RESUMO
Infantile pyknocytosis is a rare and self-limiting cause of hemolytic anemia in neonates. It can result in severe anemia and hyperbilirubinemia. The pathogenesis is unknown: a genetic origin has been discussed; however, based on the current literature it is not clear which genetic mutations should be considered. We present a case of a premature twin, in whom genetic screening was performed. Genetic mutations in 46 genes associated with hereditary hemolytic anemia and dyserythropoietic anemia were tested. No mutations were found. In infantile pyknocytosis, a genetic defect in these genes is unlikely.
Assuntos
Anemia Hemolítica Congênita/patologia , Anemia Neonatal/patologia , Doenças em Gêmeos/patologia , Eritrócitos Anormais/patologia , Marcadores Genéticos , Gravidez de Gêmeos , Anemia Hemolítica Congênita/genética , Anemia Neonatal/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Gravidez , PrognósticoRESUMO
OBJECTIVES: For new analyzers or tests, analytical evaluation is required before implementation in the clinical laboratory. We evaluated the novel Roche Cobas t711 analyzer with six newly developed coagulation assays: the activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), fibrinogen, d-dimer and anti-Xa. The evaluation included imprecision experiments, method comparison with the currently used Stago STA-R Evolution, monitoring of unfractionated heparin (UFH) with aPTT, a fast centrifugation protocol to improve turn-around time, and determination of sample stability in whole blood and plasma. DESIGN AND METHODS: Imprecision and method comparison were assessed using commercial quality control samples and patient samples, respectively. For dose monitoring of UFH with the aPTT, samples from patients treated with UFH were used. Samples from healthy volunteers were collected for evaluation of the fast centrifugation protocol (5' 2750×g) and for investigating sample stability over 6-8â¯h. RESULTS: Results for between-run precision were within the desirable specification. Method comparison showed an excellent agreement for fibrinogen, d-dimer and anti-Xa. For aPTT, PT and INR, a good correlation was found, but results were significantly lower on the t711 compared to the STA-R Evolution, which is caused by different coagulation activators. Results from the fast centrifugation protocol differed not significantly from the standard protocol (15' 2500×g). Blood and plasma samples were stable at room temperature up to 6 and 8â¯h, respectively. CONCLUSIONS: The t711 coagulation analyzer with 6 novel tests is suitable for routine use in clinical laboratories.
RESUMO
BACKGROUND: The prognostic role of the STK15 T91A polymorphism and of STK15 mRNA expression was investigated in patients with urothelial cell carcinoma (UCC). MATERIALS AND METHODS: The STK15 genotype with respect to the T91A polymorphism was assessed by restriction fragment length polymorphism in 135 patients. STK15 mRNA expression was measured in tumor tissues of 103 patients, using real-time quantitative PCR. RESULTS: The T91A polymorphism lacked any prognostic information in our patient cohort. Interestingly though, STK15 mRNA expression was increased in invasive and high-grade tumors (p-values of 0.009 and 0.0001, respectively). Additionally, patients with superficial UCC (n = 82) who had a tumor recurrence in the first year after surgery displayed elevated STK15 mRNA expression levels (p = 0.009). Kaplan-Meier survival analysis revealed an increased risk of tumor progression for patients with Ta tumors (n = 62) and high STK15 expression (log-rank p = 0.04). Furthermore, a decreased overall (log-rank p = 0.006) and UCC-specific survival (log-rank p = 0.001) were shown for patients with elevated STK15 mRNA levels. CONCLUSION: Patients with UCC and elevated levels of STK15 mRNA generally showed a more adverse disease course than patients with low levels. This may help in identifying patients in need of more aggressive treatment.
Assuntos
Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/biossíntese , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aurora Quinase A , Aurora Quinases , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Serina-Treonina Quinases/biossíntese , RNA Mensageiro/genética , Taxa de Sobrevida , Neoplasias da Bexiga Urinária/patologia , Urotélio/enzimologia , Urotélio/patologiaRESUMO
BACKGROUND: The prediction of tumor recurrence in patients with Ta urothelial cell carcinoma is inaccurate and new prognostic markers are desirable. MATERIALS AND METHODS: Surface-enhanced laser-desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) was performed on 33 primary Ta tumors (16 and 17 tumors were from patients with long and short recurrence-free periods, respectively) and data were compared to previously obtained mRNA expression profiles of 49 genes. RESULTS: The intensities of a protein peak at m/z 33331 varied most significantly between the two patient groups (p = 0.0048). This was comparable to survivin, whose mRNA expression differed most significantly (p = 0.0042) of the 49 genes. ROC analysis revealed an area under the curve for protein peak 33331 and survivin of 0.78 (95% CI, 0.62-0.94) and 0.79 (95% CI, 0.63-0.94), respectively. Protein peak 33331 and survivin identified 3 (17%) and 8 (47%) patients with a recurrence-free period of at least 4 years, respectively, without generating false-negatives. CONCLUSION: These findings indicate that SELDI-TOF MS and real-time Q-PCR analysis on the same tissue can result in the identification of markers with comparable differential expression. Such combined analyses may yield combinations of several markers that might improve disease prognosis.
Assuntos
Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Carcinoma de Células de Transição/química , Feminino , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/isolamento & purificação , Recidiva Local de Neoplasia/química , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Valor Preditivo dos Testes , Proteômica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias da Bexiga Urinária/químicaRESUMO
OBJECTIVES: The individual recurrence-free period after primary surgery of patients with Ta urothelial cell carcinoma (UCC) cannot be predicted accurately. This study aims at discriminating between patients with primary Ta UCC and long or short recurrence-free periods. METHODS: We investigated mRNA expression of 23 genes in 44 primary Ta tumours (23 and 21 tumours were from patients with long [>or=4 yr] or short [Assuntos
Carcinoma de Células de Transição/genética
, Carcinoma de Células de Transição/cirurgia
, Perfilação da Expressão Gênica
, Recidiva Local de Neoplasia/genética
, Neoplasias da Bexiga Urinária/genética
, Neoplasias da Bexiga Urinária/cirurgia
, Adulto
, Idoso
, Idoso de 80 Anos ou mais
, Feminino
, Humanos
, Masculino
, Pessoa de Meia-Idade
, Valor Preditivo dos Testes
, RNA Neoplásico/biossíntese
RESUMO
BACKGROUND: A total of 4 additional splice variants (survivin-DeltaEx3, survivin 2alpha, survivin-2B, and survivin-3B) have been described for survivin [baculoviral IAP repeat-containing protein (BIRC-5), approved gene symbol BIRC5], which has been implicated in both inhibition of apoptosis and regulation in mitosis in many tumor types. In this study, we assessed whether the survivin splice variants modulate or add to the prognostic value of total survivin in breast cancer. METHODS: With quantitative reverse transcription-PCR, we measured mRNA concentrations of survivin and all variants in tumor tissue from 275 patients with breast cancer and associated these with clinicopathologic characteristics and relapse-free survival. RESULTS: Total survivin, survivin-DeltaEx3, and survivin 2alpha mRNA levels were associated with young age and ductal histology. Total survivin and survivin-DeltaEx3 were highest in samples with advanced histological grade, whereas patients with 4-9 involved lymph nodes expressed less survivin-2B mRNA than those with 1-3 involved nodes. All variants were higher in tumors negative for steroid hormone receptors. Total survivin, survivin 2alpha, and survivin-3B were associated with poor relapse-free survival in univariate analyses. Survivin 2alpha and survivin-3B added to the prognostic value of total survivin in multivariate analyses. In addition, the prognostic value of total survivin was evident only in the presence of higher expression levels of these 2 variants. CONCLUSIONS: All variants of survivin exhibited particular associations with clinicopathologic characteristics (age, histology, grade, and steroid hormone receptor status) of breast cancer patients. Survival analyses suggest a modulating role of survivin 2alpha and survivin-3B on the biological function of total survivin.
Assuntos
Neoplasias da Mama/diagnóstico , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Splicing de RNA , Análise de Variância , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Humanos , Proteínas Inibidoras de Apoptose , Linfonodos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Recidiva Local de Neoplasia , Valor Preditivo dos Testes , RNA Mensageiro/metabolismo , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SurvivinaAssuntos
Carcinoma de Células de Transição/diagnóstico , Antígeno Ki-67/biossíntese , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/biossíntese , Neoplasias da Bexiga Urinária/diagnóstico , Bexiga Urinária/metabolismo , Urotélio/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Feminino , Humanos , Proteínas Inibidoras de Apoptose , Antígeno Ki-67/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , RNA Mensageiro/genética , Survivina , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
Accurate prediction of tumor recurrence in patients with superficial urothelial cell carcinoma (UCC) might result in a significant reduction of invasive follow-up cystoscopies. A recent study identified a panel of 26 genes from a large cDNA microarray analysis of bladder tumors that discriminated between early- and late-recurring patients with superficial Ta tumors (Dyrskjøt et al., Nat Genet 2003;33:90-6). We aimed to validate this panel of genes in 44 primary Ta UCCs (23 and 21 tumors from patients with short or prolonged recurrence-free periods, respectively), by real-time quantitative PCR. Statistical analysis showed marginal significant different mRNA expression levels between the 2 patient groups. To evaluate a supplementary effect of genes for the identification of patients with short or prolonged recurrence-free intervals, forward logistic regression analysis was applied. This revealed that a combination of the expression profiles of the genes HNRPK, LTB4DH and ANP32B resulted in the best performance, although the combination only marginally increased the predictive value of HNRPK alone. Comparing the receiver-operating-characteristic curves for HNRPK expression among patients with short or prolonged recurrence-free periods, revealed an area under the curve of 0.696 (95% CI, 0.537-0.855). Using the median HNRPK expression level as cut-off, a sensitivity of 69.6% and a specificity of 71.4% were obtained for the identification of patients with short or prolonged recurrence-free periods, respectively. In conclusion, we were not able to confirm the microarray gene expression pattern of the 26 genes shown by Dyrskjøt et al. The discovery of accurate recurrence predictive markers, therefore, remains a challenge.
Assuntos
Perfilação da Expressão Gênica/métodos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Humanos , Recidiva Local de Neoplasia/classificação , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Neoplasias da Bexiga Urinária/classificação , Urotélio/metabolismo , Urotélio/patologiaRESUMO
Expression of the anti-apoptotic protein survivin is hardly detectable or even absent in many differentiated adult tissues, but is upregulated in almost any type of cancer. Furthermore, high survivin mRNA or protein expression generally correlates with an adverse disease course. Both these important features of survivin expression have been investigated for diagnostic and prognostic purposes in many human cancers, including bladder cancer. In this review, the role of survivin in the detection of bladder tumors and the prediction of tumor recurrence in patients with superficial bladder cancer will be discussed and compared to that of other markers/tests. The most promising marker(s) will be outlined. Also, important requirements for a successful implementation of such markers in a hospital setting are discussed. Finally, future directions for the discovery of new diagnostic or prognostic candidate markers will be mentioned.
Assuntos
Biomarcadores Tumorais/sangue , Proteínas Associadas aos Microtúbulos/sangue , Proteínas de Neoplasias/sangue , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/patologia , Neoplasias da Bexiga Urinária/sangue , Neoplasias da Bexiga Urinária/diagnóstico , Animais , Forma Celular , Humanos , Proteínas Inibidoras de Apoptose , Recidiva Local de Neoplasia/genética , Prognóstico , Survivina , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
CDC91L1 (PIG-U) was recently discovered as a new oncogene in human bladder cancer and showed mRNA overexpression in 36% of primary bladder tumor tissues compared to normal urothelium. We further investigated CDC91L1 mRNA expression in 8 bladder cancer cell lines, 14 normal bladder tissues and 42 urothelial cell carcinomas by real-time quantitative PCR. The prognostic value of CDC91L1 mRNA expression was also investigated. Surprisingly, only one (2.4%) tumor tissue showed overexpression compared to normal urothelium. No significant relationship of CDC91L1 mRNA expression with increasing pathologic stage (p = 0.962) or grade (p = 0.557) was observed. Median normalized CDC91L1 mRNA expression values were 0.19 for superficial tumors (n = 21) and 0.18 for invasive tumors (n = 21). Grade I, grade II and grade III tumors had median normalized expression values of 0.26, 0.18 and 0.33, respectively. CDC91L1 mRNA expression level was not indicative of early tumor recurrence (log rank p = 0.1629), tumor progression (log rank p = 0.9307) or overall and disease-specific survival (log rank p = 0.9193 and 0.4710, respectively). Our results suggest, in contrast to those of Guo et al. (Nat Med 2004;10:374-81), that the oncogene CDC91L1 is not overexpressed at the mRNA level in urothelial cell carcinomas and cannot be used to predict the course of the disease.
Assuntos
Aciltransferases/biossíntese , Carcinoma/genética , Carcinoma/patologia , Perfilação da Expressão Gênica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Progressão da Doença , Humanos , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/biossíntese , Bexiga Urinária/fisiologia , Urotélio/fisiologiaRESUMO
For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, beta-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, beta-2-microglobin, beta-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Feminino , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Componente Principal , RNA Mensageiro/metabolismo , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosAssuntos
Biomarcadores Tumorais/análise , Carcinoma de Células de Transição/diagnóstico , Proteínas Associadas aos Microtúbulos/análise , RNA Mensageiro/análise , Neoplasias da Bexiga Urinária/diagnóstico , Bexiga Urinária/química , Biomarcadores Tumorais/genética , Carcinoma de Células de Transição/química , Dosagem de Genes , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Neoplasias da Bexiga Urinária/químicaAssuntos
Coleta de Amostras Sanguíneas/métodos , DNA/sangue , Doadores de Sangue , Feminino , Humanos , GravidezRESUMO
BACKGROUND: Previous studies suggest that the amount of circulating cell-free DNA in plasma or serum may be a promising marker for diagnosis and prediction of prognosis in patients with tumours. However, it is unclear whether DNA from plasma or from serum better reflects the clinical status. PATIENTS AND METHODS: To investigate the possible difference between serum DNA and plasma DNA levels, blood was collected from 26 patients with liver metastases and 28 age-matched healthy donors. The patients had clinical follow-up after surgery (median 23 months). DNA was quantified by two independent methods: real-time quantitative PCR (TaqMan) and fluorimetric DNA quantitation (Picogreen reagent). RESULTS: Serum DNA and plasma DNA levels did not correlate and each had a different relationship with diagnosis and prognosis. Only serum DNA was significantly associated with the presence of liver metastases, whereas only plasma DNA was predictive for recurrences. Typically, serum DNA amounts were higher than plasma in 24 out of 26 patients. CONCLUSION: This difference between plasma and serum reflects an in vitro process that occurs during clotting. High DNA increase in serum represents an indirect, yet tumour-related process. Plasma DNA levels better represent in vivo levels of circulating DNA. This has implications forfuture studies.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , DNA de Neoplasias/sangue , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/secundário , Idoso , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Humanos , Neoplasias Hepáticas/genética , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/genética , Prognóstico , Reprodutibilidade dos TestesRESUMO
We identified DD3(PCA3) as one of the most prostate cancer-specific genes at present (M. J. Bussemakers et al. Cancer Res., 59: 5975-5979, 1999). Consequently, DD3(PCA3) is an interesting candidate for use as a diagnostic and/or prognostic marker. In this study we developed a method for the accurate quantification of DD3(PCA3) mRNA, using real-time quantitative reverse transcription-PCR. DD3(PCA3) was expressed at low levels in normal prostate but not in 21 selected other normal tissues, blood, or 39 tumor samples other than prostate. The diagnostic and prognostic value of DD3(PCA3) in normal, hyperplastic, and malignant prostate tissues was determined and compared with another promising tumor marker for prostate cancer, telomerase reverse transcriptase (hTERT gene), the expression of which is related to telomerase activity. Sensitivity and specificity estimates for both genes were calculated as the area under the receiver-operating characteristics curve (AUC-ROC). DD3(PCA3) (AUC-ROC, 0.98) demonstrated better diagnostic efficacy than hTERT (AUC-ROC, 0.88). Moreover, the median increase in mRNA expression in tumor tissues compared with nonmalignant prostate tissues was much higher for DD3(PCA3) (34-fold) than for hTERT (6-fold). In tumor tissues, the median expression of DD3(PCA3) was much higher than hTERT (5849 versus 10 normalized mRNA copies). A significant relationship was observed only between tumor stage and hTERT gene expression. We conclude that expression of the DD3(PCA3) gene is a very sensitive and specific marker for the detection of prostate tumor cells in a high background of normal (prostate) cells. Consequently, DD3 measurements may be used for clinical application in prostate needle biopsies or bodily fluids such as blood, ejaculate, urine, or prostate massage fluid.
Assuntos
Antígenos de Neoplasias/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias da Próstata/metabolismo , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA , Humanos , Masculino , Neoplasias da Próstata/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Telomerase/biossíntese , Telomerase/genéticaRESUMO
Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554-559, 2002.