268 LIQUID BIOPSY IN ESOPHAGEAL ADENOCARCINOMA: ROLE OF LINE-1 HYPOMETHYLATION

2020 ◽  
Vol 33 (Supplement_1) ◽  
Author(s):  
R Alfieri ◽  
E Boldrin ◽  
M Mazza ◽  
S Realdon ◽  
M Fassan ◽  
...  
Keyword(s):  
Esophageal Adenocarcinoma ◽  
Methodological Approach ◽  
Methylation Status ◽  
Neoadjuvant Treatment ◽  
Dna Amplification ◽  
Restriction Enzymes ◽  
Clinical Staging ◽  
Low Grade ◽  
The Difference ◽  
Methylation Patterns

Abstract   Esophageal Adenocarcinoma (EADC) prognosis is still poor. Clinical staging doesn’t allow accurate prediction of response to neoadjuvant treatment and survival. Molecular characterization of EADC is an expanding field aimed to predict treatment response and prognosis. Hypomethylation of Long Interspersed Nuclear Element 1 (LINE-1) sequences recently emerged as a frequent epigenetic alteration in EADC. Aim of the study was to evaluate methylation patterns of LINE-1 that is considered a good surrogate of global DNA methylation. Methods We investigated cell free DNA (cfDNA) of 21 patients, 19 with EADC and 2 with Barrett’s esophagus (BE). To verify the possibility to use LINE-1 methylation status to predict tumor behaviour, we extended the analysis to a few longitudinal cases, one EADC and two BE. One BE patient progressed to low-grade dysplasia (LGD) and high-grade dysplasia (HGD)/EADC during surveillance, while the other remains stable. LINE-1 promoter methylation was analyzed using restriction enzymes and DNA amplification. Results Using this approach, we were able to detect the presence of hypomethylated LINE-1 sequences in EADC cfDNA. The difference in methylation level between cfDNA and constitutive DNA was statistically significant (p = 0.0001). In one EADC patient, treated with esophagectomy, LINE-1 hypomethylation was found at lower level after resection and at higher level at recurrence. In another patient affected by BE, LINE-1 hypomethylation increased during the progression from metaplastic epithelium to EADC, switching back to the level of constitutive DNA after endoscopic resection. The third patient, with stable BE during 4 years follow-up, showed non-significant variations in methylation status of LINE-1. Conclusion Our study demonstrated the feasibility of our methodological approach to detect hypomethylation events in EADC patient cfDNA. Longitudinal preliminary analysis suggested a correlation between methylation status of LINE-1 and tumor progression. Further studies on larger population are needed to confirm these encouraging data. LINE-1 methylation analysis could be useful as a new molecular assay to integrate patient monitoring.

scholarly journals Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients

2020 ◽  
Vol 21 (4) ◽  
pp. 1547 ◽  
Author(s):  
Elisa Boldrin ◽  
Matteo Curtarello ◽  
Marco Dallan ◽  
Rita Alfieri ◽  
Stefano Realdon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Esophageal Adenocarcinoma ◽  
Methodological Approach ◽  
Methylation Status ◽  
Dna Amplification ◽  
Restriction Enzymes ◽  
Poor Quality ◽  
Bisulfite Conversion ◽  
Dna Hypomethylation ◽  
Methylation Patterns

DNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett’s esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring.

High Throughput Methylation Arrays Allow for Identification of Complex Methylation Patterns in MDS.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2437-2437
Author(s):  
Ying Jiang ◽  
Christine L. OKeefe ◽  
Andrew Dunbar ◽  
Anjali Advani ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
High Throughput ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Epigenetic Silencing ◽  
Low Grade ◽  
High Grade ◽  
Methylation Patterns ◽  
Hypermethylated Genes

Abstract Genomic imprinting and epigenetic silencing determine tissue-specific methylation patterns. Altered methylation of CpG islands within gene promoters has been hypothesized as one pathogenetic mechanism operative in myelodysplastic syndrome (MDS). Promoter hypermethylation of various empirically selected tumor suppressor genes has been found in MDS prompting application of hypomethylating drugs in this disease. Identification of hypermethylated genes predicting response to these drugs would have a major impact on clinical practice. However, to date methylation-based prognostic algorithms have not been established. Global analysis of DNA methylation patterns may help to identify hypermethylated genes/promoters associated with the pathogenesis of MDS. Recently, microarray-based DNA methylation analysis platforms enabled a powerful, high-throughput analysis of the methylation status of hundreds of genes. The GoldenGate Methylation Cancer Panel I, spanning 1,536 independent CpG sites selected from 807 selected genes was applied to determine the methylation status in MDS patients (N=51; 21 low grade (RA, MDS-U, RARS or RCMD), 26 high grade (AML or RAEB) and 4 CMML). The methylation status was determined based on an internal reference and compared to healthy controls (N=22). Methylation values were averaged among the patients or analyzed separately for each patient in comparison to average values obtained in controls. Overall, controls showed a lesser degree of methylation than advanced MDS patients (average intensity 0.326 vs. 0.339, p<0.05). Subsequently, we concentrated on hypermethylated genes. There were no genes uniformly hypermethylated in all patients. For 70%, 50%, and 30% of patients with advanced MDS, 1, 26, and 85 loci were concordantly hypermethylated, while in 70%, 50% and 30% of low risk patients 5, 23 and 31 were hypermethylated, respectively. The most consistently hypermethylated genes (>50% of patients), included tumor suppressor genes (DCC, SLC22A18, FAT, TUSC3), genes involved in DNA repair (OGG1, DDB2, BCR, PARP1), cell cycle control (DBC1, SMARCB1), differentiation (MYOD1, TDGF1, FGF2, NOTCH4) and apoptosis (HDAC1, ALOX12, AXIN1). Despite the variability, the aberrant methylation spectrum in CMML, low grade MDS and high grade MDS showed significant overlap (for example FZD9, IL16, EVI2A, MBD2 and BCR), which suggests that these genes may relate to the common tumorigenesis in MDS. Certain genes show specific methylation correlating to the morphologic diagnosis and may serve as diagnostic markers. For example, the promoter of HDAC1 is hypomethylated in 81% of sAML/RAEB1/2 patients but hypermethylated in 81% of low risk cases. To assess the link between epigenetic changes and chromosomal abnormalities, we also investigated methylation pattern of MDS with del5q for selected genes at the 5q locus. Some genes that are involved in apoptosis (WNT1, TNF receptor) and proliferation (MAP3K8, CSF3) were found to be hypermethylated in comparison to controls, suggesting that epigenetic silencing may enhance the effect of haploinsuffciency for some of the genes. In sum, our study, the first application of a high-throughput microarray methylation assay in MDS, demonstrates that complex methylation patterns exist in MDS and may allow for identification for clinically relevant methylation markers.

scholarly journals Quantitative High-Resolution CpG Island Mapping with Pyrosequencing™ Reveals Disease-Specific Methylation Patterns of the CDKN2B Gene in Myelodysplastic Syndrome and Myeloid Leukemia

2007 ◽  
Vol 53 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Kai Brakensiek ◽  
Luzie U Wingen ◽  
Florian Länger ◽  
Hans Kreipe ◽  
Ulrich Lehmann
Keyword(s):  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Cpg Island ◽  
Methylation Status ◽  
Low Grade ◽  
Myeloid Malignancies ◽  
Cpg Sites ◽  
Methylation Patterns ◽  
Cdkn2b Gene ◽  
Disease Specific

Abstract Background: Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Methylation patterns are often very heterogeneous, however, presenting a serious challenge for the development of methylation assays for diagnostic purposes. Methods: We used Pyrosequencing™ technology to determine the methylation status of 68 CpG sites in the CpG island of the CDKN2B gene [cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], frequently hypermethylated in myeloid malignancies, in a series of bone marrow samples from patients with myelodysplasia and myeloid leukemia (n = 82) and from 32 controls. A total of 7762 individual methylation sites were quantitatively evaluated. Precision and reproducibility of the quantification was evaluated with several overlapping primers. Results: The use of optimized sequencing primers and the new Pyro Q-CpG™ software enabled precise and reproducible quantification with a single sequencing primer of up to 15 CpG sites distributed over ∼100 bp. Extensive statistical analyses of the whole CpG island revealed for the first time disease-specific methylation patterns of the CDKN2B gene in myeloid malignancies and small regions of differential methylation with high discriminatory power that enabled differentiation of even low-grade myelodysplastic syndrome samples from the controls, a result that was confirmed in an independent group of 9 control and 36 patient samples. Conclusion: The precise quantitative methylation mapping of whole CpG islands is now possible with Pyrosequencing software in combination with optimized sequencing primers. This method reveals disease-specific methylation patterns and enables the development of specific diagnostic assays.

scholarly journals Gene Promoter Methylation Patterns throughout the Process of Cervical Carcinogenesis

2010 ◽  
Vol 32 (1-2) ◽  
pp. 131-143
Author(s):  
Nan Yang ◽  
Esther R. Nijhuis ◽  
Haukeline H. Volders ◽  
Jasper J. H. Eijsink ◽  
Ágnes Lendvai ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Methylation Status ◽  
Gene Promoter ◽  
Low Grade ◽  
Depth Of Invasion ◽  
Cervical Carcinogenesis ◽  
Normal Epithelium ◽  
Methylation Frequency ◽  
Cervical Cancers ◽  
Methylation Patterns

Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL).Methods: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings.Results: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-β2 at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p < 0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p < 0.05, 30% vs. 58%, p < 0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p < 0.05) and lymph vascular space involvement (p < 0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p < 0.05).Conclusion: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions.

Frequent Methylation Silencing of p15INK4b(MTS2) and p16INK4a (MTS1) in B-Cell and T-Cell Lymphomas

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1773-1781 ◽  
Author(s):  
Audrey S. Baur ◽  
Phil Shaw ◽  
Nathalie Burri ◽  
Françoise Delacrétaz ◽  
Fred T. Bosman ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Methylation Status ◽  
Low Grade ◽  
B Cell Lymphomas ◽  
Reverse Transcription Pcr ◽  
Specific Pcr ◽  
T Cell Lymphomas ◽  
Exon 1 ◽  
Methylation Patterns

The methylation status of p15INK4b(MTS2), p16INK4a (MTS1) andp14ARF (p16β) was analyzed in 56 lymphomas by restriction-enzyme related polymerase chain reaction (PCR) (REP), methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS). Methylation of the p15 andp16 genes was detected, respectively, in 64% and 32% of the B-cell lymphomas, in 44% and 22% of the T-cell lymphomas, and in none of the 5 reactive lymph nodes analyzed. Both p15 andp16 genes were methylated more often in the high-grade (78% and 50%, respectively) than in the low-grade B-cell lymphomas (55% and 21%, respectively). For 5 cases, mapping of the methylated CpGs of the p16 promoter region confirmed the results of REP and MSP. In addition, a large variation in the methylation patterns ofp16 exon 1 was observed, not only from one lymphoma to another, but also within a given tumor. Methylation of p15 andp16 was associated with an absence of gene expression, as assessed by reverse transcription-PCR. The p14 gene was unmethylated and normally expressed in all 56 tumors. We found no mutations of p15, p16, or p14 in any of the 56 lymphomas. Our results suggest a role for p15 and p16gene methylation during lymphomagenesis and a possible association between p15 and p16 inactivation and aggressive transformation in B-cell and T-cell lymphomas.

Frequent Methylation Silencing of p15INK4b(MTS2) and p16INK4a (MTS1) in B-Cell and T-Cell Lymphomas

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1773-1781 ◽  
Author(s):  
Audrey S. Baur ◽  
Phil Shaw ◽  
Nathalie Burri ◽  
Françoise Delacrétaz ◽  
Fred T. Bosman ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Methylation Status ◽  
Low Grade ◽  
B Cell Lymphomas ◽  
Reverse Transcription Pcr ◽  
Specific Pcr ◽  
T Cell Lymphomas ◽  
Exon 1 ◽  
Methylation Patterns

Abstract The methylation status of p15INK4b(MTS2), p16INK4a (MTS1) andp14ARF (p16β) was analyzed in 56 lymphomas by restriction-enzyme related polymerase chain reaction (PCR) (REP), methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS). Methylation of the p15 andp16 genes was detected, respectively, in 64% and 32% of the B-cell lymphomas, in 44% and 22% of the T-cell lymphomas, and in none of the 5 reactive lymph nodes analyzed. Both p15 andp16 genes were methylated more often in the high-grade (78% and 50%, respectively) than in the low-grade B-cell lymphomas (55% and 21%, respectively). For 5 cases, mapping of the methylated CpGs of the p16 promoter region confirmed the results of REP and MSP. In addition, a large variation in the methylation patterns ofp16 exon 1 was observed, not only from one lymphoma to another, but also within a given tumor. Methylation of p15 andp16 was associated with an absence of gene expression, as assessed by reverse transcription-PCR. The p14 gene was unmethylated and normally expressed in all 56 tumors. We found no mutations of p15, p16, or p14 in any of the 56 lymphomas. Our results suggest a role for p15 and p16gene methylation during lymphomagenesis and a possible association between p15 and p16 inactivation and aggressive transformation in B-cell and T-cell lymphomas.

Dynamic Scintigraphy of the Oesophagus in the Evaluation of Reflux Oesophagitis

1990 ◽  
Vol 29 (05) ◽  
pp. 204-209
Author(s):  
B. Ugarković ◽  
D. Ivančević ◽  
D. Babić ◽  
Ž. Babić
Keyword(s):  
Reflux Oesophagitis ◽  
Oesophageal Reflux ◽  
Low Grade ◽  
Time Activity ◽  
The Difference ◽  
Asymptomatic Subjects ◽  
Oesophageal Transit ◽  
Dynamic Scintigraphy ◽  
Positive Results ◽  
Reflux Œsophagitis

A method is presented which combines gastro-oesophageal reflux quantification and oesophageal transit measurement so as to differentiate true reflux from residual oesophageal activity. A group of 33 subjects with gastro-oesophageal reflux symptoms and endoscopically confirmed reflux oesophagitis and a group of 21 asymptomatic subjects with normal oesophageal, gastric and duodenal endoscopic findings were examined. The subjects were given 37 MBq 99mTc-Sn-colloid in saline orally and then scintiscanned dynamically. The gastro-oesophageal quantification was done after transit measurement and after the oesophageal time activity (to detect residual oesophageal activity) reached its minimum. The difference in the reflux indices between the two groups was highly significant. In low-grade oesophagitis measured reflux was lower than in higher grades of disease. Only 4.7% false-positive results were observed with a specificity of 95%, indicating that this method may be superior to methods published earlier.

scholarly journals Low-grade serous epithelial ovarian cancer: a comprehensive review and update for radiologists

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sofia Amante ◽  
Filipa Santos ◽  
Teresa Margarida Cunha
Keyword(s):  
Ovarian Cancer ◽  
Serous Carcinoma ◽  
Low Grade ◽  
Resonance Imaging ◽  
Clinicopathologic Characteristics ◽  
Borderline Tumour ◽  
Molecular Features ◽  
Serous Epithelial Ovarian Cancer ◽  
The Difference

AbstractLow-grade serous carcinoma (LGSC) is an infrequent subtype of ovarian cancer, corresponding to 5% of epithelial neoplasms. This subtype of ovarian carcinoma characteristically has molecular features, pathogenesis, clinical behaviour, sensitivity to chemotherapy, and prognosis distinct to high-grade serous carcinoma (HGSC). Knowing the difference between LGSC and other ovarian serous tumours is vital to guide clinical management, which currently is only possible histologically. However, imaging can provide several clues that allow differentiating LGSC from other tumours and enable precise staging and follow-up of ovarian cancer treatment. Characteristically, LGSC appears as mixed lesions with variable papillary projections and solid components, usually in different proportions from those detected in serous borderline tumour and HGSC. Calcified extracellular bodies, known as psammoma bodies, are also a common feature of LGSC, frequently detectable within lymphadenopathies and metastases associated with this type of tumour. In addition, the characterisation of magnetic resonance imaging enhancement also plays an essential role in calculating the probability of malignancy of these lesions. As such, in this review, we discuss and update the distinct radiological modalities features and the clinicopathologic characteristics of LGSC to allow radiologists to be familiarised with them and to narrow the differential diagnosis when facing this type of tumour.

Insights into Heap Bioleaching at the Agglomerate-Scale

2017 ◽  
Vol 262 ◽  
pp. 185-188 ◽  
Author(s):  
Alison Cox ◽  
Christopher G. Bryan
Keyword(s):  
Interstitial Cell ◽  
Cell Concentration ◽  
Low Grade ◽  
Dissolved Metals ◽  
Copper Ore ◽  
Cell Numbers ◽  
Liquid Phases ◽  
Heap Bioleaching ◽  
Cell Concentrations ◽  
The Difference

Previous agglomerate-scale heap bioleaching studies have outlined the variations in cell numbers of the liquid and attached phases during colonisation of sterilised ore by a pure culture. In this study, a mixed mesophilic culture was used in agglomerate-scale columns containing non-sterilised low-grade copper ore. Over a six - month period, columns were harvested at various intervals to provide snapshots of the metal distribution and the quantity, location, and ecological variations of mineral-oxidizing microbes within the ore bed. The initial colonisation period in this experiment was dissimilar to previous work, as the indigenous community was retained within the ore-bed throughout acid agglomeration. The overall colonisation phase lasted for approximately 1,000 hours until cell concentrations stabilised. In each column, less than 0.05% of the total cells were found in the leachate, 15-20% in the interstitial phase and the remaining ~80% were attached to the mineral surface. Once cell numbers had stabilised, interstitial cell concentrations were approximately 2,000× greater than those in the leachate. This difference persisted for the duration of the experiment. Copper concentrations in the two liquid phases generally decreased over time, but were on average 50× higher in the interstitial phase. Iron concentrations were more stable, but again were 30× higher in the interstitial phase. This demonstrates that that the difference in cell concentration between the leachate and interstitial phases cannot be explained through diffusion gradients within the system as it is much greater than those observed for the dissolved metals. It also shows that the specific environmental conditions of the interstitial and attached cells are very different to those inferred through analysis of leachates alone.

Soil stripping and replacement for the rehabilitation of bauxite-mined land at Weipa. I. Initial changes to soil organic matter and related parameters

Soil Research ◽  
2000 ◽  
Vol 38 (2) ◽  
pp. 345 ◽  
Author(s):  
G. D. Schwenke ◽  
D. R. Mulligan ◽  
L. C. Bell
Keyword(s):  
Organic Matter ◽  
Soil Organic Matter ◽  
Field Experiments ◽  
Surface Soil ◽  
Soil Disturbance ◽  
Microbial Biomass C ◽  
Low Grade ◽  
Double Pass ◽  
Organic C ◽  
The Difference

At Weipa, in Queensland, Australia, sown tree and shrub species sometimes fail to establish on bauxite-mined land, possibly because surface-soil organic matter declines during soil stripping and replacement. We devised 2 field experiments to investigate the links between soil rehabilitation operations, organic matter decline, and revegetation failure. Experiment 1 compared two routinely practiced operations, dual-strip (DS) and stockpile soil, with double-pass (DP), an alternative method, and subsoil only, an occasional result of the DS operation. Other treatments included variations in stripping-time, ripping-time, fertiliser rate, and cultivation. Dilution of topsoil with subsoil, low-grade bauxite, and ironstone accounted for the 46% decline of surface-soil (0–10 cm) organic C in DS compared with pre-strip soil. In contrast, organic C in the surface-soil (0–10 cm) of DP plots (25.0 t/ha) closely resembled the pre-strip area (28.6 t/ha). However, profile (0–60 cm) organic C did not differ between DS (91.5 t/ha), DP (107 t/ha), and pre-strip soil (89.9 t/ha). Eighteen months after plots were sown with native vegetation, surface-soil (0–10 cm) organic C had declined by an average of 9% across all plots. In Experiment 2, we measured the potential for post-rehabilitation decline of organic matter in hand-stripped and replaced soil columns that simulated the DS operation. Soils were incubated in situ without organic inputs. After 1 year’s incubation, organic C had declined by up to 26% and microbial biomass C by up to 61%. The difference in organic C decline between vegetated replaced soils (Expt 1) and bare replaced soils (Expt 2) showed that organic inputs affect levels of organic matter more than soil disturbance. Where topsoil was replaced at the top of the profile (DP) and not ploughed, inputs from volunteer native grasses balanced oxidation losses and organic C levels did not decline.

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