Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA.

Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centered at ∼50 bp. We show that these "ultrashort" cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments, n = 28) than in plasma of patients with cancer (median = 14.2%, n = 21, P < 0.0001). The ultrashort cfDNA fragments map to accessible chromatin regions of blood cells, particularly in promoter regions with the potential to adopt G-quadruplex (G4) DNA secondary structures. G4-positive promoter chromatin accessibility is significantly enriched in ultrashort plasma cfDNA fragments from healthy individuals relative to patients with cancers (P < 0.0001), in whom G4-cfDNA enrichment is inversely associated with copy number aberration-inferred tumor fractions. Our findings redraw the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments. Sequencing of MB-ssDNA libraries could facilitate the characterization of gene regulatory regions and DNA secondary structures via liquid biopsy. Our data underline the diagnostic potential of ultrashort cfDNA through classification for cancer patients.

Fragmentation patterns and personalized sequencing of cell-free DNA in urine and plasma of glioma patients.

Glioma-derived cell-free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma-derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient's tumor, we detected tumor-derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10-3 , 3.1 × 10-5 , and 4.7 × 10-5 , respectively. We identified a shift in the size distribution of tumor-derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole-genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non-malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non-malignant brain disorders (P = 1.7 × 10-2 ) and healthy individuals (P = 5.2 × 10-9 ). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80-0.91) suggesting possibilities for truly non-invasive cancer detection.

CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response.

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

ctDNA monitoring using patient-specific sequencing and integration of variant reads.

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to parts per million. This resulted in median area under the curve (AUC) values of 0.98 in advanced cancers and 0.80 in early-stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that INVAR may be applied without requiring personalized sequencing panels so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

Detection of ctDNA from Dried Blood Spots after DNA Size Selection.

BACKGROUND: Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole-genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models. METHODS: We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and polymerase chain reaction (PCR). RESULTS: Analyzing a 50 µL dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment-size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood spots from 2 patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites. CONCLUSION: Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.

Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors.

BACKGROUND: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established. METHODS: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine. RESULTS: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings. CONCLUSIONS: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.

Enhanced detection of circulating tumor DNA by fragment size analysis.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

Noninvasive detection of F8 int22h-related inversions and sequence variants in maternal plasma of hemophilia carriers.

Direct detection of F8 and F9 sequence variants in maternal plasma of hemophilia carriers has been demonstrated by microfluidics digital PCR. Noninvasive prenatal assessment of the most clinically relevant group of sequence variants among patients with hemophilia, namely, those involving int22h-related inversions disrupting the F8 gene, poses additional challenges because of its molecular complexity. We investigated the use of droplet digital PCR (ddPCR) and targeted massively parallel sequencing (MPS) for maternal plasma DNA analysis to noninvasively determine fetal mutational status in pregnancies at risk for hemophilia. We designed family-specific ddPCR assays to detect causative sequence variants scattered across the F8 and F9 genes. A haplotype-based approach coupled with targeted MPS was applied to deduce fetal genotype by capturing a 7.6-Mb region spanning the F8 gene in carriers with int22h-related inversions. The ddPCR analysis correctly determined fetal hemophilia status in 15 at-risk pregnancies in samples obtained from 8 to 42 weeks of gestation. There were 3 unclassified samples, but no misclassification. Detailed fetal haplotype maps of the F8 gene region involving int22h-related inversions obtained through targeted MPS enabled correct diagnoses of fetal mutational status in 3 hemophilia families. Our data suggest it is feasible to apply targeted MPS to interrogate maternally inherited F8 int22h-related inversions, whereas ddPCR represents an affordable approach for the identification of F8 and F9 sequence variants in maternal plasma. These advancements may bring benefits for the pregnancy management for carriers of hemophilia sequence variants; in particular, the common F8 int22h-related inversions, associated with the most severe clinical phenotype.

COFFEE: control-free noninvasive fetal chromosomal examination using maternal plasma DNA.

OBJECTIVE: The aim of this study is to develop an approach for analyzing plasma DNA sequencing data for noninvasive fetal chromosomal aneuploidy testing that does not require the comparison with control samples or a series of selected genomic regions. RESULTS: We developed the control-free noninvasive fetal chromosomal examination (COFFEE) algorithm by utilizing the size differences between the fetally derived and maternally derived DNA molecules in maternal plasma. We applied COFFEE on three datasets generated in different experimental settings. COFFEE showed 100% accuracy in trisomy 21 testing on these datasets. In contrast, samples analyzed using an existing control-based z-score method would introduce a false-positive result because of batch-to-batch variation, when the tested samples were analyzed using control samples from other batches. We believe that COFFEE is useful for enhancing the cost-effectiveness of noninvasive fetal chromosomal aneuploidy testing particularly in laboratories with small caseloads. Source code and testing datasets for COFFEE are available for download at http://www.cuhk.edu.hk/med/cpy/Research/COFFEE/. CONCLUSION: Control-free noninvasive fetal chromosomal examination is demonstrated to be a versatile data analysis approach and could enhance the application of noninvasive fetal chromosomal aneuploidy detection. © 2017 John Wiley & Sons, Ltd.

Non-invasive prenatal diagnosis of thalassemias using maternal plasma cell free DNA.

Non-invasive prenatal testing (NIPT) using maternal plasma cell free DNA has already reshaped the existing prenatal care system for pregnancies screened for common chromosomal aneuploidies. On the other hand, much progress has been made in developing NIPT for monogenic diseases. Thalassemia served as a disease model to develop strategies for NIPT of monogenic traits. One approach focuses on the detection or exclusion of paternally inherited fetal mutations that are absent from the mother's genome. The assessment of maternally inherited mutations in maternal plasma requires the use of highly sensitive DNA quantification techniques. The relative mutation dosage (RMD) or the relative haplotype dosage (RHDO) approach provides a powerful tool to elucidate the fetal mutational status even in populations with lower genetic diversity, where the parents share the same mutation. With expected decrease in sequencing costs, NIPT for monogenic diseases is becoming an affordable option in prenatal care.

Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends.

Plasma DNA obtained from a pregnant woman was sequenced to a depth of 270× haploid genome coverage. Comparing the maternal plasma DNA sequencing data with the parental genomic DNA data and using a series of bioinformatics filters, fetal de novo mutations were detected at a sensitivity of 85% and a positive predictive value of 74%. These results represent a 169-fold improvement in the positive predictive value over previous attempts. Improvements in the interpretation of the sequence information of every base position in the genome allowed us to interrogate the maternal inheritance of the fetus for 618,271 of 656,676 (94.2%) heterozygous SNPs within the maternal genome. The fetal genotype at each of these sites was deduced individually, unlike previously, where the inheritance was determined for a collection of sites within a haplotype. These results represent a 90-fold enhancement in the resolution in determining the fetus's maternal inheritance. Selected genomic locations were more likely to be found at the ends of plasma DNA molecules. We found that a subset of such preferred ends exhibited selectivity for fetal- or maternal-derived DNA in maternal plasma. The ratio of the number of maternal plasma DNA molecules with fetal preferred ends to those with maternal preferred ends showed a correlation with the fetal DNA fraction. Finally, this second generation approach for noninvasive fetal whole-genome analysis was validated in a pregnancy diagnosed with cardiofaciocutaneous syndrome with maternal plasma DNA sequenced to 195× coverage. The causative de novo BRAF mutation was successfully detected through the maternal plasma DNA analysis.

Digital PCR analysis of circulating nucleic acids.

Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date.

Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies.

Recently published international guidelines recommend the clinical use of noninvasive prenatal test (NIPT) for aneuploidy screening only among pregnant women whose fetuses are deemed at high risk. The applicability of NIPT to aneuploidy screening among average risk pregnancies requires additional supportive evidence. A key determinant of the reliability of aneuploidy NIPT is the fetal DNA fraction in maternal plasma. In this report, we investigated if differences in fetal DNA fractions existed between different pregnancy risk groups. One hundred and ninety-five singleton pregnancies with male fetuses divided into 3 groups according to first trimester screening parameters were examined for fetal DNA percentage by counting Y chromosome DNA sequences using massively parallel sequencing. Fetal DNA fractions were compared between risk groups and assessed for correlations with first trimester screening parameters. There was no statistically significant difference in fetal DNA fractions across the high, intermediate and low risk groups. Fetal DNA fraction showed a strong negative correlation with maternal weight. Fetal DNA fraction also showed weak but significant correlations with gestational age, crown-rump length, multiple of medians of free ß-subunit of human chorionic gonadotropin and pregnancy-associated plasma protein A. Similar fetal DNA fractions in maternal plasma between high, intermediate and low risk pregnant women is a precondition for uniform performance of the aneuploidy NIPTs for the general population. This study thus shows that the aneuploidy screening by NIPT is likely to offer similar analytical reliability without respect to the a priori fetal aneuploidy risk.