Tumor-derived DNA can be found in the plasma of cancer patients. In this study, we explored the use of shotgun massively parallel sequencing (MPS) of plasma DNA from cancer patients to scan a cancer ...genome noninvasively.
Four hepatocellular carcinoma patients and a patient with synchronous breast and ovarian cancers were recruited. DNA was extracted from the tumor tissues, and the preoperative and postoperative plasma samples of these patients were analyzed with shotgun MPS.
We achieved the genomewide profiling of copy number aberrations and point mutations in the plasma of the cancer patients. By detecting and quantifying the genomewide aggregated allelic loss and point mutations, we determined the fractional concentrations of tumor-derived DNA in plasma and correlated these values with tumor size and surgical treatment. We also demonstrated the potential utility of this approach for the analysis of complex oncologic scenarios by studying the patient with 2 synchronous cancers. Through the use of multiregional sequencing of tumoral tissues and shotgun sequencing of plasma DNA, we have shown that plasma DNA sequencing is a valuable approach for studying tumoral heterogeneity.
Shotgun DNA sequencing of plasma is a potentially powerful tool for cancer detection, monitoring, and research.
Context:
Congenital adrenal hyperplasia (CAH) is an autosomal recessive condition that arises from mutations in CYP21A2 gene, which encodes for the steroidogenic enzyme 21-hydroxylase. To prevent ...genital ambiguity in affected female fetuses, prenatal treatment with dexamethasone must begin on or before gestational week 9. Currently used chorionic villus sampling and amniocentesis provide genetic results at approximately 14 weeks of gestation at the earliest. This means that mothers who want to undergo prenatal dexamethasone treatment will be unnecessarily treating seven of eight fetuses (males and three of four unaffected females), emphasizing the desirability of earlier genetic diagnosis in utero.
Objective:
The objective of the study was to develop a noninvasive method for early prenatal diagnosis of fetuses at risk for CAH.
Patients:
Fourteen families, each with a proband affected by phenotypically classical CAH, were recruited.
Design:
Cell-free fetal DNA was obtained from 3.6 mL of maternal plasma. Using hybridization probes designed to capture a 6-Mb region flanking CYP21A2, targeted massively parallel sequencing (MPS) was performed to analyze genomic DNA samples from parents and proband to determine parental haplotypes. Plasma DNA from pregnant mothers also underwent targeted MPS to deduce fetal inheritance of parental haplotypes.
Results:
In all 14 families, the fetal CAH status was correctly deduced by targeted MPS of DNA in maternal plasma, as early as 5 weeks 6 days of gestation.
Conclusions:
MPS on 3.6 mL plasma from pregnant mothers could potentially provide the diagnosis of CAH, noninvasively, before the ninth week of gestation. Only affected female fetuses will thus be treated. Our strategy represents a generic approach for noninvasive prenatal testing for an array of autosomal recessive disorders.
Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA ...sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using pairedend massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.
The anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients ...respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow–localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS).
•Response to the CD38-targeting antibody daratumumab is significantly associated with CD38 expression levels on the tumor cells.•Resistance to daratumumab is accompanied by increased expression of complement-inhibitory proteins.
Summary
Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently ...hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.
Itch and pain are refractory symptoms of many ocular conditions. Ocular itch is generated mainly in the conjunctiva and is absent from the cornea. In contrast, most ocular pain arises from the ...cornea. However, the underlying mechanisms remain unknown. Using genetic axonal tracing approaches, we discover distinct sensory innervation patterns between the conjunctiva and cornea. Further genetic and functional analyses in rodent models show that a subset of conjunctival-selective sensory fibers marked by MrgprA3 expression, rather than corneal sensory fibers, mediates ocular itch. Importantly, the actions of both histamine and nonhistamine pruritogens converge onto this unique subset of conjunctiva sensory fibers and enable them to play a key role in mediating itch associated with allergic conjunctivitis. This is distinct from skin itch, in which discrete populations of sensory neurons cooperate to carry itch. Finally, we provide proof of concept that selective silencing of conjunctiva itch-sensing fibers by pruritogen-mediated entry of sodium channel blocker QX-314 is a feasible therapeutic strategy to treat ocular itch in mice. Itch-sensing fibers also innervate the human conjunctiva and allow pharmacological silencing using QX-314. Our results cast new light on the neural mechanisms of ocular itch and open a new avenue for developing therapeutic strategies.
Massively parallel sequencing has recently been used in noninvasive prenatal diagnosis. The current costs of this technology are still relatively expensive, however, and sample throughput is still ...relatively low when it is used as a molecular diagnostic tool. Rather than nonselectively sequencing the genome, target enrichment provides a logical approach for more efficient and cost-effective massively parallel sequencing because it increases the proportion of informative data from the targeted region(s). Existing applications of targeted sequencing have mainly been qualitative analyses of genomic DNA. In this study, we investigated its applicability in enriching selected genomic regions from plasma DNA and the quantitative performance of this approach.
DNA was extracted from plasma samples collected from 12 pregnant women carrying female fetuses. The SureSelect Target Enrichment System (Agilent Technologies) was used to enrich for exons on chromosome X. Plasma DNA libraries with and without target enrichment were analyzed by massively parallel sequencing. Genomic DNA samples of the mother and fetus for each case were genotyped by microarray.
For the regions targeted by the enrichment kit, the mean sequence coverage of the enriched samples was 213-fold higher than that of the nonenriched samples. Maternal and fetal DNA molecules were enriched evenly. After target enrichment, the coverage of fetus-specific alleles within the targeted region increased from 3.5% to 95.9%.
Targeted sequencing of maternal plasma DNA permits efficient and unbiased detection of fetal alleles at genomic regions of interest and is a powerful method for measuring the proportion of fetal DNA in a maternal plasma sample.
We performed a high-resolution analysis of the biological characteristics of plasma DNA in systemic lupus erythematosus (SLE) patients using massively parallel genomic and methylomic sequencing. A ...number of plasma DNA abnormalities were found. First, aberrations in measured genomic representations (MGRs) were identified in the plasma DNA of SLE patients. The extent of the aberrations in MGRs correlated with anti-double–stranded DNA (anti-dsDNA) antibody level. Second, the plasma DNA of active SLE patients exhibited skewed molecular size-distribution profiles with a significantly increased proportion of short DNA fragments. The extent of plasma DNA shortening in SLE patients correlated with the SLE disease activity index (SLEDAI) and anti-dsDNA antibody level. Third, the plasma DNA of active SLE patients showed decreased methylation densities. The extent of hypomethylation correlated with SLEDAI and anti-dsDNA antibody level. To explore the impact of anti-dsDNA antibody on plasma DNA in SLE, a column-based protein G capture approach was used to fractionate the IgG-bound and non–IgG-bound DNA in plasma. Compared with healthy individuals, SLE patients had higher concentrations of IgG-bound DNA in plasma. More IgG binding occurs at genomic locations showing increased MGRs. Furthermore, the IgG-bound plasma DNA was shorter in size and more hypomethylated than the non–IgG-bound plasma DNA. These observations have enhanced our understanding of the spectrum of plasma DNA aberrations in SLE and may provide new molecular markers for SLE. Our results also suggest that caution should be exercised when interpreting plasma DNA-based noninvasive prenatal testing and cancer testing conducted for SLE patients.
Significance Through the use of massively parallel sequencing, we have demonstrated a spectrum of plasma DNA abnormalities in patients with systemic lupus erythematosus. These abnormalities include aberrant measured genomic representations, hypomethylation, and DNA fragment size shortening. The binding of anti-double–stranded DNA antibody to plasma DNA appears to be an important factor associated with these abnormalities. These findings provide valuable insights into the biology of plasma DNA in an autoimmune disease and have potential implications for the development of new molecular markers for systemic lupus erythematosus.
A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal ...diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner.
We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses.
A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia.
Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.
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