Genetic markers associated with early cancer-specific mortality following prostatectomy.

BACKGROUND: This study sought to identify novel effectors and markers of localized but potentially life-threatening prostate cancer (PCa), by evaluating chromosomal copy number alterations (CNAs) in tumors from patients who underwent prostatectomy and correlating these with clinicopathologic features and outcome. METHODS: CNAs in tumor DNA samples from 125 patients in the discovery cohort who underwent prostatectomy were assayed with high-resolution Affymetrix 6.0 single-nucleotide polymorphism microarrays and then analyzed using the Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm. RESULTS: The assays revealed 20 significant regions of CNAs, 4 of them novel, and identified the target genes of 4 of the alterations. By univariate analysis, 7 CNAs were significantly associated with early PCa-specific mortality. These included gains of chromosomal regions that contain the genes MYC, ADAR, or TPD52 and losses of sequences that incorporate SERPINB5, USP10, PTEN, or TP53. On multivariate analysis, only the CNAs of PTEN (phosphatase and tensin homolog) and MYC (v-myc myelocytomatosis viral oncogene homolog) contributed additional prognostic information independent of that provided by pathologic stage, Gleason score, and initial prostate-specific antigen level. Patients whose tumors had alterations of both genes had a markedly elevated risk of PCa-specific mortality (odds ratio = 53; 95% CI = 6.92-405, P = 1 × 10(-4)). Analyses of 333 tumors from 3 additional distinct patient cohorts confirmed the relationship between CNAs of PTEN and MYC and lethal PCa. CONCLUSIONS: This study identified new CNAs and genes that likely contribute to the pathogenesis of localized PCa and suggests that patients whose tumors have acquired CNAs of PTEN, MYC, or both have an increased risk of early PCa-specific mortality.

CBFB and MYH11 in inv(16)(p13q22) of acute myeloid leukemia displaying close spatial proximity in interphase nuclei of human hematopoietic stem cells.

To gain a better understanding of the mechanism of chromosomal translocations in cancer, we investigated the spatial proximity between CBFB and MYH11 genes involved in inv(16)(p13q22) found in patients with acute myeloid leukemia. Previous studies have demonstrated a role for spatial genome organization in the formation of tumorigenic abnormalities. The nonrandom localization of chromosomes and, more specifically, of genes appears to play a role in the mechanism of chromosomal translocations. Here, two-color fluorescence in situ hybridization and confocal microscopy were used to measure the interphase distance between CBFB and MYH11 in hematopoietic stem cells (HSCs), where inv(16)(p13q22) is believed to occur, leading to leukemia development. The measured distances in HSCs were compared with mesenchymal stem cells, peripheral blood lymphocytes, and fibroblasts, as spatial genome organization is determined to be cell-type specific. Results indicate that CBFB and MYH11 are significantly closer in HSCs compared with all other cell types examined. Furthermore, the CBFB-MYH11 distance is significantly reduced compared with CBFB and a control locus in HSCs, although separation between CBFB and the control is ∼70% of that between CBFB and MYH11 on metaphase chromosomes. HSCs were also treated with fragile site-inducing chemicals because both the genes contain translocation breakpoints within these regions. However, treatment with fragile site-inducing chemicals did not significantly affect the interphase distance. Consistent with previous studies, our results suggest that gene proximity may play a role in the formation of cancer-causing rearrangements, providing insight into the mechanism of chromosomal abnormalities in human tumors.

APOL1 Long-term Kidney Transplantation Outcomes Network (APOLLO): Design and Rationale.

INTRODUCTION: Much of the higher risk for end-stage kidney disease (ESKD) in African American individuals relates to ancestry-specific variation in the apolipoprotein L1 gene (APOL1). Relative to kidneys from European American deceased-donors, kidneys from African American deceased-donors have shorter allograft survival and African American living-kidney donors more often develop ESKD. The National Institutes of Health (NIH)-sponsored APOL1 Long-term Kidney Transplantation Outcomes Network (APOLLO) is prospectively assessing kidney allograft survival from donors with recent African ancestry based on donor and recipient APOL1 genotypes. METHODS: APOLLO will evaluate outcomes from 2614 deceased kidney donor-recipient pairs, as well as additional living-kidney donor-recipient pairs and unpaired deceased-donor kidneys. RESULTS: The United Network for Organ Sharing (UNOS), Association of Organ Procurement Organizations, American Society of Transplantation, American Society for Histocompatibility and Immunogenetics, and nearly all U.S. kidney transplant programs, organ procurement organizations (OPOs), and histocompatibility laboratories are participating in this observational study. APOLLO employs a central institutional review board (cIRB) and maintains voluntary partnerships with OPOs and histocompatibility laboratories. A Community Advisory Council composed of African American individuals with a personal or family history of kidney disease has advised the NIH Project Office and Steering Committee since inception. UNOS is providing data for outcome analyses. CONCLUSION: This article describes unique aspects of the protocol, design, and performance of APOLLO. Results will guide use of APOL1 genotypic data to improve the assessment of quality in deceased-donor kidneys and could increase numbers of transplanted kidneys, reduce rates of discard, and improve the safety of living-kidney donation.

Sonographic findings in trisomy 9.

OBJECTIVE: The purpose of this study was to identify the most common prenatal sonographic findings in fetuses with complete trisomy 9. METHODS: A retrospective review of all cases of trisomy 9 at 5 participating institutions over a 15-year interval was conducted. Indications for referral and sonographic findings in each case were reviewed to identify characteristic fetal structural anomalies. RESULTS: Six cases of trisomy 9 are presented. Most patients were referred for abnormal sonographic findings on screening examinations (66%) or advanced maternal age (33%). Fetal heart defects and central nervous system malformations were the most frequent sonographic anomalies seen. CONCLUSIONS: Sonographic findings in trisomy 9 are similar to those found in other autosomal trisomies. Because trisomy 9 is uniformly lethal and is not included as part of the standard prenatal aneuploidy screening by fluorescence in situ hybridization analysis, clinicians should be cautious in counseling patients with structurally abnormal fetuses until the full karyotype is available.

Ultrasound findings in trisomy 22.

We sought to identify the characteristic sonographic findings of fetal trisomy 22 by performing a retrospective review of nine cases of fetal trisomy 22. All cases of chromosomal mosaicism were excluded, as were first-trimester losses. Indications for sonography, gestational age, and sonographically detected fetal anomalies were analyzed. The majority of patients were referred for advanced maternal age or abnormal ultrasound findings on screening exam. Oligohydramnios was the most common sonographic finding, present in 55% of affected fetuses. Intrauterine growth restriction and increased nuchal thickness were slightly less frequent.

X-linked chronic granulomatous disease secondary to skewed X chromosome inactivation in a female with a novel CYBB mutation and late presentation.

Chronic granulomatous disease (CGD) is characterized by defects in the superoxide producing enzyme NADPH oxidase causing phagocytes to improperly clear invading pathogens. Here we report findings of a late presenting 16-year-old female with X-linked CGD. The patient presented with community-acquired pneumonia, but symptoms persisted for 2 weeks during triple antimicrobial coverage. Cultures revealed Aspergillus fumigatus which was resolved through aggressive voriconazole treatment. Neutrophil studies revealed NADPH oxidase activity and flavocytochrome b(558) levels that were 4-8% of controls and suggested carrier status of the mother. We found a null mutation in the CYBB gene (c.252insAG) predicting an aberrant gp91(phox) protein (p.Cys85fsX23) in the heterozygous state. Methylation analysis demonstrated extremely skewed X chromosome inactivation favoring the maternally inherited defective gene. In conclusion, a novel mutation in the CYBB gene and an extremely skewed X-inactivation event resulted in the rare expression of the CGD phenotype in a carrier female.

Rapid group B streptococci screening using a real-time polymerase chain reaction assay.

OBJECTIVE: To estimate the clinical performance characteristics of a real-time polymerase chain reaction (PCR) assay using vaginal/rectal swabs from antepartum (35-37 weeks of gestation) and intrapartum women. METHODS: The assay evaluated is a qualitative, automated, real-time PCR test for the detection of group B streptococci, with results available in approximately 75 minutes. Enrollment in this multicenter clinical study occurred between October 2005 and January 2006. Vaginal/rectal swabs were analyzed by nursing personnel (intrapartum tests) or by laboratory technologists (all others). Polymerase chain reaction assay results were compared with culture using standard methods, including selective broth medium, and to a predicate nucleic acid amplification test. RESULTS: Of 1,028 enrolled women, 234 were deemed ineligible, and 10 had unresolved test results. Of the 784 remaining women, valid PCR assay results were obtained on the first test attempt for 93.0%. Performance characteristics relative to culture were sensitivity 91.1%, specificity 96.0%, positive predictive value 87.8%, negative predictive value 97.1%, and accuracy 94.8%. These results exceeded those obtained using the predicate nucleic acid amplification test. CONCLUSION: Performance characteristics of the PCR assay exceed the threshold recommended by the Centers for Disease Control and Prevention when compared with culture. The test is sufficiently robust to be performed for intrapartum patients in a point-of-care setting by medical professionals. LEVEL OF EVIDENCE: II.

Deletion of a small consensus region at 6q15, including the MAP3K7 gene, is significantly associated with high-grade prostate cancers.

PURPOSE: Chromosome 6q14-21 is commonly deleted in prostate cancers, occurring in approximately 22% of all tumors and approximately 40% of metastatic tumors. However, candidate prostate tumor suppressor genes in this region have not been identified, in part due to the large and broad nature of the deleted region implicated in previous studies. EXPERIMENTAL DESIGN: We first used high-resolution Affymetrix single nucleotide polymorphism arrays to examine DNA from malignant and matched nonmalignant cells from 55 prostate cancer patients. We identified a small consensus region on 6q14-21 and evaluated the deletion status within the region among additional 40 tumors and normal pairs using quantitative PCR and fluorescence in situ hybridization. We finally tested the association between the deletion and Gleason score using the Fisher's exact test. RESULTS: Tumors with small, interstitial deletions at 6q14-21 defined an 817-kb consensus region that is affected in 20 of 21 tumors. The MAP3K7 gene is one of five genes located in this region. In total, MAP3K7 was deleted in 32% of 95 tumors. Importantly, deletion of MAP3K7 was highly associated with higher-grade disease, occurring in 61% of tumors with Gleason score >or=8 compared with only 22% of tumors with Gleason score

Detection of programmed cell death in cells exposed to genotoxic agents using a caspase activation assay.

Many environmental toxins cause DNA damage. Cells that have sustained significant DNA damage must attempt to repair the damage prior to replication, in which aberrant base incorporation can result in an irreversible mutation. If a cell cannot repair the damage, however, it may commit suicide through a genetically regulated programmed cell death (PCD) pathway. Crucial to the ultimate execution of PCD is a family of cysteine proteases called caspases. Activation of these enzymes occurs late in the PCD pathway, when a cell can no longer avoid cell death, but earlier than other PCD markers, such as morphological changes or DNA fragmentation. This protocol details a method for using fluorochrome-conjugated caspase inhibitors for the detection of activated caspases in intact cells using fluorescent microscopy.

Donor-derived myeloid sarcoma in two kidney transplant recipients from a single donor.

We report the rare occurrence of donor-derived myeloid sarcoma in two kidney transplant patients who received organs from a single deceased donor. There was no evidence of preexisting hematologic malignancy in the donor at the time of organ recovery. Both recipients developed leukemic involvement that appeared to be limited to the transplanted organ. Fluorescence in situ hybridization (FISH) and molecular genotyping analyses confirmed that the malignant cells were of donor origin in each patient. Allograft nephrectomy and immediate withdrawal of immunosuppression were performed in both cases; systemic chemotherapy was subsequently administered to one patient. Both recipients were in remission at least one year following the diagnosis of donor-derived myeloid sarcoma. These cases suggest that restoration of the immune system after withdrawal of immunosuppressive therapy and allograft nephrectomy may be sufficient to control HLA-mismatched donor-derived myeloid sarcoma without systemic involvement.

Fas-associating death domain protein overexpression induces apoptosis in lung cancer cells.

OBJECTIVES: Non-small cell lung cancers commonly develop resistance to radiation and chemotherapy, and they often present at stages beyond surgical resectability. Because current treatment modalities are inadequate, novel therapies are necessary to reduce the effects of the increasing incidence in pulmonary neoplasms. Fas-associating death domain protein is a central mediator of death receptor-initiated apoptosis that directly activates the caspase-8 protease. We hypothesized that overexpression of Fas-associating death domain protein would effectively eradicate lung cancer cells by induction of apoptosis. METHODS: Cultured A549 alveolar carcinoma and NCI-H226 squamous carcinoma cells were exposed to increasing multiplicities of infection of a replication-deficient, adenoviral vector that expresses the wild-type murine Fas-associating death domain protein gene or control virus for 4 hours. Twenty-four hours later, cells were assessed for viability by crystal violet staining and caspase activation by microscopic analysis. Protein lysates were examined by Western blotting for expression of Fas-associating death domain protein and activated caspase-8. RESULTS: Adenoviral infection with the wild-type murine Fas-associating death domain protein gene in A549 cells resulted in a dose-dependent expression of Fas-associating death domain protein and the appearance of cleaved, activated caspase-8. Increasing multiplicities of infection of the wild-type murine Fas-associating death domain protein gene, but not control adenovirus, was associated with increased cell death in A549 and NCI-H226 cells. The wild-type murine Fas-associating death domain protein gene infection of A549 cells at multiplicities of infection of 50 induced at least 10-fold increase in Fas-associating death domain protein levels and decreased viability by > 50% (n = 3; P <.001). CONCLUSION: Overexpression of Fas-associating death domain protein induced dose-dependent cell death in A549 and NCI-H226 lung epithelial cancer cells. Expression of Fas-associating death domain protein results in activation of caspases, a hallmark of apoptosis. Delivery of the wild-type murine Fas-associating death domain protein gene to drug- and radiation-resistant lung cancer may be a novel method for therapy of non-small cell lung cancer.

In vivo expression of p53 and Bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions.

Forty-four specimens of non-malignant and malignant human lung tissue, taken from patients with non-small cell lung cancer (NSCLC), were examined for the expression of wild-type p53, mutant p53, and bcl-2 and the occurrence of programmed cell death (apoptosis). Wild-type p53 expression peaked in peritumoral and metaplastic samples, whereas mutant p53, bcl-2 and apoptosis were first detected in metaplasia and increased with progression to carcinoma. Bcl-2 positive samples had lower levels of apoptosis than bcl-2 negative samples and was independent of wild-type or mutant p53 expression. These results suggest that the over-expression of wild-type p53 may be an early cellular response to an alteration in normal cellular homeostasis. The ensuing increase in apoptosis appears to be relatively independent of mutant or wild-type p53 expression, but does not occur in cells expressing bcl-2.

Detection of programmed cell death in cells exposed to genotoxic agents using a caspase activation assay.

Many toxins that individuals are exposed to cause DNA damage. Cells that have sustained DNA damage may attempt to repair the damage prior to replication. However, if a cell has sustained serious damage it cannot repair, it will commit suicide through a genetically regulated programmed cell death (PCD) pathway. Crucial to the ultimate execution of PCD is a family of cysteine proteases called caspases. Activation of these enzymes occurs late enough in the PCD pathway that a cell can no longer avoid cell death, but still earlier than PCD-associated morphological changes or DNA fragmentation. This protocol details a method for using fluorochrome-conjugated caspase inhibitors for the detection of activated caspases in intact cells. The analysis and documentation is performed using fluorescence microscopy.

p53-Dependent anticancer effects of leptomycin B on lung adenocarcinoma.

PURPOSE: Leptomycin B (LMB) and/or its derivatives are considered a novel class of cancer therapeutics through blocking chromosome maintenance region 1, which mediates p53 nuclear export. The objectives of the present study were to first evaluate the cytotoxic effects of LMB on a normal human lung epithelial cell line (BEAS-2B) and three human lung adenocarcinoma cell lines with various p53 status (wild type: A549, mutant: NCI-H522, and null: NCI-H358) and then to identify LMB-induced gene expression alterations in human p53 signaling pathway. METHODS: Cells were treated with 0.01-100 nM LMB or 0.1% ethanol (vehicle control) for 4-72 h. Gene expression analyses using gene array for 84 genes involved in p53-mediated signaling pathways were performed in A549 and NCI-H358 after treatment with 20 nM LMB or vehicle control for 24 h. RESULTS: Cytotoxic results from MTS assays revealed a significant dose- and time-dependent effect of LMB on all cell lines. However, this effect was more pronounced in cancer cells than in normal cells, and cancer cells with p53 wild type tended to be less sensitive than those with p53 mutant or null. A total of 23 genes, predominantly involved in apoptosis and cell cycle/proliferation, were significantly altered in A549 after LMB treatment, while no strong modulating effects were observed in NCI-H358. The protein expression of two selected genes, p21 and survivin, was further confirmed by Western blots. CONCLUSION: Our results suggest that LMB has anti-cancer potential and provides a new regimen of individualized therapy for lung cancer treatment.

Recurring translocation (10;17) and deletion (14q) in clear cell sarcoma of the kidney.

CONTEXT: Clear cell sarcoma of the kidney (CCSK) is a prognostically unfavorable renal neoplasm of childhood. Previous cytogenetic studies of CCSK have reported balanced translocations t(10;17)(q22;p13) and t(10;17)(q11;p12). Although the tumor suppressor gene p53 is located at the chromosome 17p13 breakpoint, p53 abnormalities are rarely present in these tumors. OBJECTIVE: To identify cytogenetic abnormalities in CCSK and correlate these findings with other clinicopathologic parameters. DESIGN: A retrospective review of CCSK patients from 1990 to 2005 was conducted at our medical center. We performed clinical and histologic review, p53 immunohistochemical and classic cytogenetics (or ploidy analysis), and p53 fluorescence in situ hybridization analyses. RESULTS: Five male patients (age range, 6 months to 4 years) were identified with cytogenetic abnormalities. Of 3 cytogenetically informative cases, one revealed a clonal balanced translocation t(10;17)(q22;p13) and an interstitial deletion of chromosome 14, del(14)(q24.1q31.1), and the other 2 patients had normal karyotypes. Fluorescence in situ hybridization for p53 in the t(10;17) case revealed no deletion. Immunohistochemical evaluation of p53 demonstrated lack of nuclear protein accumulation in all cases. CONCLUSIONS: Together with the published literature, our results indicate that translocation (10;17) and interstitial deletions of chromosome 14q are recurring cytogenetic lesions in CCSK. To date, 3 cases of CCSK or "sarcomatoid Wilms tumors" have been reported to exhibit t(10;17). One previously reported case of CCSK contained deletion 14q. Results of p53 immunohistochemistry and/or p53 fluorescence in situ hybridization in this report suggest lack of mutations or deletions of this tumor suppressor in these CCSK cases. The t(10;17) breakpoint and deletion of chromosome 14q24 suggest that other genes are involved in tumor pathogenesis.

Depletion of Bcl-2 by an antisense oligonucleotide induces apoptosis accompanied by oxidation and externalization of phosphatidylserine in NCI-H226 lung carcinoma cells.

Oxidant-induced apoptosis involves oxidation of many different and essential molecules including phospholipids. As a result of this non-specific oxidation, any signaling role of a particular phospholipid-class of molecules is difficult to elucidate. To determine whether preferential oxidation of phosphatidylserine (PS) is an early event in apoptotic signaling related to PS externalization and is independent of direct oxidant exposure, we chose a genetic-based induction of apoptosis. Apoptosis was induced in the lung cancer cell line NCI-H226 by decreasing the amount of Bcl-2 protein expression by preventing the translation of bcl-2 mRNA using an antisense bcl-2 oligonucleotide. Peroxidation of phospholipids was assayed using a fluorescent technique based on metabolic integration of an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (PnA), into cellular phospholipids and subsequent HPLC separation of cis-PnA-labeled phospholipids. We found a decrease in Bcl-2 was associated with a selective oxidation of PS in a sub-population of the cells with externalized PS. No significant difference in oxidation of cis-PnA-labeled phospholipids was observed in cells treated with medium alone or a nonsense oligonucleotide. Treatment with either nonsensc or antisense bcl-2 oligonucleotides was not associated with changes in the pattern of individual phospholipid classes as determined by HPTLC. These metabolic and topographical changes in PS arrangement in plasma membrane appear to be early responses to antisense bcl-2 exposure that trigger a PS-dependent apoptotic signaling pathway. This observed externalization of PS may facilitate the 'labeling' of apoptotic cells for recognition by macrophage scavenger receptors and subsequent phagocytic clearance.