Assessing the value of CAN-gene mutations using MALDI-TOF MS.

PURPOSE: To identify cancer-linked genes, Sjöblom et al. and Wood et al. performed a genome-wide mutation screening in human breast and colorectal cancers. 140 CAN-genes were found in breast cancer, which in turn contained overall 334 mutations. These mutations could prove useful for diagnostic and therapeutic purposes. METHODS: We used a MALDI-TOF MS 40-plex assay for testing 40 loci within 21 high-ranking breast cancer CAN-genes. To confirm mutations, we performed single-plex assays and sequencing. RESULTS: In general, the mutation rate of the analyzed loci in our sample cohort was very low. No mutation from the 40 loci analyzed could be found in the 6 cell lines. In tissue samples, a single breast cancer tissue sample showed heterozygosity at locus c.5834G>A within the ZFYVE26 gene (Zinc finger FYVE domain-containing gene 26). CONCLUSIONS: Sjöblom et al./Wood et al. already showed that the vast majority of CAN-genes are mutated at very low frequency. Due to the fact that we only found one mutation in our cohort, we therefore assume that at the selected loci, mutations might be low-frequency events and therefore, more rarely detectable. However, further evaluation of the CAN-gene mutations in larger cohorts should be the aim of further studies.

A selected pre-amplification strategy for genetic analysis using limited DNA targets.

BACKGROUND: Limited DNA resources or limited DNA targets in predominant backgrounds for genetic tests can lead to misdiagnosis. We developed a strategy to selectively increase the amount of minor targets through a specific pre-amplification procedure. METHODS: We used the model of circulating cell free (ccf) male fetal DNA as a minor target in the predominant maternal plasma DNA to evaluate the strategy. The sex determining region (SRY) locus on the Y chromosome was used to identify ccf fetal DNA, and the human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was used to identify ccf total DNA in maternal plasma. We selectively pre-amplified the minor target SRY locus using the Expand Long Template PCR system and assessed the efficiency of the pre-amplification by real-time PCR, for both SRY and GAPDH, to compare the quantities of pre-amplified fetal DNA with those of maternal total DNA without pre-amplification. RESULTS: The selected pre-amplification increased the amount of ccf fetal DNA dramatically (Wilcoxon test: p =0.000, the fold change = 11,596). After selected preamplification, a proportion of 2.19% of the ccf fetal minor part in the predominant maternal component was changed up to 25,334%. The increased amounts of ccf fetal DNA found with the pre-amplification are not correlated to the amounts found without the procedure (r=-0.017, p=0.949). CONCLUSIONS: This strategy may be useful in genetic analysis with limited DNA resources and limited DNA targets in predominant background molecules. However, this approach is not suitable for quantitative assessments, due to the fact that quantitative imbalanced amplification was observed as a result of the pre-amplification procedure.

Circulating cell-free DNA as a potential biomarker for minimal and mild endometriosis.

It has recently been reported that high concentrations of circulating cell-free (ccf) nucleic acids in plasma and serum could be used as biomarkers for non-invasive monitoring a wide variety of malignant and benign proliferations and inflammatory conditions. Endometriosis is one of the most common benign gynaecological proliferations with inflammatory activation in premenopausal women. Real-time multiplex polymerase chain reaction was used for synchronized quantification of the glyceraldehyde-3-phosphate dehydrogenase gene sequence in nuclear DNA (nDNA) and the ATP synthase-8 gene sequence in mitochondrial DNA (mtDNA). DNA was extracted from 500 microl serum and plasma of 19 cases with endometriosis to measure the total amount of ccf nDNA and ccf mtDNA. The concentration of ccf nDNA in plasma was significantly higher in the endometriosis group than in the control group (P = 0.046). The cut-off value selected by a receiver operating characteristic curve could provide a sensitivity of 70% and a specificity of 87% to discriminate between the minimal or mild cases and normal controls. The finding of significantly increased concentrations of ccf nDNA in plasma of patients with endometriosis suggests that ccf nDNA might be a potential biomarker for developing non-invasive diagnostic test in endometriosis.

Mitochondrial DNA content in paired normal and cancerous breast tissue samples from patients with breast cancer.

INTRODUCTION: We develop a multiplex quantitative real-time PCR for synchronized analysis of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) to investigate relative mtDNA abundance in paired normal and cancerous breast tissues. MATERIALS AND METHODS: The amounts of nDNA and mtDNA in 102 tissue samples were quantified for both glyceraldehype-3-phosphodehydrogenase (GAPDH) gene and mtDNA encoded ATPase (MTATP) 8 gene. The average threshold cycle (Ct) number values of the nDNA and mtDNA were used to calculate relative mtDNA content in breast tissues. RESULTS: The median delta Ct (DeltaCt) and the median mtDNA content for normal and cancerous breast tissues were 6.73 and 2.54, as well as 106.50 and 5.80 (P = 0.000, respectively). The mtDNA content was decreased in 82% of cancerous breast tissues compared with the normal ones. The changes were associated with hormone receptor status. CONCLUSION: Our finding suggests that decreased mtDNA content in breast cancer may have diagnostic and prognostic value for the disease.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for genotyping of human platelet-specific antigens.

BACKGROUND: Genotyping of single-nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, where finally tools for end users have become available to design primers and analyze SNPs of their own interest. This study investigated the potential of this technique in platelet (PLT) genotyping and developed a validated method for genotyping of clinical relevant human PLT antigens (HPAs). STUDY DESIGN AND METHODS: A multiplex assay using MALDI-TOF MS to analyze six HPA loci (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-15) simultaneously in a single reaction was applied for the genotyping of 100 DNA samples from a cohort of plateletpheresis donors and a patient population (n = 20) enriched for rare alleles. The genotyping results using MALDI-TOF MS were validated by the comparison with the results from typing by polymerase chain reaction with sequence-specific primers and conventional DNA sequencing. RESULTS: Both homozygous and heterozygous genotypes of HPA-1 to -5 and -15 of the 120 individuals were easily identified by a six-plexed assay on MALDI-TOF MS. The three approaches achieved a 100 percent concordance for the genotyping results of the six HPA loci. CONCLUSION: Compared to conventional methods, the MALDI-TOF MS showed several advantages, such as a high velocity, the ability to perform multiplexed assays in a single reaction, and automated high-throughput analysis of samples. This enables cost-efficient large-scale PLT genotyping for clinical applications.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR.

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100 percent efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

High-throughput hacking of the methylation patterns in breast cancer by in vitro transcription and thymidine-specific cleavage mass array on MALDI-TOF silico-chip.

Over the last decade, the rapidly expanding interest in the involvement of DNA methylation in developmental mechanisms, human diseases, and malignancies has highlighted the need for an accurate, quantitative, and high-throughput assay. Existing methods are limited and are often too laborious for high-throughput analysis or inadequate for quantitative analysis of methylation. Recently, a MassCLEAVE assay has been developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze base-specific methylation patterns after bisulfite conversion. To find an efficient and more cost-effective high-throughput method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple target CpG residues by using thymidine-specific cleavage mass array on matrix-assisted laser desorption/ionization time-of-flight silicon chips. We used this novel quantitative approach for the analysis of DNA methylation patterns of four tumor suppressor genes in 96 breast tissue samples from 48 patients with breast cancer. Each individual contributed a breast cancer specimen and corresponding adjacent normal tissue. We evaluated the accuracy of the approach and implemented critical improvements in experimental design.

Comparison of serum VEGF and its soluble receptor sVEGFR1 with serum cell-free DNA in patients with breast tumor.

BACKGROUND: The observed elevated levels of vascular endothelial growth factor (VEGF), its soluble receptor (sVEGFR1) and cell-free DNA (cfDNA) in the serum of patients with various cancers have attracted much attention to the possibility of using these agents as biomarkers for cancers. We wanted to find out whether VEGF, VEGFR1 or cfDNA is a biomarker for breast cancer. METHODS: In this study, we used enzyme-linked immunosorbent assay (ELISA) to examine the levels of serum VEGF and VEGFR1 in 23 patients with benign tumors, 19 patients with breast cancer and 32 age-matched healthy females. The levels of circulating cell-free DNA were measured using TaqMan real-time PCR analysis for the glyceraldehydes 3 phosphate dehydrogenase gene (GAPDH), We compared the serum levels of VEGF and its soluble receptors with those of the cell-free serum DNA. RESULTS: In terms of serum levels of either VEGF or its soluble receptors VEGFR1, we found no significant difference between healthy individuals and women with benign breast tumors and breast cancer. However, there was a significant increase in circulating cell-free DNA in women with both benign and malignant breast tumors when compared with the corresponding healthy control group. We also found a significant negative correlation between VEGF and its soluble receptor VEGFR1 (r=-0.328 and p=0.004), and a significant positive correlation between VEGF and cell-free serum DNA (r=0.241 and p=0.033). CONCLUSIONS: We can conclude that quantitative analysis of circulating cell-free DNA in serum may provide molecular biological understanding of breast tumors in women. It would also suggest that serum levels of VEGF and VEGFR1 have less significance.

Positive correlation of cell-free DNA in plasma/serum in patients with malignant and benign breast disease.

BACKGROUND: Circulating cell-free (ccf) DNA is measurable in healthy individuals and in higher concentration in patients with benign and malignant breast disease (BD). PATIENTS AND METHODS: In paired plasma and serum samples ccf DNA was extracted and quantified by real-time quantitative PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. RESULTS: The concentration of ccf DNA in serum was higher in patients with benign and malignant BD (p = 0.023/p = 0.001) compared to healthy controls, whereas ccf DNA in plasma was higher in patients with malignant BD compared to patients with benign BD or healthy controls (p = 0.012/0.007). The ccf DNA correlated significantly between plasma and serum samples in patients with benign (p = 0.01; R: 0.677) as well as malignant BD (p = 0.01; R:0.713). CONCLUSION: The positive correlation between ccf DNA in plasma and serum in patients with benign as well as malignant BD, might have a diagnostic value for discriminating between malignant and benign BD.