Search

Abstract Localized prostate cancer is frequently composed of multiple spatially distinct tumors with significant inter- and intra-tumoral molecular heterogeneity. This genomic diversity gives rise to many competing clones that may drive the biological trajectory of the disease. Previous large-scale sequencing efforts have focused on the evolutionary process in metastatic prostate cancer, revealing a potential clonal progression to castration resistance. However, the clonal origin of synchronous lymph node (LN) metastases in primary disease is still unknown. Here, we perform multi-region, targeted next generation sequencing and construct phylogenetic trees in men with prostate cancer with synchronous LN metastasis to better define the pathologic and molecular features of primary disease most likely to spread to the LNs. Collectively, we demonstrate that a combination of histopathologic and molecular factors, including tumor grade, presence of extra-prostatic extension, cellular morphology, and oncogenic genomic alterations are associated with synchronous LN metastasis.

Tomlins et al. develop an Immunotherapy Response Score (IRS) to predict clinical benefit from checkpoint inhibition across solid tumors. IRS integrates TMB and gene expression, is evaluable from FFPE material, and is predictive for real-world progression-free survival and overall survival in patients receiving PD-1/PD-L1 blockade therapy.

The diagnosis of prostate cancer brain metastasis (PCBM) has increased. Here, the authors investigate the landscape of somatic genetic alterations in brain metastases in a PCBM cohort of 51 patients with non-synchronous matched primary samples available for 20 patients.

Nearly all estrogen receptor (ER)‐positive (POS) metastatic breast cancers become refractory to endocrine (ET) and other therapies, leading to lethal disease presumably due to evolving genomic alterations. Timely monitoring of the molecular events associated with response/progression by serial tissue biopsies is logistically difficult. Use of liquid biopsies, including circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), might provide highly informative, yet easily obtainable, evidence for better precision oncology care. Although ctDNA profiling has been well investigated, the CTC precision oncology genomic landscape and the advantages it may offer over ctDNA in ER‐POS breast cancer remain largely unexplored. Whole‐blood (WB) specimens were collected at serial time points from patients with advanced ER‐POS/HER2‐negative (NEG) advanced breast cancer in a phase I trial of AZD9496, an oral selective ER degrader (SERD) ET. Individual CTC were isolated from WB using tandem CellSearch®/DEPArray™ technologies and genomically profiled by targeted single‐cell DNA next‐generation sequencing (scNGS). High‐quality CTC (n = 123) from 12 patients profiled by scNGS showed 100% concordance with ctDNA detection of driver estrogen receptor α (ESR1) mutations. We developed a novel CTC‐based framework for precision medicine actionability reporting (MI‐CTCseq) that incorporates novel features, such as clonal predominance and zygosity of targetable alterations, both unambiguously identifiable in CTC compared to ctDNA. Thus, we nominated opportunities for targeted therapies in 73% of patients, directed at alterations in phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 2 (FGFR2), and KIT proto‐oncogene, receptor tyrosine kinase (KIT). Intrapatient, inter‐CTC genomic heterogeneity was observed, at times between time points, in subclonal alterations. Our analysis suggests that serial monitoring of the CTC genome is feasible and should enable real‐time tracking of tumor evolution during progression, permitting more combination precision medicine interventions.

Background: Serum prostate-specific antigen (PSA), used in prostate cancer screening, is nonspecific for cancer and is affected by age and prostate volume. More specific biomarkers could be more accurate for early detection of prostate cancer and reduce unnecessary prostate biopsies. Objective: To evaluate the association of age and prostate volume with urinary MyProstateScore (MPS) in a screened, longitudinal cohort without evidence of prostate cancer. Design, setting, and participants: The Olmsted County Study included men aged 40–79 yr who underwent biennial prostate cancer screening. PSA ≥4.0 ng/ml or abnormal rectal examination triggered prostate biopsy, and patients with cancer were excluded. The remaining men submitted urinary specimens for PCA3 and TMPRSS2:ERG testing. Outcome measurements and statistical analysis: MPS was calculated using the validated, locked model for grade group ≥2 cancer that includes serum PSA, urinary PCA3, and urinary TMPRSS2:ERG. The associations of age and volume with biomarkers were assessed in multivariable regression models. The t statistic was used to quantify the strength of associations independent of the unit of measurement, and R2 values were used to estimate the proportion of biomarker variance explained by each factor. Results and limitations: The study included 314 screened men without evidence of cancer. In multivariable models including age and volume, PCA3 score was significantly associated with age (t = 7.51; p

In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with Crotalus atrox (CA venom) snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of CA venom. HUVEC monolayers stimulated with 10 µg/mL CA venom for 3 h displayed cellular retraction, which coincided with 53.0 ± 6.5 percent viability. In contrast, venom concentrations of 100 µg/mL produced a complete disruption of cellular adherence and viability decreased to 36.6 ± 1.0. The zinc probe Fluozin-3AM was used to detect intracellular zinc in non-stimulated controls, HUVEC stimulated with 10 µg/mL CA venom or HUVEC preincubated with TPEN for 2 h then stimulated with 10 µg/mL CA venom. Fluorescent intensity analysis returned values of 1434.3 ± 197.4 for CA venom demonstrating an increase of about two orders of magnitude in labile zinc compared to non-stimulated controls. Endothelial response to CA venom induced a 96.1 ± 3.0- and 4.4 ± 0.41-fold increase in metallothionein 1X (MT1X) and metallothionein 2A (MT2A) gene expression. Zinc chelation during CA venom stimulation significantly increased cell viability, suggesting that the maintenance of zinc homeostasis during envenomation injury improves cell survival.

Primary Central Nervous System Lymphoma (PCNSL) is a lymphoid malignancy of the brain that occurs in ~1500 patients per year in the US. PCNSL can spread to the vitreous and retina, where it is known as vitreoretinal lymphoma (VRL). While confirmatory testing for diagnosis is dependent on invasive brain tissue or cerebrospinal fluid sampling, the ability to access the vitreous as a proximal biofluid for liquid biopsy to diagnose PCNSL is an attractive prospect given ease of access and minimization of risks and complications from other biopsy strategies. However, the extent to which VRL, previously considered genetically identical to PCNSL, resembles PCNSL in the same individual with respect to genetic alterations, diagnostic strategies, and precision-medicine based approaches has hitherto not been explored. Furthermore, the degree of intra-patient tumor genomic heterogeneity between the brain and vitreous sites has not been studied. In this work, we report on targeted DNA next-generation sequencing (NGS) of matched brain and vitreous samples in two patients who each harbored VRL and PCSNL. Our strategy showed enhanced sensitivity for molecular diagnosis confirmation over current clinically used vitreous liquid biopsy methods. We observed a clonal relationship between the eye and brain samples in both patients, which carried clonal CDKN2A deep deletions, a highly recurrent alteration in VRL patients, as well as MYD88 p.L265P activating mutation in one patient. Several subclonal alterations, however, in the genes SETD2, BRCA2, TERT, and broad chromosomal regions showed heterogeneity between the brain and the eyes, between the two eyes, and among different regions of the PCNSL brain lesion. Taken together, our data show that NGS of vitreous liquid biopsies in PCNSL patients with VRL highlights shared and distinct genetic alterations that suggest a common origin for these lymphomas, but with additional site-specific alterations. Liquid biopsy of VRL accurately replicates the findings for PCNSL truncal (tumor-initiating) genomic alterations; it can also nominate precision medicine interventions and shows intra-patient heterogeneity in subclonal alterations. To the best of our knowledge, this study represents the first interrogation of genetic underpinnings of PCNSL with matched VRL samples. Our findings support continued investigation into the utility of vitreous liquid biopsy in precision diagnosis and treatment of PCNSL/VRL.

Vitreoretinal lymphoma (VRL) is an uncommon eye malignancy, and VRLs of T cell origin are rare. They are difficult to treat, and their molecular underpinnings, including actionable genomic alterations, remain to be elucidated. At present, vitreous fluid liquid biopsies represent a valuable VRL sample for molecular analysis to study VRLs. In this study, we report the molecular diagnostic workup of a rare case of bilateral T cell VRL and characterize its genomic landscape, including identification of potentially targetable alterations. Using next-generation sequencing of vitreous-derived DNA with a pan-cancer 126-gene panel, we found a copy number gain of BRAF and copy number loss of tumor suppressor DNMT3A. To the best of our knowledge, this represents the first exploration of the T cell VRL cancer genome and supports vitreous liquid biopsy as a suitable approach for precision oncology treatments.

Hepatic cirrhosis is commonly associated with hyperestrogenism. Previous studies have reported morphologic changes in benign and malignant prostate tissue exposed to estrogen or anti-androgens. To our knowledge, histopathologic features of prostatic adenocarcinoma in patients with cirrhosis have not been well-reported. We present a case of incidental, but pathologically significant, prostatic adenocarcinoma detected on autopsy in a 67-year-old male patient with cirrhosis and spider angiomata. The morphologic and immunohistochemical features (including variable ERG expression) of the prostatic adenocarcinoma were consistent with hormone exposure related changes, suggesting that cirrhosis-induced elevated estrogen-to-testosterone ratio and exogenous hormone therapy might induce similar phenotypes.

A recent phase III trial of the MET kinase inhibitor cabozantinib in men with castration-resistant prostate cancer (CRPC) failed to meet its primary survival end point; however, most men with CRPC have intact androgen receptor (AR) signaling. As previous work supports negative regulation of MET by AR signaling, we hypothesized that intact AR signaling may have limited the efficacy of cabozantinib in some of these patients. To assess the role of AR signaling on MET inhibition, we first performed an in silico analysis of human CRPC tissue samples stratified by AR signaling status (+ or −), which identified MET expression as markedly increased in AR− samples. In vitro, AR signaling inhibition in AR+ CRPC models increased MET expression and resulted in susceptibility to ligand (HGF) activation. Likewise, MET inhibition was only effective in blocking cancer phenotypes in cells with MET overexpression. Using multiple AR+ CRPC in vitro and in vivo models, we showed that combined cabozantinib and enzalutamide (AR antagonist) treatment was more efficacious than either inhibitor alone. These data provide a compelling rationale to combine AR and MET inhibition in CRPC and may explain the negative results of the phase III cabozantinib study in CRPC. Similarly, the expression of MET in AR− disease, whether due to AR inhibition or loss of AR signaling, suggests potential utility for MET inhibition in select patients with AR therapy resistance and in AR− prostate cancer.

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with 95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.

Objective: To better evaluate tertiary Gleason pattern reporting and to evaluate the impact of tertiary Gleason pattern 5 (TP5) on prostate cancer pathological features and biochemical recurrence at our large single institution. Methods: We retrospectively reviewed 1962 patients who underwent radical prostatectomy (RP) for prostate cancer; TP5 was reported in 159 cases (8.1%). Men with Gleason score (GS) 7 and GS 8 disease were divided into subgroups with and without TP5, and histopathological features were compared. Multivariate analyses were conducted to assess the impact on TP5 on biochemical-free survival (BFS). Results: Tumors possessing GS 3 + 4 with TP5 were more likely to exhibit extraprostatic extension (EPE) and had a larger tumor diameter (TD) than GS 3 + 4 alone. GS 3 + 4 with TP5 was also associated with positive surgical margins (SM), seminal vesicle involvement (SVI), and higher pre-operative prostate-specific antigen (PSA) values, but without statistical significance. GS 4 + 3 with TP5 more commonly presented with EPE, positive SM, SVI, and greater TD and pre-operative PSA level than GS 4 + 3 alone. In multivariate analysis, Gleason score, EPE, and TP5 were overall independent risk factors for PSA recurrence in this cohort. Additionally, GS 4 + 3 with TP5 was associated with shorter time to recurrence versus GS 4 + 3 alone. Conclusion: Our results emphasize the importance of TP5 and suggest that criteria for tertiary pattern reporting in prostate cancer should be standardized. Further studies are needed to evaluate the role of tertiary patterns in prognostic models.

The research community at large is expending considerable resources to sequence the coding region of the genomes of tumors and other human diseases using targeted exome capture (i.e., “whole exome sequencing”). The primary goal of targeted exome sequencing is to identify nonsynonymous mutations that potentially have functional consequences. Here, we demonstrate that whole-exome sequencing data can also be analyzed for comprehensively monitoring somatic copy number alterations (CNAs) by benchmarking the technique against conventional array CGH. A series of 17 matched tumor and normal tissues from patients with metastatic castrate-resistant prostate cancer was used for this assessment. We show that targeted exome sequencing reliably identifies CNAs that are common in advanced prostate cancer, such as androgen receptor (AR) gain and PTEN loss. Taken together, these data suggest that targeted exome sequencing data can be effectively leveraged for the detection of somatic CNAs in cancer.

TMPRSS2-ERG gene fusions occur in 50% of prostate cancers and result in the overexpression of a chimeric fusion transcript that encodes a truncated ERG product. Previous attempts to detect truncated ERG products have been hindered by a lack of specific antibodies. Here, we characterize a rabbit anti-ERG monoclonal antibody (clone EPR 3864; Epitomics, Burlingame, CA) using immunoblot analysis on prostate cancer cell lines, synthetic TMPRSS2-ERG constructs, chromatin immunoprecipitation, and immunofluorescence. We correlated ERG protein expression with the presence of ERG gene rearrangements in prostate cancertissues using a combined immunohistochemistry(IHC) and fluorescence in situ hybridization (FISH) analysis. We independently evaluated two patient cohorts and observed ERG expression confined to prostate cancer cells and high-grade prostatic intraepithelial reoplasia associated with ERG-positive cancer, as well as vessels and lymphocytes (where ERG has a known biologic role). Image analysis of 131 cases demonstrated nearly 100% sensitivity for detecting ERG rearrangement prostate cancer, with only 2 (1.5%) of 131 cases demonstrating strong ERG protein expression without any known ERG gene fusion. The combired pathology evaluation of 207 patient tumors for ERG protein expression had 95.7% sensitivity and 96.5% specificity for determining ERG rearrangement prostate cancer. Ir conclusion, this study qualifies a specific anti-ERG antibody and demonstrates exquisite association between ERG gene rearrangement and truncated ERG protein product expression. Giver the ease of performing IHC versus FISH, ERG protein expression may be useful for molecularly subtypirg prostate cancer based or ERG rearrangement status and suggests clinical utility it prostate needle biopsy evaluation.

Prostate cancer is the most common type of tumor found in American men and is the second leading cause of cancer death in males. To identify biomarkers that distinguish prostate cancer from normal, we compared multiple gene expression profiling studies. Through meta-analysis of expression array data from multiple prostate cancer studies, we identified GOLM1 (Golgi membrane protein 1, Golm 1) as consistently up-regulated in clinically localized prostate cancer. This observation was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and validated at the protein level by immunoblot assay and immunohistochemistry. Prostate epithelial cells were identified as the cellular source of GOLM1 expression using laser capture microdissection. Immunohistochemical staining localized the GOLM1 signal to the subapical cytoplasmic region, typical of a Golgi distribution. Surprisingly, GOLM1 immunoreactivity was detected in the supernatants of prostate cell lines and in the urine of patients with prostate cancer. The mechanism by which intact GOLM1 might be released from cells has not yet been elucidated. GOLM1 transcript levels were measured in urine sediments using quantitative PCR on a cohort of patients presenting for biopsy or radical prostatectomy. We found that urinary GOLM1 mRNA levels were a significant predictor of prostate cancer. Further, GOLM1 outperformed serum prostate-specific antigen (PSA) in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.622 for GOLM1 (P = .0009) versus 0.495 for serum PSA (P = .902). Our data indicating the up-regulation of GOLM1 expression and its appearance in patients' urine suggest GOLM1 as a potential novel biomarker for clinically localized prostate cancer.

TMPRSS2-ERG gene fusions are the predominant molecular subtype of prostate cancer. Here, we explored the role of TMPRSS2-ERG gene fusion product using in vitro and in vivo model systems. Transgenic mice expressing the ERG gene fusion product under androgen-regulation develop mouse prostatic intraepithelial neoplasia (PIN), a precursor lesion of prostate cancer. Introduction of the ERG gene fusion product into primary or immortalized benign prostate epithelial cells induced an invasion-associated transcriptional program but did not increase cellular proliferation or anchorage-independent growth. These results suggest that TMPRSS2-ERG may not be sufficient for transformation in the absence of secondary molecular lesions. Transcriptional profiling of ERG knockdown in the TMPPRSS2-ERG-positive prostate cancer cell line VCaP revealed decreased expression of genes over-expressed in prostate cancer versus PIN and genes overexpressed in ETS-positive versus -negative prostate cancers in addition to inhibiting invasion. ERG knockdown in VCaP cells also induced a transcriptional program consistent with prostate differentiation. Importantly, VCaP cells and benign prostate cells overexpressing ERG directly engage components of the plasminogen activation pathway to mediate cellular invasion, potentially representing a downstream ETS target susceptible to therapeutic intervention. Our results support previous work suggesting that TMPRSS2-ERG fusions mediate invasion, consistent with the defining histologic distinction between PIN and prostate cancer.

Global molecular profiling of cancers has shown broad utility in delineating pathways and processes underlying disease, in predicting prognosis and response to therapy, and in suggesting novel treatments. To gain further insights from such data, we have integrated and analyzed a comprehensive collection of “molecular concepts”" representing > 2500 cancer-related gene expression signatures from Oncomine and manual curation of the literature, drug treatment signatures from the Connectivity Map, target gene sets from genome-scale regulatory motif analyses, and reference gene sets from several gene and protein annotation databases. We computed pairwise association analysis on all 13,364 molecular concepts and identified > 290,000 significant associations, generating hypotheses that link cancer types and subtypes, pathways, mechanisms, and drugs. To navigate a network of associations, we developed an analysis platform, the Molecular Concepts Map. We demonstrate the utility of the approach by highlighting molecular concepts analyses of Myc pathway activation, breast cancer relapse, and retinoic acid treatment.

We recently reported the identification of recurrent gene fusions in the majority of prostate cancers involving the 5V untranslated region of the androgenregulated gene TMPRSS2, the ETS family members ERG, ETV1, ETV4. Here we report the noninvasive detection of these gene fusions in the urine of patients with clinically localized prostate cancer. By quantitative polymerase chain reaction, we assessed the expression of ERG, TMPRSS2:ERG transcripts in urine samples obtained after prostatic massage from 19 patients (11 prebiopsy, 8 pre-radical prostatectomy) with prostate cancer. We observed a strong concordance between ERG overexpression, TMPRSS2:ERG expression, with 8 of 19 (42%) patients having detectable TMPRSS2:ERG transcripts in their urine. Importantly, by fluorescence in situ hybridization, we confirmed the presence or the absence of TMPRSS2:ERG gene fusions in matched prostate cancer tissue samples from three of three patients with fusion transcripts in their urine, from two of two patients without fusion transcripts in their urine. These results demonstrate that TMPRSS2:ERG gene fusions can be detected in the urine of patients with prostate cancer, support larger studies on prospective cohorts for noninvasive detection of prostate cancer.

Expression profiling of clinically obtainable tumor specimens has been hindered by the need for microgram quantities of RNA. In vitro transcription (IVT)-based amplifications are most commonly used to amplify small quantities of RNA for microarray analysis. However, significant drawbacks exist with IVT-based amplification, and the need for alternative amplification methods remains. Herein, we validate whole transcriptome amplification (WTA), an exponential amplification technique that produces cDNA libraries and amplified target in 3 to 4 hours from nanogram quantities of total RNA using a combination of cDNA microarrays and quantitative polymerase chain reaction (PCR). We demonstrate that WTA material can serve as a “molecular archive” because a WTA cDNA library can be faithfully amplified through multiple rounds of PCR amplification, allowing it to serve as a bankable and distributable resource. To demonstrate applicability, WTA was combined with laser capture microdissection to profile frozen prostate tissues. Unlike most IVT-based and exponential amplification techniques, WTA does not depend on the presence of a poly-A tail. Thus, we demonstrate that WTA is compatible with artificially degraded RNA and RNA isolated from formalin-fixed paraffin-embedded tissues. Taken together, WTA represents a versatile approach to profile and archive cDNA from minute tumor samples and is compatible with partially degraded RNA.

The platelet-derived growth factor receptor (PDGFR) is a receptor tyrosine kinase overexpressed in a subset of solid tumors and therefore is the target of drugs inhibiting this function such as imatinib mesylate (Gleevec). Thus far, drug therapy has played a limited role in the treatment of localized prostate cancer (PCa). This study characterizes PDGFR-β expression in a wide spectrum of PCa samples to provide empirical data as part of a rational treatment strategy. A survey of five published prostate expression array studies, including 100 clinically localized PCa, did not identify tumors with increased PDGFR-β expression level. Protein expression of PDGFR-β, as determined by immunohistochemistry, revealed 5% of clinically localized PCa and 16% of metastatic PCa cases to show moderate or strong expression. To develop a strategy to detect patients most likely to profit from Gleevec treatment, we analyzed cDNA expression array data from 10,000 transcripts for PDGFR-β expression and divided tumors in groups based on PDGFR-β expression level. Performing a supervised analysis to identify potential comarkers of PDGFR-β in PCa, we identified a set of genes whose expression was associated with PDGFR-β status including early growth response 1 (Egri), an upstream effector of PDGF (4.2-fold upregulation), α-methylacyl-CoA racemase, as well as v-Maf and neuroblastoma suppressor of tumorigenicity (both with a 2.2-fold downregulation). Taken together, this study suggests that only a small subset of PCas may be amenable to tyrosine kinase inhibitors specific for PDGFR.