|Ahead of print publication
A study for evaluating clinical relevance of circulating cell-free DNA in cervical cancer
Anju Shrivastava1, Garima Singh2, Kumud Tiwari2, Surendra Pratap Mishra3, Satyajit Pradhan4, Lalit Mohan Agarwal1, Samarendra Kumar Singh2
1 Department of Radiotherapy and Radiation Medicine, Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
4 Mahamana Pandit Madan Mohan Malaviya Cancer Centre, Homi Bhabha Cancer Hospital, Visakhapatnam, Andhra Pradesh, India
|Date of Submission||04-Apr-2020|
|Date of Decision||12-Apr-2020|
|Date of Acceptance||22-Apr-2020|
|Date of Web Publication||26-Sep-2020|
Samarendra Kumar Singh,
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: Recent techniques available for the detection of cervical cancer (CC) are highly invasive and costly, which makes it a rate-limiting step toward early diagnosis of this fatal disease. Evaluation of circulating cell-free DNA (ccfDNA) through liquid biopsy is a minimally invasive and cost-effective method that may serve as a unique tumor marker for early detection, treatment monitoring, the status of residual disease, and distant tumor metastasis in CC patients.
Materials and Methods: In this study, initially, ccfDNA was measured in serum samples from 11 histopathologically proven cervix carcinoma patients and 8 controls. On successful screening, it was further extended to 2 more patients with a series of serum samples extracted at 3 different phases of the concurrent chemoradiotherapy (i.e., before, during, and after 6 months of follow-up).
Results: Agarose gel electrophoresis profile for ccfDNA of CC patients showed that of 11 patients, 4 patients had a comparatively higher tumor burden (ccfDNA) than the other 7 patients. Notably, during concurrent chemoradiotherapy, ccfDNA load disappeared and, after 6 months of follow-up, appeared back due to distant metastasis.
Conclusion: Hence, we propose that this method could be an affordable and reliable way to diagnose/screen CC.
Keywords: Biomarker, cervical cancer, circulating cell-free DNA, liquid biopsy, metastasis
|How to cite this URL:|
Shrivastava A, Singh G, Tiwari K, Mishra SP, Pradhan S, Agarwal LM, Singh SK. A study for evaluating clinical relevance of circulating cell-free DNA in cervical cancer. J Can Res Ther [Epub ahead of print] [cited 2021 Jan 25]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=296269
| > Introduction|| |
Cervical cancer (CC) is the fourth most common cancer among women accounting for 570,000 new cases and 311,000 deaths worldwide. 99.7% of all CCs are caused by the human papillomavirus (HPV) infection. HPV is a nonenveloped double-stranded DNA virus which acts by integrating into the host genome and, with the help of HPV oncoproteins, leads to the oncogenic transformation and progression of CC., In India, CC ranks second with 22.86% of all cancers among women and 12% of all cancer cases in both men and women. There are an estimated 96,922 new cases registered, which is 17% of the global burden and 60,078 deaths that are 19% of worldwide CC deaths in India (ICMR, 2018). The deaths and disability-adjusted life year rates in CC patients increase after the age of 30 years and drop after the age of 60 years., Majority of the cases reported in India are already in advanced stage of the disease where prognosis becomes a challenge. The reason behind this is the lack of awareness, proper screening, and high medical cost. The Advanced early detection methods which are available such as Pap smear are not very sensitive for detecting recurrent and metastatic CC. Therefore, we need a minimally invasive and more specific method to monitor the HPV-mediated CC risk and metastatic burden. Recent breakthrough understandings of cancer development and related biological processes suggest that liquid biopsy from blood-containing circulating cell-free DNA (ccfDNA) is one such method which is rapid, cost-effective, and minimally invasive. It can be very effective toward providing information about metastatic burden and therapeutic targets in cancer patients, and hence could be an affordable and efficient screening tool.
The term ccfDNA was first introduced by Mandel and Metais in the year 1948. These are the fragments of DNA discharged in the bloodstream by apoptotic or necrotic cells. The shorter half-life of an hour or less, makes ccfDNA a real-time fingerprint of the disease burden. In cancer patients, the elevated rate of necrosis increases the burden of ccfDNA in blood plasma and serum. Circulating tumor DNA (ctDNA) concentration directly depends on the circulating tumor cells (CTC) in the blood and can range from 0.1% to 90%. In the very early stages of cancer, the CTC concentration in blood is low, so do the ctDNA concentration. Therefore, to detect ctDNA load at this stage, a more sensitive assay or a higher volume of blood is required. The mechanism is still unclear that how tumor cells break off and escape into circulating blood.
This study discusses the use of ccfDNA to determine the tumor load among CC patients and also change in the profile of the ccfDNA before, during, and after the treatment among CC patients. This study can prove to be useful because, in most of the cases during chemotherapy and radiation therapy, solid biopsy could prove to be fatal for the patient, while liquid biopsy can be safely used to quantify the ccfDNA over the course of treatment and on the basis of which further confirmatory diagnosis (such as polymerase chain reaction and sequencing) and treatment regimen can be decided. Furthermore, ccfDNA burden in the serum during the follow-up can unveil any distant tumor metastasis and, to certain degree, could help in hypothesizing whether the patient will have a relapse or not. The ccfDNA can be used as a powerful means for monitoring treatment progress and, to certain level, could predict the development of resistance to the treatment.
| > Materials and Methods|| |
In this prospective study, blood samples were collected from the controls and CC patients (postoperative [P.O.], Stages I, II, III, and IV). A series of blood samples from 2 HPV-positive patients before, during, and 6-month follow-up after treatment were collected. A total of 25 samples including that of patients and controls (healthy humans) were included in this study.
This study was performed in the Department of Molecular Biology, with the collaboration of the Department of Radiotherapy and Radiation Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, between January 2018 and March 2019. Thirteen histologically confirmed CC patients referred to the Department of Radiotherapy and Radiation Medicine were enrolled in the study. The clinical staging was done by an oncologist and classified according to the International Federation of Gynecology and Obstetrics (FIGO) staging of cancer cervix. Eight healthy, age-matched females, of the same socioeconomic background, who came as bystanders of the patient were recruited as controls. All controls gave written, informed consent for their blood and its utilization for research purposes. The exclusion criteria were pregnancy (at the time of sampling), chronic diseases such as diabetes, liver disease or autoimmune disorders, acute infections (at the time of sampling), immunocompromised state, history or laboratory evidence of malignancy, and not being under any pharmacological therapy.
The study practice and patient consent procedures were permitted by the Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University (ECR/Bhu/Inst/UP/2013/Re-registration-2017 date January 31, 2017), with the Ethical Code Dean/2018/EC/329 on the date January 02, 2018. Informed written consent from all participants was obtained.
All patients were treated with concurrent chemoradiotherapy, during which they received 46–50 Gy (2 Gray/fraction) to the whole pelvis by three-dimensional conformal or modulated beam radiotherapy in 23–25 fractions over 5 weeks. They also received weekly concurrent cisplatin at a rate of 35–40 mg/m2. Further high-dose-rate intracavitary brachytherapy within 1 week of completion of external beam radiation therapy (EBRT) with iridium-192 source was also administered. Later, they were given three fractions weekly of 7.0 Gy or two weekly fractions of 9.0 Gy. The whole treatment (EBRT with CCT and brachytherapy) was completed within 45–55 days.
Blood sampling and processing
Under aseptic conditions, 5-ml random venous blood samples were drawn into Vacutainer (Becton Dickinson) from CC patients as well as controls (healthy volunteers), left the tubes to stand undisturbed for 30 min at room temperature for the retraction of the clot, and then centrifuged at 3000 rpm for 10 min at 4°C. Separated serum was carefully aspirated into screw-cap Cryovial tubes, to avoid hemolysis or contamination of the separated blood clot, aliquoted, and immediately stored at −20°C for further analysis.
From two other CC patients, blood samples were collected at three different phases of the treatment: Phase 1: before the start of therapy; Phase 2: during the course of concurrent chemoradiotherapy, i.e., after 12 fractions of EBRT; Phase 3: after 6 months of completion of the whole treatment.
Circulating cell-free DNA isolation from serum
Serum samples were thawed at 4°C, and 300 μL of serum was used for isolation of ccfDNA using GSure® serum isolation kit (GCC Biotech, India) as per the protocol mentioned in the manual. DNA was then eluted in 30-μl Tris-EDTA (pH 7.5). Isolated ccfDNA was quantified with the help of NanoDrop 2000c spectrophotometer (Thermo Scientific, USA). All handling of patient samples (blood) and its waste was in accordance with the Biomedical Waste Management Rules 2016.
Agarose gel electrophoresis
To analyze serum ccfDNA, 1% agarose gel electrophoresis was performed and visualized by ethidium bromide (10 μg/ml in the gel) staining under a ultraviolet light in Gel Doc (Bio-Rad, USA). ccfDNA from blood of a healthy person was used as a control.
| > Results|| |
The population included in the study was Asian Indian and ranged in age from 35 to 70 years. A total of 25 blood samples, 13 patients and 8 controls, were collected. Of 13 patients, 2 patient samples were collected before, during, and after treatment (as mentioned in the method). DNA was isolated from all the 25 samples, and the quality of DNA was assessed on the basis of A260/280 ratio. For control, DNA from the serum of healthy person was isolated and observed the trend of ccfDNA by agarose gel electrophoresis [Figure 1]a. The results obtained showed the band of 180–200 bp, which represents the nucleosomal DNA released in the plasma of healthy person due to day-to-day cell death and apoptosis [Figures 1b lane 1 and 2b lane 1]. As the level of ccfDNA in a healthy individual varies from person to person due to various factors such as age and time of sample collection, different intensities of DNA bands could be observed.
|Figure 1: Circulating cell-free DNA isolation and agarose gel electrophoreses imaging of cervical cancer patient samples. (a) The workflow involved in sampling and isolation of circulating cell-free DNA from patient blood sample. (b) Agarose gel profile for circulating cell-free DNA belonging to the patients from different levels of cancer metastasis. (c) Circulating cell-free DNA concentration in different stages of cervical cancer|
Click here to view
Observed ccfDNA pattern in healthy person was then used as a control for CC patient samples in which the ccfDNA load was assumed to be increased due to the imbalance in equilibrium between the release of DNA in plasma and clearing of DNA from circulation. We proceeded to the ccfDNA isolation [Figure 1]a for CC patients, and the results were analyzed by agarose gel electrophoresis [Figure 1]b. In control samples, we only observed the band for nucleosomal DNA fragment which was of ~200 bp, whereas in the CC patients, we observed smear of high-molecular-weight DNA. The smear shows the apoptotic burden of highly proliferating cells. Since the ccfDNA load varies on the basis of the CTC present in the bloodstream which implicates that higher the proliferation, higher the CTCs which lead to a higher load of ccfDNA in the sample, the higher load of ccfDNA in sample CC1, CC5, CC8, and CC9 [Figure 1]b lanes 2, 6, 9, and 10] indicates that these patients had a higher level of proliferation and metastasis in comparison to other samples. When the trend of these results was cross-checked to their respective patient case histories, it was found that all the four patients were either from Stage III or Stage IV level of metastatic CC. The lower concentration of ccfDNA in sample CC2, CC3, CC4, CC6, CC7, CC10, and CC11 shows lower invasion and metastasis among which patients belonging to sample CC2, CC4, and CC7 had Stage II cancer [Figure 1]b lanes 3, 5, and 8], whereas CC3, CC6, CC10, and CC11 [Figure 1]b lanes 3, 6, 10, and 11] were either Stage I or P.O. cases. Furthermore, the mean values (± standard deviation) of ccfDNA concentration [Figure 1]c were 5.3 ± 0.88 for P.O./Stage I cases, 7.90 ± 0.85 for Stage II cases, and 16.75 ± 2.17 for Stage III/IV cases, which follows the same pattern of CTC level in different CC stages.
After observing the varied pattern of ccfDNA in CC patients before treatment, we then wanted to see the trends of ccfDNA in three different phases: (i) before, (ii) during, and (iii) after treatment [Figure 2]a. Therefore, two patients, one from FIGO Stage III (sample 1) and another from FIGO Stage II (sample II), were followed till 6 months of follow-up after treatment. The higher ccfDNA load in “before treatment condition” [Figure 2]a lanes 2 and 5] indicates the higher proliferation and CTC in the bloodstream, as discussed earlier. Interestingly, we observed no DNA smear or the apoptotic ccfDNA load during the treatment [Figure 2]b lanes 3 and 6], which indicates a very low or negligible amount of CTC in the bloodstream. This might be due to the inhibitory effect of chemoradiotherapy on proliferating cells, and also, it could have unburdened the plasma clearance mechanism. Interestingly, in “after treatment samples,” we observed reappearance of smear DNA in the gel which indicates the recurrence of CTCs in the blood [Figure 2]b lanes 4 and 7] due to relapse or due to some kinds of other infections., Further confirmatory tests (solid biopsy and computed tomography scan) performed on these two patients showed that there was a relapse and tumor has metastasized to distant sites. We also noticed that although during treatment, the ccfDNA level was almost the same, during relapse, the ccfDNA level for Stage III CC patients increased at a much higher rate than the Stage II CC patient [Figure 2]c.
|Figure 2: Circulating cell-free DNA isolation and agarose gel electrophoreses imaging of cervical cancer patient samples at different stages of treatment. (a) The workflow involved in sampling and isolation of circulating cell-free DNA from patient blood sample. (b) Agarose gel profile for circulating cell-free DNA belonging to the patients in three different phases: before treatment, during treatment, and after 6 months of treatment. (c) Comparison of circulating cell-free DNA concentration in samples isolated from different stages of cervical cancer for three phases: before, during, and after 6 months of treatment|
Click here to view
| > Discussion|| |
CC is one of the major causes of global mortality among women. Various risk factors and markers have been recognized for this cancer., Almost all the reports on the ccfDNA advocate toward the mechanism that ccfDNA released in the circulation is due to necrosis or apoptosis, in which first the genomic DNA is cleaved into large fragments and then broken down into small fragments of nucleosomal units. DNA concentration in the normal control varies from 0 to 100 ng/ml which in cancer patients may exceed to 5 μg/ml. Several reports have also shown that higher ccfDNA load in serum of various cancer patients,,, could be due to histone modifications. This study focuses on the role of ccfDNA in CC diagnosis and effectiveness of treatment given to the patients in Indian population which can be further applied globally. In this study, we first observed ccfDNA pattern in a healthy individual which was then used as a control for the ccfDNA pattern in CC patients. Further extension of the study on two patients in three different phases, i.e., before, during, and after the treatment, shows how ccfDNA load in the serum could be used as an efficient marker of prognosis. In highly proliferating condition and metastasis, the load is so high that the ccfDNA clearing mechanism could not cope up, and hence, highly sheared DNA of higher molecular weight (genomic DNA) was observed [Figures 1b, c and 2b, c] due to the metastasized tumor cells in the circulation. The lower ccfDNA concentration in P.O. cases is probably because of the surgical removal of cancerous tissues which might reduce the apoptotic burden [Figure 1]c. The disappearance of ccfDNA load during treatment represents that the treatment regimen administered to the patients was working and clearance of ccfDNA from system is due to the reduced load of CTC. Due to distant metastasis, the tumor cells could not clear up from the system, and hence, recurrences of ccfDNA in relapse cases are obvious [Figure 2]b and c]. The higher concentration of ccfDNA after treatment for Stage III cancer shows its more aggressive behavior than that of Stage II cancer cells [Figure 2]c. This Study has shown that ccfDNA load can easily be visualized on agarose gel, which in turn can reduce the cost of the procedure and prove to be an efficient first line of diagnosis in a resource-deprived country like India. This could well establish a significant correlation between the ccfDNA pattern with stage, recurrence, metastatic ability, and effectiveness of the treatment. Just like all the methods, there are some limitations of the study. Since the very early stages and in P.O. cases, a lower number of CTC are present in the circulation; therefore, this technique in those cases cannot confirm the presence of cancer, and further histopathological tests need to be performed for the confirmation. This technique can be used efficiently for primary screening purpose.
| > Conclusion|| |
According to the WHO guidelines, every woman belonging to the age group of 21–65 years is relatively more prone to develop CC and should get screened at an interval of every 3 years for any precancerous lesions occurring. In developing country like India with limited funds and awareness, this is not feasible which contributes to the undetected progression of CC. Many CC patients die because of ineffective drug response and tumor metastasis. Due to distinct morphologic and phenotypic features of tumor cells, people respond differently to the treatments. The development of minimally invasive liquid biopsy from blood can prove to be a useful means of monitoring a patient's response to the treatment. Furthermore, during the follow-up studies of a patient, the load of ccfDNA could indicate the possible relapse of a patient in the early stages only. Since this tumor burden diagnostic technique is not confined at the local level of detection, it can also detect the level of distant metastasis. This initial study had shown some promising results, which indicate that ccfDNA detection is a reliable, cost-effective, and sensitive method and can prove to be a potential tumor marker in case of CC. We are also exploring the possibility of using this method as a reliable prognostic factor with more number of patients and longer follow-ups. Hence, on the basis of the results obtained, it can be stated that it has the potential to be widely helpful at detecting cancer burden and level of metastasis in patients.
We are sincerely thankful to the Institute of Medical Sciences, Banaras Hindu University (BHU). We are also thankful to Molecular Biology Departmental Head Prof. Sunit Kumar Singh for all the support. We also express our gratitude to Radiotherapy and Radiation Medicine Departmental Head Prof. Uday Pratap Shahi, Sir Sunderlal Hospital, BHU.
Financial support and sponsorship
SKS was supported by Ramalingaswami Grant (BT/RLF/Re-entry/43/2016) from the Department of Biotechnology (DBT), Government of India. GS was supported as JRF by DBT, Government of India, and KT was supported by an intramural grant for a Ph.D. program of Institute of Medical Sciences, BHU. The work was partially funded by an intramural grant from the Department of Radiotherapy and Radiation Medicine, Institute of Medical Sciences, BHU.
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394-424.
Moody CA, Laimins LA. Human papillomavirus oncoproteins: Pathways to transformation. Nat Rev Cancer 2010;10:550-60.
Kiran S, Dar A, Singh SK, Lee KY, Dutta A. The deubiquitinase USP46 is essential for proliferation and tumor growth of HPV-transformed cancers. Mol Cell 2018;72:823-35.
Dandona L, Dandona R, Kumar GA, Shukla DK, Paul VK, Balakrishnan K., Nations within a nation: Variations in epidemiological transition across the states of India, 1990-2016 in the Global Burden of Disease Study. Lancet 2017;390:2437-60.
Misra JS, Srivastava S, Singh U, Srivastava AN. Risk-factors and strategies for control of carcinoma cervix in India: Hospital based cytological screening experience of 35 years. Indian J Cancer 2009;46:155-9.
] [Full text]
Sreedevi A, Javed R, Dinesh A. Epidemiology of cervical cancer with special focus on India. Int J Womens Health 2015;7:405-14.
Koh W, Greer BE, Abu-Rustum NR, Apte SM, Campos SM, Chan J, et al
. Cervical cancer. JNatl Compr Canc Netw 2013;11:320-43.
Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF, et al
. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N
Engl J Med 2013;368:1199-209.
Heitzer E, Auer M, Gasch C, Pichler M, Ulz P, Hoffmann EM, et al
. Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing. Cancer Res 2013;73:2965-75.
Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l'homme. CR Seances Soc Biol Fil 1948;142:241-3.
Elshimali YI, Khaddour H, Sarkissyan M, Wu Y, Vadgama JV. The clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Int J Mol Sci 2013;14:18925-58.
Ward TH, Cummings J, Dean E, Greystoke A, Hou JM, Backen A, et al
. Biomarkers of apoptosis. Br J Cancer 2008;99:841-6.
Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al
. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 2014;6:224ra24.
Alix-Panabières C, Pantel K. Circulating tumor cells: Liquid biopsy of cancer. Clin Chem 2013;59:110-8.
Qin Z, Ljubimov VA, Zhou C, Tong Y, Liang J. Cell-free circulating tumor DNA in cancer. Chin J Cancer 2016;35:36.
Nagata S, Nagase H, Kawane K, Mukae N, Fukuyama H. Degradation of chromosomal DNA during apoptosis. Cell Death Differ 2003;10:108-16.
Fatouros IG, Jamurtas AZ, Nikolaidis MG, Destouni A, Michailidis Y, Vrettou C, et al
. Time of sampling is crucial for measurement of cell-free plasma DNA following acute aseptic inflammation induced by exercise. Clin Biochem 2010;43:1368-70.
van der Vaart M, Pretorius PJ. The origin of circulating free DNA. Clin Chem 2007;53:2215.
Volik S, Alcaide M, Morin RD, Collins C. Cell-free DNA (cfDNA): Clinical significance and utility in cancer shaped by emerging technologies. Mol Cancer Res 2016;14:898-908.
Xie J, Yang J, Hu P. Correlations of circulating cell-free DNA with clinical manifestations in acute myocardial infarction. Am J Med Sci 2018;356:121-9.
Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977;37:646-50.
Pérez-Barrios C, Nieto-Alcolado I, Torrente M, Jiménez-Sánchez C, Calvo V, Gutierrez-Sanz L, et al
. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: Impact on biomarker testing. Transl Lung Cancer Res 2016;5:665.
Anker P, Mulcahy H, Chen XQ, Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev 1999;18:65-73.
Kang Z, Stevanović S, Hinrichs CS, Cao L. Circulating cell-free DNA for metastatic cervical cancer detection, genotyping, and monitoring. Clin Cancer Res 2017;23:6856-62.
Mirtavoos-Mahyari H, Ghafouri-Fard S, Khosravi A, Motevaseli E, Esfahani-Monfared Z, Seifi S, et al
. Circulating free DNA concentration as a marker of disease recurrence and metastatic potential in lung cancer. Clin Transl Med 2019;8:14.
McAnena P, Brown JA, Kerin MJ. Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer. Cancers (Basel) 2017;9:5.
Srivastava AN, Misra JS, Srivastava S, Das BC, Gupta S. Cervical cancer screening in rural India: Status & current concepts. Indian J Med Res 2018;148:687-96.
] [Full text]
Valastyan S, Weinberg RA. Tumor metastasis: Molecular insights and evolving paradigms. Cell 2011;147:275-92.
Marusyk A, Polyak K. Tumor heterogeneity: Causes and consequences. Biochim Biophys Acta 2010;1805:105-17.
[Figure 1], [Figure 2]