Detection Of Mutations In Plasma Research Articles

Abstract 538: Cell-free whole genome sequencing for the detection of cancer in patients with germline BRCA1/2 mutations

Abstract Background: Hereditary breast and ovarian cancer syndrome (HBOC) is caused by a germline likely/pathogenic variant in BRCA1 or BRCA2, leading to increased risk of developing breast, ovarian, prostate, and pancreatic cancers. While HBOC patients often undergo regular surveillance including breast imaging, other cancers have no effective screening or biomarkers. One emerging technology is the analysis of circulating tumor DNA (ctDNA), fragments of DNA that are shed from the tumor into the bloodstream. In sporadic cancer, several studies have demonstrated the advantages and sensitivity of cell-free whole genome sequencing (cfWGS) compared to targeted panel sequencing. However, the use of cfWGS for cancer detection in HBOC has not been explored. Methods: Matched tumor, germline, and plasma were collected from 5 HBOC patients (3 breast, 2 ovarian) as part of the Canada-wide CHARM consortium (https://charmconsortium.ca). Tumor (40x), germline (20x), and plasma (20x) underwent whole genome sequencing (WGS) and subsequent variant calling pipeline using an ensemble of 6 variant callers. Insertions, deletions, and structural variants were not included in downstream analyses. Plasma tumor fraction prediction and copy number alterations were performed using ichorCNA. Detection of tumor associated mutations in plasma was compared to MRDetect software. Results: Variant calling identified an average of 11,170 tumor (7,649-16,706) and 1,215 (544-3,205) plasma SNVs per patient. Somatic BRCA1/2 mutations were detected in 0/5; whereas deletion of the intact BRCA1/2 allele was identified in 5/5 patients. An average of 24.6% (8.3%-75.9%) of plasma mutations where shared with SNVs identified in matched tumor. Targeted panel deep-sequencing (20,000X) was only able to detect mutations in TP53 in 3/5 cases (0.8% - 8.6%). Plasma derived copy number alterations correlated well with tumor derived copy number alterations in 3/5 cases and ichorCNA predicted tumor fractions ranged from 0.038 to 0.196. Conclusions: Traditionally, targeted panel sequencing has been used to identify tumor associated mutations in plasma. While this technique is sensitive, major disadvantages include the narrow breadth of the captured regions and the inability to detect deletion events such as those that occur in BRCA1/2. Here we show that plasma WGS is effective for detecting tumor associated mutations in HBOC and is more sensitive than targeted panel sequencing. We are currently expanding our cohort to an additional 20 matched tumor pairs, including patients with pre-diagnosis plasma timepoints. Citation Format: Derek Wong, Adriana Aguilar-Mahecha, Josiane Lafleur, Clarissa Chan, Kirsten Farncombe, Maia Norman, Leslie Oldfield, Prabhjit Basra, Stephanie Pederson, Johanna Wellum, Stephenie Prokopec, Mark Basik, Raymond H. Kim, Trevor Pugh. Cell-free whole genome sequencing for the detection of cancer in patients with germline BRCA1/2 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 538.

Abstract 582: Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma

Abstract Driver mutations in CTNNB1 are a hallmark of hepatoblastoma (HB) and offer a common biomarker for a liquid biopsy approach that is based on the presence of CTNNB1 circulating tumor DNA (ctDNA). Initial results 1 showed an association of CTNNB1 ctDNA variant allele frequency (VAF) identified through digital droplet PCR (ddPCR) and the levels of serum Alpha-fetoprotein (AFP, current HB biomarker) throughout the course of treatment of three patients with HB. However, applying custom probe-designed ddPCR assays may pose challenges for real-time data at diagnosis. Our primary objective is to investigate the utility of a universal next-generation sequencing (NGS) assay to detect CTNNB1 ctDNA in patients with HB. Our secondary objective is to compare the levels of: (1) NGS ctDNA, (2) ddPCR ctDNA, (3) AFP, and verify if they correlate with tumor burden and treatment response. We developed and tested a custom NGS assay covering exons 2 to 4 of CTNNB1 for detection of somatic mutations in plasma. We used the QIASeq platform which: (1) is based on a single primer extension and, thus, captures both single nucleotide variants (SNV) and structural variants (SV), and (2) integrates unique molecular indexing (UMI), thus allowing determination of ctDNA levels from VAF. Our cohort included 18 patients, 13 with Sanger sequencing confirmed CTNNB1 somatic mutations in the matched tumors: 6 missense SNVs and 7 deletions ranging from 96 to 348 base pairs. Our NGS assay was able to accurately detect mutations in 9/13 (69%, 7 SV and 2 SNV) of the Sanger confirmed cases, and SV and missense SNV in 2/5 (40%) cases pending orthogonal Sanger confirmation. Available ddPCR data for 3 patients (8 samples) 1 and corresponding NGS data showed similar ctDNA levels (deviation ranging between 1.2-8.5%). NGS CTNNB1 variants were detected in samples taken at initial diagnosis in 8/9 (89%), following neoadjuvant chemotherapy in 4/9 (44%), post-operatively for fully resected localized disease in 0/4 (0%), and metastatic recurrence in 1/5 (20%), suggesting a positive correlation between CTNNB1 variants and tumor burden. AFP levels were available for 24/27 samples, with abnormal levels detected in 23 samples. The ctDNA levels determined from NGS did not correlate with AFP levels; however, longitudinal plasma samples (n=9) showed similar dynamics of both ctDNA and AFP, reflecting the response to treatment. To conclude, we show that ctDNA detectable with our universal HB NGS assay is a good surrogate marker of tumor burden and shows correlation with treatment response. SV, prevalent in approximately 50% of cases of HB, may be inadequately captured and thereby misrepresented in probe-capture NGS platforms but were highly detectable in our assay. Further work is needed to understand the interplay of both AFP and ctDNA in HB monitoring. Reference: 1. Kahana-Edwin S, McCowage G, Cain L, et al. Exploration of CTNNB1 ctDNA as a putative biomarker for hepatoblastoma. Pediatr Blood Cancer. 2020. doi:10.1002/pbc.28594 Citation Format: Smadar Kahana-Edwin, Andre E. Minoche, Lucy E. Cain, Geoffrey McCowage, Sarah E. Woodfield, Sanjeev A. Vasudevan, Sarah Kummerfeld, Jonathan Karpelowsky. Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 582.

Comparison of two targeted ultra-deep sequencing technologies for analysis of plasma circulating tumour DNA in endocrine-therapy-resistant breast cancer patients

PurposeThere is growing interest in the application of circulating tumour DNA (ctDNA) as a sensitive tool for monitoring tumour evolution and guiding targeted therapy in patients with cancer. However, robust comparisons of different platform technologies are still required. Here we compared the InVisionSeq™ ctDNA Assay with the Oncomine™ Breast cfDNA Assay to assess their concordance and feasibility for the detection of mutations in plasma at low (< 0.5%) variant allele fraction (VAF).MethodsNinety-six plasma samples from 50 patients with estrogen receptor (ER)-positive metastatic breast cancer (mBC) were profiled using the InVision Assay. Results were compared to the Oncomine assay in 30 samples from 26 patients, where there was sufficient material and variants were covered by both assays. Longitudinal samples were analysed for 8 patients with endocrine resistance.ResultsWe detected alterations in 59/96 samples from 34/50 patients analysed with the InVision assay, most frequently affecting ESR1, PIK3CA and TP53. Complete or partial concordance was found in 28/30 samples analysed by both assays, and VAF values were highly correlated. Excellent concordance was found for most genes, and most discordant calls occurred at VAF < 1%. In longitudinal samples from progressing patients with endocrine resistance, we detected consistent alterations in sequential samples, most commonly in ESR1 and PIK3CA.ConclusionThis study shows that both ultra-deep next-generation sequencing (NGS) technologies can detect genomic alternations even at low VAFs in plasma samples of mBC patients. The strong agreement of the technologies indicates sufficient reproducibility for clinical use as prognosic and predictive biomarker.

Abstract A31: Tracking the evolution of soft tissue sarcoma and GIST using liquid biopsies

Abstract Molecular analysis of circulating tumor DNA (ctDNA) has a large potential for clinical application by capturing mutations and heterogeneity through noninvasive sampling. The CircSarc studies aims to provide new insights into the clinical utility of liquid biopsies in soft tissue sarcomas (CircSarc STS) and gastrointestinal stromal tumors (CircSarc GIST). In CircSarc STS, we have enrolled 35 high-grade soft-tissue sarcoma patients with localized disease. Plasma from each patient has been collected longitudinally throughout the course of their disease, with a follow-up of up to 5 years. To identify patient-specific somatic mutations, the patients’ tumor and germline DNA have been exome sequenced. Somatic mutations in cfDNA are being identified using ultra-low-pass WGS or targeted NGS. At time of surgery and/or progression, we detect ctDNA in 30% of the first soft-tissue sarcoma samples with higher levels in larger or metastatic tumors. In a proof-of-concept study, we have shown that that levels of tumor-specific mutations in plasma correlated to clinical manifestation of metastatic disease. In CircSarc GIST, plasma has been collected for a cohort of 30 metastatic GISTs and at time of diagnosis for 50 GISTs. Somatic mutations in the tumor have been determined by targeted sequencing or whole-exome sequencing. Somatic mutations in ctDNA are identified using targeted NGS. The levels of somatic mutations in ctDNA are being correlated with clinicopathologic features. ctDNA was detected in 36% of treatment-naïve GIST patients, including all patients with metastatic disease, and the tumor burden was the most important detection determinant. Analysis of metastatic GISTs is ongoing, but repeated plasma samples in a progressing patient have demonstrated that clonal evolution can be monitored over time. ctDNA analysis of the patient revealed multiple resistance mutations, and these were spatially distributed in the primary tumor. Our work shows that detection of mutations in plasma is particularly feasible for patients with high tumor burden. Mutational analysis by use of liquid biopsies can capture the molecular heterogeneity of the tumor and guide treatment decisions during progression. Citation Format: Heidi M. Namløs, Kjetil Boye, Seyed H Moosavi, Daniel Vodak, Nitin Sharma, Susanne Lorenz, Bodil Bjerkehagen, Stine Næss, Eivind Hovig, Nina Jebsen, Anders Ståhlberg, Ola Myklebost, Leonardo A. Meza-Zepeda. Tracking the evolution of soft tissue sarcoma and GIST using liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A31.

Highly sensitive detection of ALK resistance mutations in plasma using droplet digital PCR

BackgroundOn-target resistance mechanisms found in one-third of patients receiving anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) are secondary ALK mutations in ALK-rearranged non-small cell lung cancer (NSCLC). There are large variations in the resistant mutations, unlike the epithelial growth factor receptor (EGFR) T790 M seen with the use of EGFR-TKIs. Liquid biopsy approaches using cell-free DNA (cfDNA) are used for screening and monitoring of mutations in NSCLC. However, feasible protocol for the simultaneous detection of multiple secondary ALK mutations using droplet digital PCR (ddPCR) has not been developed. An efficient strategy using cfDNA in cancer diagnostics, the development of more accurate and cost-effective tools to identify informative multiple secondary ALK mutations is clinically required.MethodsTo establish a feasible assay to monitor ALK-TKI resistance mutations, we first evaluated the feasibility of ddPCR-based screening for cfDNA mutation detection of 10 distinct secondary ALK mutations. Positive samples were then re-analyzed using mutation-specific probes to track the growth of mutation clones with a high sensitivity.ResultsBlood samples from seven ALK-positive patients were analyzed using the ddPCR protocol. Secondary G1202R ALK mutations were identified in 2 of 7 patients by the screening assay. Using the mutation-specific probes, monitoring the resistant clone during the clinical course of the disease was well demonstrated in each of the patients.ConclusionThe protocol for ddPCR-based liquid biopsy has a feasibility for the screening of secondary ALK-TKI resistance mutations and offers a tool for a cost-effective monitoring of progression in NSCLC.

Abstract 937: Longitudinal circulating-tumor DNA profiling of EGFR-mutated non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors

Abstract Background: In EGFR-mutant non-small cell lung cancer (NSCLC), progression disease (PD) under 1st-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) is driven by the EGFR T790M mutation in about 50% of cases. This mutation is targeted with osimertinib, a specific and potent inhibitor that also showed superior efficacy to that of 1st-generation EGFR-TKIs in the 1st-line treatment EGFR+ NSCLC. Strategy: We performed a longitudinal circulating-tumor DNA (ctDNA) analysis to evaluate 1) the dynamics of sensitizing or resistant mutations over time and 2) the genomic alterations associated with EGFR-TKI resistance, in NSCLC patients treated with either 1st-generation EGFR-TKIs (n=24) or with osimertinib (n=20). ctDNA sequencing was performed using InVisionSeq™, an amplicon-based NGS (36-gene panel) assay. Results: Sensitivity was 80% and 100% for the detection of EGFR-activating mutations and the EGFR T790M mutation, respectively, at PD in ctDNA vs tissue biopsies. The type of progression appears to influence the detection of EGFR mutations in plasma. Patients (pts) with exclusively brain or thoracic PD had significantly lower allelic fractions (AFs) of EGFR-activating mutations than pts with systemic PD (P=0.006). AFs of both EGFR-activating and EGFR T790M mutations were highest in patients with progressive metastases in liver or bone. High cell-free DNA load was associated with the presence of TP53 mutations regardless of the time under treatment (n=248 samples). The total number of mutations detected in ctDNA was higher in pts treated with osimertinib vs. 1st-generation EGFR-TKIs (P=0.004), suggesting a potential increase of tumor heterogeneity over time. We observed that the AFs of mutant ctDNA were correlated with response to treatment. Complete clearance of EGFR T790M (AF detection limit: 0.01%) was observed in pts who presented a partial response to osimertinib (n=17), of these, EGFR-activating mutations were found in 4/17 pts (AF:0.05-1.03%). PD was evidenced in 36/44 pts. Emergence of mechanisms of resistance, before the confirmation of clinical PD, was evidenced in 5 and 3 pts under erlotinib and osimertinib, respectively. PD to 1st-generation EGFR-TKIs was mainly driven by EGFR T790M (86%), followed by mutations in PIK3CA or PTEN. We detected the emergence of heterogeneous potential mechanisms of resistance to osimertinib in 3/9 cases, including mutations in EGFR, KRAS, BRAF as well as HER2 and MET amplification. We did not identify genomic drivers of resistance in 16/36 pts, despite evidence of clinical PD. These pts had either exclusive brain or thoracic progression. ctDNA WES will be performed in these samples. Conclusion: Our results suggest that ctDNA sequencing might be a complementary, noninvasive tool to monitor response to treatment and heterogeneous mechanisms of resistance in NSCLC pts treated with EGFR inhibitors. Citation Format: Sandra Ortiz-Cuaran, Aurélie Swalduz, Camille Léonce, Solène Marteau, Séverine Martinez, Gilles Clapisson, Virginie Avrillon, Luc Odier, Lionel Falchero, Pierre Fournel, Emma Green, Clive Morris, Maurice Pérol, Pierre Saintingy. Longitudinal circulating-tumor DNA profiling of EGFR-mutated non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 937.

Tyrosine kinase inhibitor sensitive PDGFRΑ mutations in GIST: Two cases and review of the literature.

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal malignancies of the gastrointestinal tract. Most GISTs harbor a c-KIT (80%) or a PDGFRα (10%) mutation that leads to constitutive activation of the tyrosine kinase receptor. Response to treatment with tyrosine kinase inhibitors (TKIs) is dependent on mutational status of the tumor. The most common mutation in PDGFRα, D842V, is known to be imatinib resistant. Almost all other PDGFRα mutations are imatinib sensitive. We describe two patients with a PDGFRα exon 18 mutated GIST responding to treatment with TKIs. One of these patients has a p.M844_S847 deletion, not previously described in relation with TKI treatment response. Mutations in circulating tumor DNA were detectable with digital droplet PCR in serial plasma samples taken during treatment and correlated with treatment response of both patients. Computer 3D-modeling of the PDGFRα kinase domain of these two variants revealed no direct interference in imatinib or sunitinib binding and no effect in its activity in contrast to the reported structure of the imatinib resistant D842V mutation.An overview is given of the literature regarding the evidence of patients with different PDGFRα mutated GISTs on response to TKIs. The findings emphasize the use of mutational analysis in GIST to provide patients personalized treatment. Detection of mutations in plasma is feasible and can provide real-time information concerning treatment response. We suggest to register GIST patients with these uncommon mutations in a prospective international database to understand the tumor biology and obtain more evidence of such mutations to predict treatment response.

Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid

Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms. cfDNA from 31 patients with diverse advanced cancers and known tumor mutation status was deep sequenced using targeted hotspot panels. Forty-five of 52 expected mutations and two additional mutations (KRAS p.Q61H and EZH2 p.Y646F) were detected in plasma through a custom bioinformatics pipeline. We observed comparable cfDNA concentration/integrity between collection tubes within 16 hours of plasma separation and equal analytical performance among NGS platforms, with 1% detection sensitivity for cfDNA genotyping.

Abstract 488: Detection of somatic mutations in plasma allows for non-invasive real-time therapy response monitoring of lung cancer patients

Abstract Tumor-specific (somatic) mutations in plasma can serve as biomarkers for tumor detection, monitor tumor response to specific therapies, detect residual disease after surgery, and long-term follow-up. The intrinsic low abundance of circulating cell-free tumor DNA (cfDNA) makes the detection and quantification of such mutations in plasma a challenging task. This study aimed to establish a comprehensive strategy to be used in the detection of clinically relevant somatic mutations in plasma of lung cancer patients both at diagnosis and during follow-up of disease. Plasma samples from healthy controls were used to isolate cfDNA and to optimize the experimental approach. A custom multiplex PCR panel was designed to target 420 unique genetic variants. To cover a broad range (0.0 -100%) of variant allele fractions (VAF), as expected for somatic mutations in the plasma of cancer patients, the cfDNA samples from the controls were mixed at five different ratios and sequenced using five different levels of coverage. In order to evaluate our strategy, plasma samples obtained at different stages of disease were collected from a group of 100 lung cancer patients and used to isolate cfDNA. The mean treatment follow up time was 6 months with a minimum of 1 and a maximum of 23 months. Different enrichment strategies where tested. All amplified products were used to prepare libraries and sequenced using the Ion PGM system. The QuantStudio 3D Digital PCR System was used to confirm selected results. Our controlled data set enabled us to define VAF, allele depth and allele quality score cutoffs for a reliable detection of low VAF in cfDNA. For variants present at VAF ≥5%, a sensitivity of 100% was achieved at ≥500x coverage. At ≥1000x coverage, more than 94% of the variants present at VAF &amp;lt;5% were successfully detected with an average sequencing quality score &amp;gt;Q30. In the plasma isolated from lung cancer patients, tumor derived genetic mutations could be identified in as little as 10ng of cfDNA. Mutations identified in cfDNA mirrored the mutations identified in tumor biopsies. Mutations with VAF as low as 0.1% could be detected in cfDNA and confirmed using Digital PCR. The changes in tumor-specific mutations in cfDNA was then correlated with the clinical follow up of the patients. Response to therapy, including resistance, could be observed by tracking specific mutations in cfDNA and the detection of tumor relapse could be anticipated up to two months using cfDNA when compared to standard methodologies. In conclusion, the comprehensiveness and speed of the NGS methodology, combined with the high sensitivity of detection, delivered an experimental protocol for the detection of somatic mutations in cfDNA for non-invasive real time therapy response monitoring of cancer patients. This strategy can be implemented in any molecular pathology laboratory with clear benefits for the management of cancer patients. Citation Format: Jose L. Costa, Gabriela Fernandes, Joana Reis, Miguel Silva, Venceslau Hespanhol, José C. Machado. Detection of somatic mutations in plasma allows for non-invasive real-time therapy response monitoring of lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 488.

Abstract 3968: Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples

Abstract Background Over the past years, tumor derived alterations in plasma has emerged as a promising cancer biomarker. The clinical applications envisioned with liquid biopsies range from monitoring of treatment response to disease progression and acquired resistance. However, the analysis of circulating tumor DNA (ctDNA) is challenging owing to the small fraction of tumor specific DNA relative to background levels of non-tumor derived DNA and therefore requires highly specific and sensitive techniques. Experimental procedures The Ion AmpliSeqTM Cancer Hotspot Panel v2, covering 2,800 COSMIC mutations from 50 cancer genes was used to analyze 31 primary tumor and metastatic lesions along with 19 matched plasma samples from 13 breast cancer (BC) patients receiving neoadjuvant treatment and 6 advanced stage metastatic BC patients. Digital PCR (dPCR) experiments were set up using mutation-specific assays (Biorad) for the detection of 13 hotspot mutations in PIK3CA, TP53, PTEN, AKT1, CDKN2A and STK11 genes previously identified in the solid tumor samples using next generation sequencing (NGS). Results The specificity and sensitivity of the mutation-specific dPCR assays were tested in dilution curves using synthetic oligonucleotides and tumor samples bearing the mutation of interest. Mutations could be robustly detected in plasma samples using dPCR with a sensitivity as low as 0.1% and a sensitivity of 0.5% using NGS. All mutations previously identified using NGS were validated using dPCR in tumor (n = 27) as well as in plasma samples (n = 5). Moreover, dPCR identified 4 additional mutations in 4 plasma samples in the neoadjuvant setting and 4 in the metastatic setting, 2 in tumor and 2 in plasma samples. The mutant allelic frequencies (MAF) of these newly identified mutations were very low (range 0.2%-1.8%) close to the detection limit of the technology. Through the use of dPCR, the detection rate of plasma ctDNA increased from 8% using NGS to 38% in the neoadjuvant setting and from 67% using NGS to 100% in the metastatic setting thereby highlighting the greater sensitivity of the dPCR technology. The MAF assessed by NGS and dPCR were highly correlated both in the neoadjuvant and metastatic (Spearman ρ = 0.84 and ρ = 0.97) settings, a higher concordance between NGS and dPCR being observed when the MAF are high. Conclusions The use of dPCR allows the detection of low-frequency tumor-specific mutations in plasma with a greater sensitivity compared to NGS providing the high accuracy and sensitivity essential for liquid biopsies. Citation Format: Françoise Rothé, Ghizlane Rouas, Michelle Coureau, Marion Maetens, David Brown, Christos Sotiriou, Michail Ignatiadis. Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3968.

A tale of locust swarms, cannibals, ageing and human obesity

Liquid biopsy refers to the analysis of DNA released by tumours into the circulation of patients for the detection, prognostication, selection of therapeutic targets or monitoring of malignancies. For example, regulatory approval has been granted for the detection of epidermal growth factor receptor mutations in plasma of patients with non-small cell lung cancers as a means to stratify therapy. However, the key challenge in the diagnostics of cancer is about its early detection. Given that cancers are highly heterogeneous, our group has been developing whole genome approaches to detect the signatures of cancer via a liquid biopsy. The rationale is to extract as much of the molecular information as possible from a plasma sample to catch a glimpse of the possible presence of cancer. Genomewide approaches to detect tumour-associated copy number aberrations, molecular size profile, DNA methylation signatures and transcriptomic features from circulating nucleic acids have been developed. We have further developed an approach to pinpoint the anatomical location of malignancy, naming to determine a tissue map non-invasively. In summary, many facets of molecular information could be extracted from a liquid biopsy.

Determination of EGFR gene somatic mutations in tissues and plasma of patients with advanced non-small cell lung cancer

The presence of activating mutations in the EGFR gene influences cell proliferation, angiogenesis, and increases metastatic ability; it has a significant impact on the choice of medical therapy of non-small cell lung cancer (NSCLC). The use of targeted therapy with tyrosine kinase inhibitors requires performance of appropriate genetic tests. The aim of this study was to design a real-time PCR-based diagnostic kit for fast and cheap of EGFR mutations testing in paraffin blocks and plasma, and kit validation using samples from patients with NSCLC, and also comparative estimation of diagnostic features of real-time PCR with wild type blocking and digital PCR for mutation testing in blood plasma. The study included 156 patients with various types of adenocarcinoma differentiation. It was designed a simple and efficient real-time PCR-based method of detecting L858R activating mutation and del19 deletion in the EGFR gene for DNA isolated from paraffin blocks. Kit for EGFR mutations was validated using 411 samples of paraffin blocks. The proposed system showed high efficiency for DNA testing from paraffin blocks: a concordance with results of testing with therascreen® EGFR RGQ PCR Kit ("Qiagen", Germany) was 100%. It has been shown the possibility of using this test system for the detection of mutations in plasma.

2185 Detection of KRAS mutations in plasma from patients with metastatic colorectal cancer by the next-generation sequencing

2185 Detection of KRAS mutations in plasma from patients with metastatic colorectal cancer by the next-generation sequencing

Abstract 4930: Next generation sequencing performance for the detection of mutations in plasma cell free DNA

Abstract The identification of mutations in circulating tumor DNA (ctDNA) can serve as a biomarker for non-invasive diagnosis and real time therapeutic monitoring. Although next generation sequencing (NGS) is a promising technology, the intrinsic low abundance of ctDNA in the high background of wild-type cell free DNA (cfDNA) makes the detection and quantification of such mutations in plasma a challenging task. This study aims to measure the performance of NGS analysis when applied to the identification of somatic mutations in plasma samples. Plasma samples from two healthy controls were used to isolate cfDNA. Constitutional genomic DNA from these controls was previously genotyped using the Human OmniExpress BeadChip. A custom multiplex PCR panel was designed to target 420 unique variants. To cover a broad range (0.5%-100%) of minor allele frequencies (MAF), as expected for somatic mutations in the plasma of cancer patients, the cfDNA samples from the two controls were mixed at five different rations (7:25, 1:6, 1:8, 1:20 and 1:100). Because sequence coverage is a key driver of accuracy, 5 different coverage profiles (100x, 500x, 1000x, 2000x and 5000x) were established for each of the cfDNA pools. On total 25 libraries were generated, pooled and sequenced with the Ion PGM system. Ion Reporter software was used for data analysis. The observed MAFs were compared with expected results for all the variants of each cfDNA pool. Median exon sequencing coverage of 133x, 650x, 1312x, 2628x and 5466x was obtained for each of the 5 cfDNA pools, with &amp;gt;99% of the amplicons covered. Overall base substitution and indel detection performance was high (&amp;gt;95% of the variants were detected across the different coverage levels). A direct correlation between expected and observed MAFs for the known variants was observed. For variants present at MAF ≥5%, a sensitivity of 91.4% and 100% was achieved at 100x and ≥500x coverage levels, respectively. For variants with 5% ≤ MAF ≤ 10% at 100x of coverage, the average quality score was &amp;lt;Q20. For variants occurring at low MAF (&amp;lt;5%) the detection sensitivity and the quality score dropped as coverage decreased. At 1000x coverage and above, more than 94% of the variants present at MAF &amp;lt;5% were successfully detected with an average quality score &amp;gt;Q30. Overall, positive predictive value remained high (&amp;gt;98%), with an average of 5 false-positive calls across the full coverage range. To validate our approach we analyzed 10 pairs of primary tumour/plasma samples. All cases carried known mutations of the EGFR gene detected during routine analysis. We were able to detect the corresponding mutation in the plasma samples in all cases. MAF in plasma samples ranged from 3% to 85%. Here we demonstrate that NGS can be used to detect mutations in cfDNA. Our approach with controlled data sets also enabled us to define allele frequency, allele depth, and allele quality score cutoffs for a reliable detection of low MAF variants in ctDNA. Citation Format: Ana Justino, Gabriela Fernandes, Ana Barroso, Barbara Parente, Venceslau Hespanhol, Jose C. Machado, Jose L. Costa. Next generation sequencing performance for the detection of mutations in plasma cell free DNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4930. doi:10.1158/1538-7445.AM2015-4930

Abstract 3399: Detection of rare mutations in plasma by droplet digital PCR

Abstract Detection and quantitation of specific mutations in circulating plasma holds promise for earlier and less invasive diagnosis of disease. This presents significant analytical challenges, particularly as the biomarker may differ from its highly abundant wildtype by only a single nucleotide. Conventional methods have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Compounding this, the amount of circulating nucleic acid in plasma is low. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. We evaluated ddPCR for the detection and quantitation of several clinically important mutations in the EGFR and KRAS loci from clinical samples derived from normal and tumor plasma samples. We also demonstrate the feasibility of multiplexing of Kras and EGFR assays to improve sample processing efficiency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3399. doi:1538-7445.AM2012-3399

Detection of nucleophosmin gene mutations in plasma from patients with acute myeloid leukemia: clinical significance and implications.

Roughly one-third of acute myeloid leukemia (AML) patients exhibit mutations in the nucleophosmin (NPM1) gene, and multiple studies have linked these mutations with a more favorable clinical outcome. We developed an assay for the detection of NPM1 mutations in peripheral blood plasma, and compared the results with clinical outcomes from a single institution. Analyzing plasma from previously untreated AML patients revealed NPM1 insertion mutations in 24 of 98 (24%) patients, with greater sensitivity than existing peripheral blood cell-based tests which showed positivity in only 22 of the 24 patients. Plasma testing allowed the detection of a novel 4 bp deletion in NPM1 in one patient. Analysis of clinical data corroborated previous data linking NPM1 mutations with better clinical outcome. These data underline the significance of NPM1 in the biology and clinical behavior of AML, and demonstrate the reliability and efficacy of plasma-based testing for NPM1 mutations.

A sensitive method for detection of EGFR gene mutations in plasma

18027 Background: Somatic mutations in the tyrosine kinase (TK) domain of the EGFR gene are associated with clinical response to TK inhibitors in patients with non-small cell lung cancer. An assay that detects such mutations in the plasma would provide a noninvasive technique to assess suitability for TK inhibitor therapy. Here we describe: 1) the development of a sensitive assay to detect the E746-A750 deletion in the TK domain of EGFR in plasma; and 2) optimization of the method for plasma DNA extraction. Methods: Our assay uses Mse I to specifically digest wild-type (WT) genomic DNA (gDNA) to reduce non-specific amplification. After digestion, samples are PCR- amplified using 1 unlabeled primer and 1 FAM-labeled primer spanning the E746-A750 deletion region and fluorescence detected with an automated analyzer. Serial dilution studies were conducted using H1650 gDNA (del E746-A750 cell line) diluted in WT gDNA after Mse I digestion. To assess detection of the deletion in plasma, 3–4 mL whole blood was spiked with 1–37 ng H1650 gDNA; gDNA from the separated plasma was then extracted (silica column/2-propanol precipitation), digested with Mse I, and amplified as above. Several extraction methods were evaluated using pooled plasma samples and PBS spiked with 10–350 ng gDNA: silica column, magnetic bead, phenol:chloroform, and 2-propanol precipitation. Results: Digestion of the WT EGFR allele followed by deletion-specific fluorescent PCR allowed detection of caof ca. 1 copy of the del E746-A750 EGFR gene (10 pg gDNA) diluted to 0.001%. Furthermore, del E746-A750 was successfully detected in 1/5 the final DNA volume (5 μl) in all spiked blood samples. Magnetic bead-based DNA extraction yielded the highest percent recovery of gDNA from PBS (69% recovery of the 10-ng sample). Phenol:chloroform extraction gave the highest yield with pooled plasma samples. Conclusions: The combination of an optimized DNA extraction method, clearing the plasma DNA sample of amplifiable WT DNA by restriction digestion, and mutation-specific fluorescent PCR provides a highly sensitive assay for detection of somatic mutations in plasma. No significant financial relationships to disclose.

Enhanced detection of EGFR mutations in plasma from non-small cell lung cancer (NSCLC) patients using Scorpion primers

7682 Background: Activating epidermal growth factor receptor (EGFR) mutations identified in NSCLC patient tumors are often associated with rapid and profound response to EGFR tyrosine kinase inhibitors (TKIs). Conventional detection methods are cumbersome and may underestimate mutations frequencies due to limited sensitivity. We and others have shown that tumor-specific mutations such as KRAS can be detected in tumor DNA shed into patient plasma (Kimura, Ann N Y Acad Sci, 2004). Here we describe the performance of an allele- specific real-time PCR system utilizing Scorpion primers (kindly provided by DxS, UK) to detect EGFR mutations in plasma. Methods: DNA was extracted from archival tumor blocks, slides and plasma obtained from consenting patients. In order to determine the sensitivity of this technique both in terms of the ratio of mutant-to-wild-type genomic DNA as well as the minimum amount of DNA required for detection, a dilution experiment was conducted. Genomic DNA from cell lines containing either the exon 21 L858R point mutation or the exon 21 E746–750 deletion was diluted with wild-type genomic DNA at ratios ranging from 1:2 to 1:10,000. Clinical specimens including plasma and/or tissue from 35 advanced stage NSCLC patients treated with EGFR-TKIs were examined. Results: Mutant DNA was successfully detected when it comprised as little as 0.1% of the total sample or as low as 25 pg of mutant-positive genomic DNA in a pool of 2.5 μg of total DNA. EGFR mutations were identified by this approach in both plasma and tissue of 2 patients who were complete responders to EGFR-TKI therapy, only one of which was detectable by direct sequencing. For the 7 patients where only tissue was available, two were positive both with the Scorpion primers and direct sequencing while the rest were wild-type. Of 21 patients where only plasma was available, 6 mutations were detected using the Scorpion primers. Conclusions: Allele-specific Scorpion technology is 1) highly specific and sensitive for EGFR mutation analysis, 2) able to detect mutations that were not observable by direct sequencing in plasma and tissue, 3) capable of detecting mutations in shed tumor DNA in plasma of advanced NSCLC patients and 4) may be suitable for monitoring response or detecting recurrence. No significant financial relationships to disclose.

Differential diagnosis between pancreatic adenocarcinoma and chronic pancreatitis: Interest in the detection of K-ras mutations in plasma

Differential diagnosis between pancreatic adenocarcinoma and chronic pancreatitis: Interest in the detection of K-ras mutations in plasma

Differential diagnosis between pancreatic adenocarcinoma and chronic pancreatitis: Interest in the detection of K-ras mutations in plasma

Differential diagnosis between pancreatic adenocarcinoma and chronic pancreatitis: Interest in the detection of K-ras mutations in plasma