological staining using trichrome, hematoxylin, and eosin Y (H&E), and safranin O confirmed more collagen organization in the Disc PRP group at 21 and 84 d. Limited inflammation and recovery toward preoperative mechanical properties indicate PEU nanofiber discs as translationally relevant.

Evaluating an educational intervention to alleviate distress amongst men with newly diagnosed prostate cancer and their partners.

OBJECTIVE: To determine whether an education session alleviates distress for both patients with prostate cancer and their partners; and whether their partner's attendance at the session; and disease, treatment, and sociodemographic characteristics affect changes in distress levels. PATIENTS, SUBJECTS AND METHODS: We identified men with untreated prostate cancer at the Vancouver Prostate Centre between February 2015 and March 2016 who agreed to attend our education session. The session consisted of a didactic presentation covering the biology of prostate cancer, treatment options, and side-effects, followed by a private joint session with a urologist and radiation oncologist. We assessed distress using the Distress Thermometer (DT) and compared pre- and post-session distress, and change in distress between patients and partners using matched and unmatched t-tests, respectively. We also assessed pre-session anxiety using the seven-item Generalised Anxiety Disorder measure, and decisional certainty using the Decisional Conflict Scale. RESULTS: In all, 71 patients and 48 partners participated in the study. Attending the session led to a significant reduction in the median DT score for patients (4.0-3.0, P < 0.01) and partners (5.0-4.0, P = 0.02). Partners reported higher distress both before and after the session (4.9 vs 3.8, P = 0.03 pre-session and 4.2 vs 3.2, P = 0.03 post-session). The presence of a partner at the session did not affect patients' pre- or post-session distress or the success of the session at alleviating distress. Sociodemographic and clinical characteristics had little effect on distress levels. CONCLUSIONS: An interdisciplinary education session is equally effective at alleviating distress for both patients with prostate cancer and their female partners.

Hiding in plain view: genetic profiling reveals decades old cross contamination of bladder cancer cell line KU7 with HeLa.

PURPOSE: KU7 is a popular urothelial carcinoma cell line that was isolated from the bladder of a patient at Keio University in 1980. It has subsequently been widely used in laboratories around the world. We describe how routine cell line authentication revealed that KU7 was cross contaminated almost 30 years ago with HeLa, a cervical carcinoma cell line. MATERIALS AND METHODS: Presumed KU7 clones dating from 1984 to 1999 were provided by M.D. Anderson Cancer Center, Vancouver Prostate Centre, Kyoto University, Tokyo Medical University and Keio University. HeLa was obtained from ATCC. Genomic DNA was isolated and short tandem repeat analysis was performed at the M.D. Anderson Cancer Center Characterized Cell Line Core Facility, Johns Hopkins University Fragment Analysis Facility and RIKEN BioResource Center, Ibaraki, Japan. Comparative genomic hybridization was performed on a platform (Agilent Technologies, Santa Clara, California) at Vancouver Prostate Centre. RESULTS: The short tandem repeat profile of all KU7 clones was an exact match with that of HeLa. Comparative genomic hybridization of all samples revealed an abundance of shared chromosomal aberrations. Slight differences in some genomic areas were explained by genomic drift in different KU7 clones separated by many years. CONCLUSIONS: Our analysis identified that cross contamination of KU7 with HeLa occurred before 1984 at the source institution. All KU7 clones in the urological literature should be considered HeLa and experimental results should be viewed in this light. Our results emphasize the need to authenticate cell lines in oncological research.

Integrated genome and transcriptome sequencing identifies a novel form of hybrid and aggressive prostate cancer.

Next-generation sequencing is making sequence-based molecular pathology and personalized oncology viable. We selected an individual initially diagnosed with conventional but aggressive prostate adenocarcinoma and sequenced the genome and transcriptome from primary and metastatic tissues collected prior to hormone therapy. The histology-pathology and copy number profiles were remarkably homogeneous, yet it was possible to propose the quadrant of the prostate tumour that likely seeded the metastatic diaspora. Despite a homogeneous cell type, our transcriptome analysis revealed signatures of both luminal and neuroendocrine cell types. Remarkably, the repertoire of expressed but apparently private gene fusions, including C15orf21:MYC, recapitulated this biology. We hypothesize that the amplification and over-expression of the stem cell gene MSI2 may have contributed to the stable hybrid cellular identity. This hybrid luminal-neuroendocrine tumour appears to represent a novel and highly aggressive case of prostate cancer with unique biological features and, conceivably, a propensity for rapid progression to castrate-resistance. Overall, this work highlights the importance of integrated analyses of genome, exome and transcriptome sequences for basic tumour biology, sequence-based molecular pathology and personalized oncology.

From sequence to molecular pathology, and a mechanism driving the neuroendocrine phenotype in prostate cancer.

The current paradigm of cancer care relies on predictive nomograms which integrate detailed histopathology with clinical data. However, when predictions fail, the consequences for patients are often catastrophic, especially in prostate cancer where nomograms influence the decision to therapeutically intervene. We hypothesized that the high dimensional data afforded by massively parallel sequencing (MPS) is not only capable of providing biological insights, but may aid molecular pathology of prostate tumours. We assembled a cohort of six patients with high-risk disease, and performed deep RNA and shallow DNA sequencing in primary tumours and matched metastases where available. Our analysis identified copy number abnormalities, accurately profiled gene expression levels, and detected both differential splicing and expressed fusion genes. We revealed occult and potentially dormant metastases, unambiguously supporting the patients' clinical history, and implicated the REST transcriptional complex in the development of neuroendocrine prostate cancer, validating this finding in a large independent cohort. We massively expand on the number of novel fusion genes described in prostate cancer; provide fresh evidence for the growing link between fusion gene aetiology and gene expression profiles; and show the utility of fusion genes for molecular pathology. Finally, we identified chromothripsis in a patient with chronic prostatitis. Our results provide a strong foundation for further development of MPS-based molecular pathology.

Cobalt ions induce metabolic stress in synovial fibroblasts and secretion of cytokines/chemokines that may be diagnostic markers for adverse local tissue reactions to hip implants.

Adverse local tissue reactions (ALTRs) are a prominent cause of hip implant failure. ALTRs are characterized by aseptic necrosis and leukocyte infiltration of synovial tissue. The prevalence of ALTRs in hips with failing metal implants, with highest rates occurring in patients with metal-on-metal articulations, suggests a role for CoCrMo corrosion in ALTR formation. Although hypersensitivity reactions are the most accepted etiology, the precise cellular mechanism driving ALTR pathogenesis remains enigmatic. Here we show that cobalt ions released by failing hip implants induce mitochondrial stress and cytokine secretion by synovial fibroblasts: the presumptive initiators of ALTR pathogenesis. We found that in-vitro treatment of synovial fibroblasts with cobalt, but not chromium, generated gene expression changes indicative of hypoxia and mitophagy responses also observed in ALTRs biopsies. Inflammatory factors secreted by cobalt-exposed synovial fibroblasts were among those most concentrated in ALTR synovial fluid. Furthermore, both conditioned media from cobalt-exposed synovial fibroblasts, and synovial fluid from ALTRs patients, elicit endothelial activation and monocyte migration. Finally, we identify the IL16/CTACK ratio in synovial fluid as a possible diagnostic marker of ALTRs. Our results provide evidence suggesting that metal ions induce cell stress in synovial fibroblasts that promote an inflammatory response consistent with initiating ALTR formation. STATEMENT OF SIGNIFICANCE: We demonstrate that the cytotoxic effects of cobalt ions on the synovial cells (fibroblast) is sufficient to trigger inflammation on hip joints with metal implants. Cobalt ions affect mitochondrial function, leading to the auto phagocytosis of mitochondria and trigger a hypoxic response. The cell's hypoxic response includes secretion of cytokines that are capable of trigger inflammation by activating blood vessels and enhancing leukocyte migration. Among the secreted cytokines is IL-16, which is highly concentrated in the synovial fluid of the patients with adverse local tissue reactions and could be use as diagnostic marker. In conclusion we define the cells of the hip joint as key players in triggering the adverse reactions to hip implants and providing biomarkers for early diagnosis of adverse reactions to hip implants.

Multiomics Characterization of Low-Grade Serous Ovarian Carcinoma Identifies Potential Biomarkers of MEK Inhibitor Sensitivity and Therapeutic Vulnerability.

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry-based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. SIGNIFICANCE: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.

Identification of gene signature for treatment response to guide precision oncology in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is a common therapy resistant disease with aberrant angiogenic and immunosuppressive features. Patients with metastatic disease are treated with targeted therapies based on clinical features: low-risk patients are usually treated with anti-angiogenic drugs and intermediate/high-risk patients with immune therapy. However, there are no biomarkers available to guide treatment choice for these patients. A recently published phase II clinical trial observed a correlation between ccRCC patients' clustering and their response to targeted therapy. However, the clustering of these groups was not distinct. Here, we analyzed the gene expression profile of 469 ccRCC patients, using featured selection technique, and have developed a refined 66-gene signature for improved sub-classification of patients. Moreover, we have identified a novel comprehensive expression profile to distinguish between migratory stromal and immune cells. Furthermore, the proposed 66-gene signature was validated using a different cohort of 64 ccRCC patients. These findings are foundational for the development of reliable biomarkers that may guide treatment decision-making and improve therapy response in ccRCC patients.

Insulin receptor expression by human prostate cancers.

BACKGROUND: Although recent laboratory and population studies suggest that prostate cancer may be responsive to insulin, there is a gap in knowledge concerning the expression of insulin receptors on benign or malignant prostate tissue. METHODS: We immunostained 644 cores on tissue microarrays prepared from 29 prostate tissue samples without malignancies, 78 Gleason grade 3 cancers, 21 Gleason grade 4 cancers and 33 Gleason grade 5 cancers with antibodies against the insulin-like growth factor I receptor and the insulin receptor. RESULTS: We observed immunoreactivity with both antibodies, which implies the presence of hybrid receptors as well as IGF-I receptors and insulin receptors. Insulin receptor staining intensity was significantly (P < 0.001) higher on malignant than benign prostate epithelial cells. Analysis of information from public gene expression databases confirmed that co-expression of insulin receptor mRNA and IGF-I receptor mRNA is common in prostate cancer specimens. RT-PCR methods provided evidence for the presence of mRNA for both IR-A and IR-B insulin receptor isoforms. CONCLUSION: These observations document the presence of insulin receptors on primary human prostate cancers. The findings are relevant not only to ongoing clinical trials of drug candidates that target IGF-I and/or insulin receptors, but also to the hypothesis that obesity-associated hyperinsulinemia mediates the adverse effect of obesity on prostate cancer prognosis.

BAP1 haploinsufficiency predicts a distinct immunogenic class of malignant peritoneal mesothelioma.

BACKGROUND: Malignant peritoneal mesothelioma (PeM) is a rare and fatal cancer that originates from the peritoneal lining of the abdomen. Standard treatment of PeM is limited to cytoreductive surgery and/or chemotherapy, and no effective targeted therapies for PeM exist. Some immune checkpoint inhibitor studies of mesothelioma have found positivity to be associated with a worse prognosis. METHODS: To search for novel therapeutic targets for PeM, we performed a comprehensive integrative multi-omics analysis of the genome, transcriptome, and proteome of 19 treatment-naïve PeM, and in particular, we examined BAP1 mutation and copy number status and its relationship to immune checkpoint inhibitor activation. RESULTS: We found that PeM could be divided into tumors with an inflammatory tumor microenvironment and those without and that this distinction correlated with haploinsufficiency of BAP1. To further investigate the role of BAP1, we used our recently developed cancer driver gene prioritization algorithm, HIT'nDRIVE, and observed that PeM with BAP1 haploinsufficiency form a distinct molecular subtype characterized by distinct gene expression patterns of chromatin remodeling, DNA repair pathways, and immune checkpoint receptor activation. We demonstrate that this subtype is correlated with an inflammatory tumor microenvironment and thus is a candidate for immune checkpoint blockade therapies. CONCLUSIONS: Our findings reveal BAP1 to be a potential, easily trackable prognostic and predictive biomarker for PeM immunotherapy that refines PeM disease classification. BAP1 stratification may improve drug response rates in ongoing phases I and II clinical trials exploring the use of immune checkpoint blockade therapies in PeM in which BAP1 status is not considered. This integrated molecular characterization provides a comprehensive foundation for improved management of a subset of PeM patients.

Targeting MCT4 to reduce lactic acid secretion and glycolysis for treatment of neuroendocrine prostate cancer.

Development of neuroendocrine prostate cancer (NEPC) is emerging as a major problem in clinical management of advanced prostate cancer (PCa). As increasingly potent androgen receptor (AR)-targeting antiandrogens are more widely used, PCa transdifferentiation into AR-independent NEPC as a mechanism of treatment resistance becomes more common and precarious, since NEPC is a lethal PCa subtype urgently requiring effective therapy. Reprogrammed glucose metabolism of cancers, that is elevated aerobic glycolysis involving increased lactic acid production/secretion, plays a key role in multiple cancer-promoting processes and has been implicated in therapeutics development. Here, we examined NEPC glucose metabolism using our unique panel of patient-derived xenograft PCa models and patient tumors. By calculating metabolic pathway scores using gene expression data, we found that elevated glycolysis coupled to increased lactic acid production/secretion is an important metabolic feature of NEPC. Specific inhibition of expression of MCT4 (a plasma membrane lactic acid transporter) by antisense oligonucleotides led to reduced lactic acid secretion as well as reduced glucose metabolism and NEPC cell proliferation. Taken together, our results indicate that elevated glycolysis coupled to excessive MCT4-mediated lactic acid secretion is clinically relevant and functionally important to NEPC. Inhibition of MCT4 expression appears to be a promising therapeutic strategy for NEPC.

Stromal Gene Expression is Predictive for Metastatic Primary Prostate Cancer.

BACKGROUND: Clinical grading systems using clinical features alongside nomograms lack precision in guiding treatment decisions in prostate cancer (PCa). There is a critical need for identification of biomarkers that can more accurately stratify patients with primary PCa. OBJECTIVE: To identify a robust prognostic signature to better distinguish indolent from aggressive prostate cancer (PCa). DESIGN, SETTING, AND PARTICIPANTS: To develop the signature, whole-genome and whole-transcriptome sequencing was conducted on five PCa patient-derived xenograft (PDX) models collected from independent foci of a single primary tumor and exhibiting variable metastatic phenotypes. Multiple independent clinical cohorts including an intermediate-risk cohort were used to validate the biomarkers. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The outcome measurement defining aggressive PCa was metastasis following radical prostatectomy. A generalized linear model with lasso regularization was used to build a 93-gene stroma-derived metastasis signature (SDMS). The SDMS association with metastasis was assessed using a Wilcoxon rank-sum test. Performance was evaluated using the area under the curve (AUC) for the receiver operating characteristic, and Kaplan-Meier curves. Univariable and multivariable regression models were used to compare the SDMS alongside clinicopathological variables and reported signatures. AUC was assessed to determine if SDMS is additive or synergistic to previously reported signatures. RESULTS AND LIMITATIONS: A close association between stromal gene expression and metastatic phenotype was observed. Accordingly, the SDMS was modeled and validated in multiple independent clinical cohorts. Patients with higher SDMS scores were found to have worse prognosis. Furthermore, SDMS was an independent prognostic factor, can stratify risk in intermediate-risk PCa, and can improve the performance of other previously reported signatures. CONCLUSIONS: Profiling of stromal gene expression led to development of an SDMS that was validated as independently prognostic for the metastatic potential of prostate tumors. PATIENT SUMMARY: Our stroma-derived metastasis signature can predict the metastatic potential of early stage disease and will strengthen decisions regarding selection of active surveillance versus surgery and/or radiation therapy for prostate cancer patients. Furthermore, profiling of stroma cells should be more consistent than profiling of diverse cellular populations of heterogeneous tumors.

Comprehensive expression analysis of L-dopa decarboxylase and established neuroendocrine markers in neoadjuvant hormone-treated versus varying Gleason grade prostate tumors.

Current hormone withdrawal therapies used for treatment of advanced prostate cancer lead to androgen-independent tumor growth. Increased prostatic neuroendocrine (NE) cell density has been implicated in promoting progression of prostate cancer, but the process by which this occurs remains unclear. The aim of this study was to determine whether there is an association of increased NE differentiation with neoadjuvant hormone therapy and Gleason grade. Using adjacently sectioned tissue microarrays, the expression profile of novel and known NE markers were monitored. L-Dopa decarboxylase (DDC), a catecholamine synthesis enzyme and androgen receptor (AR) coregulator protein, was identified as an additional NE marker of prostate cancer. Immunohistochemical analysis of DDC with the established NE markers, chromogranin A and bombesin, revealed a significant increase in NE differentiation after 6 months of hormone therapy and after progression to androgen independence but no apparent correlation with Gleason grade. In addition, dual immunofluorescence analysis revealed that approximately 55% of the mixed population of DDC- and chromogranin A-expressing NE cells continue to express AR. Taken together, these results suggest that the increase of NE differentiation in prostate cancers depends specifically on duration of hormone therapy. This increase may be due to the transdifferentiation of AR-expressing epithelial-derived adenocarcinoma cells into an NE cell phenotype.

Notch signaling is significantly suppressed in basal cell carcinomas and activation induces basal cell carcinoma cell apoptosis.

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs). In some respects, HFs can be defined as 'ordered' skin appendage growths, while BCCs can be regarded as 'disordered' skin appendage growths. The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage. Human nodular BCCs, along with HFs and non­follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry. Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1). Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively. Specific molecular mechanisms were found to be involved in the process of cell self­renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways. However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs. Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation. The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs. Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering.

Metabolic heterogeneity signature of primary treatment-naïve prostate cancer.

To avoid over- or under-treatment of primary prostate tumours, there is a critical need for molecular signatures to discriminate indolent from aggressive, lethal disease. Reprogrammed energy metabolism is an important hallmark of cancer, and abnormal metabolic characteristics of cancers have been implicated as potential diagnostic/prognostic signatures. While genomic and transcriptomic heterogeneity of prostate cancer is well documented and associated with tumour progression, less is known about metabolic heterogeneity of the disease. Using a panel of high fidelity patient-derived xenograft (PDX) models derived from hormone-naïve prostate cancer, we demonstrated heterogeneity of expression of genes involved in cellular energetics and macromolecular biosynthesis. Such heterogeneity was also observed in clinical, treatment-naïve prostate cancers by analyzing the transcriptome sequencing data. Importantly, a metabolic gene signature of increased one-carbon metabolism or decreased proline degradation was identified to be associated with significantly decreased biochemical disease-free patient survival. These results suggest that metabolic heterogeneity of hormone-naïve prostate cancer is of biological and clinical importance and motivate further studies to determine the heterogeneity in metabolic flux in the disease that may lead to identification of new signatures for tumour/patient stratification and the development of new strategies and targets for therapy of prostate cancer.

Increased insulin-like growth factor I receptor expression and signaling are components of androgen-independent progression in a lineage-derived prostate cancer progression model.

Apoptosis and inhibition of mitosis are primary mechanisms mediating androgen ablation therapy-induced regression of prostate cancer (PCa). However, PCa readily becomes androgen independent, leading to fatal disease. Up-regulated growth and survival signaling is implicated in development of resistance to androgen ablation therapy. We are testing the hypothesis that insulin-like growth factor (IGF) responsiveness is required for androgen-independent (AI) progression. Using the LNCaP human PCa progression model, we have determined that IGF-I-mediated protection from apoptotic stress and enhanced mitotic activity is androgen dependent in LNCaP cells but is androgen independent in lineage-derived C4-2 cells. Both cell lines exhibit androgen-responsive patterns of IGF-I receptor (IGF-IR) expression, activation, and signaling to insulin receptor substrate-2 and AKT. However, C4-2 cells express higher levels of IGF-IR mRNA and protein and exhibit enhanced IGF-I-mediated phosphorylation and downstream signaling under androgen-deprived conditions. In comparisons of naive and AI metastatic human PCa specimens, we have confirmed that IGF-IR levels are elevated in advanced disease. Together with our LNCaP/C4-2 AI progression model data, these results indicate that increased IGF-IR expression is associated with AI antiapoptotic and promitotic IGF signaling in PCa disease progression.

The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells.

Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.

Traumatic Supraspinatus Tears in Patients Younger Than 25 Years.

Traumatic rotator cuff tears in patients younger than 25 years are rare events, with few reports in the literature. When compared with the more mature shoulder, the young, healthy supraspinatus tendon is a robust tendon that is able to absorb a significant amount of energy before tendon failure. Therefore, the diagnosis of a rotator cuff tear can be often overlooked in this population due to the patient's age. This is a report of traumatic supraspinatus repairs in patients younger than 25 years. Nine patients younger than 25 years were identified with a posttraumatic supraspinatus tear as visualized during routine diagnostic shoulder arthroscopy. These 9 patients represented 0.33% of all rotator cuff repairs during a 9-year period. Average patient age was 19.1 years (±3.7 years; range, 13 to 25 years). Magnetic resonance imaging failed to diagnose a rotator cuff tear in 50% of the patients. Mean delay from injury to surgery was 6.6 months. All tears were arthroscopically repaired. Concomitant anterior instability pathology was demonstrated among 66.7% of the patients. No complications were reported. At latest follow-up, all patients reported minimal to no shoulder pain and were tolerating strenuous work, activities, and sports without significant complaints. Even with advanced imaging, the diagnosis of a rotator cuff tear can often be missed in this patient population. Although clinical outcomes can be good, care must be taken to broaden the diagnostic differential in young patients with posttraumatic shoulder pain.

Bcl-2 predicts response to neoadjuvant chemotherapy and is overexpressed in lymph node metastases of urothelial cancer of the bladder.

PURPOSE: To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patients with urothelial cancer of the bladder (UCB). METHODS: Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelvic lymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients with UCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy (residual primary tumor [ypT+], n = 38); residual LN metastases [ypN+], n = 24) (prechemotherapy/postchemotherapy TMA cohort). Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity. RESULTS: In both TMA cohorts, Bcl-2 overexpression was significantly (P<0.05) more frequent in LN metastases than in primary tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA: ypT+7/35 [20%] vs. ypN+11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of the bladder cancer specimens showed significantly (P = 0.04) higher ypT stages and less regression in their cystectomy specimens than did the control group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathological parameters added significant prognostic information. CONCLUSIONS: Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might help to select likely nonresponders.