Aneuploidy and Tetraploidy as Distinct Patterns During Melanomagenesis.

Melanoma is characterized by a high degree of chromosome instability (CIN), the loss or gain of entire chromosomes or pieces of chromosomes. Also, CIN is likely to drive the progression of benign melanocytic lesions to malignant tumors, although very little is known about the acquisition of the mechanisms that promote CIN along this progression. Here, we describe the development of a model system to study the progression of melanomagenesis starting with normal human melanocytes followed by inactivation of the p53 and pRb tumor suppressors by addition of the E6/E7 proteins. The cells were then transduced with a growth-promoting, constitutionally-active mutant NRAS. The addition of E6/E7 and E6/E7 NRAS was found to give a growth advantage to the cells compared to normal melanocytes and a statistically significant gain of aneuploidy; aneuploidy was 24.7% in primary melanocytes, 33.8% in E6/E7 melanocytes, and 70.5% in E6/E7 NRAS melanocytes. Further, we found an increase in tetraploid cells in the cell model which was statistically significant between primary melanocytes and E6/E7, NRAS melanocytes. We also observed an increase in aneuploid cells between three population doublings in primary melanocytes, whereas this increase was not seen in the E6/E7 melanocytes. Together, these data demonstrate that this model system utilizing stepwise addition of genetic mutations driving melanomagenesis is a useful tool to study CIN and could even be used to study the mechanisms responsible for these alterations in genetic makeup.

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies.

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.

Minimally invasive prostate cancer detection test using FISH probes.

PURPOSE: The ability to test for and detect prostate cancer with minimal invasiveness has the potential to reduce unnecessary prostate biopsies. This study was conducted as part of a clinical investigation for the development of an OligoFISH(®) probe panel for more accurate detection of prostate cancer. MATERIALS AND METHODS: One hundred eligible male patients undergoing transrectal ultrasound biopsies were enrolled in the study. After undergoing digital rectal examination with pressure, voided urine was collected in sufficient volume to prepare at least two slides using ThinPrep. Probe panels were tested on the slides, and 500 cells were scored when possible. From the 100 patients recruited, 85 had more than 300 cells scored and were included in the clinical performance calculations. RESULTS: Chromosomes Y, 7, 10, 20, 6, 8, 16, and 18 were polysomic in most prostate carcinoma cases. Of these eight chromosomes, chromosomes 7, 16, 18, and 20 were identified as having the highest clinical performance as a fluorescence in situ hybridization test and used to manufacture the fluorescence in situ hybridization probe panels. The OligoFISH(®) probes performed with 100% analytical specificity. When the OligoFISH(®) probes were compared with the biopsy results for each individual, the test results highly correlated with positive and negative prostate biopsy pathology findings, supporting their high specificity and accuracy. Probes for chromosomes 7, 16, 18, and 20 showed in the receiver operator characteristics analysis an area under the curve of 0.83, with an accuracy of 81% in predicting the biopsy result. CONCLUSION: This investigation demonstrates the ease of use with high specificity, high predictive value, and accuracy in identifying prostate cancer in voided urine after digital rectal examination with pressure. The test is likely to have positive impact on clinical practice and advance approaches to the detection of prostate cancer. Further evaluation is warranted.

One-year monitoring of an oligonucleotide fluorescence in situ hybridization probe panel laboratory-developed test for bladder cancer detection.

BACKGROUND: Previously, we had developed and manufactured an oligonucleotide fluorescence in situ hybridization (OligoFISH) probe panel based on the most clinically sensitive chromosomes found in a reference set of bladder carcinoma cases. The panel was clinically validated for use as a diagnostic and monitoring assay for bladder cancer, reaching 100% correlation with the results of the UroVysion test. After 1 year of using this probe panel, we present here the comparison of cytology, cystoscopy, and pathology findings to the OligoFISH probe panel results to calculate its clinical performance. MATERIALS AND METHODS: In order to calculate clinical performance, we compared the OligoFISH results to the cytology and cystoscopy/pathology findings for 147 initial diagnoses and 399 recurrence monitorings. Finally, we compared clinical performance to published values for the UroVysion test, including both low- and high-grade tumors. RESULTS: Chromosomes 3, 6, 7, and 20 were highly involved in bladder carcinoma aneuploidy. At the initial diagnosis, we obtained 90.5% (95% confidence interval [CI]: 84.5%-94.7%) accuracy, 96.8% sensitivity (95% CI: 91.0%-99.3%), 79.2% specificity (95% CI: 65.9%-87.8%), 89.2% positive predictive value (PPV; 95% CI: 81.5%-94.5%), and 93.3% negative predictive value (NPV; 95% CI: 81.7%-97.3%). When monitoring for recurrence, we obtained 85.2% accuracy (95% CI: 81.3%-88.5%), 82.0% sensitivity (95% CI: 76.0%-87.1%), 88.4% specificity (95% CI: 83.2%-92.5%), 87.7% PPV (95% CI: 82.1%-92.0%), and 83.0% NPV (95% CI: 77.3%-87.8%). When looking at low- and high-grade tumors, the test showed 100% sensitivity for high-grade tumors (95% CI: 92.5%-100%) and 87.5% sensitivity (95% CI: 68.8%-95.5%) for low-grade tumors. All the clinical parameters for the OligoFISH panel were higher than the UroVysion test's published performance. We found significantly higher clinical sensitivity and NPV at initial diagnosis and significantly higher specificity and PPV for recurrence. CONCLUSION: The OligoFISH probe panel is a fast, easy, and reproducible test for bladder cancer diagnosis and monitoring, with excellent clinical performance and utility.

Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia.

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.