SeqFF is a robust and direct method to determine fetal DNA fraction. Furthermore, the method is applicable to both male and female pregnancies and can greatly improve the accuracy of noninvasive prenatal testing for fetal copy number variation.
DNA derived from NSCLC tumors can be detected with high sensitivity in urine and plasma, enabling diagnostic detection and monitoring of therapeutic response from these noninvasive "liquid biopsy" samples.
To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.
ObjectiveAs the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT.Study DesignThe study includes plasma samples collected from patients with high-risk pregnancies in our CLIA–licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting.ResultsNIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result.ConclusionNIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.
While a number of factors trend with fetal fraction across the cohort as a whole, they are not the sole determinants of fetal fraction. In this study, the variability for any one patient does not appear large enough to justify postponing testing to a later gestational age.
Precision oncology requires sensitive and specific clinical biomarkers. Carbohydrate Antigen 19-9 (CA19-9) is widely used in pancreatic ductal adenocarcinoma (PDA) but lacks sensitivity and specificity. Nearly all PDAs harbor somatic KRAS mutations, nominating circulating tumor DNA (ctDNA) KRAS as an alternative disease biomarker, however, variable clinical performance has limited its clinical utility. We applied an ultrasensitive, PCR mutation enrichment, next generation sequencing ctDNA KRAS assay in a large cohort of patients with unresectable PDA (N = 189) recruited to the BIOPAC study between 2008–2015. Baseline and longitudinal serum CA19-9 and plasma ctDNA KRAS were correlated with time to progression (TTP) and overall survival (OS). Baseline ctDNA KRAS detection rate was 93.7% (86.4% in patients with non-elevated CA19-9). ctDNA KRAS and CA19-9 were positively correlated yet independently associated with TTP and OS (ctDNA KRAS p = 0.0018 and 0.0014; CA19-9 p = 0.0294 and 0.0007, respectively). A generated model quantitating longitudinal ctDNA KRAS correctly assessed greater than 80% of patient responses. Quantitative detection of KRAS ctDNA is an informative prognostic biomarker, complementary to CA19-9 in patients with unresectable PDA. Longitudinal ctDNA KRAS may inform therapeutic decision making and provides a kinetically dynamic and quantitative metric of patient response.
Introduction: An estimated 90% of pancreatic cancers harbor somatic KRAS G12/G13 mutations. The presence of circulating tumor DNA (ctDNA) KRAS mutations at diagnosis has prognostic implications, however, recent studies on small numbers of patients demonstrate widely variable KRAS ctDNA sensitivity (27 – 71%). Accurate identification of both the presence of ctDNA KRAS mutations and quantification of the number of ctDNA KRAS mutant copies would be an improved therapeutic response biomarker over CA19-9 which is known to be uninformative in 5-10% of patients with pancreatic cancer. A diagnostic and prognostic tool with very high analytical and clinical sensitivity, along with quantification, is needed for therapeutic response monitoring. Patients and Methods: Pretreatment (baseline) and longitudinal plasma samples were prospectively collected from 239 patients with unresectable pancreatic cancer through the Danish Biomarkers in Patients with Pancreatic Cancer (BIOPAC) study. The median age at diagnosis was 67 years (range 42 – 89 years) and 51.9% (N = 112) of patients were male. The majority of patients were metastatic at diagnosis (79.6%; N=172). ctDNA KRAS G12A/C/D/R/S/V, and G13D mutations were PCR enriched, sequenced by next generation sequencing (NGS), quantified and standardized. This was achieved by generating standard curves from a sample set with known numbers of spike-in copies for mutant KRAS molecules. These sample sets were assayed in parallel with patient samples starting with PCR enrichment of mutant KRAS DNA followed by NGS. The number of mutant copies detected was standardized by normalizing the number of copies detected in the sample to a constant number of calculated genome equivalents (GEqs) of wild type DNA across all samples evaluated. Baseline KRAS mutant levels were assessed. Additional metrics analyzed include age, gender, stage, and CA19-9 levels. Results: Two-hundred and sixteen patients (90.4%) had baseline plasma samples which passed quality control metrics. Baseline serum CA19-9 levels were available for 160 patients and ranged from 2.5 – 608,500 U/mL (median 202 U/mL). Eighteen patients (11.3%) had serum CA19-9 levels below the diagnostic threshold (<37 U/mL), and 76 patients (47.5%) had serum CA19-9 levels below the prognostic threshold (<1000 U/mL). Overall, 87% of patients were positive for a KRAS mutation at baseline by ctDNA analysis, with a median of 88.51 copies per 100,000 GEqs. Patients presenting with metastatic disease had a significantly higher median number of KRAS mutations (104.2 versus 23.9 copies per 100,000 GEqs; p<0.0001) and serum CA19-9 levels (1702.0 versus 303.0 U/mL; p<0.0001) at baseline than those with locally advanced cancer. Conclusion: In this large, prospective dataset of 216 patients with unresectable pancreatic cancer, ctDNA analysis was KRAS positive in 87.0% of patients. This detection rate closely matches the published prevalence of KRAS in pancreatic cancer (90%), and out performs previous studies, demonstrating the superior assay sensitivity. ctDNA analysis offers a viable tissue biopsy alternative for determining KRAS mutation status, especially in late stage patients. Given that approximately 48% of patients had CA19-9 levels below the prognostic threshold, quantification of KRAS mutant copy load may provide a more informative biomarker. Other clinical variables, baseline and longitudinal KRAS mutant levels, and patient outcomes will be examined and presented. Citation Format: Inna Chen, Victoria M. Raymond, Jennifer A. Geis, Sandeep Pingle, Eric A. Collisson, Vlada Melnikova, Mark G. Erlander, Julia A. Johansen, Fernando Blanco.{Authors}. Detection and quantification of ctDNA KRAS mutations from patients with unresectable pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A20.
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