scholarly journals Comparison between Fluorimetry (Qubit) and Spectrophotometry (NanoDrop) in the Quantification of DNA and RNA Extracted from Frozen and FFPE Tissues from Lung Cancer Patients: A Real-World Use of Genomic Tests

Medicina ◽  
2021 ◽  
Vol 57 (12) ◽  
pp. 1375
Author(s):  
Katsuhiro Masago ◽  
Shiro Fujita ◽  
Yuko Oya ◽  
Yusuke Takahashi ◽  
Hirokazu Matsushita ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Success Rate ◽  
Quantitative Methods ◽  
Limit Of Detection ◽  
Pcr Amplification ◽  
Success Rates ◽  
Lung Cancer Patients ◽  
Dna And Rna ◽  
Ffpe Tissues ◽  
Panel Sequencing

Background and Objectives: Panel-based next-generation sequencing (NGS) has been carried out in daily clinical settings for the diagnosis and treatment guidance of patients with non-small cell lung cancer (NSCLC). The success of genomic tests including NGS depends in large part on preparing better-quality DNA or RNA; however, there are no established operating methods for preparing genomic DNA and RNA samples. Materials and Methods: We compared the following two quantitative methods, the QubitTM and NanoDropTM, using 585 surgical specimens, 278 biopsy specimens, and 82 cell block specimens of lung cancer that were used for genetic tests, including NGS. We analyzed the success rate of the genomic tests, including NGS, which were performed with DNA and RNA with concentrations that were outliers for the Qubit Fluorometer. Results: The absolute value for DNA concentrations had a tendency to be higher when measured with NanoDropTM regardless of the type of specimen; however, this was not the case for RNA. The success rate of DNA-based genomic tests using specimens with a concentration below the lower limit of QubitTM detection was as high as approximately 96%. At less than 60%, the success rate of RNA-based genomic tests, including RT-PCR, was not as satisfactory. The success rates of the AmpliSeqTM DNA panel sequencing and RNA panel sequencing were 77.8% and 91.5%, respectively. If at least one PCR amplification product could be obtained, then all RNA-based sequencing was performed successfully. Conclusions: The concentration measurements with NanoDropTM are reliable. The success rate of NGS with samples at concentrations below the limit of detection of QubitTM was relatively higher than expected, and it is worth performing PCR-based panel sequencing, especially in cases where re-biopsy cannot be performed.

scholarly journals A CRISPR Test for Rapidly and Sensitively Detecting Circulating EGFR Mutations

Diagnostics ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 114 ◽  
Author(s):  
Jen-Hui Tsou ◽  
Qixin Leng ◽  
Feng Jiang
Keyword(s):  
Lung Cancer ◽  
Cancer Patients ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Medical Diagnostics ◽  
Egfr Mutations ◽  
Nucleic Acid Detection ◽  
Great Promise ◽  
Plasma Samples ◽  
Lung Cancer Patients

The detection of EGFR mutations in circulating cell-free DNA can enable personalized therapy for cancer. The current techniques for detecting circulating EGFR mutations are expensive and time-consuming with moderate sensitivity. Emerging CRISPR is revolutionizing medical diagnostics and showing a great promise for nucleic acid detection. This study aims to develop CRISPR-Cas12a as a simple test to sensitively detect circulating EGFR mutations in plasma. Serially diluted samples of DNA containing heterozygous EGFR mutations (L858R and T790M) in wild-type genomic DNA are concurrently tested for the mutations by a CRISPR-Cas12a system and droplet digital PCR (ddPCR). The CRISPR-Cas12a system can detect both L858R and T790M with a limit of detection of 0.005% in less than three hours. ddPCR detects the mutations with a limit of detection of 0.05% for more than five hours. Plasma samples of 28 lung cancer patients and 20 cancer-free individuals are tested for the EGFR mutations by CRISPR-Cas12a system and ddPCR. The CRISPR-Cas12a system could detect L858R in plasma of two lung cancer patients whose tissue biopsies are positive for L858R, and one plasma sample of three lung cancer patients whose tissue biopsies are positive for T790M. ddPCR detects L858R in the same two plasm samples, however, does not detect T790M in any of the plasma samples. This proof of principle study demonstrates that the CRISPR-Cas12a system could rapidly and sensitively detect circulating EGFR mutations, and thus, has potential prognostic or therapeutic implications.

Current Topics and Practical Considerations of Cytology Practice in Lung Cancer: Reflexions from the Lung Symposium at the 42nd European Congress of Cytology, Malmö, 2019

2020 ◽  
Vol 64 (5) ◽  
pp. 463-470
Author(s):  
Sule Canberk ◽  
Verena Tischler ◽  
Marianne Engels
Keyword(s):  
Lung Cancer ◽  
Molecular Profile ◽  
Term Survival ◽  
Lung Cancer Patients ◽  
European Congress ◽  
Dna And Rna ◽  
Site Evaluation ◽  
Set Up

In June 2019, a lung symposium was held at the 42nd European Congress of Cytology in Malmö, Sweden. Due to the current importance of cytological samples in the diagnoses and molecular analysis to set up the utmost management of lung cancer patients, cytologists from different countries shared the experience of their institutions. The place of the cytological samples gains more and more importance on the potential long-term survival gain through personalized medicine and this harbors the improvement of the guidelines both in pathology and cytology field. In this symposium, the new 6-tiered reporting system for pulmonary cytology proposed by the Papanicolaou Society of Cytopathology and detailed cytomorphological approach to lung carcinoma including lookalike lesions and DNA- and RNA-based analysis of cytology material have been discussed. The cytopathologist plays a pivotal role in ensuring success of a correct triage for the cytology material to be sure of the adequacy and quality of the yield from the rapid on-site evaluation till the report which should encompass molecular profile in rational patient management.

scholarly journals Diagnostic Accuracy of Noninvasive Genotyping of EGFR in Lung Cancer Patients by Deep Sequencing of Plasma Cell-Free DNA

2015 ◽  
Vol 61 (9) ◽  
pp. 1191-1196 ◽  
Author(s):  
Junji Uchida ◽  
Kikuya Kato ◽  
Yoji Kukita ◽  
Toru Kumagai ◽  
Kazumi Nishino ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Patients ◽  
Deep Sequencing ◽  
Limit Of Detection ◽  
Detection System ◽  
Egfr Mutations ◽  
Plasma Dna ◽  
Lung Cancer Patients ◽  
Therapeutic Decisions ◽  
Exon 19

Abstract BACKGROUND Genotyping of EGFR (epidermal growth factor receptor) mutations is indispensable for making therapeutic decisions regarding whether to use EGFR tyrosine kinase inhibitors (TKIs) for lung cancer. Because some cases might pose challenges for biopsy, noninvasive genotyping of EGFR in circulating tumor DNA (ctDNA) would be beneficial for lung cancer treatment. METHODS We developed a detection system for EGFR mutations in ctDNA by use of deep sequencing of plasma DNA. Mutations were searched in >100 000 reads obtained from each exon region. Parameters corresponding to the limit of detection and limit of quantification were used as the thresholds for mutation detection. We conducted a multi-institute prospective study to evaluate the detection system, enrolling 288 non–small cell lung cancer (NSCLC) patients. RESULTS In evaluating the performance of the detection system, we used the genotyping results from biopsy samples as a comparator: diagnostic sensitivity for exon 19 deletions, 50.9% (95% CI 37.9%–63.9%); diagnostic specificity for exon 19 deletions, 98.0% (88.5%–100%); sensitivity for the L858R mutation, 51.9% (38.7%–64.9%); and specificity for L858R, 94.1% (83.5%–98.6%). The overall sensitivities were as follows: all cases, 54.4% (44.8%–63.7%); stages IA–IIIA, 22.2% (11.5%–38.3%); and stages IIIB–IV, 72.7% (60.9%–82.1%). CONCLUSIONS Deep sequencing of plasma DNA can be used for genotyping of EGFR in lung cancer patients. In particular, the high specificity of the system may enable a direct recommendation for EGFR-TKI on the basis of positive results with plasma DNA. Because sensitivity was low in early-stage NSCLC, the detection system is preferred for stage IIIB–IV NSCLC.

scholarly journals Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity

2020 ◽  
Vol 48 (6) ◽  
pp. 030006052093125
Author(s):  
Qing-qing Yi ◽  
Rong Yang ◽  
Jun-feng Shi ◽  
Nai-yan Zeng ◽  
Dong-yu Liang ◽  
...  
Keyword(s):  
Target Genes ◽  
Globin Gene ◽  
Pcr Amplification ◽  
Acid Extraction ◽  
Dna And Rna ◽  
Formalin Fixed Paraffin ◽  
Significant Difference ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Objectives This study aimed to investigate the factors affecting the quantity of DNA and RNA extractable from human formalin-fixed paraffin-embedded (FFPE) tissues stored for different lengths of time. Methods We randomly selected 20 FFPE specimens harvested from hysteromyoma patients with uterine fibroids during 2010, 2015, and 2017 at the Department of Pathology, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences. DNA and RNA extractions were performed using a DNA/RNA FFPE kit. DNA and RNA concentrations and their OD260/OD280 ratios were determined by a NanoDrop 2000 spectrophotometer. The human β-globin gene and aldehyde dehydrogenase-2 (ALDH2) gene were amplified from nucleic acids using a LightCycler 480 Real-Time PCR System, and PCR amplification products were electrophoresed on 1% agarose gels. Results Specimens that were stored for longer showed more degradation and a reduced concentration of DNA and RNA after nucleic acid extraction. However, there was no significant difference in DNA or RNA purity. β-globin and ALDH2 genes could be amplified from more than 99% of specimens. Conclusion We found that FFPE tissues stored for longer had a reduced quantity of extractable DNA and RNA. However, these tissues could be used for the analysis of some small target genes.

Total cost of lung cancer care associated with broad panel versus narrow panel sequencing.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 7077-7077
Author(s):  
Rogelio Alberto Brito ◽  
Bob Cullum ◽  
Kevin Hastings ◽  
Elisea Avalos-Reyes ◽  
Rebecca Karos ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Patients ◽  
Cancer Care ◽  
Lung Tumor ◽  
Progression Free Survival ◽  
Cost Of Care ◽  
Total Cost ◽  
Lung Cancer Patients ◽  
Genomic Test ◽  
Panel Sequencing

7077 Background: Many lung cancer patients are diagnosed late with advanced or metastatic disease. Targeted therapies can improve quality of life and increase the chances of progression-free survival versus conventional treatments. An understanding that there may be more than one driver mutation associated with a specific lung tumor is crucial for the timing and delivery of the most effective line of therapy. Broad panel sequencing (BPS) minimizes tissue use and enables personalized treatment that decreases the use of ineffective agents and unwarranted side effects, in addition to opening pathways to early clinical trials. However, many payors do not reimburse for BPS. The objective of this study was to determine if BPS leads to lower total cost of care versus narrow panel sequencing (NPS). Methods: We identified new lung cancer patients who completed BPS (Current Procedural Terminology (CPT) code 81455, 51+ genomic test) or NPS (CPT code 81445, 5-50 genomic test) using medical claims from January 1, 2018, to March 31, 2019. We defined total cost of care as allowed costs paid for medical and pharmacy claims across a six-month time period from the first gene sequencing panel. We also compared the allowed costs of BPS and NPS. A Student’s t-test was used to compare differences and results are presented as mean +/- standard deviation. Results: From January 2018 to March 2019, we identified 45 patients who underwent BPS sequencing and 399 patients who underwent NPS. The average BPS cost was $1,977 +/- $2,713 versus the average NPS lab cost $719 +/- $1,087, p < 0.0001. The average 6-month per member per month (PMPM) total cost was $11,535 +/- $9,168 among those who underwent BPS compared to $20,039 +/-19,642 in those who underwent NPS. This difference of $8,504 was statistically significant, p = 0.0022. Conclusions: BPS has been shown to optimize treatments in patients with lung cancer. These initial results of claims suggest that while lung cancer patients undergoing BPS have higher total sequencing costs than those undergoing NPS, BPS significantly reduces overall total cost of lung cancer care. Identifying the broader genomic landscape of a patient’s tumor earlier will empower oncology providers and lung cancer patients with information to make timely, precise treatment decisions that are ultimately more cost effective.

EGFR extracellular domain mutation in patients with lung cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e20532-e20532
Author(s):  
Renhua Guo ◽  
Dejian Gu ◽  
Jun Zhao ◽  
Lingjun Zhu ◽  
Mingjiu Chen ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Targeted Therapy ◽  
Kinase Domain ◽  
Egfr Mutations ◽  
Missense Mutations ◽  
Tumor Biopsy ◽  
Lung Cancer Patients ◽  
Hybridization Capture ◽  
Egfr Tkis ◽  
Panel Sequencing

e20532 Background: Missense mutations in the EGFR extracellular (EC) domain are found in 10% to 15% of glioblastomas(GBM). However, little is known about EGFR EC domain mutations in lung cancer. In this study, we retrospectively analyzed distribution of EGFR EC domain mutation in lung cancer. Methods: A total of 10100 lung cancer patients were analyzed in this study. EGFR mutations were detected by hybridization capture-based NGS panel sequencing with tumor biopsy, ctDNA or pleural effusion samples. Results: We identified gain-of-function mutations in the EC domain of EGFR in 36 (0.36%, 36/10100) patients with 23 patients carrying EGFR A289D/V/T/Y mutation, 10 patients harboring EGFR R108K/S mutation, 2 patients having EGFR T263P mutation, and 1 patient with EGFR G598R mutation. The EGFR EC domain mutation was detected in 23 patients with lung adenocarcinoma and 13 patients with other types of lung cancer (P = 0.018). Notably, 29 (80.56%) patients were detected with EGFR EC domain mutation and EGFR intracellular kinase domain (KD) mutation (P = 2.15×10-7). The incidence of L858R concomitant EC domain mutation was significantly higher compared with 19del (72.41% vs 17.24%, p = 28.64×10-5) or other EGFR KD mutations (72.41% vs 10.34%, p = 6.79×10-6). To further study whether L858R concomitant with EC domain mutation affected the efficiency of targeted therapy, we analyzed 6 patients who were treated with EGFR-TKIs. Among them, disease control rate of 2 months was 83.33% (5/6). Two patients had 10 months PFS. Three patients have been treated for 2, 5 and 12 months respectively, and are still on treatment. Conclusions: Our study reveals the distribution of EGFR EC domain mutation in lung cancer. EGFR EC domain mutation are more likely to occur concomitantly EGFR KD mutation, especial L858R. Meanwhile, our study shows that concomitant EC domain mutation and L858R may not affect the efficiency of targeted therapy.

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