Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in noninvasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids ...to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to maternal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma.
The assay used methylation-sensitive restriction enzymes to eliminate the maternal (unmethylated) fraction of the DNA sample. The undigested fetal DNA fraction was then coamplified in the presence of a synthetic oligonucleotide to permit competitive PCR. The amplification products were quantified by single-base extension and MALDI-TOF MS analysis.
Using 2 independent markers, (sex determining region Y)-box 14 (SOX14) and T-box 3 (TBX3), we measured a mean of 151 copies of fetal DNA/mL plasma and a mean fetal fraction of 0.13 in samples obtained from pregnant women. We investigated 242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensitivity and specificity for the assay of 99% and 100%, respectively.
By investigating several regions in parallel, we reduced the measurement variance and enabled quantification of circulating cell-free DNA. Our results indicate that this multiplex methylation-based reaction detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma.
Objective We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. Study design Sample multiplexing ...and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. Results In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89–100%) and 99.7% specificity (95% confidence interval, 98.5–99.9%). Conclusion Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.
Whereas target-specific drugs are available for treating ERBB2-overexpressing and hormone receptor-positive breast cancers, no tailored therapy exists for hormone receptor- and ERBB2-negative ...("triple-negative") mammary carcinomas. Triple-negative tumors account for 15% of all breast cancers and frequently harbor defects in DNA double-strand break repair through homologous recombination (HR), such as BRCA1 dysfunction. The DNA-repair defects characteristic of BRCA1-deficient cells confer sensitivity to poly(ADP-ribose) polymerase 1 (PARP1) inhibition, which could be relevant to treatment of triple-negative tumors. To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor AZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.
DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and ...after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.
We have found previously that acquired doxorubicin resistance in a genetically engineered mouse model for BRCA1-related breast cancer was associated with increased expression of the mouse multidrug ...resistance (Mdr1) genes, which encode the drug efflux transporter ATP-binding cassette B1/P-glycoprotein (P-gp). Here, we show that even moderate increases of Mdr1 expression (as low as 5-fold) are sufficient to cause doxorubicin resistance. These moderately elevated tumor P-gp levels are below those found in some normal tissues, such as the gut. The resistant phenotype could be completely reversed by the third-generation P-gp inhibitor tariquidar, which provides a useful strategy to circumvent this type of acquired doxorubicin resistance. The presence of MDR1A in drug-resistant tumors with a moderate increase in Mdr1a transcripts could be shown with a newly generated chicken antibody against a mouse P-gp peptide. Our data show the usefulness of realistic preclinical models to characterize levels of Mdr1 gene expression that are sufficient to cause resistance.
We have studied in vivo responses of "spontaneous" Brca1- and p53-deficient mammary tumors arising in conditional mouse mutants to treatment with doxorubicin, docetaxel, or cisplatin. Like human ...tumors, the response of individual mouse tumors varies, but eventually they all become resistant to the maximum tolerable dose of doxorubicin or docetaxel. The tumors also respond well to cisplatin but do not become resistant, even after multiple treatments in which tumors appear to regrow from a small fraction of surviving cells. Classical biochemical resistance mechanisms, such as up-regulated drug transporters, appear to be responsible for doxorubicin resistance, rather than alterations in drug-damage effector pathways. Our results underline the promise of these mouse tumors for the study of tumor-initiating cells and of drug therapy of human cancer.
Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent ...probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA–probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA–probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.
Multiplex detection of low-frequency mutations is becoming a necessary diagnostic tool for clinical laboratories interested in noninvasive prognosis and prediction. Challenges include the detection ...of minor alleles among abundant wild-type alleles, the heterogeneous nature of tumors, and the limited amount of available tissue. A method that can reliably detect minor variants <1% in a multiplexed reaction using a platform amenable to a variety of throughputs would meet these requirements. We developed a novel approach, UltraSEEK, for high-throughput, multiplexed, ultrasensitive mutation detection and used it for detection of mutant sequence mixtures as low as 0.1% minor allele frequency. The process consisted of multiplex PCR, followed by mutation-specific, single-base extension using chain terminators labeled with a moiety for solid phase capture. The captured and enriched products were then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. For verification, we successfully analyzed ultralow fractions of mutations in a set of characterized cell lines, and included a direct comparison to droplet digital PCR. Finally, we verified the specificity in a set of 122 paired tumor and circulating cell-free DNA samples from melanoma patients. Our results show that the UltraSEEK chemistry is a particularly powerful approach for the detection of somatic variants, with the potential to be an invaluable resource to investigators in saving time and material without compromising analytical sensitivity and accuracy.
There is no tailored therapy yet for human basal-like mammary carcinomas. However, BRCA1 dysfunction is frequently present in these malignancies, compromising homology-directed DNA repair. This ...defect may serve as the tumor's Achilles heel and make the tumor hypersensitive to DNA breaks. We have evaluated this putative synthetic lethality in a genetically engineered mouse model for BRCA1-associated breast cancer, using the topoisomerase I (Top1) poison topotecan as monotherapy and in combination with poly(ADP-ribose) polymerase inhibition by olaparib. All 20 tumors tested were topotecan sensitive, but response heterogeneity was substantial. Although topotecan increased mouse survival, all tumors eventually acquired resistance. As mechanisms of in vivo resistance, we identified overexpression of Abcg2/Bcrp and markedly reduced protein levels of the drug target Top1 (without altered mRNA levels). Tumor-specific genetic ablation of Abcg2 significantly increased overall survival of topotecan-treated animals (P < 0.001), confirming the in vivo relevance of ABCG2 for topotecan resistance in a novel approach. Despite the lack of ABCG2, a putative tumor-initiating cell marker, none of the 11 Abcg2(-/-);Brca1(-/-);p53(-/-) tumors were eradicated, not even by the combination topotecan-olaparib. We find that olaparib substantially increases topotecan toxicity in this model, and we suggest that this might also happen in humans.
Germ-line heterozygosity of the BRCA2 gene in women predisposes to breast and ovarian cancers. Successful therapies targeted specifically at these neoplasms have thus far remained elusive. Recent ...studies in mice have shown that inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) targets cells lacking Brca2 and xenografts derived from BRCA2-deficient ES cells or Chinese hamster ovary cells. We set out to develop a more relevant preclinical model that will inform and accelerate translation into the clinic. As such, we conditionally deleted Brca2 and p53 within murine mammary epithelium and treated the resulting tumors in situ with a highly potent PARP-1 inhibitor (AZD2281) alone or in combination with carboplatin. Daily exposure to AZD2281 for 28 days caused significant regression or growth inhibition in 46 of 52 tumors. This response was shown to be specific to tumors lacking both Brca2and p53. AZD2281/carboplatin combination therapy for 28 days showed no advantage over carboplatin monotherapy. However, if PARP inhibitor treatment was continued, this significantly increased the time to tumor relapse and death in these mice. This preclinical study is the first to show in vivo hypersensitivity of spontaneously arising Brca2-deficient mammary tumors to PARP-1 inhibition monotherapy or combination therapy. As such, our data add substantial weight to the argument for the use of PARP inhibitors as therapeutic agents against human breast cancers in which BRCA2 is mutated. Moreover, the specificity that we have shown further suggests that PARP inhibitors will be generally effective against tumors caused by dysregulation of components of the homologous recombination pathway.
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