Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer patients.

BACKGROUND: MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC. METHODS: Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE HpaII digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR. RESULTS: Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1 ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965 (95% CI: 0.94, 0.99). The statistically optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity, 95% CI: 0.45, 0.95). A tiered-based cutoff (20, 50, 80% percentile based) was applied to distinguish CRC samples with different methylation level. CONCLUSIONS: Our study demonstrated that the liquid biopsy assay for MLH1 promoter methylation detection using purely quantitative ddPCR is a simple and highly sensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.

Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples.

Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/µL with a range of 768-7510 copies/µL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.

A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment.

Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5' and 3' termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.

The cytogenetics of polar bodies: insights into female meiosis and the diagnosis of aneuploidy.

Female meiosis is comprised by two cell divisions, meiosis I (MI) and II (MII) and two different stages at which the development of the oocyte is temporarily halted. In the case of MI, this pause can potentially last for four to five decades. This added layer of complexity distinguishes female gametogenesis from its male counterpart. The single most important genetic factor impacting human reproductive success is aneuploidy. Aneuploid embryos may undergo permanent arrest during preimplantation development, fail to implant or spontaneously abort. Most aneuploidies originate during female meiosis and become increasingly common with advancing maternal age. To shed further light on the nature of aneuploidy in human oocytes, we utilized comparative genomic hybridization (CGH) to provide a detailed cytogenetic analysis of 308 first and second polar bodies (PBs). These were biopsied from fertilized oocytes, generated by 70 reproductively older women (average maternal age of 40.8 years). The total oocyte abnormality rate was 70%, and MII anomalies predominated over MI (50% aneuploidy rate versus 40.3%). Both whole chromosome non-disjunction and unbalanced chromatid predivision were seen, but the latter was the dominant MI aneuploidy-causing mechanism. Chromosome losses occurred more frequently than chromosome gains, especially during MI. Chromosomes of all sizes were found to participate in aneuploidy events, although errors involving smaller chromosomes were more common. These data reveal the spectrum of aneuploidies arising after each meiotic division, indicating that oocyte-derived abnormalities present at conception differ from those observed in established pregnancies. It is also clear that advancing maternal age had a significant adverse effect on female meiosis, and that this effect is most pronounced in MII. Indeed, our data suggest that MII may be more susceptible to age-related errors than MI.

Preimplantation genetic diagnosis of single-gene disorders: experience with more than 200 cycles conducted by a reference laboratory in the United States.

OBJECTIVE: To evaluate trends and outcomes from preimplantation genetic diagnosis (PGD) cycles. DESIGN: Retrospective data review. SETTING: A reference laboratory specializing in the provision of PGD services. PATIENT(S): One hundred sixty-two patients at risk of transmitting a serious monogenic disorder to their children. INTERVENTION(S): In vitro fertilization and PGD. MAIN OUTCOME MEASURE(S): Results of PGD cycles. RESULT(S): Two hundred twenty-four PGD cycles were referred by 59 different IVF centers. Forty-six different disorders were diagnosed, including several not previously diagnosed at the preimplantation stage. Cystic fibrosis was the most common reason for referral (73 cases). A diagnosis was obtained for 84.4% of tested embryos, with results available 6 to 36 hours from sample receipt. Only 10.7% of cycles had no transfer. The pregnancy rate per cycle with ET was 43.4%. CONCLUSION(S): Unlike previous reports of multiple PGD cycles, all of the cases in this study involved shipping of biopsied cells to a specialist reference laboratory for diagnosis. This approach, sometimes referred to as "transport PGD," accounts for the vast majority of PGD cycles in the United States. Preimplantation genetic diagnosis was shown to be an effective alternative to prenatal diagnosis for patients with an ethical or a religious objection to pregnancy termination and for infertile patients carrying a genetic disorder. Demand for this service at our center doubled in each of the last 4 years. Pregnancy rates per ET were encouraging, almost half of all patients undergoing their first PGD cycle achieving a birth or ongoing pregnancy.

A versatile strategy for preimplantation genetic diagnosis of haemophilia A based on F8-gene sequencing.

Preimplantation genetic diagnosis (PGD) of hemophilia A (HA) and other X-linked diseases through sex selection implies that male embryos will be systematically discarded, even though 50% are unaffected. The objective of the present work was to develop a PGD protocol for direct mutation identification that could be applied to first polar bodies (1PBs) in several HA clinical cases. Single buccal cells from controls and patients, and 1PBs were subjected to primer extension preamplification (PEP) PCR followed by amplification of F8 gene coding and intronic flanking regions, and direct sequencing. Moreover, multiplex fluorescent amplification of four short tandem repeats was adapted to a single cell preamplification in order to rule out contamination and allele drop-out, and for confirmatory indirect diagnosis. A couple at risk of HA transmission, with a familial mutation characterized as a 41-bp duplication in exon 14 of the F8 gene, was selected for the first clinical study. After optimizing the protocol, the complete F8 gene coding sequence was obtained from single cells to demonstrate the sensitivity of our methodology although in any clinical case only the relevant region, not the whole gene, must be amplified. The woman enrolled in the first clinical case has completed the first in-vitro fertilization cycle, and seven oocytes were analyzed with concordant results by both linkage analysis and direct sequencing method. Only one oocyte, among those diagnosed as mutation free, developed to embryo at day 3. It was transferred but pregnancy was not achieved. This PGD procedure enables non-affected and noncarrier embryo selection in families with any point or small-range mutation in the F8 gene, without the need for further custom-made modifications.

Multiplex fluorescent analysis of four short tandem repeats for rapid haemophilia A molecular diagnosis.

Indirect molecular diagnosis of hemophiliaA (HA) is carried out by analyzing intragenic polymorphic markers described along the coagulation factorVIII (FVIII) gene. Several studies have demonstrated that the two commonly used intronic short tandem repeats (STR13 and STR22) located in the FVIII gene are highly informative for this task. Two extragenic markers closely linked to FVIII (DXS1073 and DXS1108) have also been described as valuable tools for gene tracking. The objective of the present work was to develop a rapid, single-tube automated method to simultaneously analyze these four STRs. Consistent amplification was achieved by quadruplex fluorescent PCR and the products were analyzed by capillary electrophoresis. Validation of the method included DNA analysis of 88 individuals from a control population, 45 HA patients and 32 individuals from 10 HA-affected families. Statistical study showed that the STR13, STR22 and DXS1108 loci were in significant linkage disequilibrium, whereas DXS1073 was not. Nevertheless, the combination of the four markers offered a high heterozygosity rate (>90%) that improved tracing of FVIII gene inheritance. Optimal results with application to single cells in a HA preimplantation genetic diagnosis (PGD) protocol demonstrated the sensitivity of the technique. In conclusion, the automated fluorescent method described is an extremely rapid, simple and highly informative one that is easy to standardize and allows direct comparison of results among different groups working with genetic counseling, prenatal diagnosis and PGD in HA-affected families.

Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes.

BACKGROUND: Preimplantation Genetic Diagnosis (PGD) using FISH to analyze up to nine chromosomes to discard chromosomally abnormal embryos has resulted in an increase of pregnancy rates in certain groups of patients. However, the number of chromosomes that can be analyzed is a clear limitation. We evaluate the reliability of using comparative genomic hybridization (CGH) to detect the whole set of chromosomes, as an alternative to PGD using FISH. METHODS AND RESULTS: We have analysed by CGH both, first polar bodies (1PBs) and metaphase II (MII) oocytes from 30 oocytes donated by 24 women. The aneuploidy rate was 48%. Considering two maternal age groups, a higher number of chromosome abnormalities were detected in the older group of oocytes (23% versus 75%, P < 0.02). About 33% of the 1PB-MII oocyte doublets diagnosed as aneuploid by CGH would have been misdiagnosed as normal if FISH with nine chromosome probes had been used. CONCLUSION: We demonstrate the reliability of 1PB analysis by CGH, to detect almost any chromosome abnormality in oocytes as well as unbalanced segregations of maternal translocations in a time frame compatible with regular in vitro fertilization (IVF). The selection of euploid oocytes could help to increase implantation and pregnancy rates of patients undergoing IVF treatment.