<p>Detailed results of Digital Sequencing and clinical ddPCR comparison. Results shown in individual 2x2 tables for each biomarker tested. *Note, all discordances were below DFCI ddPCR's reportable range. Detailed clinical information for each is presented in Supplemental Table 3.</p>
<p>Comparisons of CA 19-9 concentration and cfDNA mutant allele fractions in plasma. (A-E) cfDNA mutant allele fractions ("cfDNA percentage") versus CA 19-9 units per milliliter (U/mL) were determined at similar times for five patients with three or more serial blood draws. cfDNA percentage represents the mutant allele fraction of the most abundant cfDNA mutation, as determined at time zero, for each patient. (F) cfDNA mutant allele fractions and CA 19-9 marker measurements for patient 56 over the course of four time points while on therapy (gemcitabine nab-paclitaxel). Note that draw number 1 occurred 17 days after diagnosis.</p>
Current microbiological techniques used for multi-analyte detection, necessary for medical diagnostics, involve expensive and time consuming methods based on sandwich immunoassays. Described below is the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a micro-channel, which can be used to detect target antigens electrically (label-free format) in real-time. As a proof of principle, we have successfully demonstrated real-time detection of target antigens by measuring instantaneous changes in ionic impedance. We have also demonstrated the selectivity of our sensors in responding to target antigens while remaining irresponsive to non-target antigens. Using this technique, an array of these sensors can be multiplexed onto a biochip and used to detect the various biomarkers present in a complex mixture.
LBA11501 The full, final text of this abstract will be available at abstracts.asco.org at 7:30 AM (EDT) on Saturday, June 4, 2016, and in the Annual Meeting Proceedings online supplement to the June 20, 2016, issue of the Journal of Clinical Oncology. Onsite at the Meeting, this abstract will be printed in the Monday edition of ASCO Daily News.
3572 Background: Serial ctDNA can measure dynamic changes in disease burden over time, however utility of serial profiling to detect changes in actionable alterations remains unclear. Methods: We evaluated 501 patients with ≥3 serial Guardant360 assays performed between 09/2016 and 11/2020 and compared MSI, fusion, amplification and single nucleotide variant (SNV) detection over time. This comprised 2147 assays with a median of 4 assays per patient (min 3, max 18) occurring an average of 163 days apart (+/- SD of 147 days). Maximum detected variant allele frequency in samples (maxVAF) was assessed for relation to changes in detected alterations as a surrogate for tumor volume. Results: Among 406 patients with assays assessable for MSI-status, 17 (4.2%) had MSI detected. New MSI detection on a subsequent assay always occurred with a rising maxVAF (3/3) that was also ≥0.7%, while loss of detectable MSI between assays always associated with falling maxVAFs (7/7) with 6/7 occurring when maxVAF fell below 0.4%. Fusions were noted in 9/501 (2%) patients. Among 3 patients who lost a detectable fusion, maxVAF decreased in 1 patient and changed ≤0.2% between assays in 2, while 2/3 patients with new fusions had rising maxVAFs and 1 patient had a falling maxVAF. Amplifications were detected in 242/501 patients (48%). While most genes had highly variable amplification detection between assays (9% serially detected), ERBB2 amplifications were more consistent and serially detected in 39% of detected cases (P < 0.0001). New detection of amplifications occurred more commonly in cases with rising maxVAF (OR 11.70, 95% CI 7.61-18.00, P < 0.0001) and loss of detectable amplifications occurred more between samples with falling maxVAF (OR 12.37, 95% CI 8.35-18.66, P < 0.0001). Change in maxVAF correlated with change in number of detected amplifications (r = 0.62, P < 0.0001), but only partially explained changes seen (R 2 = 0.39). Between serial assays, SNVs changed a median of 0 variants (IQR -1 to 1), however some patients had significant changes (max gain 21/max loss 18). Among 1646 serial time points, 454 (28%) had no change in SNVs, 674 (41%) gained SNVs, and 518 (31%) lost SNVs on subsequent assays. Gains were more common in samples with rising maxVAF (OR 7.76, 95% CI 6.18-9.73, P < 0.0001) while losses were more common when maxVAF fell (OR 6.90, 95% CI 5.47-8.66, P < 0.0001). The correlation between maxVAF change and SNV change was significant (r = 0.29, P < 0.0001), but minimally explained SNV changes (R 2 = 0.086) and was a much weaker association than noted for amplification changes. Conclusions: We noted significant differences in detection of actionable alterations across serial ctDNA assays. Increased ctDNA volume (higher maxVAF) due to tumor progression may explain some variation over time, but variability also occurs outside these changes, likely reflecting clonal evolution following therapy.
Abstract Background: ctDNA has the potential to identify patients (pts) with early stage cancer; however, current assays are challenged by limited sensitivity (~50%), reliance on a single analyte (e.g. somatic mutation detection), and/or the need for tumor tissue or genomic DNA sequencing to interpret ctDNA results. Recent studies have demonstrated that ctDNA can be detected using other biomarkers including DNA methylation. We developed a technology in which both somatic mutations and epigenomic alterations can be analyzed in a single assay. Methods: Using a large database of cell-free DNA (cfDNA) profiles generated from advanced cancer patients, we designed a targeted sequencing assay that detects somatic variants, methylation alterations, and other epigenomic variations at transcription factor binding sites associated with CRC. Total cfDNA was extracted, partitioned based on methylation level, and analyzed. Data were then filtered using a variant classifier to differentiate tumor- from non-tumor-derived alterations without a priori knowledge of tissue or germline sequencing results. A machine learning model was trained on 111 cfDNA samples from 38 late stage and 10 early stage CRC pts and 63 age-matched cancer-free controls. For the independent test set, plasma samples (4-5mL) were collected from 72 pts with stage I-IV CRC prior to and 4 weeks after (N = 50, total of 122 samples) surgical resection. 35 age-matched cancer-free controls were similarly analyzed in the test set. Results: Of the 72 pts, 62.5% were male, and median age at CRC diagnosis was 61.5 years (range 36-85). Stage distribution was 52.8% stage I/II, 40.3% stage III, and 6.9% stage IV. In the 50 pts with post-surgical samples, clinical follow-up was available for 49 (median post-surgery follow-up: 314 days; range 15-472). Utilizing this assay, pre-surgery ctDNA detection rate was 94% (68/72); 97% in stage I/II, 90% in stage III, and 100% in stage IV. Epigenomic analysis significantly enhanced ctDNA detection relative to somatic mutational analysis alone (94% vs. 56%; p<0.0001). Specificity in age-matched cancer-free controls was 94%. Discussion: Utilizing a plasma-only sequencing assay incorporating somatic genomic variant detection, epigenomic analysis, and a bioinformatic classifier to filter non-tumor derived variants, ctDNA detection rate in early stage CRC (I-III) is 94% (63/67; 95% confidence interval 86%;98%) with 94% specificity, far outperforming the detection rate of somatic sequence variant detection alone. Clinical follow-up is ongoing to evaluate post-surgery ctDNA detection rate and disease recurrence. These results have significant implications for the clinical utility of ctDNA in early stage cancer management. Citation Format: Seung-Tae Kim, Victoria M. Raymond, Joon Oh Park, Elena Zotenko, Young Suk Park, Matthew Schultz, Won Ki Kang, Oscar Westesson, Hee-Cheol Kim, Yupeng He, Justin I. Odegaard, Stefanie A. Mortimer, William J. Greenleaf, Ariel Jaimovich, Jeeyun Lee, AmirAli Talasaz. Combined genomic and epigenomic assessment of cell-free circulating tumor DNA (ctDNA) improves assay sensitivity in early-stage colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 916.
Abstract Background MBC is an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Patients with treatment-refractory MBC may benefit from tissue genomic evaluation using next-genomic sequencing (NGS). Furthermore, circulating DNA fragments with tumor-specific sequence alterations (ctDNA) found in the blood of patients with advanced disease offer the possibility of non-invasive molecular monitoring. ctDNA detects actionable mutations with the advantage of serial evaluation and allowing capture of inter- and intra-tumor heterogeneity. Methods This is a retrospective evaluation of 28 patients with MBC who failed standard therapies and had baseline plasma analyzed for ctDNA and tissue analysis (NGS) before starting new therapy. We were interested in the performance of the ctDNA test and the concordance rate of genomic alterations detected in the two tests. Selection criteria: progression of disease after standard therapies, need to detect novel molecular abnormalities for possible therapeutic targeting, or confirmation of persistence of genomic abnormalities already demonstrated in tissue or blood analysis. Guardant360™(Guardant Health) involves comprehensive sequencing of a panel of 54 gene associated with solid tumors using single-molecule digital sequencing technology. FoundationOne®(Foundation Medicine) performed the NGS on tissue evaluates the entire coding sequence of 315 cancer-related genes. Results All patients had biopsy-proved metastatic disease, and had ctDNA and NGS performed. 93% of patients had ctDNA alterations detected (with 0.1%-27.8% circulating tumor fraction), and 9 patients had serial ctDNA. Overall, for the patients we detected mutations in ctDNA and tissue, 89% of patients had a specific alteration on ctDNA that matched the NGS analysis. Among all mutations detected in tumors which are in overlapped genes, 71% of alterations were common (83% excluding gene amplifications). Interestingly, for patients who had both tests done within 8 weeks, 70% of had additional alterations in the ctDNA that were not found on NGS, such as ERBB2 mutations. Conclusions Genomic analysis using ctDNA and NGS detects genomic abnormalities in all patients with MBC with high concordance. However, each method was able to detect alterations that the other did not, suggesting the two methods can be complementary in detecting actionable mutations and expanding therapeutic options. Intriguingly, this occurred more frequently with the ctDNA, demonstrating its utility as an adjunct to tissue sampling which permits capture of the inter- and intra-tumor heterogeneity of the disease, and warrants further investigation prospectively. Citation Format: Laura K. Austin, Tiffany Avery, Rebecca Jaslow, Paolo Fortina, Dragan Sebisanovic, LaiMun Siew, Aubrey Zapanta, AmirAli Talasaz, Massimo Cristofanilli. Concordance of circulating tumor DNA (ctDNA) and next-generation sequencing (NGS) as molecular monitoring tools in metastatic breast cancer (MBC). [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 4918. doi:10.1158/1538-7445.AM2015-4918
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