Discovery and Characterization of Recurrent Gene Fusions in Prostate Cancer.

Recurrent chromosomal rearrangements have been well characterized in hematologic and mesenchymal malignancies, but not in common carcinomas. A novel bioinformatics algorithm termed Cancer Outlier Profile Analysis (COPA) was developed to analyze DNA microarray data for genes markedly over-expressed (“outliers”) in a subset of cases. COPA identified the ETS family members ERG and ETV1 as high-ranking outliers in multiple prostate cancer profiling studies. In cases with outlier expression of ERG or ETV1, recurrent gene fusions of the 5’ untranslated region of the prostate-specific, androgen-induced gene TMPRSS2 to the respective ETS family member were identified. In vitro studies in cancer cell lines demonstrated that androgen-responsive promoter elements of TMPRSS2 mediate the aberrant ETS family member over-expression. Subsequent interrogation of all ETS family members in prostate cancer profiling studies identified outlier expression of ETV4 in two of 98 cases. In one such case, ETV4 over-expression was confirmed and a fusion of the TMPRSS2 and ETV4 loci was identified. A large scale profiling and integrated molecular concepts analysis demonstrated that ETS rearrangement-positive and -negative tumors have distinct transcriptional programs, with loss at 6q21 as a possible defining genetic event in ETS negative prostate cancers. While TMPRSS2:ERG fusions are predominant, fewer TMPRSS2:ETV1 cases were identified than would be expected based on the frequency of ETV1 outlier expression. Through characterizing additional ETV1 outlier cases, novel 5’ fusion partners defining distinct functional classes of ETS gene rearrangements were identified. These include fusions involving androgen-stimulated, androgen-repressed and androgen-insensitive 5’ partners. As the commonality of ETS rearrangements is aberrant over-expression, in vitro and in vivo recapitulation demonstrated that ETV1 or ERG over-expression in benign prostate cells and the mouse prostate confers neoplastic phenotypes. Together, this work suggests a pathogenetically important role for recurrent chromosomal rearrangements in a common epithelial tumor and has important implications in the molecular diagnosis and treatment of prostate cancer. Deregulation of ETS family member expression through gene fusions appears to be a generalized mechanism for prostate cancer development in the majority of cases. Additionally, other common epithelial tumors may be driven by uncharacterized gene rearrangements.Ph.D.Molecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57601/2/tomlinss_1.pd

Informing a risk prediction model for binary outcomes with external coefficient information

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146799/1/rssc12306-sup-0001-SupInfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146799/2/rssc12306_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146799/3/rssc12306.pd

Histotripsy of the Prostate in a Canine Model: Characterization of Post-Therapy Inflammation and Fibrosis

Introduction: Histotripsy is a nonthermal, noninvasive, pulsed ultrasound technology that homogenizes tissue within the targeted volume. From previous experiments, it appeared that the resultant fibrotic response from histotripsy was limited compared with the typical tissue response seen after thermoablation. The objective of this study was to characterize the inflammatory response and quantify patterns of collagen deposition 6 weeks after in vivo canine prostate histotripsy. Methods: Histotripsy was applied to the left half of eight canine prostates to produce an intraparenchymal zone of tissue homogenization. Six weeks after treatment, prostates were harvested, sectioned, and stained with hematoxylin and eosin for histologic evaluation, CD3, CD20, and Mac387 immunohistochemistry to characterize the inflammatory components, and picrosirius red staining to identify collagen. Results: Seven of eight treated prostates exhibited only minimal residual inflammation. Visual microscopic analysis of picrosirius red slides revealed a band of dense collagen (0.5?mm wide) immediately adjacent to the cavity produced by histotripsy. This was surrounded by a second band (1?mm wide) of less dense collagen interspersed among glandular architecture. A lobar distribution of epithelial atrophy and basal cell hyperplasia reminiscent of periurethral glands and ducts was apparent surrounding the margin of the treatment cavities. Tissue loss (-31%) was apparent on the treated side of all prostates while four demonstrated a net decrease in collagen content. Conclusions: In vivo histotripsy of canine prostate produced a decrease in prostate volume coupled with a limited inflammatory and fibrotic response. A narrow (1.5?mm) band of fibrosis around the empty, reepithelialized treatment cavity was observed 6 weeks after treatment. In four cases, an overall reduction in collagen content was measured. Further studies are planned to correlate these histologic findings with alteration in mechanical tissue properties and to explore histotripsy strategies for treatment of benign prostatic hyperplasia that optimize tissue volume removal with minimization of fibrosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140079/1/end.2014.0585.pd

Identification of a novel germline SPOP mutation in a family with hereditary prostate cancer

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106871/1/pros22818.pd

Role of dutasteride in pre‐clinical ETS fusion‐positive prostate cancer models

BACKGROUND Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discovered that gene fusions between the 5′‐untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression. METHODS Here, we evaluated the expression levels of 5α‐reductase isoenzymes in prostate cancer cell lines and tissues. We tested the effect of dutasteride, a 5α‐reductase inhibitor, in TMPRSS2–ERG fusion‐positive VCaP cell proliferation and cell invasion. We also evaluated the effect of dutasteride on the TMPRSS2–ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti‐androgen in VCaP cell xenografts tumor model. RESULTS Our data showed that 5α‐reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre‐treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutasteride in combination with the anti‐androgen bicalutamide significantly decreased tumor burden in VCaP cell xenograft model. CONCLUSIONS Our findings suggest that dutasteride can inhibit ERG fusion‐positive cell growth and in combination with anti‐androgen, significantly reduce the tumor burden. Our study suggests that anti‐androgens used in combination with dutasteride could synergistically augment the therapeutic efficacy in the treatment of ETS‐positive prostate cancer. Prostate 72:1542–1549, 2012. © 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93574/1/22509_ftp.pd

At the intersection of primary pulmonary myxoid sarcoma and pulmonary angiomatoid fibrous histiocytoma: observations from three new cases

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/107555/1/his12354.pd

Comprehensive serial molecular profiling of an “N of 1” exceptional non-responder with metastatic prostate cancer progressing to small cell carcinoma on treatment

Abstract Importance Small cell carcinoma/neuroendocrine prostate cancer (NePC) is a lethal, poorly understood prostate cancer (PCa) subtype. Controversy exists about the origin of NePC in this setting. Objective To molecularly profile archived biopsy specimens from a case of early-onset PCa that rapidly progressed to NePC to identify drivers of the aggressive course and mechanisms of NePC origin and progression. Design, setting, and participants A 47-year-old patient presented with metastatic prostatic adenocarcinoma (Gleason score 9). After a 6-month response to androgen deprivation therapy, the patient developed jaundice and liver biopsy revealed exclusively NePC. Targeted next generation sequencing (NGS) from formalin-fixed paraffin-embedded (FFPE)-isolated DNA was performed from the diagnostic prostate biopsy and the liver biopsy at progression. Intervention Androgen deprivation therapy for adenocarcinoma followed by multiagent chemotherapy for NePC. Main outcomes and measures Identification of the mutational landscape in primary adenocarcinoma and NePC liver metastasis. Whether the NePC arose independently or was derived from the primary adenocarcinoma was considered based on mutational profiles. Results A deleterious somatic SMAD4 L535fs variant was present in both prostate and liver specimens; however, a TP53 R282W mutation was exclusively enriched in the liver specimen. Copy number analysis identified concordant, low-level alterations in both specimens, with focal MYCL amplification and homozygous PTEN, RB1, and MAP2K4 losses identified exclusively in the NePC specimen. Integration with published genomic profiles identified MYCL as a recurrently amplified in NePC. Conclusions and relevance NGS of routine biopsy samples from an exceptional non-responder identified SMAD4 as a driver of the aggressive course and supports derivation of NePC from primary adenocarcinoma (transdifferentiation).http://deepblue.lib.umich.edu/bitstream/2027.42/113670/1/13045_2015_Article_204.pd

Impact of the SPOP Mutant Subtype on the Interpretation of Clinical Parameters in Prostate Cancer.

Purpose: Molecular characterization of prostate cancer, including The Cancer Genome Atlas, has revealed distinct subtypes with underlying genomic alterations. One of these core subtypes, SPOP (speckle-type POZ protein) mutant prostate cancer, has previously only been identifiable via DNA sequencing, which has made the impact on prognosis and routinely used risk stratification parameters unclear. Methods: We have developed a novel gene expression signature, classifier (Subclass Predictor Based on Transcriptional Data), and decision tree to predict the SPOP mutant subclass from RNA gene expression data and classify common prostate cancer molecular subtypes. We then validated and further interrogated the association of prostate cancer molecular subtypes with pathologic and clinical outcomes in retrospective and prospective cohorts of 8,158 patients. Results: The subclass predictor based on transcriptional data model showed high sensitivity and specificity in multiple cohorts across both RNA sequencing and microarray gene expression platforms. We predicted approximately 8% to 9% of cases to be SPOP mutant from both retrospective and prospective cohorts. We found that the SPOP mutant subclass was associated with lower frequency of positive margins, extraprostatic extension, and seminal vesicle invasion at prostatectomy; however, SPOP mutant cancers were associated with higher pretreatment serum prostate-specific antigen (PSA). The association between SPOP mutant status and higher PSA level was validated in three independent cohorts. Despite high pretreatment PSA, the SPOP mutant subtype was associated with a favorable prognosis with improved metastasis-free survival, particularly in patients with high-risk preoperative PSA levels. Conclusion: Using a novel gene expression model and a decision tree algorithm to define prostate cancer molecular subclasses, we found that the SPOP mutant subclass is associated with higher preoperative PSA, less adverse pathologic features, and favorable prognosis. These findings suggest a paradigm in which the interpretation of common risk stratification parameters, particularly PSA, may be influenced by the underlying molecular subtype of prostate cancer

Molecular profiling of human prostate tissues: insights into gene expression patterns of prostate development during puberty

Testosterone production surges during puberty and orchestrates massive growth and reorganization of the prostate gland, and this glandular architecture is maintained thereafter throughout adulthood. Benign prostatic hyperplasia (BPH) and prostate adenocarcinoma (PCA) are common diseases in adulthood that do not develop in the absence of androgens. Our objective was to gain insight into gene expression changes of the prostate gland at puberty, a crucial juncture in prostate development that is androgen dependent. Understanding the role played by androgens in normal prostate development may provide greater insight into androgen involvement in prostatic diseases. Benign prostate tissues obtained from pubertal and adult age group cadaveric organ donors were harvested and profiled using 20,000 element cDNA microarrays. Statistical analysis of the microarray data identified 375 genes that were differentially expressed in pubertal prostates relative to adult prostates including genes such as Nkx3.1, TMEPAI, TGFBR3, FASN, ANKH, TGFBR2, FAAH, S100P, HoxB13, fibronectin, and TSC2 among others. Comparisons of pubertal and BPH expression profiles revealed a subset of genes that shared the expression pattern between the two groups. In addition, we observed that several genes from this list were previously demonstrated to be regulated by androgen and hence could also be potential in vivo targets of androgen action in the pubertal human prostate. Promoter searches revealed the presence of androgen response elements in a cohort of genes including tumor necrosis factor‐α induced adipose related protein, which was found to be induced by androgen. In summary, this is the first report that provides a comprehensive view of the molecular events that occur during puberty in the human prostate and provides a cohort of genes that could be potential in vivo targets of androgenic action during puberty.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154303/1/fsb2fj042415fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154303/2/fsb2fj042415fje-sup-0001.pd

Evidence for a functional role of the second C5a receptor C5L2

During experimental sepsis in rodents after cecal ligation and puncture (CLP), excessive C5a is generated, leading to interactions with C5aR, loss of innate immune functions of neutrophils, and lethality. In the current study, we have analyzed the expression of the second C5a receptor C5L2, the putative â defaultâ or nonsignaling receptor for C5a. Rat C5L2 was cloned, and antibody was developed to C5L2 protein. After CLP, blood neutrophils showed a reduction in C5aR followed by its restoration, while C5L2 levels gradually increased, accompanied by the appearance of mRNA for C5L2. mRNA for C5L2 increased in lung and liver during CLP. Substantially increased C5L2 protein (defined by binding of 125Iâ antiâ C5L2 IgG) occurred in lung, liver, heart, and kidney after CLP. With the use of serum ILâ 6 as a marker for sepsis, infusion of antiâ C5aR dramatically reduced serum ILâ 6 levels, while antiâ C5L2 caused a nearly fourfold increase in ILâ 6 when compared with CLP controls treated with normal IgG. When normal blood neutrophils were stimulated in vitro with LPS and C5a, the antibodies had similar effects on release of ILâ 6. These data provide the first evidence for a role for C5L2 in balancing the biological responses to C5a.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154410/1/fsb2fj043424fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154410/2/fsb2fj043424fje-sup-0040.pd