Cell-specific Gene Expression: Pylorus Morphogenesis and Hedgehog-regulated Enhancers.

The precise spatiotemporal control of gene expression is integral to the survival of all organisms. Inappropriate gene expression can lead to developmental defects in newborns, such as Infantile Hypertrophic Pyloric Stenosis, in which hypertrophy of pyloric sphincter smooth muscle leads to gastric outlet obstruction. This thesis work analyzes the mechanisms and consequences of cell-specific gene expression in three systems: establishment of the epithelial gastro-duodenal (pyloric) border, development of smooth muscle structures at the pylorus, and transcriptional response to Hedgehog (Hh) signaling in Drosophila. Microarray is used to characterize the antral, pyloric, and duodenal transcriptomes at embryonic days (E) 14.5 and 16.5. At E16.5, hundreds of genes are upregulated specifically in duodenal epithelium. This event is termed intestinalization because the activated genes are associated with intestinal function. Several transcription factors (i.e., Tcfec, Creb3l3, and Hnf4gamma) are upregulated in duodenal epithelium and levels of Hh signaling are downregulated in duodenal mesenchyme. In addition, novel pyloric genes are identified, including Gata3, which encodes a zinc finger transcription factor. A role for Gata3 during pylorus development is elucidated using a genetic model of Gata3 insufficiency. Gata3 and the homeodomain transcription factor Nkx2-5 co-localize with molecular markers of pyloric smooth muscle and are expressed in novel bilateral smooth muscle structures at the pylorus (i.e., the ventral pyloric cords). Loss of Gata3 alters the shape of the pylorus and attenuates the pyloric constriction. The ventral pyloric cords and outer longitudinal smooth muscle at the pylorus are absent in Gata3 null embryos. Gata3 does not control Nkx2-5 expression at the pylorus. An in silico approach identifies Hh-regulated enhancers in Drosophila. Binding sites for the Hh transcriptional effector cubitus interruptus (Ci) are significantly clustered in the genomes of two divergent Drosophila species, but mutant Ci sites are not. Putative Hh-regulated enhancers are identified by the comparison of orthologous regions of significant Ci clustering. Two of these enhancers (inv and rdx) are active in Hh-responsive cells of the Drosophila larval imaginal wing disc. These studies reveal novel gene expression patterns during pylorus morphogenesis and suggest an approach to identifying direct transcriptional targets of signaling pathways.Ph.D.Cell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91524/1/udager_1.pd

A novel RNA in situ hybridization assay for the long noncoding RNA SChLAP1 predicts poor clinical outcome after radical prostatectomy in clinically localized prostate cancer.

Long noncoding RNAs (lncRNAs) are an emerging class of oncogenic molecules implicated in a diverse range of human malignancies. We recently identified SChLAP1 as a novel lncRNA that demonstrates outlier expression in a subset of prostate cancers, promotes tumor cell invasion and metastasis, and associates with lethal disease. Based on these findings, we sought to develop an RNA in situ hybridization (ISH) assay for SChLAP1 to 1) investigate the spectrum of SChLAP1 expression from benign prostatic tissue to metastatic castration-resistant prostate cancer and 2) to determine whether SChLAP1 expression by ISH is associated with outcome after radical prostatectomy in patients with clinically localized disease. The results from our current study demonstrate that SChLAP1 expression increases with prostate cancer progression, and high SChLAP1 expression by ISH is associated with poor outcome after radical prostatectomy in patients with clinically localized prostate cancer by both univariate (hazard ratio = 2.343, P = .005) and multivariate (hazard ratio = 1.99, P = .032) Cox regression analyses. This study highlights a potential clinical utility for SChLAP1 ISH as a novel tissue-based biomarker assay for outcome prognostication after radical prostatectomy

Current and Proposed Molecular Diagnostics in a Genitourinary Service Line Laboratory at a Tertiary Clinical Institution

Abstract: The idea that detailed knowledge of molecular oncogenesis will drive diagnostic, prognostic, and therapeutic clinical decision making in an increasingly multidisciplinary practice of oncologic care has been anticipated for many years. With the recent rapid advancement in our understanding of the molecular underpinnings of genitourinary malignancies, this concept is now starting to take shape in the fields of prostate, kidney, bladder, testicular, and penile cancer. Such breakthroughs necessitate the development of robust clinical-grade assays that can be quickly made available for patients to facilitate diagnosis in challenging cases, risk-stratify patients for subsequent clinical management, select the appropriate targeted therapy from among increasingly diverse and numerous options, and enroll patients in advanced clinical trials. This rapid translation of basic and clinical cancer research requires a streamlined, multidisciplinary approach to clinical assay development, termed here the molecular diagnostics service line laboratory. In this review, we summarize the current state and explore the future of molecular diagnostics in genitourinary oncology to conceptualize a genitourinary service line laboratory at a tertiary clinical institution. Key Words: Prostate cancer, kidney cancer, bladder cancer, testicular cancer, penile cancer, immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), in situ hybridization (ISH), whole-genome sequencing (Cancer J 2014;20: 29Y42) T he genomic era is rapidly revolutionizing the practice of health care, and in almost no area is this more apparent than oncology, where molecular data are increasingly driving patient care in terms of diagnosis, prognosis, and therapeutics. The promise of personalized medicine, wherein the therapeutic options for an individual patient are tailored to his/her specific tumor genetics and biology, requires robust clinical assays for the biomarker(s) of interest. Our evolving understanding of the molecular underpinnings of urologic malignancies provides an emerging role for molecular testing in the treatment of these common neoplasms. Here, we envision the concept of a genitourinary service line laboratory at a tertiary clinical institution, and using an organ-based approach, we review the current state and explore the future of molecular diagnostics in genitourinary oncology. Genitourinary Service Line Laboratory: Concept and Services With the advent of widespread whole-genome sequencing of tumors, going forward, we expect the pace of molecular discoveries to quicken rather than abate. This trend will undoubtedly be associated with the need to bring advances made in the laboratory rapidly into the realm of routine clinical practice. We believe there are several ways this is already happening and will further develop in the future. As the cost of sequencing decreases, one such possibility is routine whole-genome sequencing of clinical tumor specimens, with the selection of therapeutics based on the prevalent targetable molecular alterations. A clinical sequencing pilot project, termed MI-ONCOSEQ, has already been established at the University of Michigan Health System (UMHS) for patients with advanced tumors that are resistant to conventional histologybased therapies. 1 Based on the results of this novel project, it seems clear to us that there are tumors that would benefit from this whole-genome sequencing approach. It is also evident that cancers arising in different organsVas well as different cancer subtypes within the same organ systemVare not uniformly similar but instead house different genetic drivers. In this age of translational medicine, it is imperative that discoveries from technologies such as clinical sequencing or traditional clinical cancer research be quickly incorporated into the development of clinical-grade assays in a CLIA (Clinical Laboratory Improvement Amendments)Ycertified environment. Broader availability of such assays will facilitate patient enrollment in a new generation of clinical trials that incorporate these rapid molecular advances and will expand the available pool of clinical sites beyond specialized academic centers. Hence, we propose the concept of dedicated molecular diagnostic service line laboratories, initially purposed for and centered around specific organ systems (i.e., genitourinary, pulmonary, etc.

Single-Center Prospective Cohort Study on the Histopathology, Genotype, and Postsurgical Outcomes of Patients With Primary Aldosteronism

Unilateral forms of primary aldosteronism are usually surgically treated to remove the source of aldosterone excess. After adrenalectomy, aldosteronism persists in some patients indicating abnormal aldosterone production from the unresected gland. Our objective was to investigate histopathology, genotype, and postsurgical outcomes in a 3-year prospective cohort of surgically treated patients for primary aldosteronism (from 2016 to 2018). The cohort comprised 60 consecutively operated patients categorized with classical or nonclassical histopathologic findings of unilateral primary aldosteronism. In the classical group were 45 solitary aldosterone-producing adenomas or dominant aldosterone-producing nodules; in the nonclassical group were 15 cases of multiple aldosterone-producing micronodules or nodules (12 cases) or aldosterone-producing diffuse hyperplasia (3 cases). The classical group displayed higher baseline plasma aldosterone concentrations (262 versus 155 pg/mL, P=0.008) and an increased aldosterone-to-renin ratio (81 versus 42, P=0.002). A high proportion of the classical group achieved complete biochemical success (97.6% versus 66.7% in the nonclassical group, P=0.002). The nonclassical versus classical group displayed an increased ratio of absolute aldosterone concentration in the contralateral adrenal vein to peripheral vein at adrenal venous sampling (3.8 versus 2.0, P=0.004). Variants in aldosterone-driver genes were identified in 85% of 41 aldosterone-producing adenomas and were excluded in the remaining 15% by CYP11B2 guided next-generation sequencing. There were no differences in clinical or biochemical outcomes in patients with a solitary aldosterone-producing adenoma categorized by KCNJ5 mutation status. In conclusion, adrenals with a nonclassical histopathology of unilateral primary aldosteronism are associated with a higher incidence of postsurgical disease persistence and increased aldosterone production from the unresected adrenal

Genital verruciform xanthoma: lessons from a contemporary multi‐institutional series

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163435/2/his14198.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163435/1/his14198_am.pd

Clinical Utility and Concordance of Upper Urinary Tract Cytology and Biopsy in Predicting Clinicopathologic Features of Upper Urinary Tract Urothelial Carcinoma

5% of urothelial carcinoma occurs in the upper urinary tract (UUT), a challenging location to biopsy. We aim to evaluate concordance between biopsy, cytology, and resection specimens in a large upper tract urothelial carcinoma (UTUC) cohort.117 UTUC resections with UUT biopsy and/or cytology specimens from 2000–2016 were retrieved; pathologic material was re-reviewed, evaluated for concordance, and correlated with clinical information. 14% pre-operative biopsies, including 8 from renal pelvis and 6 from ureter, lacked neoplastic diagnoses. 77% diagnostic biopsies included subepithelial tissue; 11% demonstrated reclassification of grade and 30% demonstrated reclassification of invasion status. 26% of renal pelvis UTUC and 36% ureter UTUC were invasive only on resection. Of 18 UTUC reclassified from noninvasive high-grade papillary urothelial carcinoma (HGPUC) to invasive HGPUC, 39% had prior radical cystectomy (versus 8% invasive UTUC and 11% noninvasive UTUC with concordant biopsies). Most high-grade UTUC (88%) and some low-grade UTUC (58%) resections had abnormal cytology results. Biopsy-resection pairs with concordant invasion status and pairs with discordant invasion status showed similar rates of recurrence (38% versus 38%) and metastasis (25% versus 27%). 14% of UUT biopsies lacked diagnostic neoplastic material. Grade concordance between biopsy and resection was high (89%), but 30% of cases showed invasion only on resection. Subepithelial tissue was less commonly present in ureter biopsies, particularly from mid or proximal ureter. UTUC in patients with prior cystectomy were more likely to show invasion on resection but not biopsy

Somatic mutations of CADM1 in aldosterone-producing adenomas and gap junction-dependent regulation of aldosterone production

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production

Serial monitoring of genomic alterations in circulating tumor cells of ER-positive/HER2-negative advanced breast cancer: feasibility of precision oncology biomarker detection.

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

Metastatic renal cell carcinoma, clear cell type, of the parotid gland

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109307/1/dc23103.pd