| Background and ObjectiveThe health burden of cancer is increasing in China, with more than 1.6 million people being diagnosed and 1.2 million people dying of the disease each year. As in most other countries, breast cancer is now the most common cancer in Chinese women. Increased levels of plasma DNA have frequently been noticed in the blood plasma of cancer patients. Nucleic acids can be released by various pathological and normal physiological mechanisms, such as active or passive release from living or dying cells. cfDNA can be found in a variety of extracellular media including the blood, cerebral spinal fluid, sputum, stool and urine, among others. It has been suggested that dead or physically damaged cells represent the predominant source of cfDNA in the blood. The apoptotic process generates DNA fragments of approximately 180 bp in length as a consequence of a programmed digestion of the genomic DNA. ctDNA can be detected in a range of different solid malignancies and levels have been shown to increase with disease stage. The analysis of ctDNA is challenging and requires highly sensitive techniques due to the small fraction of tumor specific DNA present within background levels of normal cfDNA.Trisomy 21, Down’s syndrome, occurs in about 1 out of every 800 live births. Trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) are the most clinically significant autosomal trisomies besides trisomy 21. Trisomy 18 occurs in 1 in every 6,000 live births and the incidence of trisomy 13 is estimated to be about 1 out of every 10,000 newborns. Detection of such common fetal chromosomal aberrations is a significant indication for prenatal diagnosis. Conventional prenatal testing approaches, for example, sampling of fetal genetic materials by amniocentesis or chorionic villus sampling, are invasive, and carry potential risks to the fetus. Besides these invasive methods mentioned above, noninvasive screening approaches by ultrasound scanning and maternal serum markers are useful for identifying high-risk cases, but have limited sensitivity and specificity. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma in 1977, it has drawn much attention and opens up many new approaches for non-invasive prenatal testing (NIPT) with a reduced risk of complications such as miscarriage compared with invasive procedures. Massively parallel sequencing of circulating fetal DNA in the plasma of pregnant women is a common method for noninvasive prenatal testing of fetal trisomy 13,18, and 21, and has recently changed the clinical paradigm of prenatal screening. Using this technology, the sensitivities for the detection of fetal trisomies 21 and 18 are, on average,99% and 96%, respectively, with specificities of 99% to 100%. Many professional societies have recommended that noninvasive prenatal testing can be offered to pregnant women at high risk for having a fetus with autosomal aneuploidy. Although noninvasive prenatal testing performs well, it is an advanced screen, not a diagnostic test, some cases are discordant with the direct karyotype. The reason for this distinction is that the cfDNA in the plasma of pregnant women is a mixture of placental (used as a proxy for the fetus) and maternal DNA. The cffDNA is present in a wide background of maternally-derived DNAs, and any increment in the total DNA amount (fetal and maternal) of target chromosome DNA molecules will be diluted by contributions from the pregnancy. However, circulating DNA is not restricted to pregnant women, with increased levels of plasma DNA also frequently detected in the plasma of cancer patients.The diagnosis of cancer during pregnancy is relatively uncommon, with an incidence of about 1 in 1000 gestation. Among pregnant women whose noninvasive prenatal testing results were inconsistent with the fetal karyotype, a small number of patients have subsequently been diagnosed with a previously undetected malignancy. The most common malignancies observed in pregnant women are breast and cervical cancers, Hodgkin and non-Hodgkin lymphomas, malignant melanoma, leukemia, ovarian cancer, and colorectal cancer. However, the extent to which circulating tumor DNA (ctDNA) affects the results of noninvasive prenatal testing is still unclear.Materials and MethodsA total of 36 patients diagnosed with breast tumors were included in this study. Research protocols were approved by the institutional review board of Nanfang Hospital Ethics Committee. Written informed consent was obtained from all participants. Trisomy 13,18, and 21 early miscarriage tissues were supplied from Guangzhou Darui Biotechnology Co. Ltd, Guangdong Province, China. Samples were examined by comparative genomic hybridization for karyotype confirmation. Genomic DNA was extracted and sheared using the Covaris S2 ultrasonicator (Covaris, Woburn, MA, USA). After shearing,140-200 bp fragmented DNA was purified by Agencourt XP beads (Beckman Colter, Brea, CA, USA) and quantified using the Qubit 3.0 (Life Technologies, Carlsbad, CA, USA). A total of 1,252 pregnant women who underwent NIPT after being diagnosed as high risk for fetal aneuploidies by chemistry and ultrasound screening between May 2015 and September 2015 at Guangzhou Darui Biotechnology Co. Ltd were enrolled in this study. A total of 99 non-pregnant healthy women were recruited for use as a control population.Thirty-six patients diagnosed with breast tumors at Nanfang Hospital, Southern Medical University (Guangzhou, Guangdong Province, China) between May 2015 and July 2015 were recruited prior to tumor resection. All diagnoses were pathologically confirmed by biopsy. Peripheral venous blood samples (5-10 mL) were collected in sterile tubes containing EDTA, with all DNA extraction initiated within 48 h of sample collection; 5 mL peripheral blood were collected from non-pregnant healthy women. Plasma was separated within 24 h by centrifugation at 1,600 × g for 10 min, then transferred to Eppendorf tubes. Tubes were then centrifuged again at 16,000 × g for 15 min and plasma aliquots transferred to fresh Eppendorf tubes. Purified plasma samples were stored at -20℃. DNA was extracted from 700 μL plasma using a commercial blood DNA kit (GenMag Circulating DNA from Plasma Kit, GenMag Biotech, Beijing, China), following the manufacturer’s instructions. DNA concentrations were quantified using a Qubit 3.0 and stored at-20℃ before use.Positive reference samples were generated by adding 10% fetal trisomy 13,18, or 21 DNA to the plasma DNA of non-pregnant healthy controls. Plasma DNA of women with breast tumors were divided into four equal aliquots (～700 μL each). Test samples were then generated by adding 13% fetal trisomy 13,18, or 21 cfDNA to plasma DNA extracted from women with breast tumors, respectively.DNA samples and artificial DNA mixtures were subjected to library construction using the Ion Plus Fragment Library Kit according to a modification of the protocol of the Ion Xpress Plus gDNA Fragment Library Preparation User Guide (Life Technology). Briefly, DNA fragments were end-repaired using a series of enzymes, and linker oligonucleotides were ligated to both ends of each fragment, one of which included a short sequence that effectively barcodes the DNA from that sample. Primer mixes were introduced using a 10-cycle PCR reaction. Various concentrations of AMPure XP beads were used to purify the library during all construction procedure (i.e., end repair, adapter-ligation, and amplification). Afterwards, libraries were pooled and amplified by emulsion PCR using the Ion OneTouch 2 System, which enables automated delivery of templated Ion Sphere particles (ISP). Template-positive ISPs were enriched using the Ion OneTouch enrichment system and loaded immediately onto the Ion semiconductor chip. Sequencing was performed using an Ion Proton sequencer at 300 flows, according to the manufacturer’s instructions.Taken together, in present study, we examined serum from 36 non-pregnant women with breast tumors using standard noninvasive prenatal tests. These samples were then added to sera containing trisomy 13,18, and 21 fetal DNA to figure out the extent to which maternal tumors can interrupt the result of noninvasive prenatal testing in pregnant women with benign or malignant breast tumors, respectively. ResultThe mean concentration of plasma DNA in the sera of pregnant women, breast tumor patients, and non-pregnant healthy controls were 6.76 ng,6.44 ng, and 4.18 ng, respectively. Plasma DNA concentrations were significantly higher in both pregnant women and patients with breast tumors group compared with healthy non-pregnant women (P< 0.05). In contrast, the concentration of plasma DNA in pregnant women was not significantly different from that of breast tumor patients (P=0.277). In an analysis of noninvasive prenatal testing result of 36 breast tumor cases, several chromosomal abnormalities were detected in a subset of plasma DNA samples obtained from breast tumor patients, particularly chromosome 8. Z-scores for chromosome 8 were significantly higher than those of other chromosomes in sample 17 and sample 32 (5.56%). Among these two patients, elevated z-scores were also seen for chromosomes 13 (4.09 and 10.19),18 (5.26 and 3.72), and 21 (8.61 and 3.72) in patients 17 and 32, respectively, indicating that genomic copy number variations had occurred. Interestingly, these two aneuploidy-positive patients had both been diagnosed with infiltrating ductal carcinoma prior to surgery. These two carcinomas were both triple-negative for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER-2/neu), as determined by immunohistochemical staining. The mean z-scores and SDs were 20.10±11.51, 15.35±7.32, and 11.22±5.58 for trisomy 13,18, and 21 in the pregnancy-cancer co-occurrence group, compared with 15.03±2.60,13.70±1.36, and 11.70±1.71, respectively, for simulated positive pregnancy alone. SDs for the co-occurrence group were significantly higher than those seen in cancer-free controls. While it would be expected that the mean z-score would be higher among pregnancy-cancer co-occurrence samples due to the higher starting percentage for trisomy DNA, however, the mean z-score for chromosome 21 in this group (11.22) was less than that in the simulated positive pregnancy alone group (11.70). This effect is likely due to high concentrations of ctDNA exceeding normal background levels of maternal cfDNA, which may have limited the detection of cffDNA. Indeed, obvious decreases in z-scores were also seen for trisomy of chromosomes 13 and 18 in the pregnancy-cancer co-occurrence group relative to controls (Fig 4). Among the 36 samples evaluated, the z-scores of 6 fell within the equivocal range for either trisomy 13 (n=3) or 18 (n=3), with an absolute value close to 3. Furthermore, two samples produced false negative results for trisomy 21.ConclusionTaken together, the data presented here show for the first time that ctDNA is able to affect the result of noninvasive prenatal testing in two ways. First, ctDNA can lead to false positive results due to the detection of genomic copy number variations in tumor DNA. Alternatively, ctDNA can increase the likelihood of a false negative by decreasing the proportion of circulating fetal DNA in serum. These results highlight important limitations to existing methods and may be useful for improving both detection rates and reliability of noninvasive prenatal testing. |
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