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Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
Department of Gynecology and Obstetrics, Erlangen University Hospital, University Endometriosis Center for Franconia, Friedrich-Alexander University Erlangen-Nürnberg, Germany
High levels of ccfDNA can be isolated from plasma of endometriosis patients.
•
Different methods for ccfDNA isolation can influence the output and integrity.
•
Quantities of ccfDNA could be sufficient for further downstream applications.
Abstract
Research question
Which is the optimal extraction method for isolating and quantifying circulating cell-free DNA (ccfDNA) from patients with endometriosis? Endometriosis is a common benign disease, associated with pain, infertility and reduced quality of life. Endometriosis is also a known risk factor for various cancers. Robust biomarkers for early detection and prediction of prognosis, however, are lacking. CcfDNA is an easy to obtain biomarker associated with prognosis of cancer patients and enables non-invasive analysis of somatic mutations. Recently, elevated levels of ccfDNA were detected in patients with endometriosis.
Design
Two different ccfDNA extraction methods were compared: Maxwell RSC ccfDNA plasma kit (Maxwell) and QiAamp minElute ccfDNA mini kit (QIAamp). The ccfDNA and circulating mitochondrial DNA (mtDNA) quantities from 34 patients diagnosed with endometriosis were analysed. Fluorometric measurement and quantitative reverse transcription polymerase chain reaction (qRT-PCR) of short and long ALU and mtDNA fragments were used to quantiy ccfDNA.
Results
The yield of ccfDNA isolated with the Maxwell method was significantly higher compared with the QIAamp method (P < 0.0001). Integrity of ccfDNA was significantly higher in the QIAamp isolate (P < 0.0001). Recovered mtDNA was not significantly different between both extraction methods used.
Conclusions
The choice of extraction method can significantly influence the ccfDNA output and integrity. Both methods, however, enabled isolation of sufficient ccfDNA for further downstream applications. With this approach, isolation of ccfDNA could enable the non-invasive detection and analysis of somatic mutation within endometriosis tissue.
Endometriosis is a common oestrogen-dependent benign disease, which is defined by the presence of endometrial-like tissue outside of the uterus. Clinical features associated with endometriosis are chronic pelvic pain, pain during intercourse, dysmenorrhoea and infertility (
). Endometriosis affects the receptivity of the endometrium and the development of the oocyte and embryo, leading to significant lower pregnancy rates by natural conception and by IVF (
). The cause of endometriosis is still largely unknown, although some key molecular features are similar to processes present in malignancies. Those characteristics are, for example, progressive and invasive growth, oestrogen-dependent proliferation and recurrence (
Over the past 2 years, knowledge of somatic alterations associated with endometriosis and endometriosis-associated cancer has increased. About 80% of endometriosis cases harbor somatic mutations and about 26% of cases have known cancer driver mutations (
). The biology and molecular mechanisms that lead to endometriosis and its malignant transformation, however, are still largely unknown, and clinically validated biomarkers identifying patients who have, or will develop, endometriosis and its associated cancer are lacking (
). Therefore, finding novel, non-invasive biomarkers for endometriosis to facilitate an early diagnosis and treatment of the disease, and defining subgroups of patients with a high risk of developing endometriosis-associated cancer, is of crucial importance (
Recently, elevated levels of circulating cell-free DNA (ccfDNA) were detected in patients diagnosed with endometriosis compared with healthy individuals (
). Most of the ccfDNA can be assigned to the ALU repeat family. The ALU sequences are normally about 300 nucleotides long and account for more than 10% of the human genome (
). Because of the short length, ALUs are classified as short, interspersed elements. With a copy number of 1.4 × 106 per genome, ALU elements are the most abundant short, interspersed elements and the most abundant of all mobile elements (
). The integrity of ccfDNA is defined as the ratio between long and short ALU repeats. Increased ALU ccfDNA concentrations and reduced levels of ccfDNA integrity were associated with the presence of primary and metastatic breast cancer (
). Furthermore, circulating tumour DNA levels showed an even better correlation with changes in metastatic breast cancer tumour burden than the established tumour marker CA 15-3 (
In addition to the quantification of ALU repeats and ccfDNA integrity, human mitochondrial DNA (mtDNA) in the plasma of cancer patients has been reported as a novel non-invasive biomarker (
). Furthermore, somatic mtDNA mutations are frequently present in diseased cells or cells with tumours. Also, in cancer patients, for example, adjacent, healthy appearing cells resulted in a high number of released, circulating mutated mtDNA (
). On the basis of these data, it could be assumed that somatic mutations within mtDNA released from endometriosis lesions might also be present and could be relevant for future biomarker studies. Biomarkers in endometriosis could be helpful in certain situations, for classification and prognosis and after the discovery of endometriosis, to predict the recurrence and the response to treatment.
To develop a sound protocol for isolating and analysing high-quality ccfDNA, circulating mtDNA and high levels of isolated cffDNA, robust results of ccfDNA integrity and high concentrations of mtDNA fragments (hmito) should be obtained. The aim of the present study was, therefore, to compare two methods for the isolation of ccfDNA from patients diagnosed with endometriosis, including mtDNA and quantified isolated fragments by quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Materials and methods
Patients
This prospective study was carried out between March 2018 and October 2019 at the University-Endometriosis Center for Franconia at University Hospital Erlangen, Erlangen, Germany. Patients were enrolled in the International Endometriosis Evaluation Programme (
). Blood samples were collected from patients scheduled for laparoscopy at Erlangen University Hospital. The inclusion criteria were age 18–48 years and scheduled for diagnostic or therapeutic laparoscopy. Patients with a previous diagnosis of endometriosis, previous lower abdominal operations (excluding caesarean section or appendectomy) and those with comorbidities that could potentially influence pain levels, e.g. irritable bowel syndrome, interstitial cystitis or a history of pelvic inflammatory disease, were excluded.
A total of 40 patients with suspected endometriosis were recruited. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Friedrich-Alexander University Erlangen-Nurenberg, medical faculty (protocol code 255_18B, date of approval: 17 July 2018). All participants provided written informed consent.
One patient was excluded because insufficient blood samples were provided; one patient was excluded as they had not undergone surgery and four patients were excluded because no final diagnosis of endometriosis was made during laparoscopy, resulting in 34 patients for final analysis of ccfDNA.
Clinical data were available for all 34 patients (Table 1). The percentage of missing values in each variable was 0%, except for cycle length and whether patients had ever used oral contraceptives (14.7% each), whether patients were currently using oral contraceptives (8.8%) and revised American Society for Reproductive Medicine classification (2.9%).
Table 1PATIENT CHARACTERISTICS OF ENDOMETRIOSIS CASES
All patients completed a standardized questionnaire, including questions about pregnancy history, previous use of hormonal contraceptives, medical history, family history and lifestyle. Trained medical staff reviewed the questionnaires and any unanswered questions were completed by contacting the patients.
Sample collection and plasma preparation
Plasma samples were collected from patients with suspected endometriosis treated at the University Clinic Erlangen. Written consent was obtained from all participants. Blood samples (10 ml) were collected in cell-free DNA blood collection tubes (Streck, La Vista, NE, USA) and stored at room temperature overnight. Tubes were centrifuged for 10 min at 1600 x g to separate the lymphocytes from plasma. Supernatant was removed into new 15-ml tubes without disturbing the buffy coat and centrifuged at 14,000 x g for 10 min to remove remaining debris. Plasma was then immediately used for ccfDNA extraction.
Circulating cell-free DNA extraction
Two different commercially available Kits were used to extract CcfDNA from the same plasma sample according to the manufacturer's instructions: Maxwell RSC ccfDNA plasma kit (Promega, Madison, WI, USA) (catalogue number AS1480) and QiAamp minElute ccfDNA mini kit (Qiagen, Hilden, Germany) (catalogue 55284) (Table 2). After extraction, ccfDNA was stored at –20°C until further use.
Table 2SPECIFICATIONS OF CIRCULATING CELL-FREE DNA EXTRACTION KITS USED
Specifications
Maxwell RSC ccfDNA Plasma Kit
QIAamp MinElute ccfDNA Mini Kit (50)
Manufacturer
Promega
Qiagen
Method
Magnetic beads, automatic
Magnetic beads, clean-up of the resulting pre-eluate on QIAamp MinElute columns, manual
Input volume, ml
0.2–1.0 (in this project: 1.0)
1–4 (in this project: 1.0)
Elution Volume, µl
60 (in this project: 70)
20–80 (in this project:70)
Recovered volume, µl
In this project: 60
In this project: 60–65
Hands-on time, min
10
25
Incubation/centrifugation time, min
–
25
Automated runtime, min
90
–
Total runtime, min
100
50
–, Dashes are presented if the specification is not applicable for the respective extraction kit used.
Concentrations of total isolated ccfDNA were quantified by Quantus fluorometer (Promega, Walldorf, Germany) and the QuantiFluorRNA System kit (Promega Corporation, Madison, WI, USA) (catalogue number E3310) and presented as ng/ml plasma.
Quantification of ccfDNA by ALU-based quantitiative real-time polymerase chain reaction
The amount of ccfDNA was determined by fragment length-dependent quantitative real time polymerase chain reaction (qRT-PCR) of ALU DNA repeats (Figure 1) using Step One real-time PCR System (Applied Biosystems, Waltham, MA, USA). For the qRT-PCR, two primer sets were designed to amplify the consensus ALU sequence. The primer set for the 60-bp amplicon (ALU_60) amplifies both shorter (truncated by apoptosis) and longer DNA fragments of circulating DNA, representing the total amount of ccfDNA. The second primer set (ALU_247) was designed to amplify a 247-bp amplicon only from longer DNA fragments representing released DNA from non-apoptotic cells. The sequences of the ALU_60 primer set (60-bp) were forward: 5‘-GGAGGCTGAGGCAGGAGAA-3’ and reverse: 5‘-ATCTCGGCTCACTGCA ACCT-3‘. The sequences of the ALU_247 primer set were forward: 5‘-GTGGCTCACGCCTGTAATC-3‘ and reverse: 5‘-CAGGCTGGAGTGCA GTGG-3‘. DNA integrity was calculated as the ratio of copies per µl ccfDNA (copies of 247-bp fragments/copies of 60-bp fragments) determined by qRT-PCR. Because the annealing sites of ALU_60 are within the ALU_247 annealing sites, the DNA integrity is 1.0 when the template DNA is not truncated; and 0.0 when all template DNA is completely truncated into fragments smaller than 247bp (
Alu repeat sequences are present in increased proportions compared to a unique gene in plasma/serum DNA: evidence for a preferential release from viable cells?.
Figure 1Experimental design. Blood was drawn from 34 patients diagnosed with endometriosis and collected in STRECK circulating cell-free DNA (ccfDNA) tubes. Maxwell RSC ccfDNA plasma kit (Maxwell) or QiAamp minElute ccfDNA mini kit (QiAamp) extraction method was used to extract ccfDNA; QuantusTM Fluorometer (Quantas) was used to measure ccfDNA concentrations or alternatively copy numbers of ALU or mitochondrial DNA (hmito) fragments. qRT-PCR, quantitative real-time-polymerase chain reaction.
The reaction volume for each qRT-PCR was 12.5 µl consisting of 3.2 µl of extracted template DNA and 9.3 µl Mastermix (SYBR Select Master Mix [QIAGEN], 0.25 µM forward and reverse primer and RNase-free water). The qRT-PCR was carried out at 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 1min.
The number of ccfDNA copies were calculated by a standard curve with serial dilutions (10 ng/µl-0.01 ng/µl) of specially prepared gBlocks Gene Fragments from Integrated DNA Technologies (USA) (ALU) (Table 3). Concentration of gBlocks was converted into copy numbers per µl with the following formular: (concentration) x (molecular weight) x (1 × 10–15 mol/fmol) x (Avogadro's number) = copy number/µl. A negative control and an internal control was used in each qRT-PCR assay. Each reaction was carried out in triplicates.
Approximate concentrations based on qRT-PCR analysis was calculated by a standard curve with serial dilutions of known germline DNA concentrations using ALU_60 primer sets.
Quantification of mtDNA by quantitative real-time polymerase chain reaction
The amount of mitochondrial ccfDNA was assessed by quantification of a unique mitochondrial DNA fragment (hmito). The primer set used to amplify a 64-bp amplicon was forward: 5‘-CTTCTGGCCACAGCACTTAAAC-3‘ and reverse: 5‘-GCTGGTGTTAGG GTTCTTTGTTTTT-3‘ (
Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.
). qRT-PCR was carried out equally to ALU based qRT-PCR. The number of mtDNA copies were calculated by a standard curve with serial dilutions (10 ng/µl-0.01 ng/µl) of specially prepared gBlocks Gene Fragments from Integrated DNA Technologies (USA) (hmito) equally as described above for ALU fragements (Table 3). Negative and internal controls were carried out in each plate and mean values were calculated from triplicate reactions.
Statistical considerations
Mean values between two groups were compared using Student's t test. P < 0.05 was considered significant. Statistical relation between two continuous variables was analysed using Pearson's correlation coefficient (r). Figures were plotted using Graph Pad Prism (Version 8.3.0).
Results
Circulating cell-free DNA isolation methods
STRECK tubes were collected from all patients and ccfDNA was isolated using the Maxwell RSC ccfDNA Plasma Kit (Maxwell) and the QIAamp MinElute ccfDNA Mini Kit (QIAamp) (Table 2 and Figure 1). The Maxwell kit uses an automated magnetic beads method, whereas the QIAamp kit is based on a mix between a manual magnetic beads and column method (Table 2). Comparing the specifications of those two methods reveals a higher total runtime for the Maxwell protocol compared with the QIAamp protocol, but a lower hands-on time because of the automatic procedure of the Maxwell system (Table 2). Although the Maxwell kit allows the input of plasma as low as 0.2 ml per protocol, the QIAamp kit can handle volumes up to 4 ml (Table 2).
Circulating cell-free DNA concentration
To calculate copy numbers of ALU_60, ALU_247 and hmito ccfDNA fragments, gBlocks sequences were used as a reference with known molecular weight and copy number per µl. qRT-PCR of ALU_247, ALU_60 und hmito fragments was tested for reaction efficiency (E) using gBlock fragments. A sufficient E between 82 and 98% was obtained for all qRT-PCR set-ups (Figure 2a,2b and 2c). Equations for each simple linear regression were used for quantification of copy numbers. For quantification of ccfDNA integrity, short and long ALU fragments were analysed. For example, a high integrity value (close to 1) represents ccfDNA with large fragments (200–400 bp) originating from necrosis (Figure 2d), whereas lower integrity means ccfDNA originates from apoptotic cells (<185bp).
Figure 2Quantification of circulating cell-free DNA (ccfDNA) by quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR of (A) ALU_247 and (B) ALU_60 fragments was tested for reaction efficiency (E) using gBlocks; (C) qRT-PCR of mitochondrial DNA fragments (hmito) was tested for reaction efficiency (E) using gBlocks; (D) schematic localization of ALU_247 and ALU_60 primers and formula for calculation of ccfDNA integrity.
Concentration of freshly isolated ccfDNA was measured using the Quantus fluorometer. The mean concentration of ccfDNA isolated by Maxwell was 7.37ng/ml plasma and by QIAamp 4.45 ng/ml plasma (Figure 3a). The Maxwell procedure had a significant higher ccfDNA yield (P < 0.0001). The concentration of Maxwell eluate correlated with the QIAamp concentration (r = 0.6995; 95% CI 0.4688 to 0.8409; P < 0.0001) (Figure 3b). Only for three cases, higher ccfDNA concentrations were obtain by QIAamp compared with Maxwell isolation (Figure 3c). Concentrations measured by Quantus fluorometer were correlated to concentrations calculated by ALU_60 qPCR (Figure 3d). For both Maxwell and QIAamp, ccfDNA concentrations measured by Quantus and qRT-PCR correlated significantly (r = 0.677q, P < 0.0001 and r = 0.8158, P < 0.0001, respectively) (Figure 3d and 3e).
Figure 3Concentration of isolated circulating cell-free DNA (ccfDNA). Concentrations of isolated ccfDNA were measured by Quantus TM Fluorometer (Quantas); (A) Quantus concentrations from ccfDNA isolated by Maxwell RSC ccfDNA plasma kit (Maxwell) versus QiAamp minElute ccfDNA mini kit (QIAamp) were correlated; (B) measured ccfDNA concentrations of Maxwell and QIAamp eluates per patients were presented. Solid line represents simple linear regression. The dotted line represents the bisector; (C) concentrations were additionally calculated using quantitative real-time-polymerase chain reaction (qRT-PCR) of ALU_60 fragments and a standard curve of known germline DNA concentrations for (D) Maxwell eluates and (E) QIAamp eluates. *, P < 0.001; #, Maxwell sample was excluded due to failed concentration measurement.
Copy numbers of ALU_60 and ALU_247 fragments per ml plasma were quantified by qRT-PCR (Figure 4). QIAamp eluates had significant lower mean copy numbers per ml plasma of short ALU_60 (Figure 4a) (P < 0.0001) and of long ALU_247 fragments (Figure 4b) (P < 0.0060). To unmask the effect of lower overall ccfDNA levels isolated by the QIAamp system, the copy numbers of ALU fragments per ng ccfDNA were calculated: comparison of copies per ng ccfDNA revealed no significant differences between Maxwell and QIAamp extraction methods for either ALU_60 or ALU_247 (Figure 4c and 4d, respectively). Interestingly, the calculated integrity of ccfDNA isolated by the QIAamp system (mean 0.6444) was significantly higher compared with the Maxwell (mean 0.5197) ccfDNA (Figure 4e) (P < 0.0001). Only six out of 34 cases revealed a higher integrity of ccfDNA from the Maxwell eluate compared with the QIAamp one (Figure 4f). ccfDNA concentration and integrity was additionally evaluated for each group of patients as defined by the revised American Society for Reproductive Medicine classification scores I, II, III or IV separately (Table 1 and Table 4).
Figure 4Integrity of circulating cell-free DNA (ccfDNA). Copies per ml plasma of short (A) ALU (ALU_60) and (B) long ALU (ALU_247) fragments were calculated by quantitative real-time-polymerase chain reaction (qRT-PCR) analysis. Copies of (C) ALU_60 and (D) ALU_247 per ng ccfDNA were calculated; (E) integrity was presented as the ratio between copy numbers of ALU_247 and copy numbers of ALU_60; (F) integrities of ccfDNA isolated by Maxwell RSC ccfDNA plasma kit and QiAamp minElute ccfDNA mini kit were presented per patient. *, P < 0.001; ⁎⁎, P = 0.006. Quantas, QuantusTM Fluorometer.
Concentration measured by Quantus. Maxwell, Maxwell RSC ccfDNA plasma kit; QiAMP, QiAamp minElute ccfDNA mini kit; rASRM, revised American Society of Reproductive Medicine.
Concentration measured by Quantus. Maxwell, Maxwell RSC ccfDNA plasma kit; QiAMP, QiAamp minElute ccfDNA mini kit; rASRM, revised American Society of Reproductive Medicine.
(ng/ml plasma) Mean (SD)
Maxwell integrity Mean (SD)
QIAamp integrity Mean (SD)
Minimal endometriosis
I
13
7.08 (2.30)
4.08 (2.43)
0.52 (0.12)
0.68 (0.15)
Mild endometriosis
II
8
6.90 (3.06)
4.16 (2.17)
0.47 (0.10)
0.68 (0.13)
Moderate endometriosis
III
6
9.03 (3.95)
5.67 (4.08)
0.55 (0.17)
0.59 (0.17)
Severe endometriosis
IV
6
7.69 (3.56)
4.92 (2.86)
0.56 (0.26)
0.58 (0.18)
a One patient with adenomyosis uteri and no rASRM classification.
b Concentration measured by Quantus.Maxwell, Maxwell RSC ccfDNA plasma kit; QiAMP, QiAamp minElute ccfDNA mini kit; rASRM, revised American Society of Reproductive Medicine.
Copy numbers of hmito fragments representing circulating mtDNA in plasma of endometriosis patients were quantified by qPCR (Figure 5). The detected copy numbers per ml plasma were significantly higher in the Maxwell eluates compared with the QIAamp ones (Figure 5a) (P = 0.018). In addition, the copy numbers of hmito fragments per ng ccfDNA were calculated. Lower levels of hmito ccfDNA were still obtained by the QIAamp isolation; however, those were not significantly different (Figure 5b).
Figure 5Copies of hmito circulating cell-free DNA (ccfDNA). (A) Copies per ml plasma of circulating mitochondrial DNA fragments (hmito) were calculated by quantitative real-time polymerse chain reaction (qRT-PCR) analysis; (B) measured copies of hmito by qRT-PCR were presented as copies per ng total ccfDNA. ⁎⁎, P = 0.0016. Maxwell, Maxwell RSC ccfDNA plasma kit; QiAmp, QiAamp minElute.
In this prospective experimental study, as far as we know, different methods of ccfDNA isolation and quantification (the Maxwell and QIAamp method) were compared for the first time in patients with endometriosis.
Little is known about the amount and integrity of ccfDNA in patients with endometriosis. An earlier publication used real-time multiplex polymerase chain reaction to quantify the glyceraldehyde-3-phosphate dehydrogenase gene sequence (GAPDH) in nuclear ccfDNA and the ATP synthase-8 gene (ATP8) sequence in circulating mtDNA (
). The total amount of ccfDNA was compared between an endometriosis and a control group. The concentration of ccfDNA was significantly higher in the endometriosis cohort and could be used to discriminate between endometriosis and normal controls with a sensitivity of 70% and a specificity of 87% (
). ccfDNA was isolated from peripheral blood samples for coagulant serum and from peripheral blood samples for EDTA plasma by using an automated MagNA PureTM LC Instrument and MagNA Pure LC DNA Isolation Kit, and eluted into 100 µl elution buffer (
). This approach was different to the approach that we used. In the present study, we used STRECK tubes for blood collection and both the Maxwell and QIAamp method for ccfDNA isolation. It was shown earlier that STRECK and EDTA tubes have a similar performance in preserving ccfDNA for up to 6 h at room temperature, but also that STRECK tubes are more consistent for stabilizing ccfDNA at 48 h (
). Therefore, STRECK tubes are preferred, especially in multi-centre studies in which shipment of blood tubes before processing is mandatory. STRECK tubes are currently the gold standard for preserving ccfDNA or ctDNA. Therefore, within the presented work, we focused on STRECK blood collection tubes only.
isolated and calculated a mean GAPDH genome equivalent of 660 per ml for nuclear ccfDNA. As GAPDH genome equivalents were used for quantification of total ccfDNA, results from
cannot be compared with our results. The quantification of both ALU and GAPDH sequences have been shown to be sensitive indicators for diagnosis and prediction of various cancer cases (
). Quantification of ALU copy numbers, however, gained more importance during the past 2 years owing to the prognostic significance of ccfDNA integrity (
A novel Alu-based real-time PCR method for the quantitative detection of plasma circulating cell-free DNA: sensitivity and specificity for the diagnosis of myocardial infarction.
). DNA integrity enables the measurement of ccfDNA fragmentation and has been postulated to be a sensitive and specific biomarker for cancer diagnosis and prediction of therapy response, which takes the ccfDNA amount into account and also the ratio of ccfDNA from apoptotic, e.g. healthy and cancer patients, or necrotic cells (only cancer patients) (
). Therefore, we aimed to quantify ALU copy numbers and to investigate the ccfDNA integrity.
The integrity of ccfDNA was mainly investigated in cancer patients and compared with either healthy controls or different stages of cancer. The mean reported integrity of healthy individuals varied between 0.13 and 0.15 (
). Interestingly, for our endometriosis patients, we detected a mean ccfDNA integrity of 0.5197 ± 0.1624 (0.3300–1.110) and 0.6444 ± 0.1629 (0.3400–0.9700), when isolated with the Maxwell or QIAamp system, respectively. The high integrity levels of patients diagnosed with endometriosis indicates that ccfDNA integrity might be a promising biomarker for further investigation. An increased integrity represents ccfDNA derived from both apoptotic and necrotic cells as ccfDNA from necrosis can reach up to several kbp of length (
In comparing different ccfDNA isolation methods (Maxwell and QIAamp), we detected statistically relevant differences in the obtained amount of total ccfDNA and the respective ccfDNA integrity. The QIAamp procedure resulted in an overall lower ccfDNA output for almost all samples. Two studies compared different extraction methods for the isolation of ccfDNA beforehand. The results varied between all studies. Some showed a superiority of QIAamp kit compared with the Maxwell Kit (
A comparison of four commercial kits used for isolating circulating cell-free DNA: QuickGeneMINI8L (Kurabo), Maxwell RSC cfDNA Plasma Kit (Promega), cfKapture 21 Kit (MagBio), and QIAamp MinElute ccfDNA Kit (Qiagen).
). Even though we detected lower recovery rates with the QIAamp method (by both direct fluorometric and qRT-PCR measurement), neither the short (ALU_60) nor the long (ALU_247) fragments were recovered differently when normalized to total ng ccfDNA. Consequently, both methods seem to be equally able to recover small as well as large fragments. Interestingly, however, the calculated integrity was statistically significantly different between both methods. Even though the mean number of ALU_60 and ALU_247 copies per ng total ccfDNA were similar, small individual differences resulted in increased integrity levels obtained from the QIAamp eluate. This is in line with other reports, stating that one limitation of ccfDNA purification is that it can lead to a decrease of DNA yield (dependent on fragment size and method used), which would affect ccfDNA integrity values (
For circulating mtDNA, similar results were obtained. The copy numbers of hmito were significantly lower in the QIAamp eluate compared with Maxwell. After normalization by total ng ccfDNA, however, the differences were below statistical significance. High numbers of hmito copies, however, could be recovered from endometriosis patients. An earlier publication compared genomic equivalents of mtDNA from endometriosis patients with those isolated from healthy controls (
). Nevertheless, isolation of mtDNA should be considered for future endometriosis trials owing to the potential as a biomarker for malignant transformation or the analysis of somatic alterations (
). Among others, PTEN and KRAS were reported as endometriosis susceptibility genes, and mutations in TP53 and PIK3CA have been associated with malignant transformation and cancer progression (
). Somatic mtDNA mutations have also been widely reported to be associated with neoplasma and inflammatory diseases. In addition, somatic mtDNA alterations were discussed to be a risk factor of endometriosis (
). To detect certain somatic mutations with a sensitivity of less than 0.1%, approximately more than 3.6 ng ccfDNA is necessary if an ultrasensitive method is used (
). For endometriosis patients, we recovered 7.37 and 4.45 ng ccfDNA per ml plasma by Maxwell and QIAamp extraction method, respectively. Therefore, both extraction methods allow isolation of sufficient ccfDNA for further downstream analyses, if at least 2-ml plasma is used. This could enable testing of somatic mutations or variations in ccfDNA or circulating mtDNA as a novel biomarker for endometriosis, endometriosis recurrence, possible response to treatment or malignant transformation.
The present study, however, has some limitations. First, the number of study participants was quite low (n = 34); second, the study solely focused on patients diagnosed with endometriosis without including any healthy controls. Therefore, our data do not provide information about which methodical approach best differentiates healthy controls from endometriosis patients.
In conclusion, we present novel data on the output of different methods used for isolation and quantification of ccfDNA from patients suffering with endometriosis. It further provides first insights into the ccfDNA quantities available for potential analysis of somatic alterations or association studies of ccfDNA and risk of progress or disease. This encouraging initial data suggest that ccfDNA of patients diagnosed with endometriosis might be a suitable biosample for future biomarker studies and for more in-depth analysis of the associations between levels of ccfDNA and clinical conditions.
Acknowledgments
We would like to thank Bettina Knoerr for technical assistance.
References
Afrifa J.
Zhao T.
Yu J.
Circulating mitochondria DNA, a non-invasive cancer diagnostic biomarker candidate.
A comparison of four commercial kits used for isolating circulating cell-free DNA: QuickGeneMINI8L (Kurabo), Maxwell RSC cfDNA Plasma Kit (Promega), cfKapture 21 Kit (MagBio), and QIAamp MinElute ccfDNA Kit (Qiagen).
A novel Alu-based real-time PCR method for the quantitative detection of plasma circulating cell-free DNA: sensitivity and specificity for the diagnosis of myocardial infarction.
Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.
Alu repeat sequences are present in increased proportions compared to a unique gene in plasma/serum DNA: evidence for a preferential release from viable cells?.
Stefanie Burghaus, MD, is a specialist in Erlangen, Germany. She has over 10 years’ experience in the conservative and surgical treatment of patients with endometriosis. She has published over 20 papers and is lead coordinator in updating the German-language guideline for diagnosis and treatment of endometriosis.
Key message
Different extraction methods can influence circulating cell-free DNA (ccfDNA) output and integrity. The magnetic-beads-based ccfDNA extraction method was superior compared with the magnetic-beads (column) method of ccfDNA recovery. Both methods enabled isolation of sufficient ccfDNA from endometriosis patients; therefore, ccfDNA could be an easy to obtain biomarker for future studies.
Article info
Publication history
Published online: August 10, 2021
Accepted:
August 3,
2021
Received in revised form:
July 13,
2021
Received:
March 1,
2021
Declaration: The authors report no financial or commercial conflicts of interest.
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