RESUMEN
PURPOSE: Despite suboptimal sensitivity urine cytology is often performed as an adjunct to cystoscopy for bladder cancer diagnosis. We aimed to develop a noninvasive, fast molecular diagnostic test for bladder cancer detection with better sensitivity than urine cytology while maintaining adequate specificity. MATERIALS AND METHODS: Urine specimens were collected at 18 multinational sites from subjects prior to cystoscopy or tumor resection, and from healthy and other control subjects without evidence of bladder cancer. The levels of 10 urinary mRNAs were measured in a training cohort of 483 subjects and regression analysis was used to identify a 5-mRNA model to predict cancer status. The performance of the GeneXpert® Bladder Cancer Assay, an assay labeled for investigational use only to detect the 5 mRNAs ABL1, CRH, IGF2, ANXA10 and UPK1B, was evaluated in an independent test cohort of 450 participants. RESULTS: In the independent test cohort the assay ROC curve AUC was 0.87 (95% CI 0.81-0.92). At an example cutoff point of 0.4 overall sensitivity was 73% while specificity was 90% and 77% in the hematuria and surveillance patient populations, respectively. CONCLUSIONS: We developed a 90-minute, urine based test that is simple to perform for the detection of bladder cancer. The test can help guide physician decision making in the management of bladder cancer. Additional evaluation in a prospective study is needed to establish the clinical usefulness of this assay.
Asunto(s)
Carcinoma de Células Transicionales/orina , Cistoscopía/métodos , ARN Neoplásico/orina , Neoplasias de la Vejiga Urinaria/orina , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/orina , Carcinoma de Células Transicionales/diagnóstico , Carcinoma de Células Transicionales/genética , Femenino , Estudios de Seguimiento , Marcadores Genéticos/genética , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Tiempo , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/genética , Adulto JovenRESUMEN
BACKGROUND: In patients with haematuria, a fast, noninvasive test with high sensitivity (SN) and negative predictive value (NPV), which is able to detect or exclude bladder cancer (BC), is needed. A newly developed urine assay, Xpert Bladder Cancer Detection (Xpert), measures five mRNA targets (ABL1, CRH, IGF2, UPK1B, and ANXA10) that are frequently overexpressed in BC. OBJECTIVE: To validate the performance of Xpert in patients with haematuria. DESIGN, SETTING, AND PARTICIPANTS: Voided precystoscopy urine specimens were prospectively collected at 22 sites from patients without prior BC undergoing cystoscopy for haematuria. Xpert, cytology, and UroVysion procedures were performed. Technical validation was performed and specificity (SP) was determined in patients without BC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Test characteristics were calculated based on cystoscopy and histology results, and compared between Xpert, cytology, and UroVysion. RESULTS AND LIMITATIONS: We included 828 patients (mean age 64.5 yr, 467 males, 401 never smoked). Xpert had an SN of 78% (95% confidence interval [CI]: 66-87) overall and 90% (95% CI: 76-96) for high-grade (HG) tumours. The NPV was 98% (95% CI: 97-99) overall. The SP was 84% (95% CI: 81-86). In patients with microhaematuria, only one HG patient was missed (NPV 99%). Xpert had higher SN and NPV than cytology and UroVysion. Cytology had the highest SP (97%). In a separate SP study, Xpert had an SP of 89% in patients with benign prostate hypertrophy and 92% in prostate cancer patients. CONCLUSIONS: Xpert is an easy-to-use, noninvasive test with improved SN and NPV compared with cytology and UroVysion, representing a promising tool for identifying haematuric patients with a low likelihood of BC who might not need to undergo cystoscopy. PATIENT SUMMARY: Xpert is an easy-to-perform urine test with good performance compared with standard urine tests. It should help identify (micro)haematuria patients with a very low likelihood to have bladder cancer.
Asunto(s)
ARN Mensajero/análisis , Urinálisis , Neoplasias de la Vejiga Urinaria , Cistoscopía , Femenino , Hematuria/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Vejiga Urinaria/diagnósticoRESUMEN
BACKGROUND: Identification of urinary biomarkers for detection of bladder cancer recurrence would be beneficial to minimize the frequency of cystoscopy. Our objective was to determine the usability of urine content of mRNA in the detection and prediction of bladder cancer recurrence. METHODS: We analyzed 123 prospectively cross-sectional collected urine samples from 117 patients with bladder cancer (12 incident cancers and 111 control visits). We used biopsies from cystoscopies as diagnostic criteria for recurrence, and followed the patients for a median time of 28.5 months (range 0-44 months). We measured the levels of hTERT, SENP1, PPP1CA, and MCM5 mRNA in urine by q-RT- PCR. RESULTS: We found significant differences in urinary content of hTERT (p < 0.001), SENP1 (p < 0.001), MCM5 (p < 0.001), and PPP1CA (p < 0.001) transcripts, when comparing urine samples from patients with and without tumor present in the bladder. We obtained sensitivity and specificity values for hTERT: 63/73, SENP1: 56/78, MCM5: 63/66, and PPP1CA: 69/63, respectively. Including follow-up data resulted in sensitivity and specificity values for hTERT: 62/84, SENP1:53/84, MCM5: 61/73, and PPP1CA: 65/66. Interestingly, at non-tumor visits the urinary content of especially hTERT (p = 0.0001) and MCM5 (p = 0.02) were significantly associated with subsequent tumour recurrence. Combining the markers with cytology improved the detection. The best combination was hTERT and cytology with a sensitivity of 71% and a specificity of 86% after follow-up. Further prospective validation or registration studies needs to be carried out before clinical use. CONCLUSIONS: We could use the urinary content of hTERT, SENP1, PPP1CA, and MCM5 to detect bladder cancer recurrence. All markers showed a higher sensitivity than cytology. The detection rate improved when including cytology results, but also the combination of hTERT and MCM5 increased the detection rate. Furthermore, hTERT and MCM5 levels predicted subsequent tumor recurrences.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Endopeptidasas/genética , Recurrencia Local de Neoplasia , Proteína Fosfatasa 1/genética , ARN Mensajero/orina , Telomerasa/genética , Neoplasias de la Vejiga Urinaria/diagnóstico , Anciano , Biopsia , Estudios Transversales , Cisteína Endopeptidasas , Cistoscopía , Dinamarca , Femenino , Humanos , Masculino , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Factores de Tiempo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/orinaRESUMEN
BACKGROUND: A fast, noninvasive test with high sensitivity (SN) and a negative predictive value (NPV), which is able to detect recurrences in bladder cancer (BC) patients, is needed. A newly developed urine assay, Xpert Bladder Cancer Monitor (Xpert), measures five mRNA targets (ABL1, CRH, IGF2, UPK1B, and ANXA10) that are frequently overexpressed in BC. OBJECTIVE: To validate Xpert characteristics in patients previously diagnosed with non-muscle-invasive BC. DESIGN, SETTING, AND PARTICIPANTS: Voided precystoscopy urine samples were prospectively collected at 22 sites. Xpert, cytology, and UroVysion were performed. If cystoscopy was suspicious for BC, a histologic examination was performed. Additionally, technical validation was performed and specificity was determined in patients without a history or clinical evidence of BC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Test characteristics were calculated based on cystoscopy and histology results, and compared between Xpert, cytology, and UroVysion. RESULTS AND LIMITATIONS: Of the eligible patients, 239 with a history of BC had results for all assays. The mean age was 71 yr; 190 patients were male, 53 never smoked, and 64% had previous intravesical immunotherapy (35%) or chemotherapy (29%). Forty-three cases of recurrences occurred. Xpert had overall SN of 74% (95% confidence interval [CI]: 60-85) and 83% (95% CI: 64-93) for high-grade (HG) tumors. The NPV was 93% (95% CI: 89-96) overall and 98% (95% CI: 94-99) for HG tumors. Specificity was 80% (95% CI: 73-85). Xpert SN and NPV were superior to those of cytology and UroVysion. Specificity in non-BC individuals (n=508) was 95% (95% CI: 93-97). CONCLUSIONS: Xpert has an improved NPV compared with UroVysion and cytology in patients under follow-up for BC. It represents a promising tool for excluding BC in these patients, reducing the need for cystoscopy. PATIENT SUMMARY: Xpert is an easy-to-perform urine test with good performance compared with standard urine tests. It should help optimize the follow-up of recurrent bladder cancer patients.
Asunto(s)
Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/orina , Vigilancia de la Población/métodos , ARN Mensajero/orina , Neoplasias de la Vejiga Urinaria/orina , Adulto , Anciano , Anciano de 80 o más Años , Anexinas/genética , Biopsia , Hormona Liberadora de Corticotropina/genética , Cistoscopía , Femenino , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Valor Predictivo de las Pruebas , Estudios Prospectivos , Proteínas Proto-Oncogénicas c-abl/genética , Urinálisis , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/genética , Uroplaquina Ib/genética , Adulto JovenRESUMEN
As benzene has been linked with elevated risk of both acute myeloid leukemia and lymphoma, we explored the effect of benzene exposure on levels of t(8;21), t(15;17), and t(14;18) translocations. Circulating lymphocytes of normal individuals also often contain t(14;18). Quantitative polymerase chain reaction analysis showed that 37 workers with benzene exposure had a decreased level of t(14;18) in their blood with only 16.2% having 10 or more copies of the t(14;18) BCL-2/IgH fusion gene/microg DNA, as opposed to 55% of 20 controls (P = .0063 by Fisher's exact test). This decline may be related to the immunotoxicity to specific subtypes of circulating B-lymphocytes, but the data do not support the use of t(14;18) as a biomarker of increased lymphoma risk in benzene-exposed populations. None of 88 individuals (31 controls and 57 exposed) exhibited detectable t(8;21) transcripts, and while t(15;17) transcripts were detected in two individuals, the result is inconclusive as one was exposed and the other was unexposed.
Asunto(s)
Benceno/toxicidad , Carcinógenos/toxicidad , Cromosomas Humanos/genética , Linfoma/inducido químicamente , Linfoma/genética , Exposición Profesional , Translocación Genética/efectos de los fármacos , Adulto , Estudios de Casos y Controles , Femenino , Genes bcl-2/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.
Asunto(s)
Cartilla de ADN , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Genotipo , Humanos , Temperatura de TransiciónRESUMEN
BACKGROUND: The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples. METHODS AND FINDINGS: This study evaluated the assay's analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51-97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163-302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs. CONCLUSION: In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/diagnóstico , Tamizaje Masivo/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Chlorocebus aethiops , Ebolavirus/fisiología , Genes Virales/genética , Fiebre Hemorrágica Ebola/sangre , Fiebre Hemorrágica Ebola/virología , Interacciones Huésped-Patógeno , Humanos , ARN Viral/sangre , ARN Viral/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo , Células Vero , Inactivación de VirusRESUMEN
The ideal technology for screening single-nucleotide polymorphisms requires high throughput with minimal cost per sample, minimal usage of valuable DNA resources, and maximal flexibility for assessment of new polymorphisms. We demonstrate here the feasibility of kinetic allele-specific PCR with DNA pooling (S. Germer et al., Genome Res., 10: 258-266, 2000) in a population study that satisfies all of the mentioned criteria and offers a powerful new tool for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage dysequilibrium scans. Three individuals prepared pooled DNA samples from 269 individuals separated into three racial/ethnic groups: Caucasians (n = 56), African-Americans (n = 86), and Hispanics (n = 127). We used kinetic allele-specific PCR to determine the allele frequencies of the common paraoxonase 1 polymorphism, PON1 Q191R, in these pools. Paraoxonase 1 is a critical enzyme for inactivating neurotoxic intermediates in the metabolism of organophosphates. In a blinded test of the technology, these nine pooled DNA samples were sent to Roche for genotyping by kinetic allele-specific PCR. The allele frequencies found were 0.266 +/- 0.011, 0.386 +/- 0.011, and 0.617 +/- 0.010, respectively, which were comparable to the frequencies of 0.269, 0.403, and 0.622 determined by PCR-restriction fragment length polymorphism analysis. These same samples were genotyped on two kinetic PCR platforms from different manufacturers, using three different DNA polymerases. The results were comparable between both platforms and among all three polymerases. The results demonstrate a powerful new technology for determining frequencies of single-nucleotide polymorphisms in an epidemiological study.
Asunto(s)
ADN/genética , Frecuencia de los Genes , Enfermedades Genéticas Congénitas/genética , Reacción en Cadena de la Polimerasa/métodos , Alelos , Secuencia de Bases , ADN/análisis , Femenino , Pruebas Genéticas/métodos , Humanos , Datos de Secuencia Molecular , Embarazo , Atención Prenatal , Muestreo , Sensibilidad y EspecificidadRESUMEN
We have evaluated the use of allele-specific PCR (AS PCR) on DNA pools as a tool for screening inherited genetic variants that may be associated with risk of adult acute myeloid leukemia (AML). Two DNA pools were constructed, one of 444 AML cases, and another of 823 matched controls. The pools were validated using individual genotyping data for GSTP1 and LTalpha variants. Allele frequencies for variants in GSTP1 and LTalpha were estimated using quantitative AS PCR, and when compared to individual genotyping data, a high degree of concordance was seen. AS primer pairs were designed for nine candidate genetic variants in DNA repair and cell cycle/apoptotic regulatory genes, including Cyclin D1 [codon 870 splice site variant (A>G)]; BRCA1, P871L; ERCC2, K751Q; FAS -1377 (G>A); hMLH1 -93 (G>A) and V219I; p21, S31R; and the XRCC1 R194W and R399Q variants. For six of these assays, there was at least 95% concordance between AS PCR genotyping and an alternative approach carried out on individual samples. Furthermore, these six AS PCR assays all accurately estimated allele frequencies in the pools that had been calculated using individual genotyping data. A significant disease association was seen with AML for the -1377 variant in FAS (odds ratio 1.76, 95% confidence interval 1.26-2.44). These data suggest that quantitative AS PCR can be used as an efficient screening technique for disease associations of genetic variants in DNA pools made from case-control studies.
Asunto(s)
Variación Genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Estudios de Casos y Controles , Intervalos de Confianza , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Oportunidad Relativa , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
A reproducibility study was designed to assess within-assay, between-day, and interlaboratory variability of three real-time PCR assays targeting HPV 16, HPV 18, and the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes. Fifteen HPV 16 and fifteen HPV 18 cervical swab samples were amplified in triplicate by GAPDH and HPV 16 and by GAPDH and HPV 18 assays, respectively. All samples were amplified undiluted and at a 1:10 dilution on 2 separate days in the same laboratory, and the same samples were amplified in a separate laboratory. HPV 16 and HPV 18 normalized viral load is reported as the number of HPV genomes per 20000 GAPDH copies. The analytic specificity of the HPV 16 and 18 assays was 100 and 97%, respectively. The intraclass correlation coefficients (ICC) were 0.99, 0.97, and 0.98 for HPV 16, HPV 18, and GAPDH, respectively, indicating that the variability due to experimental error was very low. Ten-fold differences in viral load could be readily discriminated across a six order of magnitude dynamic range (ca. 5-5x10(6) copies). Power of discrimination was increased at higher target concentrations (>5000 copies). The correlation of normalized HPV 16 and 18 viral load was high between the two laboratories (Spearman rho (rho)=0.96 and 0.87, respectively). These HPV 16 and HPV 18 quantitative PCR assays with GAPDH normalization are reproducibly quantitative over a broad linear dynamic range allowing for application in epidemiologic studies for measurement of viral load.
Asunto(s)
ADN Viral/análisis , Papillomaviridae/fisiología , Reacción en Cadena de la Polimerasa/métodos , Carga Viral , Cuello del Útero/virología , ADN Viral/aislamiento & purificación , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Papillomaviridae/clasificación , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Polimerasa TaqRESUMEN
Life-long exposure to estrogen is an established risk factor for breast cancer development. The underlying mechanism has been suggested to be the binding of estrogen-to-estrogen receptors in mammary tissue, which in turn promotes the proliferation and differentiation of breast tissue. Polymorphisms and haplotypes in estrogen receptor alpha (ESR1) have been reportedly associated with breast cancer risk; however, the results are not fully consistent. In this study, we investigated breast cancer risk associated with genotypes and haplotypes resulting from four ESR1 single nucleotide polymorphisms (SNPs), rs746432, rs2234693, rs9340799, and rs1801132. Genotyping has been performed on 393 breast cancer cases and 790 randomly selected controls in 1,183 Caucasian women over age 65 from the Study of Osteoporotic Fractures (SOF). We observed an allelic protective effect for SNP rs9340799 with an estimated odds ratio (OR) of 0.82 (95% CI = 0.68-1.00; P = 0.04) after adjustment for age, BMI and hip BMD. A protective effect of this SNP has been reported before in several different studies. We did not replicate the previously reported C-C-A-G haplotype association to breast cancer-the C-C-A-G haplotype from these SNPs was rare in this study (estimated frequency below 0.001% in cases and controls). No other statistically significant associations were observed between ESR1 haplotypes from the same four SNPs and the risk of breast cancer in older Caucasian women.
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Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Haplotipos/genética , Polimorfismo de Nucleótido Simple/genética , Población Blanca/genética , Anciano , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Estudios Prospectivos , Factores de RiesgoRESUMEN
BACKGROUND: The effort to eliminate transfusion complications associated with WBCs has led to the widespread use of filters able to reduce WBC concentrations to Asunto(s)
Recuento de Leucocitos
, Reacción en Cadena de la Polimerasa/métodos
, Sistemas de Computación
, ADN/sangre
, Filtración
, Humanos
, Cinética
, Recuento de Leucocitos/normas
, Reproducibilidad de los Resultados
, Sensibilidad y Especificidad