Generation Sequencing Of Cell-free DNA Research Articles

Machine learning-based somatic variant calling in cell-free DNA of metastatic breast cancer patients using large NGS panels

Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants. We used cfDNA of 70 MBC patients profiled with both the small targeted Oncomine breast panel (Thermofisher) and the much larger Qiaseq Human Breast Cancer Panel (Qiagen). The model was trained on the panels’ common regions using Oncomine hotspot mutations as ground truth. Applied to Qiaseq data, it achieved 35% sensitivity and 36% precision, outperforming basic filtering. For 20 patients we used germline DNA to filter for somatic variants and obtained 245 variants in total, while our model found seven variants, of which six were also detected using the germline strategy. In ten tumor-free individuals, our method detected in total one (potentially germline) variant, in contrast to 521 variants detected without our model. These results indicate that our model largely detects somatic variants.

Blood-Based Liquid Biopsy for Comprehensive Cancer Genomic Profiling Using Next-Generation Sequencing: An Emerging Paradigm for Non-invasive Cancer Detection and Management in Dogs.

This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can non-invasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites. Eleven dogs with a variety of confirmed cancer diagnoses (including localized and disseminated disease) who were scheduled for surgical resection, and five presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects. All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to non-invasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs. In five of the nine cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the three patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for non-invasive detection of minimal residual disease following curative-intent treatment. Liquid biopsy allows for non-invasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity.

Emerging Considerations for Noninvasive Prenatal Testing

Approaches to prenatal screening for common fetal chromosomal aneuploidies are undergoing a dynamic transformation in response to a greater understanding surrounding advances in the clinical utilities and limitations of noninvasive prenatal testing (NIPT).10 NIPT has been clinically adopted as a screening tool for aneuploidies, such as Down, Edwards, and Patau syndromes, and methodologies are primarily based on next generation sequencing of cell-free DNA (cfDNA) from maternal plasma. The cfDNA comprises maternal DNA fragments as well as placental DNA fragments that serve as a fetal surrogate marker. While NIPT was initially recommended as a screening option for high-risk women from about 10 weeks of gestation, recent clinical studies demonstrate that NIPT outperforms conventional screening approaches (e.g., first trimester combined test) regardless of the maternal age spectrum. The American College of Medical Genetics and Genomics updated recommendations in 2016 to include informing all pregnant women that NIPT is a screening option for conventionally screened aneuploidies. However, the potential expansion of NIPT utilization in prenatal care practices is faced with evolving challenges. Depending upon the laboratory, test methodology, and bioinformatics processes used, NIPT result reporting is not standardized. The decision of whether to use NIPT screening is ultimately that of the informed patient. However, it will also be driven by the ability to clearly communicate the risks and benefits of screening approaches by the clinical care team. This highlights the need for multidisciplinary collaboration in the clinical implementation of NIPT. To address these exciting advancements and emerging considerations, we invited a group of experts and early adopters of NIPT screening from multiple disciplines (genetic counseling, obstetrics, genomics, ethics, and clinical chemistry) to share their views on this topic. Does NIPT have a role beyond screening for fetal chromosomal aneuploidies in high-risk pregnancies? Judith Jackson: NIPT plays an important role in screening for …