Search

Comprehensive genetic profiling is increasingly important for the clinical workup of hematologic tumors, as specific alterations are now linked to diagnostic characterization, prognostic stratification and therapy selection. To characterize relevant genetic and genomic alterations in myeloid malignancies maximally, we utilized a comprehensive strategy spanning fluorescence in situ hybridization (FISH), classical karyotyping, Chromosomal Microarray (CMA) for detection of copy number variants (CNVs) and Next generation Sequencing (NGS) analysis. In our cohort of 569 patients spanning the myeloid spectrum, NGS and CMA testing frequently identified mutations and copy number changes in the majority of genes with important clinical associations, such as TP53, TET2, RUNX1, SRSF2, APC and ATM. Most importantly, NGS and CMA uncovered medically actionable aberrations in 75.6% of cases normal by FISH/cytogenetics testing. NGS identified mutations in 65.5% of samples normal by CMA, cytogenetics and FISH, whereas CNVs were detected in 10.1% cases that were normal by all other methodologies. Finally, FISH or cytogenetics, or both, were abnormal in 14.1% of cases where NGS or CMA failed to detect any changes. Multiple mutations and CNVs were found to coexist, with potential implications for patient stratification. Thus, high throughput genomic tumor profiling through targeted DNA sequencing and CNV analysis complements conventional methods and leads to more frequent detection of actionable alterations.

The development of targeted therapies based on specific genomic alterations has altered the treatment and management of lung and colorectal cancers. Chromosomal microarray (CMA) has allowed identification of copy number variations (CNVs) in lung and colorectal cancers in great detail, and next-generation sequencing (NGS) is used extensively to analyze the genome of cancers for molecular subtyping and use of molecularly guided therapies. The main objective of this study was to evaluate the utility of combining CMA and NGS for a comprehensive genomic assessment of lung and colorectal adenocarcinomas, especially for detecting drug targets. We compared the results from NGS and CMA data from 60 lung and 51 colorectal tumors. From CMA analysis, 33% were amplified, 89% showed gains, 75% showed losses and 41% demonstrated loss of heterozygosity; pathogenic variants were identified in 81% of colon and 67% lung specimens through NGS. KRAS mutations commonly occurred with loss in TP53 and there was significant loss of BRCA1 and NF1 among male patients with lung cancer. For clinically actionable targets, 23% had targetable CNVs when no pathogenic variants were detected by NGS. The data thus indicate that combining the two approaches provides significant benefit in a routine clinical setting not available by NGS alone.

BACKGROUND Mutations in the p53 tumor suppressor gene may correlate with an increased risk of recurrence and disease progression in patients with bladder carcinoma. The ability to accurately and sensitively detect p53 mutations in cytology specimens may be of benefit in the treatment of bladder carcinoma patients with superficial, minimally invasive disease. METHODS Genomic DNA was isolated from 49 cases, each of which was comprised of matched bladder tumor tissue, bladder wash, and voided urine specimens obtained concurrently at a single institution. The genomic DNA was analyzed for mutations in the p53 tumor suppressor gene using a p53 mutation detection assay. Automated dideoxy sequencing of mutant specimens also was performed. RESULTS Of the 49 cases, 29 (59%) showed no evidence of p53 mutations in the tumor, bladder wash, or voided urine specimens. Of the remaining 20 cases, 19 showed evidence of mutations in the tumor. Of these 19 p53 mutant bladder tumors, 16 (84%) were detected in the matched bladder wash and 16 (84%) were detected in the matched voided urine specimens. One case resulted in the detection of mutant p53 in the voided urine and the bladder wash, but not in the tumor. Analysis of the results between tumor tissue and bladder wash or tumor and voided urine showed 84.2% sensitivity, 96.8% specificity, and 91.8% accuracy. Sequence analysis of the mutant cases showed that the mutations detected in the tumor tissue were the same mutations detected in the bladder wash and the voided urine specimens. CONCLUSIONS Both voided urine and bladder wash specimens from patients with bladder carcinoma were found to provide a high rate of clinical accuracy for the determination of the p53 gene status in patients with bladder tumors. Cancer 2001;91:2127–35. © 2001 American Cancer Society.

Prostate-specific antigen (PSA) has emerged as the most useful marker for management of patients with prostate cancer. The regulation of this glycoprotein in vivo has important clinical implications. Indirect evidence indicates that the PSA glycoprotein might be regulated by androgens, and previous studies in this laboratory have demonstrated that PSA mRNA is upregulated by androgens. The current work reports a detailed study of PSA glycoprotein expression as influenced by steroid hormones in a human prostatic adenocarcinoma cell line, LNCaP. First, we have examined the steroid binding specificity of the androgen receptor in this cell line. In comparison with wild-type rat androgen receptor in prostate, the receptor in LNCaP cells has altered affinity for a number of steroids or analogs such as progesterone (R5020), antiprogesterone (RU486), two antiandrogens (cyperoterone acetate and hydroxyflutamide), and an androgen metabolite (epitestosterone). However, its affinity for androgens (mibolerone, dihydrotestosterone, and testosterone) is not changed. The receptor does not bind to the synthetic glucocorticoids (triaminolone acetonide and dexamethasone) nor to a synthetic estrogen DES (diethylstilbestrol). The change of the steroid binding specificity of the receptor is correlated with a single mutation (A----G at nucleotide #876 relative to the initiation codon) of the steroid binding domain of the receptor. The mutation and alteration of steroid-binding specificity of the androgen receptor is also correlated with PSA glycoprotein expression affected by different ligands tested. We have demonstrated that the PSA glycoprotein is upregulated by androgens and is affected by neither epidermal growth factor nor basic fibroblast growth factor. Moreover, PSA glycoprotein could be induced by R5020, estradiol, and epitestosterone; but neither glucocorticoids nor DES had any effect on PSA induction. Interestingly, although the antiandrogen, cyperotone acetate, had the ability to induce PSA, both RU486 and hydroxyflutamide could block androgen and progesterone induction of PSA glycoprotein. Therefore, we conclude that the PSA glycoprotein expression is influenced predominantly by androgens via its receptor, and the mutation of the receptor can affect the expression of this cellular gene by the steroids other than androgens.

Prostate-specific antigen (PSA) mRNA was detected by in situ hybridization utilizing a 428 base pair [35S]-labelled cDNA probe from the 3' noncoding region of the PSA gene. Thirty six fresh surgical specimens were collected from patients undergoing radical retropubic prostatectomy for carcinoma of the prostate. Quantitative analysis of the levels of PSA mRNA in both the benign and malignant tissues was performed using an IBAS 2000 Image Analysis System. The results of this study demonstrated that there is a significant decrease in the expression of PSA mRNA in the carcinoma tissue when compared to the benign epithelium. The average binding (number of silver grains/1 x 10(4) microns. 2) for 20 specimens of malignant epithelium was 475 +/- 161 and 586 +/- 140 for 16 specimens of benign epithelium (p less than 0.05). Eleven patients had both benign and malignant tissue from the same surgical specimen available for study. From these paired specimens, the PSA mRNA expression was also significantly reduced in the malignant epithelium when compared to the benign epithelium, 445 +/- 162 and 588 +/- 135 respectively (p less than 0.005). The PSA protein was detected using a monoclonal antibody to PSA with an immunohistochemical staining technique. The PSA protein expression paralleled the expression of the PSA mRNA in the majority of the tissue sections. Many of the tumor specimens showed a heterogeneous expression of PSA, whereas all of the benign epithelium had a uniform high level of PSA expression. In conclusion, PSA mRNA and protein are located only within the glandular epithelial tissue, the expression of PSA protein parallels that of the PSA mRNA, and both the PSA protein and PSA mRNA are significantly decreased in the malignant epithelium when compared to benign prostatic epithelium.

Background: NCCN guidelines and a continuously expanding collection of high-impact publications recommend interrogating hematologic neoplasms for biomarkers that yield diagnostic, prognostic, and/or therapeutic information. The advent of comprehensive, high-throughput genomic profiling technologies has enabled the detection of multiple genomic alterations in an efficient and cost effective manner, and provides insights into disease initiation, progression, therapy response and identification of therapeutic targets not available through conventional methods. Design: Based on NCCN guidelines and literature review, we designed a genomic profiling strategy composed of a targeted amplicon-based next generation sequencing (NGS) assay and cytogenomic microarray (CGA) to identify clinically actionable genomic alterations. In the current context, "actionable" was defined as helping establish a diagnosis through demonstration of "clonal hematopoiesis" as recommended by NCCN guidelines, informing prognosis, and/or providing a potential therapeutic target. These assays complement the routine work-up of hematologic tumors, which include flow cytometry, morphologic evaluation and FISH/cytogenetic analyses. Initial data from implementation of this testing strategy in our broad community-based practice are presented. Results: 865 patient samples were analyzed, which included the following: AML (168), ALL (24), MDS (194), MDS/MPN (34), CLL (82), MPN (156), samples with suspicion of a myeloid stem cell disorder (107) and others. Of the cases evaluated by NGS and CGA, genomic aberrations were detected in 70% and 48%, respectively. Conventional cytogenetic analyses revealed abnormalities in 38% of the cases for which conclusive results were obtained; abnormal FISH results were observed in 44.7%. In cases where conventional cytogenetics and FISH tests were negative, 70% were abnormal by either NGS or CGA (~81% in cases with evidence of a myeloid stem cell disorder or acute leukemia) (Figure 1). Importantly, 12% of 51 cases with normal FISH, cytogenetics and NGS results were abnormal by CGA, and 60% of 111 cases with normal FISH, cytogenetics and CGA results had actionable mutations detected by NGS. CGA and NGS aberrations were frequently detected in MPN or MDS/MPN cases with negative cytogenetics and FISH results. For example, among 156 MPN cases, CGA and NGS abnormalities were observed in 42% and 65% of cases respectively, while only 11% of cases had abnormal cytogenetics results and 10% of cases were FISH positive (Figure 2). In contrast, in cases without actionable mutations detected by NGS and CGA, which also had been analyzed by FISH/cytogenetics, 11% of 89 were cytogenetically abnormal, and 36% of 75 had genomic alterations detected by FISH. Conclusions: High throughput genomic tumor profiling through targeted DNA sequencing and analysis of copy number alterations complements conventional methods of tumor interrogation and leads to more frequent detection of actionable alterations. This is especially apparent in the context of morphologic and/or clinical suspicion of a myeloid stem cell disorder. These data indicate that integrating multiple strategies to identify informative biomarkers can enhance diagnosis, prognosis and/or therapy in hematologic disorders. Most importantly, we have observed changes in clinical management decisions across different disease states including AML, ALL, other myeloid malignancies, and CLL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ma: PathGroup: Employment. Gottwals:PathGroup: Employment. Ho:PathGroup: Employment. Fasig:PathGroup: Employment. Rietz:PathGroup: Employment. Hartley:PathGroup: Employment. Kravstov:PathGroup: Employment. Spence:PathGroup: Employment. Connor:PathGroup: Employment. Turnicky:PathGroup: Employment. Prescott:PathGroup: Employment. Lennon:PathGroup: Employment. Sathanoori:PathGroup: Employment. Flinn:Celgene Corporation: Research Funding. Berdeja:Celgene: Research Funding; Takeda: Research Funding; Curis: Research Funding; Abbvie: Research Funding; Onyx: Research Funding; MEI: Research Funding; Acetylon: Research Funding; Novartis: Research Funding; Array: Research Funding; Janssen: Research Funding; BMS: Research Funding. Wheeler:PathGroup: Employment. Coldren:PathGroup: Employment. Chandra:PathGroup: Employment. Mukherjee:PathGroup: Employment. Casey:PathGroup: Employment.

e19022 Background: A NGS program was launched in 10/2012 at a single community practice in middle Tennessee. This program was designed to discover molecular alterations with proven/potential therapeutic significance for patients (pts) with advanced cancers. We report the lung cancer cohort findings. Methods: Pts with advanced lung cancer who were candidates for systemic treatment (ECOG ≤ 2) were consented for molecular profiling. Tissue specimens were tested by NGS with 1,000X coverage in a CLIA/CAP-certified lab. Oncogenic hotspot mutations in 35 genes were tested. Results were reported to the treating M.D. 1 mutation: 55 (28%) with si...

2534 Background: With an increasing number of clinical trials testing new molecularly targeted drugs in pts with specific molecular alterations, there is a growing need for comprehensive molecular ...

Androgens are required for the development and function of the prostate. In a normal human prostate, androgens control the synthesis of proteins such as prostate-specific antigen and human glandular kallikrein. The prostate secretes these proteins as well as a number of other compounds to form the prostatic fluid. Using differential display PCR to detect novel androgen-regulated genes, clathrin heavy chain expression was identified as potentially being up-regulated by androgens in the prostate cancer cell line LNCaP. We report here that the clathrin heavy chain and light chain genes are regulated by androgens. Clathrin heavy chain messenger RNA was up-regulated by androgens in a concentration- and time-specific manner in the LNCaP cell line. Translation of clathrin heavy chain messenger RNA was stimulated by androgens. Steady state levels of clathrin light chains a and b were up-regulated in the presence of androgen in LNCaP cells. Clathrin gene expression was examined in normal rat prostates, and similar results were found. Clathrin heavy chain protein levels in the rat prostate are also affected by the androgen status of the animal. We hypothesize that clathrin may be involved in the exocytosis of androgen-regulated secretory proteins such as prostate-specific antigen and human glandular kallikrein.

BACKGROUND. The prostate is dependent on androgens for development and maintenance of its differentiated phenotype. We have applied the technique of differential display PCR to the androgen-sensitive prostate cancer cell line LNCaP to isolate androgen-responsive genes. METHODS. The technique of DD-PCR was applied to androgen-stimulated LNCaP cell RNA

11102 Background: Despite growing interest and need, molecular profiling of tumor samples is largely unavailable in community cancer centers, where nearly 80% of cancer patients (pts) are treated. In 10/12, Sarah Cannon Research Institute (SCRI) launched a community-based molecular profiling program to: 1) better understand the molecular constituency of cancer patients, 2) identify appropriate pts for phase I and II clinical trials of targeted agents, and 3) identify pts with molecular abnormalities responsive to FDA-approved agents. Methods: Eligible pts consented to testing of available biospecimens, which were interrogated for alterations in 35 cancer-related genes using NGS (1000X average coverage) in a CLIA/CAP laboratory. Results were reported to the treating physician within 14 days and stored in a database to enable correlation with clinical outcomes. Results: As of 1/13, 209 pts had been enrolled with 84% having sufficient material for assay. At least 1 mutation was detected in 46% of tumors. Results in the 3 most commonly assayed tumor types are summarized (Table). Mutations for which there are FDA-approved targeted agents were found in 14 off-label tumors (EGFR 4, KIT 3, SMO 3, BRAF 2, HER2 2). 40 pts (27%) were subsequently enrolled in clinical trials; in 19 of these, assay results influenced clinical trial selection. Conclusions: This program provides molecular profiling data to community oncologists for clinical decision making. Experience to date indicates this information can be provided in a timely manner for incorporation into clinical practice. Profiling results will enable: 1) selection of pts with appropriate tumor targets for investigational targeted agents, 2) enhanced study enrollment, 3) evaluation of FDA approved targeted agents in off-label tumor types, and 4) correlation of treatment outcomes with patterns of tumor molecular abnormalities. [Table: see text]

Androgenic hormones regulate the development and growth of many normal and tumor cells. It is clear that the androgen receptor (AR) plays a key role in the mechanism of androgen action. Activation of the androgen receptor by binding of the male hormone (either testosterone or dihydrotestosterone) results in the differential regulation of both mRNA and protein synthesis in target cells (1-5). Cloning of the androgen receptor cDNA, (the last of the known steroid receptor genes) has been achieved in several laboratories, including our own (6-10). Sequence comparison with the other steroid hormone receptor genes has revealed that the androgen receptor gene contains two highly conserved regions. Functional analysis of the estrogen- (11), progesterone- (12), and glucocorticoid receptor cDNA’s (13) has demonstrated that these two discrete regions correspond to DNA-binding and steroid-binding, respectively.

The recent cloning of human androgen receptor (AR) cDNAs in this and other laboratories has provided valuable probes for investigating the structure and function of the AR at the molecular level. We now report the overexpression of a region of the human AR containing both the DNA- and hormone-binding domains in E. coli, which provides a means to produce large amounts of AR for analysis and use in functional studies. Under isopropyl-beta-D-thiogalactopyranoside induction, a tripartite protein, consisting of beta-galactosidase, a collagenase recognition site, and AR polypeptide, was produced in E. coli JM109 using pSS20 a as a vector. About 1 mg of the fused AR could be recovered per liter bacterial culture. The induced protein could readily be detected in a sodium dodecyl sulfate-polyacrylamide gel by Coomassie blue staining. Its identity was confirmed by Western blot analysis using antibodies to both beta-galactosidase and the AR. Scatchard analysis of the androgen-binding activity of the hybrid AR revealed high affinity binding to the synthetic androgen, Mibolerone (Kd, approximately 1.2 nM). Competition studies demonstrated the fusion protein's specificity for androgens. The hybrid receptor formed immune complexes with human anti-AR serum that sedimented at about 19S in 10-50% linear sucrose gradients containing 0.4 M KCl. Gel band shift assays revealed that the hybrid receptor protein forms specific complexes with a synthetic steroid response element derived from the mouse mammary tumor virus long terminal repeat region. These results demonstrate that the recombinant AR expressed in E. coli possesses many of the functional properties characteristic of DNA- and steroid-binding domains of the native AR.

The physicochemical and immunological properties of androgen receptors from kidney and brain of testicular-feminized (Tfm) mutant mice and wild-type mice were compared. Analysis by gel filtration and sucrose density gradients revealed that the mol wt of the mutant receptor was 66K (38A; 3.8S) which was significantly smaller than the 110K (53A; 4.6S) size of the wild-type androgen receptor (P less than 0.05). Mixing experiments failed to demonstrate any role for differential proteolysis in the size differences between these receptors. Interaction of the mutant androgen receptor with specific polyclonal antiandrogen receptor antibodies produced significantly smaller immune complexes than that formed with wild-type receptor (12S vs. 17S; P less than 0.01). This confirmed the smaller size of the Tfm mutant androgen receptor and suggested that it contained fewer epitopes. The Tfm kidney cytosols also demonstrated a decreased concentration of androgen receptor-binding activity relative to that of the wild type. Together, these results suggest that the androgen insensitivity associated with the Tfm phenotype is due to a deficiency of androgen receptor in target tissues and a qualitative defect in the androgen receptor protein itself.