A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs all revealed the presence of a NAB2-STAT6 gene fusion. Using RT-PCR and sequencing, we detected this fusion in 51 of 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.
Fusions of androgen-regulated genes and v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) occur in ~50% of prostate cancers, encoding a truncated ERG product. In prostatectomy specimens, ERG-rearrangements are >99% specific for prostate cancer or high grade prostatic intraepithelial neoplasia (HGPIN) adjacent to ERG-rearranged prostate cancer by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC).
To evaluate ERG staining by IHC on needle biopsies, including diagnostically challenging cases.
Biopsies from a retrospective cohort (n=111) enriched in cores requiring diagnostic IHC and a prospective cohort from all cases over 3 months (n=311) were stained with an anti-ERG antibody (clone EPR3864).
Amongst evaluable cores (n=418), ERG staining was confined to cancerous epithelium (71/160 cores, 44%), HGPIN (12/68 cores, 18%) and atypical foci (3/28 cores, 11%), with staining in only 2/162 (1%) cores diagnosed as benign. ERG was expressed in ~5 morphologically benign glands across 418 cores, and was uniformly expressed by all cancerous glands in 70/71 cores.
ERG staining is more prostate cancer-specific than alpha-methylacyl-CoA racemase (AMACR), and staining in an atypical focus supports a diagnosis of cancer if HGPIN can be excluded. Thus, ERG staining shows utility in diagnostically challenging biopsies and may be useful in molecularly subtyping prostate cancer and risk stratifying isolated HGPIN.
MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1α signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.
Histone methyltransferases (HMTases), as chromatin modifiers, regulate the transcriptomic landscape in normal development as well in diseases such as cancer. Here, we molecularly order two HMTases, EZH2 and MMSET that have established genetic links to oncogenesis. EZH2, which mediates histone H3K27 trimethylation and is associated with gene silencing, was shown to be coordinately expressed and function upstream of MMSET, which mediates H3K36 dimethylation and is associated with active transcription. We found that the EZH2-MMSET HMTase axis is coordinated by a microRNA network and that the oncogenic functions of EZH2 require MMSET activity. Together, these results suggest that the EZH2-MMSET HMTase axis coordinately functions as a master regulator of transcriptional repression, activation, and oncogenesis and may represent an attractive therapeutic target in cancer.
In an effort to address the variable correspondence problem across large sample cohorts common in metabolomic/metabonomic studies, we have developed a pre-alignment protocol that aims to generate spectral segments sharing a common target spectrum. Under the assumption that a single reference spectrum will not correctly represent all spectra of a data set, the goal of this approach is to perform local alignment corrections on spectral regions which share a common ‘most similar’ spectrum. A natural beneficial outcome of this procedure is the automatic definition of spectral segments, a feature that is not common to all alignment methods. This protocol is shown to specifically improve the quality of alignment in 1H NMR data sets exhibiting large inter-sample compositional variation (e.g. pH, ionic strength). As a proof-of-principle demonstration, we have utilized two recently developed alignment algorithms specific to NMR data, recursive segment-wise peak alignment and interval correlated shifting and applied them to two data sets comprised of 15 aqueous cell line extract and 20 human urine 1H NMR profiles. Application of this protocol represents a fundamental shift from current alignment methodologies that seek to correct misalignments utilizing a single representative spectrum, with the added benefit that it can be appended to any alignment algorithm.
Metabolomic; alignment; NMR; urine
Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.
We sought to develop a clinical algorithm combining serum PSA with detection of TMPRSS2:ERG fusion and PCA3 in urine collected after digital rectal exam (post-DRE urine) to predict prostate cancer on subsequent biopsy.
Materials and Methods
Post-DRE urine was collected in 48 consecutive patients before prostate biopsy at two centers; qRT-PCR was used to detect PCA3 and TMPRSS2:ERG fusion transcript expression. Serum PSA was measured by clinical assay. The performance of TMPRSS2:ERG fusion, PCA3, and serum PSA as biomarkers predicting prostate cancer at biopsy was measured; a clinically practical algorithm combining serum PSA with TMPRSS2:ERG and PCA3 in post-DRE urine to predict prostate cancer was developed.
Post-DRE urine sediment provided informative RNA in 45 patients; prostate cancer was present on subsequent biopsy in 15. TMPRSS2:ERG in post-DRE urine was associated with prostate cancer (OR = 12.02; p< 0.001). PCA3 had the highest sensitivity in predicting prostate cancer diagnosis (93%), whereas TMPRSS2:ERG had the highest specificity (87%). TMPRSS2:ERG had the greatest discriminatory value in predicting prostate cancer (AUC = 0.77 compared to 0.65 for PCA3 and 0.72 for serum PSA alone). Combining serum PSA, PCA3 and TMPRSS2:ERG in a multivariable algorithm optimized for clinical utility improved cancer prediction (AUC = 0.88; specificity = 90% at 80% sensitivity).
A clinical algorithm specifying biopsy for all patients with PSA ≥10ng/ml, while restricting biopsy among those with PSA <10ng/ml to only those with detectable PCA3 or TMPRSS2:ERG in post-DRE urine, performed better than the individual biomarkers alone in predicting prostate cancer.
Screening; DRE; Biomarkers; Cancer Detection; Gene Fusion
Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.
Transcriptional repressors and corepressors play a critical role in cellular homeostasis and are frequently altered in cancer. C-terminal binding protein 1 (CtBP1), a transcriptional corepressor that regulates the expression of tumor suppressors and genes involved in cell death, is known to play a role in multiple cancers. In this study, we observed the overexpression and mislocalization of CtBP1 in metastatic prostate cancer and demonstrated the functional significance of CtBP1 in prostate cancer progression. Transient and stable knockdown of CtBP1 in prostate cancer cells inhibited their proliferation and invasion. Expression profiling studies of prostate cancer cell lines revealed that multiple tumor suppressor genes are repressed by CtBP1. Furthermore, our studies indicate a role for CtBP1 in conferring radiation resistance to prostate cancer cell lines. In vivo studies using chicken chorioallantoic membrane assay, xenograft studies, and murine metastasis models suggested a role for CtBP1 in prostate tumor growth and metastasis. Taken together, our studies demonstrated that dysregulated expression of CtBP1 plays an important role in prostate cancer progression and may serve as a viable therapeutic target.
Bone is the most common metastatic site for prostate cancer, and osseous metastases are the leading cause of morbidity from this disease. Recent autopsy studies prove that 100% of men who die of prostate cancer have bone involvement. Understanding the biology of prostate cancer and its evolution to an incurable androgen independent phenotype requires an understanding of the genetic and cellular alterations that lead to the seeding and proliferation of tumor foci in bone, as well as the microenvironment in which these metastases arise. No intensive studies, however, have been conducted on osseous metastatic tissues from patients with metastatic prostate cancer due to lack of access to such tissues for profiling and other research.
We demonstrate, for the first time, a reproducible methodology to obtain high quality clinical tumor tissues metastatic to the bone. This technique allowed the procurement of viable metastatic tumor tissue from involved bones in 13 recent autopsies conducted at the University of Michigan, and analyzed the gene expression of these tissues using real time PCR and microarrays.
We present here the discovery of non-ossified bone metastases from multiple patients with advanced prostate cancer and their subsequent characterization and comparison to non-osseous metastases from the same patients
This represents a versatile and practical approach that may be employed to characterize the steps in metastasis and the phenotypic characteristics of osseous metastasis of prostate cancer and to profile RNA, DNA and cDNA from tumor samples metastatic to the bone.
Bone marrow; tumor; metastatic prostate cancer
Breast cancer is a heterogeneous disease, exhibiting a wide range of molecular aberrations and clinical outcomes. Here we employed paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers harbor an array of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving microtubule associated serine-threonine kinase (MAST) and Notch family genes. Both MAST and Notch family gene fusions exerted significant phenotypic effects in breast epithelial cells. Breast cancer lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and over-expression of MAST1 or MAST2 gene fusions had a proliferative effect both in vitro and in vivo. These findings illustrate that recurrent gene rearrangements play significant roles in subsets of carcinomas and suggest that transcriptome sequencing may serve to identify patients with rare, actionable gene fusions.
Recurrent fusions of ETS genes are considered driving mutations in a diverse array of cancers, including Ewing’s sarcoma, acute myeloid leukemia, and prostate cancer. We investigate the mechanisms by which ETS fusions mediate their effects, and find that the product of the predominant ETS gene fusion, TMPRSS2:ERG, interacts in a DNA-independent manner with the enzyme poly(ADP-ribose) polymerase 1 (PARP1) and the catalytic subunit of DNA protein kinase (DNA-PKcs). ETS gene-mediated transcription and cell invasion require PARP1 and DNA-PKcs expression and activity. Importantly, pharmacological inhibition of PARP1 inhibits ETS positive, but not ETS negative, prostate cancer xenograft growth. Finally, overexpression of the TMPRSS2:ERG fusion induces DNA damage, which is potentiated by PARP1 inhibition in a manner similar to that of BRCA1/2-deficiency.
Prostate; Rearrangement; Gene Fusion; TMPRSS2; ERG; DNA-PKcs; PARP1
The TMPRSS2/ERG (T/E) fusion gene is present and thought to be an oncogenic driver of approximately half of all prostate cancers. Fusion of the androgen regulated TMPRSS2 promoter to the ERG oncogene results in constitutive high level expression of ERG which promotes prostate cancer invasion and proliferation. Here we report the characterization of multiple alternatively spliced T/E fusion gene isoforms which have differential affects on invasion and proliferation. We found that T/E fusion gene isoforms differentially increase NF-κB mediated transcription, which may explain in part the differences in biological activities of the T/E fusion isoforms. This increased activity is due to phosphorylation of NF-κB p65 on Ser536. Tissue microarray immunochemistry revealed that p65 phospho-Ser536 is present in the majority of prostate cancers where it is associated with ERG protein expression. The T/E fusion gene isoforms differentially increase expression of a number of NF-κB associated genes including PAR1, CCL2, FOS, TLR3 and TLR4 (Toll-like receptor 4). TLR4 activation is known to promote p65 Ser536 phosphorylation and knockdown of TLR4 with ShRNA decreases Ser536 phosphorylation in T/E fusion gene expressing cells. TLR4 can be activated by proteins in the tumor microenvironment and lipopolysacharide from Gram (−) bacteria. Our findings suggest that bacterial infection of the prostate and/or endogenous microenvironment proteins may promote progression of high-grade prostatic intraepithelial neoplasia and/or prostate cancers that express the T/E fusion gene, where the NF-κB pathway might be targeted as a rational therapeutic approach.
prostate cancer; NF-κB; ERG; fusion gene
More than 1,000,000 men undergo prostate biopsy each year in the United States, most for “elevated” serum prostate specific antigen (PSA). Given the lack of specificity and unclear mortality benefit of PSA testing, methods to individualize management of elevated PSA are needed. Greater than 50% of PSA-screened prostate cancers harbor fusions between the transmembrane protease, serine 2 (TMPRSS2) and v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) genes. Here, we report a clinical-grade, transcription-mediated amplification assay to risk stratify and detect prostate cancer noninvasively in urine. The TMPRSS2:ERG fusion transcript was quantitatively measured in prospectively collected whole urine from 1312 men at multiple centers. Urine TMPRSS2:ERG was associated with indicators of clinically significant cancer at biopsy and prostatectomy, including tumor size, high Gleason score at prostatectomy, and upgrading of Gleason grade at prostatectomy. TMPRSS2:ERG, in combination with urine prostate cancer antigen 3 (PCA3), improved the performance of the multivariate Prostate Cancer Prevention Trial risk calculator in predicting cancer on biopsy. In the biopsy cohorts, men in the highest and lowest of three TMPRSS2:ERG+PCA3 score groups had markedly different rates of cancer, clinically significant cancer by Epstein criteria, and high-grade cancer on biopsy. Our results demonstrate that urine TMPRSS2:ERG, in combination with urine PCA3, enhances the utility of serum PSA for predicting prostate cancer risk and clinically relevant cancer on biopsy.
High-throughput sequencing of polyA+ RNA (RNA-Seq) in human cancer shows remarkable potential to identify both novel markers of disease and uncharacterized aspects of tumor biology, particularly non-coding RNA (ncRNA) species. We employed RNA-Seq on a cohort of 102 prostate tissues and cells lines and performed ab initio transcriptome assembly to discover unannotated ncRNAs. We nominated 121 such Prostate Cancer Associated Transcripts (PCATs) with cancer-specific expression patterns. Among these, we characterized PCAT-1 as a novel prostate-specific regulator of cell proliferation and target of the Polycomb Repressive Complex 2 (PRC2). We further found that high PCAT-1 and PRC2 expression stratified patient tissues into molecular subtypes distinguished by expression signatures of PCAT-1-repressed target genes. Taken together, the findings presented herein identify PCAT-1 as a novel transcriptional repressor implicated in subset of prostate cancer patients. These findings establish the utility of RNA-Seq to identify disease-associated ncRNAs that may improve the stratification of cancer subtypes.
prostate cancer; transcriptome; next generation sequencing; non-coding RNA; EZH2
As hypoxia is believed to play an important role in the development and progression of prostate cancer, we evaluated whether 18F-labelled Fluoroazomycin Arabinoside (18F-FAZA) would be useful to identify tumor hypoxia in resectable prostate cancer.
PET/CT was performed on 14 patients with untreated localized primary prostate cancer 3 hours post injection of approximately 390 MBq of 18F-FAZA using forced diuresis to decrease radioactivity in the urinary bladder. Anatomical transpelvic coil and pre- and post-contrast 1.5 T MRI with endorectal coil were performed on the same day. Patients underwent radical prostatectomy and ex-vivo 3T MRI of the prostatectomy specimen within 14 days following in-vivo imaging. Imaging results were verified by whole mount histopathology plus tissue microarray (TMA) immunohistochemical (IHC) analysis for carbonic anhydrase IX (CAIX) and HIF-1α. Registration of in-vivo imaging with histology was achieved using mutual information software and performing ex-vivo MRI of the prostatectomy specimen and whole mount sectioning with block face photography as intermediate steps.
Whole mount histology identified 43 tumor nodules, 19 of them larger than 1 ml as determined on coregistered volumes featuring 18F-FAZA, MRI and histological 3D image information. None of these lesions was found to be positive for CAIX or visualized by 18F-FAZA PET/CT while IHC for HIF-1α showed variable staining of tumor tissues. Accordingly, no correlation was found between 18F-FAZA uptake and Gleason scores.
Our data based on 18F-FAZA PET/CT and CAIX IHC do not support the presence of clinically relevant hypoxia in localized primary prostate cancer including high-grade disease. Activation of HIF-1α may be independent on tissue hypoxia in primary prostate cancer.
Hypoxia; Fluoroazomycin Arabinoside; 18F-FAZA; PET/CT; PET/MRI fusion; HIF-1α; CAIX; MIB-1/Ki67; Immunohistochemistry; Prostate cancer
Patients with advanced prostate cancer (PCa) are initially susceptible to androgen withdrawal (AW), but ultimately develop resistance to this therapy (castration-resistant PCa, CRPC). Here we show that AW can promote CRPC development by increasing the levels of the receptor tyrosine kinase (RTK) ErbB3 in androgen-dependent PCa, resulting in AW-resistant cell cycle progression and increased androgen receptor (AR) transcriptional activity. CRPC cell lines and human prostate cancer tissue overexpressed ErbB3, whereas downregulation of ErbB3 prevented CRPC cell growth. Investigation of the mechanism by which AW augments ErbB3, using normal prostate derived pRNS-1-1 cells, and androgen-dependent PCa lines LNCaP, PC346C and CWR22 mouse xenografts, revealed that the AR suppresses ErbB3 protein levels, while AW relieves this suppression, demonstrating for the first time negative regulation of ErbB3 by AR. We show that AR activation promotes ErbB3 degradation in androgen-dependent cells, and that this effect is mediated by AR-dependent transcriptional upregulation of Nrdp1, an E3 ubiquitin ligase that targets ErbB3 for degradation but whose role in PCa has not been previously examined. Therefore, AW decreases Nrdp1 expression, promoting ErbB3 protein accumulation, and leading to AR-independent proliferation. However, in CRPC sublines of LNCaP and CWR22 which strongly overexpress the AR, ErbB3 levels remain elevated due to constitutive suppression of Nrdp1, which prevents AR regulation of Nrdp1. Our observations point to a model of CRPC development where progression of PCa to castration-resistance is associated with the inability of AR to transcriptionally regulate Nrdp1, and predict that inhibition of ErbB3 during AW may impair CRPC development.
EGFR; HER2; HER3; Androgen Receptor; FLRF; RNF41
While recurrent gene fusions involving ETS family transcription factors are common in prostate cancer, their products are considered “undruggable” by conventional approaches. Recently, rare “targetable” gene fusions (involving the ALK kinase), have been identified in 1–5% of lung cancers1, suggesting that similar rare gene fusions may occur in other common epithelial cancers including prostate cancer. Here we employed paired-end transcriptome sequencing to screen ETS rearrangement negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF and ESRP1-RAF1 gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and MEK inhibitors. Screening a large cohort of patients, we found that although rare (1–2%), recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers, and melanoma. Taken together, our results emphasize the importance of RAF rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion harboring solid tumors, and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.
Rationale and Objectives
PET is actively investigated to aid in target volume definition for radiation therapy (RT). Our objective was to apply an automatic computer algorithm to compute the target volume and validate the algorithm using histological data from real human prostate cancer.
Materials and Methods
Various modalities of prostate were collected. In-vivo imaging included T2 3T MRI and 11C-Choline PET. Ex-vivo imaging included 3T MRI, histology, and block face photos of the prostate specimen. A novel registration method based on mutual information and thin-plate spline was applied to all modalities. Once PET is registered with histology, a voxel by voxel comparison between PET and histology is possible. A thresholding technique based on a various fractions of the maximum standardized uptake value (SUVmax) in the tumor was applied and the respective computed threshold PET volume was compared with histological truth.
Sixteen patients whose primary tumor volumes ranged from 1.2 cm3 to 12.6 cm3 were tested. PET has low spatial resolution, thus only tumors larger than 4 cm3 were considered. Four cases met this criterion. A threshold value of 60% of the 11C-Choline SUVmax resulted in highest volume overlap between threshold PET volume and histology. Medial axis distances between threshold PET volume and histology showed following errors, 7.7± 5.2 mm (mean error ± SD).
This is a proof of concept paper demonstrating for the first time that histology-guided PET thresholding can delineate tumor volumes in real human prostate cancer.
automatic target volume definition; image registration; prostate cancer; histology; thresholding; PET; MRI; multi-modality image fusion
Although prostate-specific antigen (PSA) serum level is currently the standard of care for prostate cancer screening in the United States, it lacks ideal specificity and additional biomarkers are needed to supplement or potentially replace serum PSA testing. Emerging evidence suggests that monitoring the noncoding RNA transcript PCA3 in urine may be useful in detecting prostate cancer in patients with elevated PSA levels. Here, we show that a multiplex panel of urine transcripts outperforms PCA3 transcript alone for the detection of prostate cancer. We measured the expression of seven putative prostate cancer biomarkers, including PCA3, in sedimented urine using quantitative PCR on a cohort of 234 patients presenting for biopsy or radical prostatectomy. By univariate analysis, we found that increased GOLPH2, SPINK1, and PCA3 transcript expression and TMPRSS2:ERG fusion status were significant predictors of prostate cancer. Multivariate regression analysis showed that a multiplexed model, including these biomarkers, outperformed serum PSA or PCA3 alone in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.758 for the multiplexed model versus 0.662 for PCA3 alone (P = 0.003). The sensitivity and specificity for the multiplexed model were 65.9% and 76.0%, respectively, and the positive and negative predictive values were 79.8% and 60.8%, respectively. Taken together, these results provide the framework for the development of highly optimized, multiplex urine biomarker tests for more accurate detection of prostate cancer.
The aim of the study was to assess whether 11C-choline PET/CT could identify high-risk primary adenocarcinoma of the prostate.
11C-choline PET/CT and transpelvic MR imaging were performed in 14 patients with untreated localized primary adenocarcinoma of the prostate followed by radical prostatectomy as form of primary monotherapy within 14 days of in vivo imaging. In order to allow accurate co-registration of whole mount histology with in vivo imaging, additional ex vivo MR images of the prostatectomy specimen were obtained. Nonlinear 3D image deformations were employed for registrations of PET/CT, MR imaging and histology. Volumes of interest from tumor and benign tissue were defined based on histology and were transferred into co-registered 11C-choline PET/CT volumes to calculate the mean (T(mean)/B) and maximum (T(max)/B) tumor-to-benign prostate background ratio. We assessed whether of 11C-choline uptake correlated with local Gleason score and tumor proliferation based on MIB-1/Ki-67 expression in tumor tissues represented on a tissue microarray.
Histology confirmed 42 tumor nodules with Gleason scores between 3+2 and 4+4, with volumes ranging from 0.03 to 12.6 cm3. T(mean)/B (p < 0.01) and T(max)/B (p < 0.001) ratios were significantly increased in high Gleason score (≥4+3) lesions vs. 3+4 and lower disease, but failed to distinguish between 3+4 disease vs. 3+3 and lower. T(mean)/B and T(max)/B ratios were significantly increased in tumors with MIB-1/Ki-67 labeling index ≥ 5% (p < 0.01).
Based on our preliminary data utilizing tumor-to-benign prostate background ratios, 11C-choline preferentially identified aggressive primary prostate cancer.
11C-Choline; PET/CT; PET/MRI; Primary prostate cancer; Image fusion
We previously showed that nuclear localization of the actin-binding protein FilaminA (FlnA) corresponded to hormone-dependence in prostate cancer (Oncogene, 2007, 26:6061-6070). Intact FlnA (280kDa, cytoplasmic) cleaved to a 90kDa fragment which translocated to the nucleus in hormone-naïve cells, whereas in hormone-refractory cells, FlnA was phosphorylated, preventing its cleavage and nuclear translocation. We now examined whether FlnA localization determines a propensity to metastasis in advanced androgen independent prostate cancer.
We examined, by immunohistochemistry, FlnA localization in paraffin-embedded human prostate tissue representing different stages of progression. Results were correlated with in vitro studies in a cell model of prostate cancer.
Nuclear FlnA was significantly higher in benign prostate (0.6612±0.5888), PIN (0.6024±0.4620) and clinically localized cancers (0.69134±0.5686), compared to metastatic prostate cancers (0.3719±0.4992, p=0.0007). Cytoplasmic FlnA increased from benign prostate (0.0833±0.2677), PIN (0.1409±0.2293), localized cancers (0.3008±0.3762, p=0.0150), to metastases (0.7632±0.4414, p<0.00001). Logistic regression of metastatic vs non-metastatic tissue yielded the area-under-ROC curve as 0.67 for nuclear-FlnA, 0.79 for cytoplasmic-FlnA and 0.82 for both, indicating that metastasis correlates with cytoplasmic-to-nuclear translocation. In vitro studies showed that cytoplasmic localization of FlnA induced cell invasion whereas nuclear translocation of the protein inhibited it. FlnA dephosphorylation with the PKA inhibitor H-89 facilitated FlnA nuclear translocation, resulting in decreased invasiveness and AR transcriptional activity, and induced sensitivity to androgen withdrawal in hormone-refractory cells.
The data presented in this study indicate that in prostate cancer, metastasis correlates with cytoplasmic localization of FlnA and may be prevented by cleavage and subsequent nuclear translocation of this protein.
Filamin A; immunohistochemistry; hormone refractory; metastasis; prostate cancer
FYN is a member of the SRC family of kinases (SFKs), functionally distinct from other SFKs. It interacts with FAK and paxillin (PXN)- regulators of cell morphology and motility. We hypothesized that FYN is upregulated in prostate cancer (CaP).
Patients and Methods
Through datamining in Oncomine; cell line profiling with immunoblotting and quantitative RT-PCR; and immunohistochemical analysis, we describe FYN expression in CaP. This analysis included 32 cases of CaP, 9 prostatic intraepithelial neoplasia (PIN), and 19 normal. Samples were scored for the percentage of stained glands and intensity of staining (from 0-3). Each sample was assigned a composite score generated by multiplying percentage and intensity.
Datamining showed an 8-fold increase in FYN expression in CaP compared to normal tissue. This was specific to FYN and not present for other SFKs. Expression of FYN in CaP cell lines (LNCaP, 22Rv1, PC3, DuPro) was detected using quantitative RT-PCR and immunoblot. Expression of FYN and its signaling partners FAK and PXN was demonstrated in human tissue. Comparing normal to cancer, there was a 2.1-fold increase in median composite score for FYN (p<0.001) 1.7-fold increase in FAK (p<0.001), and a 2-fold increase in PXN (p<0.05). There was a 1.7-fold increase in FYN (p<0.05), a 1.6-fold increase in FAK (p<0.01) in CaP as compared to PIN.
These studies support the hypothesis that the FYN and its related signaling partners are upregulated in CaP and supports further investigation into the role of the FYN as a therapeutic target.
FYN; SRC; prostate cancer; paxillin; FAK
Prostate cancer (PCa) cell tethering and rolling on microvascular endothelium has been proposed to promote the extravasation of PCa cells. We have shown that these adhesive events are mediated through binding interactions between endothelial (E)-selectin and Lewis carbohydrates on PCa cells. Prior data indicate that E-selectin-mediated rolling of bone-metastatic PCa MDA PCa 2b (MDA) cells is dependent on sialyl Lewis X (sLeX)-bearing glycoproteins. To explore the molecular basis of sLeX synthesis and E-selectin ligand (ESL) activity on PCa cells, we compared and contrasted the expression level of glycosyltransferases, characteristically involved in sLeX and ESL synthesis, in ESL+ MDA cells among other ESL− metastatic PCa cell lines. We also created and examined ESLhi and ESLlo variants of MDA cells to provide a direct comparison of the glycosyltransferase expression level. We found that normal prostate tissue and all metastatic PCa cell lines expressed glycosyltransferases required for sialo-lactosamine synthesis, including N-acetylglucosaminyl-, galactosyl-, and sialyltransferases. However, compared with expression in normal prostate tissue, ESL+ MDA cells expressed a 31- and 10-fold higher level of α1,3 fucosyltransferases (FT) 3 and 6, respectively. Moreover, FT3 and FT6 were expressed at 2- to 354-fold lower levels in ESL− PCa cell lines. Consistent with these findings, ESLhi MDA cells expressed a 131- and 51-fold higher level of FT3 and FT6, respectively, compared with expression in ESLlo MDA cells. We also noted that α1,3 FT7 was expressed at a 5-fold greater level in ESLhi MDA cells. Furthermore, ESLlo MDA cells did not display sLeX on glycoproteins capable of bearing sLeX, notably P-selectin glycoprotein ligand-1. These results implicate the importance of α1,3 FT3, FT6, and/or FT7 in sLeX and ESL synthesis on metastatic PCa cells.
E-selectin; fucosyltransferase; metastasis; prostate cancer; sialyl Lewis X
Prostate cancer (PCa) cell tethering and rolling on microvascular endothelium has been proposed to promote the extravasation of PCa cells. We have shown that these adhesive events are mediated through binding interactions between endothelial (E)-selectin and Lewis carbohydrates on PCa cells. Prior data indicate that E-selectin-mediated rolling of bone-metastatic PCa MDA PCa 2b (MDA) cells is dependent on sialyl Lewis X (sLeX)-bearing glycoproteins. To explore the molecular basis of sLeX synthesis and E-selectin ligand (ESL) activity on PCa cells, we compared and contrasted the expression level of glycosyltransferases, characteristically involved in sLeX and ESL synthesis, in ESL+ MDA cells among other ESL− metastatic PCa cell lines. We also created and examined ESLhi and ESLlo variants of MDA cells to provide a direct comparison of glycosyltransferase expression level. We found that normal prostate tissue and all metastatic PCa cell lines expressed glycosyltransferases required for sialo-lactosamine synthesis, including N-acetylglucosaminyl-, galactosyl-, and sialyltransferases. However, compared with expression in normal prostate tissue, ESL+ MDA cells expressed a 31- and 10-fold higher level of α1,3 fucosyltransferases (FT) 3 and 6, respectively. Moreover, FT3 and FT6 were expressed at 2 to 354-fold lower levels in ESL− PCa cell lines. Consistent with these findings, ESLhi MDA cells expressed a 131- and 51-fold higher level of FT3 and FT6, respectively, compared with expression in ESLlo MDA cells. We also noted that α1,3 FT7 was expressed at a 5-fold greater level in ESLhi MDA cells. Furthermore, ESLlo MDA cells did not display sLeX on glycoproteins bearing sLeX, notably P-selectin glycoprotein ligand-1 (PSGL-1). These results implicate the importance of α1,3 FT3, FT6 and/or FT7 in sLeX and ESL synthesis on metastatic PCa cells.
E-selectin; fucosyltransferase; metastasis; prostate cancer; sialyl Lewis X