Jeff Simko

TMPRSS2-ERG Fusion Prostate Cancer: An Early Molecular Event Associated With Invasion

Prostate cancer (PCA) is one of the most prevalent cancers and a major leading cause of morbidity and mortality in the Western world. The TMPRSS2-ERG fusion was recently identified as a common recurrent chromosomal aberration in this malignancy. In our study, we interrogated a broad spectrum of benign, precursor, and malignant prostatic lesions to assess the TMPRSS2-ERG fusion status using a multicolor interphase fluorescence in situ hybridization assay. Samples from hospital-based cohorts consisted of 237 clinically localized PCA, 34 hormone naive metastases, 9 hormone refractory metastases, 26 high grade prostatic intraepithelial neoplasia lesions, 15 samples of benign prostatic hyperplasia, 38 of proliferative inflammatory atrophy, and 47 of benign prostatic tissue. The TMPRSS2-ERG fusion was present in 48.5% of clinically localized PCA, 30% of hormone naive metastases, 33% of hormone refractory metastases, and in 19% of high grade prostatic intraepithelial neoplasia lesions in intermingling to cancer foci. Almost all these fusion positive cases show a homogenous distribution of the fusion pattern. In contrast, none of the other samples harbored this genetic aberration. If we consider the high incidence of PCA and the high frequency of this gene fusion, TMPRSS2-ERG is the most common genetic aberration so far described in human malignancies. Furthermore, its clinical application as a biomarker and ancillary diagnostic test is promising given its high specificity.

Changes in prostate gene expression in men undergoing an intensive nutrition and lifestyle intervention.

Epidemiological and prospective studies indicate that comprehensive lifestyle changes may modify the progression of prostate cancer. However, the molecular mechanisms by which improvements in diet and lifestyle might affect the prostate microenvironment are poorly understood. We conducted a pilot study to examine changes in prostate gene expression in a unique population of men with low-risk prostate cancer who declined immediate surgery, hormonal therapy, or radiation and participated in an intensive nutrition and lifestyle intervention while undergoing careful surveillance for tumor progression. Consistent with previous studies, significant improvements in weight, abdominal obesity, blood pressure, and lipid profile were observed (all P < 0.05), and surveillance of low-risk patients was safe. Gene expression profiles were obtained from 30 participants, pairing RNA samples from control prostate needle biopsy taken before intervention to RNA from the same patients 3-month postintervention biopsy. Quantitative real-time PCR was used to validate array observations for selected transcripts. Two-class paired analysis of global gene expression using significance analysis of microarrays detected 48 up-regulated and 453 down-regulated transcripts after the intervention. Pathway analysis identified significant modulation of biological processes that have critical roles in tumorigenesis, including protein metabolism and modification, intracellular protein traffic, and protein phosphorylation (all P < 0.05). Intensive nutrition and lifestyle changes may modulate gene expression in the prostate. Understanding the prostate molecular response to comprehensive lifestyle changes may strengthen efforts to develop effective prevention and treatment. Larger clinical trials are warranted to confirm the results of this pilot study.

Characterization of TMPRSS2-ERG fusion high-grade prostatic intraepithelial neoplasia and potential clinical implications.

Purpose: More than 1,300,000 prostate needle biopsies are done annually in the United States with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer on subsequent needle biopsies in prostate-specific antigen–screened populations. In contemporary series, prostate cancer is detected in ∼20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histologic subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in prostate cancer has also been shown to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion prostate cancer is associated with a more aggressive clinical course. Experimental Design: In this study, we assessed a series of HGPIN lesions and paired prostate cancer for the presence of TMPRSS2-ERG gene fusion. Results: Fusion-positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances, the matching cancer shared the same fusion pattern. Sixty percent of TMPRSS2-ERG fusion prostate cancer had fusion-negative HGPIN. Conclusions: Given the more aggressive nature of TMPRSS2-ERG prostate cancer, the findings of this study raise the possibility that gene fusion-positive HGPIN lesions are harbingers of more aggressive disease. To date, pathologic, molecular, and clinical variables do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion prostate cancer in subsequent biopsies.

Microfluidic-Based Multiplex qRT-PCR Identifies Diagnostic and Prognostic microRNA Signatures in the Sera of Prostate Cancer Patients

Recent prostate-specific antigen-based screening trials indicate an urgent need for novel and noninvasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Noncoding microRNAs (miRNA) in the serum and plasma have been shown to have potential as noninvasive markers for physiologic and pathologic conditions. To identify serum miRNAs that diagnose and correlate with the prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR method involving the purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA-deficient) mouse ES cells as the benchmark, we confirmed the validity of our technique and uncovered a considerable lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA scores, we identified miRNA signatures that allow us to diagnose cancer patients and correlate with a prognosis. These serum signatures include oncogenic and tumor-suppressive miRNAs, suggesting functional roles in prostate cancer progression.

von Hippel-Lindau Gene Status and Response to Vascular Endothelial Growth Factor Targeted Therapy for Metastatic Clear Cell Renal Cell Carcinoma

PURPOSE The von Hippel-Lindau (VHL) gene is often inactivated (by mutation or promoter hypermethylation) in renal cell carcinoma but the relation to therapeutic outcome is unclear. MATERIALS AND METHODS Patients with metastatic clear cell renal cell carcinoma with available baseline tumor samples who received vascular endothelial growth factor targeted therapy were included in analysis. Patient characteristics, VHL gene status and clinical outcome were documented. Our primary end point was to test for response rate in relation to VHL inactivation. Progression-free survival and overall survival in relation to VHL status were investigated as secondary end points. RESULTS A total of 123 patients were evaluable. Response rate, median progression-free survival and median overall survival were 37% (95% CI 28-46), 10.8 (95% CI 7.7-14.8) and 29.8 (CI not estimable) months, respectively. Patients with VHL inactivation had a response rate of 41% vs 31% for those with wild-type VHL (p = 0.34). Patients with loss of function mutations (frameshift, nonsense, splice and in-frame deletions/insertions) had a 52% response rate vs 31% with wild-type VHL (p = 0.04). On multivariate analysis the presence of a loss of function mutation remained an independent prognostic factor associated with improved response. Progression-free survival and overall survival were not significantly different based on VHL status. CONCLUSIONS To our knowledge this is the largest analysis investigating the impact of VHL inactivation on the outcome of vascular endothelial growth factor targeted agents in metastatic renal cell carcinoma. We did not find a statistically significant increase in response to vascular endothelial growth factor targeted agents in patients with VHL inactivation. Loss of function mutations identified a population of patients with a greater response. Investigation of downstream markers is under way.

Morphological features of TMPRSS2–ERG gene fusion prostate cancer†

The TMPRSS2–ETS fusion prostate cancers comprise 50–70% of the prostate‐specific antigen (PSA)‐screened hospital‐based prostate cancers examined to date, making it perhaps the most common genetic rearrangement in human cancer. The most common variant involves androgen‐regulated TMPRSS2 and ERG, both located on chromosome 21. Emerging data from our group and others suggests that TMPRSS2–ERG fusion prostate cancer is associated with higher tumour stage and prostate cancer‐specific death. The goal of this study was to determine if this common somatic alteration is associated with a morphological phenotype. We assessed 253 prostate cancer cases for TMPRSS2–ERG fusion status using an ERG break‐apart FISH assay. Blinded to gene fusion status, two reviewers assessed each tumour for presence or absence of eight morphological features. Statistical analysis was performed to look for significant associations between morphological features and TMPRSS2–ERG fusion status. Five morphological features were associated with TMPRSS2–ERG fusion prostate cancer: blue‐tinged mucin, cribriform growth pattern, macronucleoli, intraductal tumour spread, and signet‐ring cell features, all with p‐values < 0.05. Only 24% (n = 30/125) of tumours without any of these features displayed the TMPRSS2–ERG fusion. By comparison, 55% (n = 38/69) of cases with one feature (RR = 3.88), 86% (n = 38/44) of cases with two features (RR = 20.06), and 93% (n = 14/15) of cases with three or more features (RR = 44.33) were fusion positive (p < 0.001). To our knowledge, this is the first study that demonstrates a significant link between a molecular alteration in prostate cancer and distinct phenotypic features. The strength of these findings is similar to microsatellite unstable colon cancer and breast cancer involving BRCA1 and BRCA2 mutations. The biological effect of TMPRSS2–ERG overexpression may drive pathways that favour these common morphological features that pathologists observe daily. These features may also be helpful in diagnosing TMPRSS2–ERG fusion prostate cancer, which may have both prognostic and therapeutic implications. Copyright

The Relationship Between Prostate Specific Antigen Change and Biopsy Progression in Patients on Active Surveillance for Prostate Cancer

PURPOSE We assessed whether an association exists between a change in prostate specific antigen and biopsy progression in men on active surveillance. MATERIALS AND METHODS A cohort of patients undergoing active surveillance for prostate cancer was identified from the urological oncology database at our institution. Multivariate logistic regression was performed to determine whether prostate specific antigen velocity, defined as the change in ln(prostate specific antigen) per year, is associated with biopsy progression, defined as a Gleason upgrade or volume progression on repeat biopsy within 24 months of diagnosis. RESULTS A total of 241 men with a mean ± SD age of 61 ± 7 years and mean prostate specific antigen 4.9 ± 2.2 ng/ml met study inclusion criteria. Median time to repeat biopsy was 10 months (IQR 6-13). Biopsy progression developed in 55 men (23%), including a Gleason score upgrade in 46 (19%), greater than 33% positive cores in 11 (5%) and greater than 50% maximum single core positive in 12 (5%). The median prostate specific antigen ratio per year was 1.0 (IQR 0.95-1.03), although 1 man had a ratio of greater than 1.26 (doubled over 3 years) and 7 had a ratio of less than 1/1.26 (halved over 3 years). On multivariate analysis prostate specific antigen doubling within 3 years was associated with a 1.4-fold increase in the odds of biopsy progression (OR 1.4, 95% CI 0.6-3.4, p = 0.46). CONCLUSIONS There is little change in prostate specific antigen during the first 24 months of surveillance in men with well staged, low risk prostate cancer. We believe that these findings highlight the importance of repeat biopsy during surveillance.

A multi-marker assay to distinguish malignant melanomas from benign nevi

The histopathological diagnosis of melanoma can be challenging. No currently used molecular markers accurately distinguish between nevus and melanoma. Recent transcriptome analyses have shown the differential expression of several genes in melanoma progression. Here, we describe a multi-marker diagnostic assay using 5 markers (ARPC2, FN1, RGS1, SPP1, and WNT2) overexpressed in melanomas. Immunohistochemical marker expression was analyzed in 693 melanocytic neoplasms comprising a training set (tissue microarray of 534 melanomas and nevi), and 4 independent validation sets: tissue sections of melanoma arising in a nevus; dysplastic nevi; Spitz nevi; and misdiagnosed melanocytic neoplasms. Both intensity and pattern of expression were scored for each marker. Based on the differential expression of these 5 markers between nevi and melanomas in the training set, a diagnostic algorithm was obtained. Using this algorithm, the lesions in the validation sets were diagnosed as nevus or melanoma, and the results were compared with the known histological diagnoses. Both the intensity and pattern of expression of each marker were significantly different in melanomas compared to nevi. The diagnostic algorithm exploiting these differences achieved a specificity of 95% and a sensitivity of 91% in the training set. In the validation sets, the multi-marker assay correctly diagnosed a high percentage of melanomas arising in a nevus, Spitz nevi, dysplastic nevi, and misdiagnosed lesions. The multi-marker assay described here can aid in the diagnosis of melanoma.

Activated Lymphocyte Recruitment Into the Tumor Microenvironment Following Preoperative Sipuleucel-T for Localized Prostate Cancer

Background Sipuleucel-T is a US Food and Drug Administration–approved immunotherapy for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Its mechanism of action is not fully understood. This prospective trial evaluated the direct immune effects of systemically administered sipuleucel-T on prostatic cancer tissue in the preoperative setting. Methods Patients with untreated localized prostate cancer were treated on an open-label Phase II study of sipuleucel-T prior to planned radical prostatectomy (RP). Immune infiltrates in RP specimens (posttreatment) and in paired pretreatment biopsies were evaluated by immunohistochemistry (IHC). Correlations between circulating immune response and IHC were assessed using Spearman rank order. Results Of the 42 enrolled patients, 37 were evaluable. Adverse events were primarily transient, mild-to-moderate and infusion related. Patients developed T cell proliferation and interferon-γ responses detectable in the blood following treatment. Furthermore, a greater-than-three-fold increase in infiltrating CD3+, CD4+ FOXP3-, and CD8+ T cells was observed in the RP tissues compared with the pretreatment biopsy (binomial proportions: all P < .001). This level of T cell infiltration was observed at the tumor interface, and was not seen in a control group consisting of 12 concurrent patients who did not receive any neoadjuvant treatment prior to RP. The majority of infiltrating T cells were PD-1+ and Ki-67+, consistent with activated T cells. Importantly, the magnitude of the circulating immune response did not directly correlate with T cell infiltration within the prostate based upon Spearman’s rank order correlation. Conclusions This study is the first to demonstrate a local immune effect from the administration of sipuleucel-T. Neoadjuvant sipuleucel-T elicits both a systemic antigen-specific T cell response and the recruitment of activated effector T cells into the prostate tumor microenvironment.

Isolation and genomic analysis of circulating tumor cells from castration resistant metastatic prostate cancer

BackgroundThe number of circulating tumor cells (CTCs) in metastatic prostate cancer patients provides prognostic and predictive information. However, it is the molecular characterization of CTCs that offers insight into the biology of these tumor cells in the context of personalized treatment.MethodsWe developed a novel approach to isolate CTCs away from hematopoietic cells with high purity, enabling genomic analysis of these cells. The isolation protocol involves immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS). To evaluate the feasibility of isolation of CTCs by IE/FACS and downstream genomic profiling, we conducted a pilot study in patients with metastatic castration resistant prostate cancer (CRPC). Twenty (20) sequential CRPC patients were assayed using CellSearch™. Twelve (12) patients positive for CTCs were subjected to immunomagnetic enrichment and fluorescence activated cell sorting (IE/FACS) to isolate CTCs. Genomic DNA of CTCs was subjected to whole genome amplification (WGA) followed by gene copy number analysis via array comparative genomic hybridization (aCGH).ResultsCTCs from nine (9) patients successfully profiled were observed to have multiple copy number aberrations including those previously reported in primary prostate tumors such as gains in 8q and losses in 8p. High-level copy number gains at the androgen receptor (AR) locus were observed in 7 (78%) cases. Comparison of genomic profiles between CTCs and archival primary tumors from the same patients revealed common lineage. However, high-level copy number gains in the AR locus were observed in CTCs, but not in the matched archival primary tumors.ConclusionsWe developed a new approach to isolate prostate CTCs without significant leukocyte admixture, and to subject them to genome-wide copy number analysis. Our assay may be utilized to explore genomic events involved in cancer progression, e.g. development of castration resistance and to monitor therapeutic efficacy of targeted therapies in clinical trials in a relatively non-invasive manner.