Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Cancers (Basel) ; 15(24)2023 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-38136324

RESUMO

BACKGROUND: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. METHODS: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. RESULTS: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90-232 days). CONCLUSIONS: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring.

2.
JCO Precis Oncol ; 7: e2300389, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37883729

RESUMO

PURPOSE: Surveillance after primary melanoma treatment aims to detect early signs of low-volume systemic disease. The current standard of care, surveillance imaging, is costly and difficult to access. We therefore sought to develop methylated DNA markers (MDMs) as promising alternatives for disease surveillance. METHODS: We used reduced representation bisulfite sequencing (RRBS) to identify MDMs in DNA samples obtained from metastatic melanoma, benign nevi, and normal skin tissues. The identified MDMs underwent validation in an independent cohort of tissue and buffy coat DNA samples. Subsequently, we tested the validated MDMs in the plasma DNA of patients with metastatic melanoma undergoing surveillance with total body imaging and compared them with cancer-free controls. To estimate the overall predictive accuracy of the MDMs, we used random forest modeling with bootstrap cross-validation. RESULTS: Forty MDMs demonstrated discrimination between melanoma cases and controls consisting of benign nevi and normal skin. Nine MDMs passing biological validation in tissue were run on 77 plasma samples from individuals with a history of metastatic melanoma, 49 of whom had evidence of disease detected by imaging at the time of blood draw, and 100 cancer-free controls. The cross-validated sensitivity of the panel for imaging-positive disease was 80% with a specificity of 100% in cancer-free controls, resulting in an overall AUC of 0.88 (95% CI, 0.81 to 0.96). The survival estimates for patients with melanoma who tested positive for the panel at 6 months and 1 year were 67% and 56%, respectively, while those who tested negative had survival rates of 100% and 92%. CONCLUSION: MDMs identified by RRBS demonstrate a high degree of concordance with imaging results in the plasma of patients with metastatic melanoma. Further prospective studies in larger intended use cohorts are needed to confirm these findings.


Assuntos
Melanoma , Nevo , Humanos , Marcadores Genéticos , Estudos Prospectivos , Melanoma/diagnóstico , Melanoma/genética , DNA
3.
Gynecol Oncol ; 165(3): 568-576, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35370009

RESUMO

OBJECTIVE: Aberrant DNA methylation is an early event in carcinogenesis which could be leveraged to detect ovarian cancer (OC) in plasma. METHODS: DNA from frozen OC tissues, benign fallopian tube epithelium (FTE), and buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify OC MDMs. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent OC and FTE FFPE tissues. MDMs were tested using Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) assays in pre-treatment plasma from women newly diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. RESULTS: Thirty-three MDMs showed marked methylation fold changes (10 to >1000) across all OC subtypes vs FTE. Eleven MDMs (GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, RIPPLY3, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (73 (80%) high-grade serous (HGS)) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95% CI, 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86-0.96)). Among the 5 stage I/II HGS OCs included, all were correctly identified. CONCLUSIONS: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded candidate MDMs for OC that performed with high sensitivity and specificity in plasma. Larger plasma-based OC MDM studies, including testing of pre-diagnostic specimens, are warranted.


Assuntos
Metilação de DNA , Neoplasias Ovarianas , Biomarcadores Tumorais/genética , Proteínas CELF/genética , Carcinoma Epitelial do Ovário/diagnóstico , Carcinoma Epitelial do Ovário/genética , Estudos de Viabilidade , Feminino , Marcadores Genéticos , Humanos , Proteínas do Tecido Nervoso/genética , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Transaminases/genética
4.
Cancers (Basel) ; 13(23)2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34884994

RESUMO

Analysis of circulating tumor cells (CTCs) from blood samples provides a non-invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon-related mRNA transcripts (USH1C and CKMT1A). Cancer cell transcripts are detected by RT-qPCR using proprietary Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) technology. We validate each step of the workflow using colorectal cancer (CRC) cell lines spiked into blood and compare this with antibody-based cell detection. USH1C and CKMT1A are expressed in healthy colon tissue and CRC cell lines, while only low-level expression can be detected in healthy white blood cells (WBCs). The qPCR reaction shows a near-perfect amplification efficiency for all primer targets with minimal interference of WBC cDNA. Spike-in of 10 cancer cells in 3 mL blood can be detected and statistically separated from control blood using the RT-qPCR assay after rVAR2 capture (p < 0.01 for both primer targets, Mann-Whitney test). Our results provide a validated workflow for highly sensitive detection of magnetically enriched cancer cells.

5.
Gastrointest Endosc ; 94(3): 498-505, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33857451

RESUMO

BACKGROUND AND AIMS: We previously identified a 5 methylated DNA marker (MDM) panel for the detection of nonendoscopic Barrett's esophagus (BE). In this study, we aimed to recalibrate the performance of the 5 MDM panel using a simplified assay in a training cohort, validate the panel in an independent test cohort, and explore the accuracy of an MDM panel with only 3 markers. METHODS: Participants were recruited from 3 medical centers. The sponge on a string device (EsophaCap; CapNostics, Concord, NC, USA) was swallowed and withdrawn, followed by endoscopy, in BE cases and control subjects. A 5 MDM panel was blindly assayed using a simplified assay. Random forest modeling analysis was performed, in silico cross-validated in the training set, and then locked down, before test set analysis. RESULTS: The training set had 199 patients: 110 BE cases and 89 control subjects, and the test set had 89 patients: 60 BE cases and 29 control subjects. Sensitivity of the 5 MDM panel for BE diagnosis was 93% at 90% specificity in the training set and 93% at 93% specificity in the test set. Areas under the receiver operating characteristic curves were .96 and .97 in the training and test sets, respectively. Model accuracy was not influenced by age, sex, or smoking history. Multiple 3 MDM panels achieved similar accuracy. CONCLUSIONS: A 5 MDM panel for BE is highly accurate in training and test sets in a blinded multisite case-control analysis using a simplified assay. This panel may be reduced to only 3 MDMs in the future. (Clinical trial registration number: NCT02560623.).


Assuntos
Esôfago de Barrett , Neoplasias Esofágicas , Esôfago de Barrett/diagnóstico , Estudos de Casos e Controles , Estudos de Coortes , Marcadores Genéticos , Humanos , Curva ROC
6.
Clin Cancer Res ; 27(9): 2523-2532, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33593879

RESUMO

PURPOSE: We have previously identified tissue methylated DNA markers (MDMs) associated with pancreatic ductal adenocarcinoma (PDAC). In this case-control study, we aimed to assess the diagnostic performance of plasma MDMs for PDAC. EXPERIMENTAL DESIGN: Thirteen MDMs (GRIN2D, CD1D, ZNF781, FER1L4, RYR2, CLEC11A, AK055957, LRRC4, GH05J042948, HOXA1, PRKCB, SHISA9, and NTRK3) were identified on the basis of selection criteria applied to results of prior tissue experiments and assays were optimized in plasma. Next, 340 plasma samples (170 PDAC cases and 170 controls) were assayed using target enrichment long-probe quantitative amplified signal method. Initially, 120 advanced-stage PDAC cases and 120 healthy controls were used to train a prediction algorithm at 97.5% specificity using random forest modeling. Subsequently, the locked algorithm derived from the training set was applied to an independent blinded test set of 50 early-stage PDAC cases and 50 controls. Finally, data from all 340 patients were combined, and cross-validated. RESULTS: The cross-validated area under the receiver operating characteristic curve (AUC) for the training set was 0.93 (0.89-0.96) for the MDM panel alone, 0.91 (95% confidence interval, 0.87-0.96) for carbohydrate antigen 19-9 (CA19-9) alone, and 0.99 (0.98-1) for the combined MDM-CA19-9 panel. In the test set of early-stage PDAC, the AUC for MDMs alone was 0.84 (0.76-0.92), CA19-9 alone was 0.87 (0.79-0.94), and combined MDM-CA19-9 panel was 0.90 (0.84-0.97) significantly better compared with either MDMs alone or CA19-9 alone (P = 0.0382 and 0.0490, respectively). At a preset specificity of 97.5%, the sensitivity for the combined panel in the test set was 80% (28%-99%) for stage I disease and 82% (68%-92%) for stage II disease. Using the combined datasets, the cross-validated AUC was 0.9 (0.86-0.94) for the MDM panel alone and 0.89 for CA19-9 alone (0.84-0.93) versus 0.97 (0.94-0.99) for the combined MDM-CA19-9 panel (P ≤ 0.0001). Overall, cross-validated sensitivity of MDM-CA19-9 panel was 92% (83%-98%), with an observed specificity of 92% at the preset specificity of 97.5%. CONCLUSIONS: Plasma MDMs in combination with CA19-9 detect PDAC with significantly higher accuracy compared with either biomarker individually.


Assuntos
Biomarcadores Tumorais , Antígeno CA-19-9/sangue , Metilação de DNA , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/etiologia , Estudos de Casos e Controles , Comorbidade , Biologia Computacional/métodos , Feminino , Humanos , Masculino , Estadiamento de Neoplasias , Neoplasias Pancreáticas/sangue , Curva ROC
7.
Clin Cancer Res ; 27(1): 141-149, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33028593

RESUMO

PURPOSE: We aimed to assess the concordance of colorectal cancer-associated methylated DNA markers (MDM) in primary and metastatic colorectal cancer for feasibility in detection of distantly recurrent/metastatic colorectal cancer in plasma. EXPERIMENTAL DESIGN: A panel of previously discovered colorectal cancer-associated MDMs was selected. MDMs from primary and paired metastatic colorectal cancer tissue were assayed with quantitative methylation-specific PCR. Plasma MDMs were measured blindly by target enrichment long-probe quantitative-amplified signal assays. Random forest modeling was used to derive a prediction algorithm of MDMs in archival plasma samples from primary colorectal cancer cases. This algorithm was validated in prospectively collected plasma samples from recurrent colorectal cancer cases. The accuracy of the algorithm was summarized as sensitivity, specificity, and area under the curve (AUC). RESULTS: Of the 14 selected MDMs, the concordance between primary and metastatic tissue was considered moderate or higher for 12 MDMs (86%). At a preset specificity of 95% (91%-98%), a panel of 13 MDMs, in plasma from 97 colorectal cancer cases and 200 controls, detected stage IV colorectal cancer with 100% (80%-100%) sensitivity and all stages of colorectal cancer with an AUC of 0.91 (0.87-0.95), significantly higher than carcinoembryonic antigen [AUC, 0.72 (0.65-0.79)]. This panel, in plasma from 40 cases and 60 healthy controls, detected recurrent/metastatic colorectal cancer with 90% (76%-97%) sensitivity, 90% (79%-96%) specificity, and an AUC of 0.96 (0.92-1.00). The panel was positive in 0.30 (0.19-0.43) of 60 patients with no evidence of disease in post-operative patients with colorectal cancer. CONCLUSIONS: Plasma assay of novel colorectal cancer-associated MDMs can reliably detect both primary colorectal cancer and distantly recurrent colorectal cancer with promising accuracy.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Metilação de DNA , Recidiva Local de Neoplasia/diagnóstico , Conduta Expectante/métodos , Idoso , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Neoplasias Colorretais/terapia , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/prevenção & controle , Curva ROC , Reprodutibilidade dos Testes
8.
Cancer Epidemiol Biomarkers Prev ; 29(8): 1570-1576, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32467348

RESUMO

BACKGROUND: Emerging colorectal cancer trends demonstrate increased incidence and mortality in younger populations, prompting consideration of average-risk colorectal cancer screening initiation at age 45 versus 50 years. However, screening test performance characteristics in adults 45-49 years have been minimally described. To inform the biologic rationale for multi-target stool DNA (mt-sDNA) screening in younger patients, we analyzed and compared tissue levels of methylation (BMP3, NDRG4) and mutation (KRAS) markers included in the FDA-approved, mt-sDNA assay (Cologuard; Exact Sciences Corporation). METHODS: Within 40-44, 45-49, and 50-64 year age groups, archived colorectal tissue specimens were identified for 211 sporadic colorectal cancer cases, 123 advanced precancerous lesions (APLs; adenomas >1 cm, high-grade dysplasia, ≥25% villous morphology, or sessile serrated polyp; 45-49 and 50-64 age groups only), and 204 histologically normal controls. Following DNA extraction, KRAS, BMP3, and NDRG4 were quantified using QuARTS assays, relative to ACTB (reference gene). RESULTS: None of the molecular marker concentrations were significantly associated with age (P > 0.05 for all comparisons), with the exception of NDRG4 concentration in APL samples (higher in older vs. younger cases; P = 0.008). However, NDRG4 levels were also statistically higher in APL case versus normal control samples in both the 45-49 (P < 0.0001) and 50-64 (P < 0.0001) year age groups. CONCLUSIONS: Overall, these findings support the potential for earlier onset of average-risk colorectal cancer screening with the mt-sDNA assay. IMPACT: These novel data address an identified knowledge gap and strengthen the biologic basis for earlier-onset, average-risk screening with the mt-sDNA assay.


Assuntos
Neoplasias Colorretais/epidemiologia , Fatores Etários , Detecção Precoce de Câncer , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
9.
Clin Gastroenterol Hepatol ; 18(3): 676-683.e3, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31323382

RESUMO

BACKGROUND & AIMS: Precursors of pancreatic cancer arise in the ductal epithelium; markers exfoliated into pancreatic juice might be used to detect high-grade dysplasia (HGD) and cancer. Specific methylated DNA sequences in pancreatic tissue have been associated with adenocarcinoma. We analyzed these methylated DNA markers (MDMs) in pancreatic juice samples from patients with pancreatic ductal adenocarcinomas (PDACs) or intraductal papillary mucinous neoplasms (IPMNs) with HGD (cases), and assessed their ability to discriminate these patients from individuals without dysplasia or with IPMNs with low-grade dysplasia (controls). METHODS: We obtained pancreatic juice samples from 38 patients (35 with biopsy-proven PDAC or pancreatic cystic lesions with invasive cancer and 3 with HGD) and 73 controls (32 with normal pancreas and 41 with benign disease), collected endoscopically from the duodenum after secretin administration from February 2015 through November 2016 at 3 medical centers. Samples were analyzed for the presence of 14 MDMs (in the genes NDRG4, BMP3, TBX15, C13orf18, PRKCB, CLEC11A, CD1D, ELMO1, IGF2BP1, RYR2, ADCY1, FER1L4, EMX1, and LRRC4), by quantitative allele-specific real-time target and signal amplification. We performed area under the receiver operating characteristic curve analyses to determine the ability of each marker, and panels of markers, to distinguish patients with HGD and cancer from controls. MDMs were combined to form a panel for detection using recursive partition trees. RESULTS: We identified a group of 3 MDMs (at C13orf18, FER1L4, and BMP3) in pancreatic juice that distinguished cases from controls with an area under the receiver operating characteristic value of 0.90 (95% CI, 0.83-0.97). Using a specificity cut-off value of 86%, this group of MDMs distinguished patients with any stage of pancreatic cancer from controls with 83% sensitivity (95% CI, 66%-93%) and identified patients with stage I or II PDAC or IPMN with HGD with 80% sensitivity (95% CI, 56%-95%). CONCLUSIONS: We identified a group of 3 MDMs in pancreatic juice that identify patients with pancreatic cancer with an area under the receiver operating characteristic value of 0.90, including patients with early stage disease or advanced precancer. These DNA methylation patterns might be included in algorithms for early detection of pancreatic cancer, especially in high-risk cohorts. Further optimization and clinical studies are needed.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/diagnóstico , DNA , Detecção Precoce de Câncer , Humanos , Suco Pancreático , Neoplasias Pancreáticas/diagnóstico
10.
Clin Cancer Res ; 25(24): 7396-7404, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31527170

RESUMO

PURPOSE: The burden of esophageal cancer continues to rise, and noninvasive screening tools are needed. Methylated DNA markers (MDM) assayed from plasma show promise in detection of other cancers. For esophageal cancer detection, we aimed to discover and validate MDMs in tissue, and determine their feasibility when assayed from plasma. EXPERIMENTAL DESIGN: Whole-methylome sequencing was performed on DNA extracted from 37 tissues (28 EC; 9 normal esophagus) and 8 buffy coat samples. Top MDMs were validated by methylation specific PCR on tissue from 76 EC (41 adeno, 35 squamous cell) and 17 normal esophagus. Quantitative allele-specific real-time target and signal amplification was used to assay MDMs in plasma from 183 patients (85 EC, 98 controls). Recursive partitioning (rPART) identified MDM combinations predictive of esophageal cancer. Validation was performed in silico by bootstrapping. RESULTS: From discovery, 23 candidate MDMs were selected for independent tissue validation; median area under the receiver operating curve (AUC) for individual MDMs was 0.93. Among 12 MDMs advanced to plasma testing, rPART modeling selected a 5 MDM panel (FER1L4, ZNF671, ST8SIA1, TBX15, ARHGEF4) which achieved an AUC of 0.93 (95% CI, 0.89-0.96) on best-fit and 0.81 (95% CI, 0.75-0.88) on cross-validation. At 91% specificity, the panel detected 74% of esophageal cancer overall, and 43%, 64%, 77%, and 92% of stages I, II, III, and IV, respectively. Discrimination was not affected by age, sex, smoking, or body mass index. CONCLUSIONS: Novel MDMs assayed from plasma detect esophageal cancer with moderate accuracy. Further optimization and clinical testing are warranted.


Assuntos
Biomarcadores Tumorais/sangue , Metilação de DNA , Detecção Precoce de Câncer/métodos , Epigenoma , Neoplasias Esofágicas/sangue , Neoplasias Esofágicas/diagnóstico , Idoso , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Neoplasias Esofágicas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Fatores de Troca de Nucleotídeo Guanina Rho/sangue , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Sialiltransferases/sangue , Sialiltransferases/genética , Proteínas com Domínio T/sangue , Proteínas com Domínio T/genética , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/genética
11.
Am J Gastroenterol ; 114(9): 1539-1549, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31306149

RESUMO

OBJECTIVES: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy. In pancreatic tissue and cyst fluid (CF) from PCLs, we sought to identify and validate novel methylated DNA markers (MDMs) that discriminate HGD/PC from low-grade dysplasia (LGD) or no dysplasia (ND). METHODS: From an unbiased whole-methylome discovery approach using predefined selection criteria followed by multistep validation on case (HGD or PC) and control (ND or LGD) tissues, we identified discriminant MDMs. Top candidate MDMs were then assayed by quantitative methylation-specific polymerase chain reaction on archival CF from surgically resected PCLs. RESULTS: Of 25 discriminant MDMs identified in tissue, 13 were selected for validation in 134 CF samples (21 cases [8 HGD, 13 PC], 113 controls [45 ND, 68 LGD]). A tree-based algorithm using 2 CF-MDMs (TBX15, BMP3) achieved sensitivity and specificity above 90%. Discrimination was significantly better by this CF-MDM panel than by mutant KRAS or carcinoembryonic antigen, with areas under the receiver operating characteristic curve of 0.93 (95% confidence interval: 0.86-0.99), 0.71 (0.57-0.85), and 0.72 (0.60-0.84), respectively. Cutoffs for the MDM panel applied to an independent CF validation set (31 cases, 56 controls) yielded similarly high discrimination, areas under the receiver operating characteristic curve = 0.86 (95% confidence interval: 0.77-0.94, P = 0.2). DISCUSSION: Novel MDMs discovered and validated in tissue accurately identify PCLs harboring HGD/PC. A panel of 2 MDMs assayed in CF yielded results with potential to enhance current risk prediction algorithms. Prospective studies are indicated to optimize and further evaluate CF-MDMs for clinical use.


Assuntos
Carcinoma Ductal Pancreático/genética , Cistadenoma Seroso/genética , Metilação de DNA/genética , Cisto Pancreático/genética , Neoplasias Intraductais Pancreáticas/genética , Neoplasias Pancreáticas/genética , Lesões Pré-Cancerosas/genética , Idoso , Proteína Morfogenética Óssea 3/genética , Antígeno Carcinoembrionário/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patologia , Líquido Cístico/metabolismo , Cistadenoma Seroso/diagnóstico , Cistadenoma Seroso/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Cisto Pancreático/diagnóstico , Cisto Pancreático/patologia , Neoplasias Intraductais Pancreáticas/diagnóstico , Neoplasias Intraductais Pancreáticas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas com Domínio T/genética
12.
Clin Gastroenterol Hepatol ; 17(5): 914-921.e5, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29775793

RESUMO

BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs), including ulcerative colitis and Crohn's disease, are at increased risk for colorectal cancer (CRC). Analyses of DNA methylation patterns in stool samples have been reported to detect CRC in patients with IBD. We sought to validate these findings in larger cohorts and assess the accuracy of analysis of DNA methylation patterns in stool for detection of CRC and high-grade dysplasia (HGD) normalized to methylation level at ZDHHC1. METHODS: We obtained buffered, frozen stool samples from a US case-control study and from 2 European surveillance cohorts (referral or population based) of patients with chronic ulcerative colitis (n = 248), Crohn's disease (n = 82), indeterminate colitis (n = 2), or IBD with primary sclerosing cholangitis (n = 38). Stool samples were collected before bowel preparation for colonoscopy or at least 1 week after colonoscopy. Among the study samples, stools from individuals with IBD but without neoplasia were used as controls (n = 291). DNA was isolated from stool, exposed to bisulfite, and then assayed by multiplex quantitative allele-specific real-time target and signal amplification. We analyzed methylation levels of BMP3, NDRG4, VAV3, and SFMBT2 relative to the methylation level of ZDHHC1, and compared these between patients with CRC or HGD and controls. RESULTS: Levels of methylation at BMP3 and VAV3, relative to ZDHHC1 methylation, identified patients with CRC and HGD with an area under the curve value of 0.91 (95% CI, 0.77-1.00). Methylation levels at specific promotor regions of these genes identified 11 of the 12 patients with CRC and HGD, with 92% sensitivity (95% CI, 60%-100%) and 90% specificity (95% CI, 86%-93%). The proportion of false-positive results did not differ significantly among the case-control, referral cohort, and population cohort studies (P = .60) when the 90% specificity cut-off from the whole sample set was applied. CONCLUSIONS: In an analysis of stool samples from 3 independent studies of 332 patients with IBD, we associated levels of methylation at 2 genes (BMP3 and VAV3), relative to level of methylation at ZDHHC1, with detection of CRC and HGD. These methylation patterns identified patients with CRC and HGD with more than 90% specificity, and might be used in CRC surveillance.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Metilação de DNA , DNA/análise , Fezes/química , Doenças Inflamatórias Intestinais/complicações , Patologia Molecular/métodos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade
13.
Hepatology ; 69(3): 1180-1192, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30168613

RESUMO

Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues. Candidate MDMs were confirmed by quantitative methylation-specific PCR in DNA from independent tissues (74 HCC, 29 controls). A phase I plasma pilot incorporated quantitative allele-specific real-time target and signal amplification assays on independent plasma-extracted DNA from 21 HCC cases and 30 controls with cirrhosis. A phase II plasma study was then performed in 95 HCC cases, 51 controls with cirrhosis, and 98 healthy controls using target enrichment long-probe quantitative amplified signal (TELQAS) assays. Recursive partitioning identified best MDM combinations. The entire MDM panel was statistically cross-validated by randomly splitting the data 2:1 for training and testing. Random forest (rForest) regression models performed on the training set predicted disease status in the testing set; median areas under the receiver operating characteristics curve (AUCs; and 95% confidence interval [CI]) were reported after 500 iterations. In phase II, a six-marker MDM panel (homeobox A1 [HOXA1], empty spiracles homeobox 1 [EMX1], AK055957, endothelin-converting enzyme 1 [ECE1], phosphofructokinase [PFKP], and C-type lectin domain containing 11A [CLEC11A]) normalized by beta-1,3-galactosyltransferase 6 (B3GALT6) level yielded a best-fit AUC of 0.96 (95% CI, 0.93-0.99) with HCC sensitivity of 95% (88%-98%) at specificity of 92% (86%-96%). The panel detected 3 of 4 (75%) stage 0, 39 of 42 (93%) stage A, 13 of 14 (93%) stage B, 28 of 28 (100%) stage C, and 7 of 7 (100%) stage D HCCs. The AUC value for alpha-fetoprotein (AFP) was 0.80 (0.74-0.87) compared to 0.94 (0.9-0.97) for the cross-validated MDM panel (P < 0.0001). Conclusion: MDMs identified in this study proved to accurately detect HCC by plasma testing. Further optimization and clinical testing of this promising approach are indicated.


Assuntos
DNA de Neoplasias/sangue , Neoplasias Hepáticas/sangue , Carcinoma Hepatocelular , Metilação de DNA , DNA de Neoplasias/metabolismo , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Método Simples-Cego
14.
Clin Cancer Res ; 24(22): 5724-5734, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29844130

RESUMO

Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls.Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case-control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls.Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71-95) of gastric adenocarcinomas at 95% specificity.Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724-34. ©2018 AACR.


Assuntos
Biomarcadores Tumorais , Metilação de DNA , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácidos Nucleicos Livres , Estudos de Coortes , Feminino , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Projetos Piloto , Prognóstico , Reprodutibilidade dos Testes , Neoplasias Gástricas/diagnóstico
15.
Anal Chem ; 81(21): 8949-56, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19874056

RESUMO

RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of approximately 75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array composed of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/análise , Ressonância de Plasmônio de Superfície/métodos , Sequência de Bases , Sítios de Ligação , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Oligonucleotídeos/química
16.
J Clin Virol ; 40(2): 129-34, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17728179

RESUMO

We report the use of a prototype Invader Plus method (Third Wave Technologies, Inc., Madison, WI) for the qualitative detection of varicella-zoster virus (VZV) and differentiation of wild-type and Oka vaccine VZV. The analytical sensitivity of the VZV Invader Plus reagents is at 10 copies per reaction. A total of 174 skin and mucous swab specimens were used to validate the assay's performance. The sensitivity and specificity were 98.3% and 98.1%, respectively, in comparison to a PCR-EIA assay. A perfect 100% agreement was obtained when VZV wild-type and vaccine differentiation was performed on 54 VZV-positive swab specimens against an allele-specific FRET real-time assay. The Invader Plus method provides another reliable tool for qualitative detection of VZV and differentiation of wild-type and vaccine virus.


Assuntos
Vacina contra Varicela/isolamento & purificação , Varicela/diagnóstico , Herpes Zoster/diagnóstico , Herpesvirus Humano 3/isolamento & purificação , Alelos , Varicela/virologia , Vacina contra Varicela/classificação , Diagnóstico Diferencial , Herpes Zoster/virologia , Herpesvirus Humano 3/classificação , Humanos , Mucosa/virologia , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Pele/virologia , Vacinas Atenuadas/classificação , Vacinas Atenuadas/isolamento & purificação
17.
J Clin Microbiol ; 44(9): 3443-7, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16954297

RESUMO

We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.0% and 98.6%, respectively, when the results of the method were validated against the results obtained with a PCR colorimetric microtiter plate system by use of clinical CSF specimens.


Assuntos
Líquido Cefalorraquidiano/virologia , Transferência Ressonante de Energia de Fluorescência/métodos , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Viroses do Sistema Nervoso Central/diagnóstico , Viroses do Sistema Nervoso Central/virologia , Sondas de DNA , DNA Viral/análise , DNA Viral/líquido cefalorraquidiano , Transferência Ressonante de Energia de Fluorescência/instrumentação , Herpes Simples/diagnóstico , Herpes Simples/virologia , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/classificação , Herpesvirus Humano 2/genética , Sensibilidade e Especificidade , Timidina Quinase/genética
18.
RNA ; 10(7): 1153-61, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15208450

RESUMO

The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.


Assuntos
MicroRNAs/análise , MicroRNAs/genética , Sequência de Bases , Linhagem Celular , Técnicas Genéticas , Células HeLa , Humanos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química
19.
J Mol Biol ; 328(3): 537-54, 2003 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-12706715

RESUMO

Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex and were used as distance restraints in MD simulations. The final structure suggests that the substrate's 5' flap interacts with the enzyme's helical arch and that the helix-hairpin-helix motif interacts with the template strand in the downstream duplex eight base-pairs from the cleavage site. This model suggests specific interactions between the 3' end of the upstream oligonucleotide and the enzyme. The proposed structure presents the first detailed description of substrate recognition by structure-specific 5' nucleases.


Assuntos
DNA/química , DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Modelos Moleculares , Sequência de Bases , Simulação por Computador , Metilação de DNA , Análise Mutacional de DNA , Endonucleases Flap , Compostos Organofosforados/química , Pyrococcus furiosus/enzimologia , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...