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BACKGROUND: Colorectal (CRC) and lung adenocarcinoma share many genetic and pathological similarities. A circulating tumor DNA (ctDNA) test for CRC may also be useful for detection of lung adenocarcinoma. This study determined if a methylated BCAT1/IKZF1 ctDNA test for CRC can be used for detection of lung adenocarcinoma. PATIENTS AND METHODS: Circulating cell free DNA (ccfDNA) was extracted from plasma collected prospectively from healthy controls, patients in remission from CRC, patients with lung adenocarcinoma, and patients with isolated metastatic CRC lung lesions. Plasma ccfDNA was bisulfite converted and assessed for methylated BCAT1/IKZF1 by quantitative real-time PCR. Comparisons between the different patient groups for a positive ctDNA test (BCAT1 and/or IKZF1) and ctDNA levels (% of total ccfDNA), as well as any associations with clinicopathological and demographic features, were assessed. RESULTS: Methylated BCAT1/IKZF1 ctDNA was detected in 18/39 (46.2 %) patients with lung adenocarcinoma, which was significantly (p < 0.001) higher compared to healthy controls (49/606; 8.1 %) and patients in remission from CRC (22/171, 12.9 %). Patients with stage III/IV lung adenocarcinoma had higher BCAT1/IKZF1 ctDNA positivity compared to stage I/II cases (68.2 % vs 17.7 %, p < 0.01), where a significantly higher proportion tested positive for methylated IKZF1 ctDNA alone (54.6 % vs 5.9 %, p < 0.001). There was no difference in BCAT1/IKZF1 ctDNA test positivity between patients with stage IV primary lung adenocarcinoma (n = 17) compared to lung-metastasising CRC cases (n = 17; 70.6 % v 64.3 %). CONCLUSION: A ctDNA test measuring methylated BCAT1/IKZF1 can sensitively detect lung adenocarcinoma and may be a promising aid for detection of advanced disease. CLINICAL TRIAL REGISTRATIONS: Australian and New Zealand Clinical Trials Registry, www.anzctr.org.au, ACTRN12616001138471, ACTRN12611000318987.
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Adenocarcinoma de Pulmão , Biomarcadores Tumorais , Metilação de DNA , Fator de Transcrição Ikaros , Neoplasias Pulmonares , Humanos , Fator de Transcrição Ikaros/genética , Masculino , Feminino , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/sangue , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/diagnóstico , Pessoa de Meia-Idade , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Idoso , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Estudos Prospectivos , Adulto , Estudos de Casos e Controles , TransaminasesRESUMO
Background: Colorectal cancer (CRC) has a high rate of recurrence, in particular for advanced disease, but prognosis based on staging and pathology at surgery can have limited efficacy. The presence of circulating tumor DNA (ctDNA) at diagnosis could be used to improve the prediction for disease recurrence. Objectives: To assess the impact of detecting methylated BCAT1/IKZF1 ctDNA at diagnosis in combination with demographic, lifestyle, clinical factors and tumor pathology, to assess predictive value for recurrence. Design: A retrospective cohort study. Methods: The cohort included 180 patients (36 with recurrent CRC), who had undergone complete treatment and surveillance for a minimum of 3 years. Participant clinical details and ctDNA methylated BCAT1/IKZF1 results were compared between those with and without recurrence, and cox regression analysis assessed each factor on disease-free survival. Results: Clinical factors independently associated with reduced disease-free survival included nodal involvement (HR = 3.83, 95% CI 1.56-9.43, P = .003), M1 stage (HR = 4.41, 95% CI 1.18-16.45, P = .027), a resection margin less than 2 mm (HR = 4.60, 95% CI 1.19-17.76, P = .027), perineural involvement (HR = 2.50, 95% CI 1.01-6.17, P = .047) and distal tumors (HR = 3.13, 95% CI 1.07-9.18, P = .037). Methylated BCAT1/IKZF1 was detected in 51.7% (93/180) of pre-treatment plasma samples. When a positive ctDNA finding was considered in combination with these clinical prognostic factors, there was improved predictive power of recurrence for patients with perineural involvement (HR = 4.44, 95% CI 1.92-10.26, P < .001), and it marginally improved the predictive factor for M1 stage (HR = 7.59, 95% CI 2.30-25.07, P = .001) and distal tumors (HR = 5.04, 95% CI 1.88-13.49, P = .001). Conclusions: Nodal invasion, metastatic disease, distal tumor site, low resection margins and perineural invasion were associated with disease recurrence. Pre-treatment methylated ctDNA measurement can improve the predictive value for recurrence in a subset of patients, particularly those with perineural involvement. Registration: Australian and New Zealand Clinical Trials Registry #12611000318987.
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BACKGROUND: The risk of recurrence after completion of curative-intent treatment of colorectal cancer (CRC) is hard to predict. Post-treatment assaying for circulating tumor DNA (ctDNA) is an encouraging approach for stratifying patients for therapy, but the prognostic value of this approach is less explored. This study aimed to determine if detection of methylated BCAT1 and IKZF1 following completion of initial treatment identified patients with a poorer recurrence-free survival (RFS). METHODS: 142 CRC stage I-III cases with at least 2 years of follow up (unless recurrence was evident sooner) and a methylated BCAT1/IKZF1 test result between 2 weeks and 12 months after completion of initial treatment were eligible for study inclusion. The association between BCAT1/IKZF1 and RFS was assessed by the log-rank (Mantel-Cox) method. Cox proportional hazard regression analysis was used for multivariable survival analysis. RESULTS: Thirty-three (23.2%) had recurrence at a median 1.6y (interquartile range: 0.8-2.4). Methylated BCAT1/IKZF1 was detected in 19 of the 142 patients (13.4%) and was associated with a significant risk of recurrence (hazard ratio [HR] 5.7, 95%CI: 1.9-17.3, p = 0.002). Three-year RFS for patients with or without detectable methylated BCAT1/IKZF1 was 56.5% and 83.3%, respectively. Multivariable analysis showed that detection of methylated BCAT1/IKZF1 (HR = 2.6, p = 0.049) and site of the primary tumor (HR = 4.2, p = 0.002) were the only significant prognostic indicators of poor RFS. CONCLUSIONS: BCAT1/IKZF1 methylation testing after curative-intent treatment is an independent prognostic indicator for RFS and identifies a subgroup at high risk. Personalized surveillance is warranted for patients with these ctDNA biomarkers detectable after curative-intent treatment.
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Neoplasias Colorretais , Humanos , Prognóstico , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Recidiva Local de Neoplasia/genética , Fator de Transcrição Ikaros/genética , Biomarcadores Tumorais/genética , Transaminases/genéticaRESUMO
BACKGROUND: A blood assay measuring methylated BCAT1 and IKZF1 can detect recurrent colorectal cancer (CRC) with high sensitivity but suboptimal specificity. This study aimed to establish an upper reference limit (URL) of these biomarkers in a reference population without CRC, apply that threshold to detecting clinical recurrence in patients who had undergone definitive therapy for CRC, and compare the performance of the biomarkers with carcinoembryonic antigen (CEA). METHODS: The level of methylation was reported as the aggregate methylated BCAT1 and IKZF1 expressed as a percentage of total plasma DNA. A reference population of patients confirmed to have no colorectal neoplasia (n = 857) was used to determine the URL. Test accuracy for clinical recurrence was determined in a post-treatment surveillance population (n = 549; 77 recurrence cases). RESULTS: A methylation level of 0.07%, corresponding to the 98th percentile in the reference population, was set as the URL. In the surveillance population, 60 patients had methylation levels above 0.07%, and 81.7% of these had recurrence. In comparison with no minimum threshold being applied, assay sensitivity with a URL of 0.07% yielded similar sensitivity (63.6% [CI, 51.9%-74.3%] vs 64.9% [CI, 53.8%-74.7%]; P = .87) and higher specificity (97.7% [CI, 95.9%-98.8%] vs 91.3% [CI, 88.4%-93.5%]; P < .001). The BCAT1/IKZF1 test was 2.5-fold more sensitive than CEA for detecting recurrences considered amenable to surgery with curative intent (50.0% vs 20.8%; P = .016). CONCLUSIONS: Applying a threshold for positivity to the methylated BCAT1/IKZF1 blood assay improved the specificity for CRC recurrence without compromising sensitivity. Both the sensitivity and the specificity were superior to those of CEA.
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DNA Tumoral Circulante , Neoplasias Colorretais , Biomarcadores Tumorais/genética , Antígeno Carcinoembrionário , DNA Tumoral Circulante/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA , Humanos , Fator de Transcrição Ikaros/genética , Recidiva , TransaminasesRESUMO
BACKGROUND: Detection of circulating cell-free DNA (ccfDNA) methylated in BCAT1 and IKZF1 is sensitive for detection of colorectal cancer (CRC), but it is not known if these biomarkers are present in other common adenocarcinomas. OBJECTIVE: Compare methylation levels of BCAT1 and IKZF1 in tissue and plasma from breast, prostate, and colorectal cancer patients. METHODS: Blood was collected from 290 CRC, 32 breast and 101 prostate cancer patients, and 606 cancer-free controls. Tumor and matched normal tissues were collected at surgery: 26 breast, 9 prostate and 15 CRC. DNA methylation in BCAT1 and IKZF1 was measured in blood and tissues. RESULTS: Either biomarker was detected in blood from 175/290 (60.3%) of CRC patients. The detection rate was higher than that measured in controls (48/606 (8.1%), OR = 18.2, 95%CI: 11.1-29.0). The test positivity rates in breast and prostate cancer patients were 9.4% (3/32) and 6.9% (7/101), respectively, and not significantly different to that measured in gender-matched controls (8.0% (33/382) females (OR = 0.84, 95%CI: 0.23-3.1) and 7.6% (26/318) males (OR = 0.86, 95%CI: 0.65-2.1). In tumor and non-neoplastic tissues, 93.5% (14/15) of CRC tumors were methylated in BCAT1 and/or IKZF1 (p< 0.004). Only 11.5% (3/26) and 44.4% (4/9) (p= 0.083) of breast and prostate tumors were hypermethylated in these two genes. CONCLUSIONS: Detection of circulating DNA methylated in BCAT1 and IKZF1 is sensitive and specific for CRC but not breast or prostate cancer.
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Neoplasias Colorretais , Neoplasias da Próstata , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA , Metilação de DNA , Feminino , Humanos , Fator de Transcrição Ikaros/genética , Masculino , Neoplasias da Próstata/genética , Transaminases/genéticaRESUMO
Failure of colorectal cancer (CRC) treatment is due to residual disease, and its timely identification is critical for patient survival. Detecting CRC-associated mutations in patient circulating cell-free DNA is confounded by tumor mutation heterogeneity, requiring primary tumor sequencing to identify relevant mutations. In this study, we assessed BCAT1 and IKZF1 methylation levels to quantify circulating tumor DNA (ctDNA) and investigated whether this method can be used to assess tumor burden and efficacy of therapy. In 175 patients with CRC who were ctDNA-positive pretreatment, ctDNA levels were higher with advancing stage (P < 0.05) and correlated with tumor diameter (r = 0.35, P < 0.001) and volume (r = 0.58, P < 0.01). After completion of treatment (median of 70 days [IQR 49-109] after surgery, +/- radiotherapy, +/- chemotherapy), ctDNA levels were reduced in 98% (47/48) and were undetectable in 88% (42/48) of patients tested. For those with incomplete adjuvant chemotherapy after surgery, roughly half remained ctDNA-positive (11/21, 52.4%). The presence of ctDNA after treatment was associated with disease progression (HR 9.7, 95%CI 2.5-37.6) compared to no ctDNA. Assaying blood for ctDNA methylated in BCAT1/IKZF1 has the potential for identifying residual disease due to treatment failure, informing a potential need for therapy adjustment in advanced disease.
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DNA Tumoral Circulante , Neoplasias Colorretais , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Metilação de DNA/genética , Humanos , Fator de Transcrição Ikaros/genética , Transaminases/genética , Carga TumoralRESUMO
BACKGROUND: Differentially-methylated regions (DMRs) are characteristic of colorectal cancer (CRC) and some occur more frequently than common mutations. This study aimed to evaluate the clinical utility of assaying circulating cell-free DNA for methylation in BCAT1, IKZF1 and IRF4 for detection of CRC. METHODS: A multiplexed real-time PCR assay targeting DMRs in each of the three genes was developed. Assay accuracy was explored in plasma specimens banked from observational cross-sectional trials or from volunteers scheduled for colonoscopy or prior to CRC surgery. RESULTS: 1620 specimens were suitable for study inclusion including 184 and 616 cases with CRC and adenomas, respectively, and 820 cases without neoplasia (overall median age, 63.0 years; 56% males). Combining the PCR signals for all targeted DMRs returned the best sensitivity for CRC (136/184, 73.9%, 95% CI 67.1-79.7), advanced adenomas (53/337, 15.7%, 95% CI 12.0-20.1) and high-grade dysplastic (HGD) adenomas (9/35, 25.7%, 95% CI 14.0-42.3) with a 90.1%, specificity for neoplasia (739/820, 95% CI 87.9-92.0, p < 0.01). Detection of methylation in all three genes were more likely in CRC cases than those without it (OR 28.5, 95% CI 7.3-121.2, p < 0.0001). Of the 81 positive cases without neoplasia, 62 (76.5%) were positive by a single PCR replicate only and predominantly due to detection of methylated BCAT1 (53.2%). Single replicate positivity was significantly higher than that in CRC (26/136, 19.1%, p < 0.0001), and single BCAT1 replicate positivity was more likely in cases without neoplasia than in CRC (OR 17.7, 95% CI 6.6-43.3, p < 0.0001). When a positive result was limited to those with ≥ 1 PCR replicate positive for either IKZF1 or IRF4, or at least two replicates positive for BCAT1, the multi-panel test maintained a high sensitivity for CRC (131/184, 71.2%, 95% CI 64.3-77.3) and HGD adenomas (8/35, 22.9%, 95% CI 11.8-39.3, p = 0.029) but improved specificity significantly (772/820, 94.1%, 95% CI 92.3-95.6, p < 0.0001 vs. any PCR replicate positive). CONCLUSION: The multi-panel methylation assay differentiates cases with CRC from those without it and does so with high specificity when criteria for BCAT1 detection are applied. The marker panel is flexible and studies in those at average risk for CRC are now warranted to determine which panel configuration best suits screening goals. TRIAL REGISTRATION: ACTRN12611000318987. Registered 25 March 2011, https://www.anzctr.org.au/ ACTRN12611000318987.
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Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Metilação de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Programas de Rastreamento/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Most recurrences of early-stage colorectal cancer detected with current surveillance measures are widespread and incurable. Circulating tumor DNA (ctDNA) may facilitate earlier diagnosis of recurrent colorectal cancer and improve cancer-related outcomes. METHODS: Plasma from patients undergoing standard surveillance after definitive treatment for stage II/III colorectal cancer was assayed with COLVERA and carcinoembryonic antigen (CEA) at a single time point. Results were correlated with radiographic imaging. Assay performance, including sensitivity and specificity for recurrence, were compared. Impact of potentially confounding variables was also explored. RESULTS: 322 patients were included in the final analysis, and 27 recurrences were documented over a median follow-up period of 15 months. Sensitivity for recurrence was 63% [confidence interval (CI), 42.4-80.6] and 48% (CI, 28.7-68.1) for COLVERA and CEA (≥5 ng/mL), respectively (P = 0.046), while specificity was 91.5% (CI, 87.7-94.4) and 96.3% (CI, 93.4-98.1), respectively (P = 0.016). Smoking and age were independent predictors of CEA but not COLVERA positivity. CONCLUSIONS: COLVERA was more sensitive but less specific than CEA in detecting recurrent colorectal cancer. Short median follow-up may have been responsible for apparent false positives in COLVERA. Studies with serial sampling and longer follow-up are needed to assess whether earlier detection of colorectal cancer recurrence translates into clinical benefit. IMPACT: This prospective study showed that COLVERA (a two-gene ctDNA assay) was more sensitive for detection of recurrence in a cohort of patients undergoing surveillance after definitive therapy for stages II and III colorectal cancer.
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DNA Tumoral Circulante/metabolismo , Fator de Transcrição Ikaros/metabolismo , Transaminases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de NeoplasiasRESUMO
Salmonella Infantis is one of the five serovars most frequently causing human salmonellosis in Europe, mainly associated with poultry. A clone harbouring a conjugative plasmid of emerging S. Infantis (pESI)-like megaplasmid, carrying multidrug resistant (MDR) and extended-spectrum beta-lactamases (ESBL) genes, has spread in the Italian broiler chicken industry also causing human illness. This work is aimed at elucidating the molecular epidemiology of S. Infantis and pESI-like in Europe using whole-genome sequencing and bioinformatics analysis, and to investigate the genetic relatedness of S. Infantis clones and pESI-like from animals, meat, feed and humans provided by institutions of nine European countries. Two genotyping approaches were used: chromosome or plasmid SNP-based analysis and the minimum spanning tree (MST) algorithm based on core-genome multilocus sequence typing (cgMLST). The European S. Infantis population appeared heterogeneous, with different genetic clusters defined at core-genome level. However, pESI-like variants present in 64.1â% of the isolates were more genetically homogeneous and capable of infecting different clonal lineages in most of the countries. Two different pESI-like with ESBL genes (n=82) were observed: blaCTX-M-1-positive in European isolates and blaCTX-M-65-positive in American isolates (study outgroup). Both variants had toxin-antitoxin systems, resistance genes towards tetracyclines, trimethoprim, sulphonamides and aminoglycosides, heavy metals (merA) and disinfectants (qacEΔ). Worryingly, 66â% of the total isolates studied presented different gyrA chromosomal point mutations associated with (fluoro)quinolone resistance (MIC range 0.125-0.5 mg/L), while 18â% displayed transferable macrolide resistance mediated by mph, mef and erm(B) genes. Proper intervention strategies are needed to prevent further dissemination/transmission of MDR S. Infantis and pESI-like along the food chain in Europe.
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Tipagem de Sequências Multilocus/métodos , Plasmídeos/genética , Infecções por Salmonella/epidemiologia , Salmonella/classificação , Sequenciamento Completo do Genoma/métodos , Ração Animal/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Conjugação Genética , Farmacorresistência Bacteriana Múltipla , Europa (Continente)/epidemiologia , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Carne/microbiologia , Epidemiologia Molecular , Filogenia , Filogeografia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Salmonella/genética , Salmonella/isolamento & purificação , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: The sensitive detection of recurrent colorectal cancer (CRC) by the measurement of circulating tumor DNA (ctDNA) might improve the chance of a cure. This study compared a quantitative methylated ctDNA test with carcinoembryonic antigen (CEA) in the setting of surveillance for recurrence. METHODS: Blood samples collected either during surveillance or within 12 months of the confirmation of recurrence were assayed for ctDNA (methylated branched-chain amino acid transaminase 1 [BCAT1]/Ikaros family zinc-finger 1 protein [IKZF1]) and CEA. The optimal ctDNA threshold was determined by receiver operating characteristic analysis, and the test performance for the detection of recurrence was compared with CEA (5 ng/mL threshold). RESULTS: The study cohort comprised 144 eligible patients and included 50 recurrence events. The sensitivity of the methylated ctDNA test for recurrence was 66.0% (95% confidence interval [CI], 57.1%-69.3%), which was significantly higher than the sensitivity of CEA (31.9%; 95% CI, 22.8%-36.6%; P < .001). The sensitivity for resectable recurrence (n = 20) was also higher (ctDNA, 60.0%; CEA, 20.0%; P = .01). The specificity did not differ between the tests (ctDNA, 97.9%; 95% CI, 93.2%-99.6%; CEA, 96.4%; 95% CI, 91.4%-99.0%). When adjustments were made for other predictors of the presence of recurrence, a positive ctDNA test was an independent predictor (odds ratio, 155.7; 95% CI, 17.9-1360.6; P < .001), whereas CEA was not (odds ratio, 2.5; 95% CI, 0.3-20.6; P = .407). CONCLUSIONS: The quantitative ctDNA test showed superior sensitivity in comparison with CEA without a difference in the specificity for detecting recurrent CRC. Longitudinal studies are warranted to further assess the utility (specifically the survival benefit) of methylated BCAT1/IKZF1 ctDNA in the surveillance of patients with CRC.
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Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/análise , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Antígeno Carcinoembrionário/sangue , Epigênese Genética , Feminino , Humanos , Fator de Transcrição Ikaros/sangue , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Transaminases/sangueRESUMO
Suboptimal participation is commonly observed in colorectal cancer screening programs utilizing fecal tests. This randomized controlled trial tested whether the offer of a blood test as either a "rescue" strategy for fecal test nonparticipants or an upfront choice, could improve participation. A total of 1,800 people (50-74 years) were randomized to control, rescue, or choice groups (n = 600/group). All were mailed a fecal immunochemical test (FIT, OC-Sensor, Eiken Chemical Company) and a survey assessing awareness of the screening tests. The rescue group was offered a blood test 12 weeks after FIT nonparticipation. The choice group was given the opportunity to choose to do a blood test (Colvera, Clinical Genomics) instead of FIT at baseline. Participation with any test after 24 weeks was not significantly different between groups (control, 37.8%; rescue, 36.9%; choice, 33.8%; P > 0.05). When the rescue strategy was offered after 12 weeks, an additional 6.5% participated with the blood test, which was greater than the blood test participation when offered as an upfront choice (1.5%; P < 0.001). Awareness of the tests was greater for FIT than for blood (96.2% vs. 23.1%; P < 0.0001). In a population familiar with FIT screening, provision of a blood test either as a rescue of FIT nonparticipants or as an upfront choice did not increase overall participation. This might reflect a lack of awareness of the blood test for screening compared with FIT.
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Análise Química do Sangue , Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer/métodos , Sangue Oculto , Participação do Paciente/estatística & dados numéricos , Idoso , Análise Química do Sangue/métodos , Análise Química do Sangue/psicologia , Análise Química do Sangue/estatística & dados numéricos , Comportamento de Escolha , Neoplasias Colorretais/psicologia , Detecção Precoce de Câncer/psicologia , Detecção Precoce de Câncer/estatística & dados numéricos , Feminino , Humanos , Masculino , Programas de Rastreamento/métodos , Programas de Rastreamento/psicologia , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Aceitação pelo Paciente de Cuidados de Saúde/psicologia , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Fatores Socioeconômicos , Austrália do Sul/epidemiologiaRESUMO
Gastrointestinal cancers, including oesophageal, gastric and colorectal cancers (CRC) have high rates of disease recurrence despite curative resection. There are a number of recent studies that have investigated the use of circulating tumor DNA (ctDNA) for prognostic value in these cancers. We reviewed studies that had been published prior to March 2018 that assessed the prognostic values of ctDNA in patients with oesophageal and gastric cancers, gastrointestinal stromal tumors (GIST) and CRC. We identified 63 eligible clinical studies that focussed on recurrence and survival. Studies assessed investigated various ctDNA biomarkers in patients with different stages of cancer undergoing surgical resection, chemotherapy and no treatment. For oesophageal squamous cell carcinoma and oesophageal adenocarcinoma, methylation of certain genes such as APC and DAPK have been highlighted as promising biomarkers for prognostication, but these studies are limited and more comprehensive research is needed. Studies focusing on gastric cancer patients showed that methylation of ctDNA in SOX17 and APC were independently associated with poor survival. Two studies demonstrated an association between ctDNA and recurrence and survival in GIST patients, but more studies are needed for this type of gastrointestinal cancer. A large proportion of the literature was on CRC which identified both somatic mutations and DNA methylation biomarkers to determine prognosis. ctDNA biomarkers that identified somatic mutations were more effective if they were personalized based on mutations found in the primary tumor tissue, but ctDNA methylation studies identified various biomarkers that predicted increased risk of recurrence, poor disease free survival and overall survival. While the use of non-invasive ctDNA biomarkers for prognosis is promising, larger studies are needed to validate the clinical utility for optimizing treatment and surveillance strategies to reduce mortality from gastrointestinal cancers.
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PURPOSE: Methylation in IKZF1 and BCAT1 are common events in colorectal cancer (CRC). They are often detected in blood as circulating tumor DNA (ctDNA) at diagnosis and disappear after surgery in most CRC patients. A prospective study was conducted to determine the relationship between detection of these markers following surgery and risk for residual disease and for recurrence. METHODS: ctDNA status with methylated BCAT1 and IKZF1 was determined within 12 months of surgical resection of CRC, and was related to presence of or risk for residual disease (margins involved, metastases present or nature of node involvement), and to recurrence-free survival. RESULTS: Blood was collected from 172 CRC patients after surgery and 28 (16%) were ctDNA positive. Recurrence was diagnosed in 23 of the 138 with clinical follow-up after surgery (median follow-up 23.3 months, IQR 14.3-29.5). Multivariate modeling indicated that features suggestive of residual disease were an independent predictor of post-surgery ctDNA status: cases with any of three features (close resection margins, apical node involved, or distant metastases) were 5.3 times (95% CI 1.5-18.4, p = 0.008) more likely to be ctDNA positive. Multivariate analysis showed that post-surgery ctDNA positivity was independently associated with an increased risk of recurrence (HR 3.8, 1.5-9.5, p = 0.004). CONCLUSIONS: CRC cases positive for methylated ctDNA after surgery are at increased risk of residual disease and subsequently recurrence. This could have implications for guiding recommendations for adjuvant therapy and surveillance strategies. Randomized studies are now indicated to determine if monitoring cases with these biomarkers leads to survival benefit.
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DNA Tumoral Circulante/genética , Neoplasias Colorretais/genética , Metilação de DNA , Idoso , Idoso de 80 Anos ou mais , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Intervalo Livre de Doença , Feminino , Predisposição Genética para Doença , Humanos , Fator de Transcrição Ikaros/genética , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Estadiamento de Neoplasias , Neoplasia Residual , Estudos Prospectivos , RNA Longo não Codificante/genéticaRESUMO
Background: Cell-free circulating tumour-derived DNA (ctDNA) can be detected by testing for methylated BCAT1 and IKZF1 DNA, which has proven sensitivity for colorectal cancer (CRC). A prospective correlative biomarker study between presence of methylated BCAT1 and IKZF1 in tissue and blood was conducted in cases with CRC to explore how detection of such ctDNA biomarkers relates to cancer characteristics, methylation in tissue and surgical resection of the primary cancer. Methods: Enrolled patients with invasive CRC had blood collected at diagnosis, prior to any treatment or surgery (peri-diagnostic sample). A subgroup of patients also had cancer and adjacent non-neoplastic tissue collected at surgical resection, as well as a second blood sample collected within 12 months of surgery (post-surgery sample). DNA was extracted from all samples and assayed for methylated BCAT1 and IKZF1 to determine the degree of methylation in tissue and the presence of ctDNA in blood. Results: Of 187 cases providing peri-diagnostic blood samples, tissue was available in 91, and 93 provided at least one post-surgery blood sample for marker analysis. Significant methylation of either BCAT1 or IKZF1 was seen in 86/91 (94.5%) cancer tissues, with levels independent of stage and higher than that observed in adjacent non-neoplastic specimens (P < 0.001). ctDNA methylated in BCAT1 or IKZF1 was detected in 116 (62.0%) cases at diagnosis and was significantly more likely to be detected with later stage (P < 0.001) and distal tumour location (P = 0.004). Of the 91 patients who provided pre-and post-surgery blood samples, 47 patients were ctDNA-positive at diagnosis and 35 (74.5%) became negative after tumour resection. Conclusion: This study has shown that BCAT1 and IKZF1 methylation are common events in CRC with almost all cancer tissues showing significant levels of methylation in the two genes. The presence of ctDNA in blood is stage-related and show rapid reversion to negative following surgical resection. Monitoring methylated BCAT1 and IKZF1 levels could therefore inform adequacy of surgical resection. Trial registration: Australian New Zealand Clinical Trial Registry number 12611000318987. Registered 25 March 2011.
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DNA Tumoral Circulante/genética , Neoplasias Colorretais/genética , Fator de Transcrição Ikaros/genética , Transaminases/genética , Biomarcadores Tumorais/genética , Colo/química , Colo/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/cirurgia , Metilação de DNA , Epigênese Genética , Feminino , Humanos , Masculino , Estadiamento de Neoplasias , Estudos Prospectivos , Reto/química , Reto/patologiaRESUMO
The genes BCAT1 and IKZF1 are hypermethylated in colorectal cancer (CRC), but little is known about how this relates to gene expression. This study assessed the relationship between methylation and gene expression of BCAT1 and IKZF1 in CRC and adjacent non-neoplastic tissues. The tissues were obtained at surgery from 36 patients diagnosed with different stages of CRC (stage I n = 8, stage II n = 13, stage III n = 10, stage IV n = 5). Methylated BCAT1 and IKZF1 were detected in 92% and 72% CRC tissues, respectively, with levels independent of stage (P > .05). In contrast, only 31% and 3% of non-neoplastic tissues were methylated for BCAT1 and IKZF1, respectively (P < .001). The IKZF1 messenger RNA (mRNA) expression was significantly lower in the cancer tissues compared with that of non-neoplastic tissues, whereas the BCAT1 mRNA levels were similar. The latter may be due to the BCAT1 polymerase chain reaction assay detecting more than 1 mRNA transcript. Further studies are warranted to establish the role of the epigenetic silencing of IKZF1 in colorectal oncogenesis.
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Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.
Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Proteínas de Escherichia coli/genética , Plasmídeos/genética , Salmonella/efeitos dos fármacos , Salmonella/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/microbiologia , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas de Membrana , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Multiplex , Plasmídeos/metabolismo , Salmonella/isolamento & purificação , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
BACKGROUND: Colvera™ is a test that detects circulating tumor-derived DNA in patients with colorectal cancer by assaying for the presence of methylated BCAT1 and IKZF1 in blood. This study describes the analytical and clinical performance characteristics of the test. METHODS: Validation was performed in accordance with ISO15189 and National Pathology Accreditation Advisory Council requirements. Spiked samples including 264 plasma and 120 buffer samples were randomized, divided into 8 batches of 48 samples, and processed over 8 days using 2 equipment lines (each line consisting of a QIAsymphony SP/AS, QIACube HT, and LC480); 2 reagent batches; and 2 operators to determine limit of detection, selectivity/specificity, precision, reproducibility, ruggedness, and susceptibility to commonly known interfering substances. Clinical performance was validated by assaying 222 archived plasma samples from subjects (n = 26 with cancer) enrolled in a previous prospective trial. RESULTS: The limit of detection for Colvera was 12.6 pg/mL (95% CI, 8.6-23.9 pg/mL), which equates to 2 diploid genome copies per milliliter plasma. No statistically significant difference was determined between testing days (n = 8), instrumentation, operators, or reagent batches in precision studies for the methylation-specific assays. The assay performance was unaffected by 9 commonly known interference substances, variations in bisulfite conversion, or quantitative PCR settings (cycling temperatures, incubation times, and oligonucleotide concentrations). For this clinical cohort, sensitivity and specificity estimates for Colvera were 73.1% (19 of 26; 95% CI, 52.2-88.4) and 89.3% (175 of 196; 95% CI, 84.1-93.2), respectively. CONCLUSION: Colvera is a robust test and suitable for detection of circulating tumor-derived DNA by measuring levels of methylated BCAT1 and IKZF1 in human blood plasma.
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Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.
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Recurrence will develop in 30-50% of colorectal cancer (CRC) cases despite apparent clearance following treatment. Carcinoembryonic antigen (CEA) is the only guideline-recommended blood test for monitoring cases for recurrence, but its sensitivity and specificity are suboptimal. This observational study compared a novel 2-gene (methylated BCAT1 and IKZF1 DNA) blood test with CEA for detection of recurrent CRC. We conducted a paired comparison of the BCAT1/IKZF1 test with CEA (cut-off 5 ng/mL) in blood from patients in remission after treatment for primary CRC and undergoing surveillance. Blood collected in the 12 months prior to or 3 months after complete investigational assessment of recurrence status were assayed and the results compared by McNemar's test. Of 397 patients enrolled, 220 underwent satisfactory assessment for recurrence and 122 had blood testing performed within the prescribed period. In 28 cases with recurrent CRC, CEA was positive in 9 (32%; 95% CI 16-52%) compared to 19 (68%; 95% CI 48-84%) positive for methylated BCAT1/IKZF1 (P = 0.002). All samples that were CEA positive were also BCAT1/IKZF1 positive. In 94 patients without clinically detectable recurrence, CEA was positive in 6 (6%, 95% CI 2-13%) and BCAT1/IKZF1 in 12 (13%, 95% CI 7-21%), P = 0.210. The odds ratio of a positive CEA test for recurrence was 6.9 (95% CI 2-22) compared to 14.4 (5-39) for BCAT1/IKZF1. The BCAT1/IKZF1 test was more sensitive for recurrence than CEA and the odds of recurrence given a positive test was twice that of CEA. The BCAT1/IKZF1 test should be further considered for monitoring cases for recurrence.
Assuntos
Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/diagnóstico , Metilação de DNA , Fator de Transcrição Ikaros/sangue , Recidiva Local de Neoplasia/diagnóstico , Transaminases/sangue , Idoso , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Estudos Transversais , Feminino , Humanos , Fator de Transcrição Ikaros/genética , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/genética , Sensibilidade e Especificidade , Transaminases/genética , Conduta ExpectanteRESUMO
OBJECTIVES: To compare the performance of a new blood test for colorectal cancer (CRC) to an established fecal immunochemical test (FIT) in a study population with the full range of neoplastic and non-neoplastic pathologies encountered in the colon and rectum. METHODS: Volunteers were asked to complete a FIT prior to colonoscopy. Blood was collected after bowel preparation but prior to colonoscopy, and plasma was assayed for the presence of methylated BCAT1 and IKZF1 DNA using a multiplex real-time PCR assay. Sensitivity and specificity estimates for the blood test were calculated from true- and false-positive rates for neoplasia and compared with FIT at a range of fecal hemoglobin (Hb) concentration positivity thresholds. RESULTS: In total, 1,381 volunteers (median age 64 years; 49% male) completed both tests prior to colonoscopy. Estimated sensitivity of the BCAT1/IKZF1 blood test for CRC was 62% (41/66; 95% confidence interval 49-74%) with a specificity of 92% (1207/1315; 90-93%). FIT returned the same specificity at a cutoff of 60 µg Hb/g, at which its corresponding sensitivity for cancer was 64% (42/66; 51-75%). In the range of commonly used FIT cutoffs, respective cancer sensitivity and specificity estimates with FIT were: 59% (46-71%) and 93% (92-95%) at 80 µg Hb/g, and 79% (67-88%) and 81% (78-83%) at 10 µg Hb/g. Although estimated sensitivities were not significantly different between the two tests for any stage of cancer, FIT showed a significantly higher sensitivity for advanced adenoma at the lower cutoffs. Specificity of FIT, but not of the BCAT1/IKZF1 blood test, deteriorated substantially in people with overt blood in the feces. When combining FIT (cutoff 10 µg Hb/g) with the BCAT1/IKZF1 blood test, sensitivity for cancer was 89% (79-96%) at 74% (72-77%) specificity. CONCLUSIONS: A test based on detection of methylated BCAT1/IKZF1 DNA in blood has comparable sensitivity but better specificity for CRC than FIT at the commonly used positivity threshold of 10 µg Hb/g. Further evaluation of the new test relative to FIT in the population screening context is now required to fully understand the potential advantages and disadvantages of these biomarkers in screening.