Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

Pfitzner, Claudia; Schröder, Isabel; Scheungraber, Cornelia; Doğan, Aşkın; Runnebaum, Ingo B.; Häfner, Norman

doi:10.1038/srep03970

Cited by 26 publications

(22 citation statements)

References 32 publications

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“…Additionally, given the possibility of non-tumor epithelial cells in blood, as well as the possibility of ectopic transcription of epithelial genes by these cells, it will be important to identify additional tumor-specific transcripts 94 . Wholegenome amplification has been a challenge for single-cell analysis, due to amplification bias and the potential for illegitimate transcription.…”

Section: Genomic Analysismentioning

confidence: 99%

Opportunities and Challenges for Pancreatic Circulating Tumor Cells

et al. 2016

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Sensitive and reproducible platforms have been developed for detection, isolation, and enrichment of circulating tumor cells (CTCs)-rare cells that enter the blood from solid tumors, including those of the breast, prostate gland, lung, pancreas, and colon. These might be used as biomarkers in diagnosis or determination of prognosis. CTCs are no longer simply detected and quantified; they are now used in ex vivo studies of anticancer agents and early detection. We review what we have recently learned about CTCs from pancreatic tumors, describing advances in their isolation and analysis and challenges to their clinical utility. We summarize technologies used to isolate CTCs from blood samples of patients with pancreatic cancer, including immunoaffinity and label-free physical attribute-based capture. We explain methods of CTC analysis and how findings from these studies might be used to detect cancer at earlier stages, monitor disease progression, and determine prognosis. We review studies that have expanded CTCs for testing of anticancer agents and how these approaches might be used to personalize treatment. Advances in the detection, isolation, and analysis of CTCs have increased our understanding of the dissemination and progression of pancreatic cancer. However, standardization of methodologies and prospective studies are needed for this emerging technology to have a significant effect on clinical care.

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Section: Genomic Analysismentioning

confidence: 99%

Opportunities and Challenges for Pancreatic Circulating Tumor Cells

et al. 2016

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“…The most prevalent RNA quantification method is based on reverse-transcription quantitative polymerase chain reaction (RT-qPCR), in which the reverse transcriptase first transforms RNA into complementary DNA (cDNA) before the cDNA is PCR amplified and monitored in real-time in order to perform post-amplification quantitative analysis. Although digital PCR is capable of enumerating rare RNA targets in one-step when combined with a reverse transcription process 11 12 13 14 , the nonlinear enzymatic reverse transcription process is known to introduce quantification bias 15 16 . In this regard, a non-reverse transcription based method may provide a better alternative for RNA quantification.…”

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confidence: 99%

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

Guan

Chen

Rane

et al. 2015

Sci Rep

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We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

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“…We consequently adopted HPV genes as markers of cancer cells to evaluate the authenticity of CTC isolated in the present study. Several reports have carried out PCR amplification of HPV DNA or mRNA from peripheral blood cells to detect CTC in cervical cancer . In older studies, PBMC fractions were directly subjected to analyses .…”

Section: Discussionmentioning

confidence: 99%

“…Out of a total of 13 cervical cancer patients, 3 were positive for HPV mRNA (23%). In another study, digital RT‐PCR analysis was carried out for HPV genes to estimate the presence of CTC . Of 10 cervical cancer patients, 3 were positive.…”

Section: Discussionmentioning

confidence: 99%

“…Several reports have carried out PCR amplification of HPV DNA or mRNA from peripheral blood cells to detect CTC in cervical cancer. [35][36][37][38][39] In older studies, PBMC fractions were directly subjected to analyses. [35][36][37] Existence of HPV DNA was associated with overall survival or poor prognosis in these studies.…”

Section: Hpv Infection Is Associated With Carcinogenesis In Cervical mentioning

confidence: 99%

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Detection of circulating tumor cells in cervical cancer using a conditionally replicative adenovirus targeting telomerase‐positive cells

et al. 2017

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Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase‐positive cells, which was enabled to infect coxsackievirus‐adenovirus receptor‐negative cells and to reduce false‐positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti‐CD45 and anti‐pan cytokeratin antibodies. GFP (+)/CD45 (−) cells were isolated and subjected to whole‐genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC‐positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV‐infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker‐dependent systems do not have the capacity to detect these cells in cervical cancer patients.

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Digital-Direct-RT-PCR: a sensitive and specific method for quantification of CTC in patients with cervical carcinoma

Cited by 26 publications

References 32 publications

Opportunities and Challenges for Pancreatic Circulating Tumor Cells

Opportunities and Challenges for Pancreatic Circulating Tumor Cells

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

Detection of circulating tumor cells in cervical cancer using a conditionally replicative adenovirus targeting telomerase‐positive cells

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