Comparison of Native Aspirates and Cytological Smears Obtained by EUS-Guided Biopsies for Effective DNA/RNA Marker Testing in Pancreatic Cancer

Benešová, Lucie; Halkova, Tereza; Bunganič, Bohuš; Belšánová, Barbora; Zavoral, Miroslav; Traboulsi, Eva; Minárik, Marek

doi:10.1007/s12253-018-0490-9

Cited by 5 publications

(9 citation statements)

References 28 publications

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“…Thus, it has been hypothesized that analysis of cytological smears from EUS-FNA would be a better approach for the determination of miRNA levels as this would allow a precise evaluation of the fraction and representation of tumor cells. As mentioned before, Benesova et al[15] showed how the overall amount of RNA extracted from air-dried cytological smears was lower compared to tissue acquired with EUS-FNA and put in RNALater, but gave reliable results with clinical validity ( i.e ., prognostic role of miR-21).…”

Section: Feasibility and Clinical Utility Of Pancreatic Eus-fna/fnb Fmentioning

confidence: 99%

“…Nevertheless, although EUS-FNA allows the extraction of DNA/RNA from the pancreatic sample, the absence of a pre-evaluation of this sample by a cytologist does not allow certainty regarding tumor cellularity in the sample. Some studies[15] have tried to overcome this limitation by performing parallel FNA and cytological evaluation of the same samples. Benesova et al[15] extracted DNA and RNA from FNA-acquired tissue (put in RNALater) and FN cytological (FNC) air-dried smear, with a selected area trimmed out, of same patients undergoing EUS-FNA with a 22 G needle.…”

Section: Introductionmentioning

confidence: 99%

“…Some studies[15] have tried to overcome this limitation by performing parallel FNA and cytological evaluation of the same samples. Benesova et al[15] extracted DNA and RNA from FNA-acquired tissue (put in RNALater) and FN cytological (FNC) air-dried smear, with a selected area trimmed out, of same patients undergoing EUS-FNA with a 22 G needle. The overall amount of isolated DNA/RNA from EUS-FNC samples was lower compared to EUS-FNA samples (10 ng vs 147 ng, respectively, for DNA; 164 vs 642 ng, respectively, for RNA); however, the KRAS-mutant detection frequency in EUS-FNC samples was 90% compared to 78% in EUS-FNA samples.…”

Section: Introductionmentioning

confidence: 99%

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New era for pancreatic endoscopic ultrasound: From imaging to molecular pathology of pancreatic cancer

Testoni

Redegalli

Petrone

et al. 2019

WJGO

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With recent advances in molecular pathology and the development of new chemotherapy regimens, the knowledge of the molecular alterations of pancreatic ductal adenocarcinoma (PDAC) is becoming appealing for stratifying patients for prognosis and response to a defined treatment. Archival formalin-fixed, paraffin-embedded samples are a useful source of genomic deoxyribonucleic acid; nevertheless, most studies employed formalin-fixed, paraffin-embedded samples deriving from surgical specimens, which are therefore representative of <20% of PDAC patients. Indeed, the development of a reliable methodology for endoscopic ultrasound-guided tissue acquisition, stabilization, and analysis is crucial for the development of molecular markers for clinical use in order to achieve “personalized medicine”. With the development of new needles, this technique is able to retrieve a high quantity and quality of PDAC tissue that can be used not only for diagnosis but also for mutational and transcriptome evaluations and for the development of primary cell or tissue cultures. In the present editorial, we discuss the current knowledge regarding the use of endoscopic ultrasound as a tool to obtain samples for molecular analyses, its possible pitfalls, and its use for the development of disease models such as xenografts or organoids.

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Section: Feasibility and Clinical Utility Of Pancreatic Eus-fna/fnb Fmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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New era for pancreatic endoscopic ultrasound: From imaging to molecular pathology of pancreatic cancer

Testoni

Redegalli

Petrone

et al. 2019

WJGO

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“…While pre‐analytical steps for molecular tests on formalin‐fixed paraffin‐embedded (FFPE) tissue samples are relatively standardized, the variety of cytological sample preparation techniques poses a challenge for standardized molecular testing. Numerous studies already compared different extraction methods of nucleic acids from various cytological samples, 3–5 however there is limited data for extraction of nucleic acids from fresh unfixed cell samples 6–8 …”

Section: Introductionmentioning

confidence: 99%

Optimization of pre‐analytical and analytical steps for DNA and RNA analysis of fresh cytology samples

et al. 2022

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Background: Different cytology preparations can be used for molecular diagnostics, however the influence of pre-analytical and analytical steps on the results are not yet well defined. We aimed to determine optimal steps for efficient extraction of DNA and RNA from fresh cells for molecular diagnostics.Methods: MCF7 and FaDu human cell lines, were used as a model to determine fresh cells storage conditions (temperature: 25°C, 4°C, −20°C, −80°C; duration: 0 h, 4 h, 12 h, 24 h, 48 h) and optimal nucleic acids extraction method. Besides, the minimal number of total cells and minimal percentage of mutated cells needed for successful extraction of nucleic acids and subsequent determination of present mutation were evaluated.Results: Extraction of nucleic acids using spin columns yielded the highest quantity and quality of nucleic acids. Isolation of nucleic acids was feasible in all storage conditions, however higher temperature and longer duration of fresh cells storage were associated with lower quality of isolated nucleic acids and similar quantification cycle of housekeeping genes. Successful molecular testing was feasible with least 10 4 cells, while specific mutation was detected in as low as 5% of mutated cells. Conclusions:Our cell line model, mimicking fresh cytology samples, showed that quantity of extracted either DNA or RNA declined with higher temperatures and longer duration of storage but regardless of the storage conditions, we successfully detected both housekeeping genes and mutated gene using qPCR.

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“…Denaturing capillary electrophoresis (DCE) has been used for routine mutation detection in a variety of solid cancers including colon and rectum [24][25][26], lung [27,28], pancreas [29,30] or brain [31]. The technique is based on heteroduplex formation with subsequent electrophoretic separation to visualize presence of mutant alleles in an abundance of nonmutated wildtype alleles [32].…”

Section: Introductionmentioning

confidence: 99%

Detection and Quantification of ctDNA for Longitudinal Monitoring of Treatment in Non-Small Cell Lung Cancer Patients Using a Universal Mutant Detection Assay by Denaturing Capillary Electrophoresis

Benešová¹,

Ptackova²,

Halkova³

et al. 2022

Pathol. Oncol. Res.

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Background: Observation of anticancer therapy effect by monitoring of minimal residual disease (MRD) is becoming an important tool in management of non-small cell lung cancer (NSCLC). The approach is based on periodic detection and quantification of tumor-specific somatic DNA mutation in circulating tumor DNA (ctDNA) extracted from patient plasma. For such repetitive testing, complex liquid-biopsy techniques relying on ultra-deep NGS sequencing are impractical. There are other, cost-effective, methods for ctDNA analysis, typically based on quantitative PCR or digital PCR, which are applicable for detecting specific individual mutations in hotspots. While such methods are routinely used in NSCLC therapy prediction, however, extension to cover broader spectrum of mutations (e.g., in tumor suppressor genes) is required for universal longitudinal MRD monitoring.Methods: For a set of tissue samples from 81 NSCLC patients we have applied a denaturing capillary electrophoresis (DCE) for initial detection of somatic mutations within 8 predesigned PCR amplicons covering oncogenes and tumor suppressor genes. Mutation-negative samples were then subjected to a large panel NGS sequencing. For each patient mutation found in tissue was then traced over time in ctDNA by DCE.Results: In total we have detected a somatic mutation in tissue of 63 patients. For those we have then prospectively analyzed ctDNA from collected plasma samples over a period of up to 2 years. The dynamics of ctDNA during the initial chemotherapy therapy cycles as well as in the long-term follow-up matched the clinically observed response.Conclusion: Detection and quantification of tumor-specific mutations in ctDNA represents a viable complement to MRD monitoring during therapy of NSCLC patients. The presented approach relying on initial tissue mutation detection by DCE combined with NGS and a subsequent ctDNA mutation testing by DCE only represents a cost-effective approach for its routine implementation.

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Comparison of Native Aspirates and Cytological Smears Obtained by EUS-Guided Biopsies for Effective DNA/RNA Marker Testing in Pancreatic Cancer

Cited by 5 publications

References 28 publications

New era for pancreatic endoscopic ultrasound: From imaging to molecular pathology of pancreatic cancer

New era for pancreatic endoscopic ultrasound: From imaging to molecular pathology of pancreatic cancer

Optimization of pre‐analytical and analytical steps for DNA and RNA analysis of fresh cytology samples

Detection and Quantification of ctDNA for Longitudinal Monitoring of Treatment in Non-Small Cell Lung Cancer Patients Using a Universal Mutant Detection Assay by Denaturing Capillary Electrophoresis

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