scholarly journals A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting

Cancers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 290 ◽  
Author(s):  
Laure Sorber ◽  
Karen Zwaenepoel ◽  
Koen De Winne ◽  
Kaat Van Casteren ◽  
Elien Augustus ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Transit Time ◽  
Multicenter Study ◽  
Clinical Setting ◽  
Liquid Biopsy ◽  
High Speed ◽  
Mutational Analysis ◽  
Mutational Status ◽  
Ctdna Analysis ◽  
Gdna Contamination

A multicenter study was performed to determine an optimal workflow for liquid biopsy in a clinical setting. In total, 549 plasma samples from 234 non-small cell lung cancer (NSCLC) patients were collected. Epidermal Growth Factor Receptor (EGFR) circulating cell-free tumor DNA (ctDNA) mutational analysis was performed using digital droplet PCR (ddPCR). The influence of (pre-) analytical variables on ctDNA analysis was investigated. Sensitivity of ctDNA analysis was influenced by an interplay between increased plasma volume (p < 0.001) and short transit time (p = 0.018). Multistep, high-speed centrifugation both increased plasma generation (p < 0.001) and reduced genomic DNA (gDNA) contamination. Longer transit time increased the risk of hemolysis (p < 0.001) and low temperatures were shown to have a negative effect. Metastatic sites were found to be strongly associated with ctDNA detection (p < 0.001), as well as allele frequency (p = 0.034). Activating mutations were detected in a higher concentration and allele frequency compared to the T790M mutation (p = 0.003, and p = 0.002, respectively). Optimization of (pre-) analytical variables is key to successful ctDNA analysis. Sufficient plasma volumes without hemolysis or gDNA contamination can be achieved by using multistep, high-speed centrifugation, coupled with short transit time and temperature regulation. Metastatic site location influenced ctDNA detection. Finally, ctDNA levels might have further value in detecting resistance mechanisms.

scholarly journals Translational Application of Circulating DNA in Oncology: Review of the Last Decades Achievements

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1251 ◽  
Author(s):  
Tuaeva ◽  
Falzone ◽  
Porozov ◽  
Nosyrev ◽  
Trukhan ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Liquid Biopsy ◽  
Prognostic Significance ◽  
Circulating Tumor Dna ◽  
Molecular Techniques ◽  
Unknown Primary ◽  
Diagnostic Strategies ◽  
Early Diagnosis Of Cancer ◽  
Mutational Status ◽  
Ctdna Analysis

In recent years, the introduction of new molecular techniques in experimental and clinical settings has allowed researchers and clinicians to propose circulating-tumor DNA (ctDNA) analysis and liquid biopsy as novel promising strategies for the early diagnosis of cancer and for the definition of patients’ prognosis. It was widely demonstrated that through the non-invasive analysis of ctDNA, it is possible to identify and characterize the mutational status of tumors while avoiding invasive diagnostic strategies. Although a number of studies on ctDNA in patients’ samples significantly contributed to the improvement of oncology practice, some investigations generated conflicting data about the diagnostic and prognostic significance of ctDNA. Hence, to highlight the relevant achievements obtained so far in this field, a clearer description of the current methodologies used, as well as the obtained results, are strongly needed. On these bases, this review discusses the most relevant studies on ctDNA analysis in cancer, as well as the future directions and applications of liquid biopsy. In particular, special attention was paid to the early diagnosis of primary cancer, to the diagnosis of tumors with an unknown primary location, and finally to the prognosis of cancer patients. Furthermore, the current limitations of ctDNA-based approaches and possible strategies to overcome these limitations are presented.

scholarly journals A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wendell Jones ◽  
Binsheng Gong ◽  
Natalia Novoradovskaya ◽  
Dan Li ◽  
Rebecca Kusko ◽  
...  
Keyword(s):  
Quality Control ◽  
Allele Frequency ◽  
Liquid Biopsy ◽  
Limit Of Detection ◽  
Reference Sample ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Analytical Accuracy ◽  
And Performance ◽  
Reference Samples

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

scholarly journals Chronic Lymphocytic Leukemia in Kuwait

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5467-5467
Author(s):  
Salem Alshemmari ◽  
Ramesh Pandita ◽  
Abdulaziz Hamadah ◽  
Ahmad Alhuraiji
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Board Of Directors ◽  
Mutational Analysis ◽  
Staging System ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Western Countries ◽  
Mutational Status ◽  
Study Results ◽  
History Of

Background :Chronic lymphocytic leukemia (CLL) is common malignancy in Western countries. However, little known about this disease entity in our area. This study exploring the biology in out patients' population. Method:Patients with confirmed CLL under IGHV and TP53 mutational analysis at presentation or during follow up. We also integrated other clinical and biological parameter in this study. Results: A total of 137 cases were analyzed, median age 61 years (range:34-89); 30% of the cases age was<55 years at presentation. There was 108 males vs. 29 females M:F ratio 3.7. Two patients gave a family history of CLL, while 1 patient gave a history of other lymphoproliferative disorders. Binet staging system available in 134 cases, A: 109 (81.3%), B: 12 (9%), C:13 (9.7%). B2 macroglobulin elevated in 40/112 (36%) cases and 10/103 (10%) had M-spike. CD38 positivity reported in 37/112 (33%) of cases. Cytogenetics data evaluable in 85 cases: isolated del(13q): 35%, isolated trisomy 12 (16.5%), del(11q) (4.5%), del(17p)(2.4%). IGHV mutational status mutated vs unmutated: 40% vs 60%. Cases with available treatment information on 132 cases. Fifty cases required treatment due to disease progression. First line treatment Bendamustine-Rituximab (BR) 3 cases, Fludarabine Cyclophosphamide Rituximab (FCR) 30 cases and Chlorambucil with anti-CD 20 antibody 6 cases. At the time of review, 3 cases on ibrutinib (2 in 3rdline and 1 case in the 4thline). Conclusion: This is the first study to shed light on CLL in our area. There are biological differences between our patients' population and the western countries. Disclosures Pandita: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Identification of a Clinical Cutoff Value for Multiplex KRASG12/G13 Mutation Detection in Colorectal Adenocarcinoma Patients Using Digital Droplet PCR, and Comparison with Sanger Sequencing and PNA Clamping Assay

2020 ◽  
Vol 9 (7) ◽  
pp. 2283
Author(s):  
Kyung Ha Lee ◽  
Tae Hee Lee ◽  
Min Kyung Choi ◽  
In Sun Kwon ◽  
Go Eun Bae ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Mutant Allele ◽  
Sanger Sequencing ◽  
Colorectal Adenocarcinoma ◽  
Diagnostic Sensitivity ◽  
Absolute Quantitation ◽  
Tissue Samples ◽  
Mutant Allele Frequency ◽  
Mutational Status

KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) is a major predictive marker for anti-epidermal growth factor receptor treatment, and determination of KRAS mutational status is crucial for successful management of colorectal adenocarcinoma. More standardized and accurate methods for testing KRAS mutation, which is vital for therapeutic decision-making, are required. Digital droplet polymerase chain reaction (ddPCR) is an advanced digital PCR technology developed to provide absolute quantitation of target DNA. In this study, we validated the clinical performance of ddPCR in determination of KRAS mutational status, and compared ddPCR results with those obtained by Sanger sequencing and peptide nucleic acid-clamping. Of 81 colorectal adenocarcinoma tissue samples, three repeated sets of KRASG12/G13 mutation were measured by ddPCR, yielding high consistency (ICC = 0.956). Receiver operating characteristic (ROC) curves were constructed to determine KRASG12/G13 mutational status based on mutant allele frequency generated by ddPCR. Using the best threshold cutoff (mutant allele frequency of 7.9%), ddPCR had superior diagnostic sensitivity (100%) and specificity (100%) relative to the two other techniques. Thus, ddPCR is effective for detecting the KRASG12/G13 mutation in colorectal adenocarcinoma tissue samples. By allowing definition of the optimal cutoff, ddPCR represents a potentially useful diagnostic tool that could improve diagnostic sensitivity and specificity.

scholarly journals Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

2020 ◽  
Vol 10 (8) ◽  
pp. 2895 ◽  
Author(s):  
Giuseppa De Luca ◽  
Sonia Lastraioli ◽  
Romana Conte ◽  
Marco Mora ◽  
Carlo Genova ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Kinase Inhibitors ◽  
Limit Of Detection ◽  
Variant Calling ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Coverage Metrics ◽  
Free Dna ◽  
Mutational Status ◽  
Nsclc Patients

Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p < 0.0001) and sequencing performance (p < 0.0001). In addition, 0.1% limit of detection could be achieved even when < 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories.

KRAS Mutation: Should We Test for It, and Does It Matter?

2013 ◽  
Vol 31 (8) ◽  
pp. 1112-1121 ◽  
Author(s):  
Patrick J. Roberts ◽  
Thomas E. Stinchcombe
Keyword(s):  
Lung Cancer ◽  
Monoclonal Antibodies ◽  
Predictive Value ◽  
Clinical Utility ◽  
Mutational Analysis ◽  
Small Cell ◽  
Small Cell Lung ◽  
Kras Mutations ◽  
Mutational Status ◽  
Egfr Tki

Lung cancer is the leading cause of cancer mortality in the United States and worldwide. Previously, lung cancer was simplistically divided into non–small-cell lung cancer (NSCLC) and small-cell lung cancer. However, in the last decade, we have gone from a simplistic binary system of classifying lung cancer to defining NSCLC on the basis of molecular subsets. KRAS mutations represent the most common molecular change in NSCLC. The presence of KRAS mutation has been shown to be associated with a poor prognosis in NSCLC, but this is of little clinical utility. More important is determining the clinical utility of KRAS mutational analysis for predicting benefit of chemotherapy, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), anti-EGFR monoclonal antibodies, or other novel therapeutics. Current data does not support the routine use of KRAS mutational analysis for predicting chemotherapy benefit. Additionally, there was significant interest in using KRAS status to select patients for EGFR TKI and anti-EGFR monoclonal antibodies. However, the EGFR mutational status has demonstrated significant predictive value in the selection of patients for EGFR TKI therapy and is now the preferred test. An association between KRAS mutational status and benefit of anti-EGFR monoclonal antibodies has not been demonstrated in NSCLC. Here we review, in the context of NSCLC, the underlying biology of KRAS mutations, the predictive value of KRAS mutations for therapeutic intervention, and the integration of KRAS mutational testing into our current clinical paradigms.

TA-B5 short transit-time photoconductive detectors for high-speed optical communications

1980 ◽  
Vol 27 (11) ◽  
pp. 2187-2187
Author(s):  
J.C. Gammel ◽  
G.M. Metze ◽  
H. Ohno ◽  
J.M. Ballantyne
Keyword(s):  
Transit Time ◽  
Optical Communications ◽  
High Speed ◽  
Photoconductive Detectors

IgH Rearrangements and Mutational Status in Class-Switched IgG B-CLL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2793-2793 ◽  
Author(s):  
James A.L. Fenton ◽  
David Gonzalez ◽  
Andy C. Rawstron ◽  
Gareth J. Morgan ◽  
Andrew S. Jack ◽  
...  
Keyword(s):  
B Cells ◽  
Mutational Analysis ◽  
Somatic Hypermutation ◽  
Gene Segment ◽  
Specific Antigen ◽  
Distinct Pattern ◽  
Published Data ◽  
Molecular Features ◽  
Mutational Status ◽  
Igh Genes

Abstract Analysis of immunoglobulin heavy chain (IgH) rearrangements in B-CLL differentiates subgroups of patients with significantly different clinical outcomes. Cases can be categorised according to mutational status of the variable (V) segment with unmutated VH regions linked to a worse prognosis. A restricted pattern of use of specific VH, DH and JH gene segments has also been reported in B-CLL. It has been hypothesised that B-CLL originates as a clonal expansion of B-cells that have been selected and activated by contact with self or foreign antigens, leading to those small clones to proliferate, mutate their IGH genes, acquire genetic lesions and eventually become malignant. B-CLL cells normally express low levels of Ig on the surface, normally IgM, although a proportion of patients express IgG or IgA, following the class-switch recombination (CSR) process. We have analysed the pattern of SHM and gene segment usage in this particular subgroup for 44 patients with IgG B-CLL. Successful PCR amplification of recombined Smu-Sgamma switch region DNA was achieved in 40 patients, confirming the presence of IgG class-switching. Mutational analysis of IgH V genes revealed 80% of patients contained more than 2% somatic hypermutation (SHM), with 63% of samples having a greater than 5% SHM rate. For VH gene segment usage, a significant predominance of the VH4 family was seen in 22 cases (50%), followed by VH3 in 17 cases (39%), while VH1 family was found in only 3 of 44 samples, this differs from classical IgM B-CLL where VH3 family usage predominates. Overall, VH4-34 was the most frequently used gene segment (34%), followed by VH3-07 (14%) and VH4-39 (9%). DH6-13 was the most frequently used DH segment (21%), followed by DH6-19 (13%). JH gene segment usage did not differ from normal B-cells, with JH4 being the most frequently used, followed by JH6 and JH5. There was a significant association between VH4-39, DH6-13 and JH5 in three samples all containing unmutated sequence. Together this data reveals a distinct pattern of IGH VDJ rearrangements in IgG B-CLL compared to classical IgM B-CLL. Firstly, the rate of SHM in IgG B-CLL (80%) is significantly higher than the 50% observed in IgM B-CLL. Secondly, VH segment usage pattern differs between the two subgroups with a significant under-representation of VH1 as well as an over-representation of VH4 family members in the IgG subgroup. Finally, there is a striking association between VH4-39 and DH6-13/JH5 in the very few unmutated rearrangements. This could be indicative of a different clonal history of these particular B cells in B-CLL. Together with recent published data, this latter finding suggests that this is a further sub-category exclusive to IgG B-CLL, where selection of a specific antigen receptor may have lead to B-CLL development in such cases. We conclude that class-switched IgG B-CLL contains different molecular features in the IgH genes compared with classical IgM B-CLL, and other B-cell malignancies. The clinical implications of these differences, especially the relationship between the mutational status of the VH genes and outcome in IgG B-CLL, will be further investigated.

Sign in / Sign up

Export Citation Format

Share Document