Chromosomal microarray provides enhanced targetable gene aberration detection when paired with next generation sequencing panel in profiling lung and colorectal tumors.

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.

Clinical sensitivity of p53 mutation detection in matched bladder tumor, bladder wash, and voided urine specimens.

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.

Isolation and androgen regulation of the human homeobox cDNA, NKX3.1.

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 to detect and isolate androgen-responsive genes. RESULTS: The human homeobox gene NKX3.1, the homologue to mouse Nkx3.1, recently isolated by Beiberich et al. [J Biol Chem 1996;271:31779-31782], was detected and cloned. NKX3.1 is induced by androgens in a time- and concentration-dependent manner. NKX3.1 is induced 6- to 7-fold in 12 hr, with a significant induction seen in 2 hr. This regulation is at the level of transcription, as androgens increase the number of new NKX3.1 transcripts, and de novo protein synthesis is not required. In humans, NKX3.1 is expressed most highly in the prostate, with a much lower level of expression seen in the testis. No other tissue examined showed detectable levels of NKX3.1 expression. CONCLUSIONS: NKX3.1 is an androgen-regulated, prostate-specific homeobox gene. We hypothesize that it may play a role in the development and differentiation of the prostate.

Clathrin gene expression is androgen regulated in the prostate.

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.

The androgen receptor in the testicular feminized (Tfm) mouse may be a product of internal translation initiation.

Androgen insensitivity in the testicular feminized (Tfm) mouse is caused by frame-shift mutation in the androgen receptor (AR) mRNA, which results in a stop codon in the amino terminus. Despite this mutation, a smaller sized protein corresponding to the DNA- and steroid-binding domain of the AR can be synthesized from the cloned Tfm AR cDNA by in vitro translation. The Tfm AR construct was demonstrated to express a protein capable of binding androgen with an affinity similar to the cloned wild-type AR. Although the Tfm AR product failed to transactivate mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter when low concentrations (100 ng) of Tfm AR vector were cotransfected, higher concentrations (5000 ng) resulted in a residual amount of transactivation, suggesting lower level transactivating capabilities. By cotransfecting the Tfm AR expression vector with the wild-type receptor, it was demonstrated that the product of the Tfm AR gene is capable of inhibiting the transactivation activity of the wild-type receptor. These data suggest that although the Tfm AR mRNA fails to produce a full-length AR because of the frame-shift mutation, a smaller protein capable of binding both steroid and DNA can be produced by translation from an internal initiation codon. This product could account for the low levels of androgen-binding activity detected previously in the Tfm mouse.

Amino acid changes in the peptide binding site have structural consequences at the surface of class I glycoproteins.

Structural changes on the surface of the class I Ag binding domain resulting from point mutations localized inside the Ag binding cleft of the H-2Kb and Kf glycoproteins were revealed using mAb. Both the loss and gain of antibody binding sites found among naturally occurring K glycoproteins resulted from single amino acid substitutions at a variety of different positions buried within the Ag binding groove. Each of the amino acid replacements analyzed represented naturally occurring diversity known to exist among the functional class I Ag-presenting molecules of the mouse. The binding of the affected mAb was not significantly altered in Kb molecules expressed by transfected T2 cells. Because T2 cells have been shown to express Kb molecules that are either largely devoid of bound peptides or bind a vastly different set of low affinity peptides, it is unlikely that the detected structural changes were caused by alterations in the spectrum of peptides bound by the class I variant glycoproteins. Similarly, a class I point mutant, Kb-97R, that also has been shown previously to bind a very different set of peptides in comparison to the parental Kb molecule also displays normal antibody binding properties. We conclude from these studies that structural diversity within the Ag binding cleft indirectly influences the external surface of the Ag-presenting domain of the class I H chain. Significantly, this surface is the interface between the T cell receptor and MHC molecules and may make contributions to the fine specificity of allorecognition.

Hormonal regulation of prostate-specific antigen (PSA) glycoprotein in the human prostatic adenocarcinoma cell line, LNCaP.

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.

Overexpression of a partial human androgen receptor in E. coli: characterization of steroid binding, DNA binding, and immunological properties.

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.

In situ hybridization of prostate-specific antigen mRNA in human prostate.

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.

The androgen receptor of the testicular-feminized (Tfm) mutant mouse is smaller than the wild-type receptor.

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.

Monoclonal antibodies against the androgen receptor: recognition of human and other mammalian androgen receptors.

Monoclonal antibodies against the androgen receptor (AR) will provide useful probes for elucidating both the structure and function of this important regulatory protein. Recently, human autoimmune anti-AR sera have been described. The purpose of the current work was to immortalize lymphocytes from the blood of patients with high titer anti-AR antibodies and to produce monoclonal antibodies against the receptor in vitro. Human serum samples (10 microliters) were incubated in high ionic strength buffer (400 mM KCl) for 16 h at 0 C with [3H]Mibolerone-labeled cytosol (100-200 fmol AR) from Dunning tumors. Receptor-antibody complexes were precipitated with goat antihuman immunoglobulin (Ig) antibody. From our 1005 serum samples examined, 5 specimens were detected which precipitated greater than 40% of the AR. These antibodies recognized the AR from human, rat, mouse, dog, steer, chicken, and hamster, but did not recognize estrogen, progesterone, or glucocorticoid receptors. By sucrose gradient analysis in high salt (0.4 M KCl) 1 of the antisera shifted the 4.4S monomeric receptor to 8S, and the others shifted the receptor to 18S. However, all of the antibodies were shown to be IgG class by immunoprecipitation with class-specific second antibodies. Peripheral blood lymphocytes donated by these patients were isolated by histopaque density gradient sedimentation, activated in vitro, transformed with Epstein-Barr virus, and seeded into 96-well plates. From 263 million human lymphocytes plated in 96-well dishes, 1215 wells gave rise to Epstein-Barr virus-transformed lymphoblastoid cells, and 8 of these wells were determined to be anti-AR positive. Cells from 2 of the positive wells were cloned and designated CB54 and UA67, both of which secreted IgG class antibodies against the AR. These 2 monoclonal antibodies have been shown to be highly specific for the AR and to cross-react with the AR from human, rat, and hamster. Studies with the monomeric form of the AR and its proteolytic fragment using sucrose density gradients have suggested that the 2 antibodies recognize different epitopes on the monomeric AR molecule. Furthermore, by Western blot analysis the antibodies have identified the AR as an 118K protein on a sodium dodecyl sulfate gel, which is consistent with our previous findings of the mol wt of the AR.