Research ArticleExperimental Studies
Open Access

Diagnostic Value of Circulating Cell-free DNA in Patients With Papillary Thyroid Cancer

RAIMONDA KLIMAITE, MINTAUTE KAZOKAITE, AISTE KONDROTIENE, DALIA DAUKSIENE, RASA VERKAUSKIENE, BIRUTE ZILAITIENE and ALBERTAS DAUKSA
Anticancer Research May 2022, 42 (5) 2289-2299; DOI: https://doi.org/10.21873/anticanres.15708

Abstract

Aim: We investigated whether the occurrence and development of papillary thyroid cancer (PTC) might be predicted using levels of circulating cell-free DNA (cfDNA). Materials and Methods: The peripheral blood samples were collected from 68 patients with PTC, 31 patients with nodular goiter (NG), and 86 healthy controls (HC). The concentration of cfDNA was measured by qPCR using three primer sets: β-actin99, β-actin394 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in plasma samples. Results: It was demonstrated that plasma β-actin99 and β-actin394 in the PTC group were significantly higher compared to HC (p<0.05 and p<0.001, respectively). The cfDNA integrity index was significantly higher in the PTC patients compared to HC and NG (p<0.001, p<0.05, respectively). The cfDNA concentration in the NG group was significantly higher than in the PTC (p<0.05 and p<0.001, respectively). Moreover, in most PTC patients with suppressed thyroglobulin, the β-actin394 and cfDNA integrity index was significantly decreased after surgery (p<0.05 and p<0.001, respectively). ROC analysis revealed that cfDNA integrity index can be used as a potential marker in distinguishing PTC from HC (AUC 0.901, p<0.001) and NG (AUC 0.629, p<0.05). Conclusion: Increased concentration of cfDNA β-actin99 and β-actin394 may be a valuable biomarker that differentiates PTC patients from HC. Also, an increased cfDNA integrity index may be a suitable parameter which differentiates PTC patients from NG and HC.

Key Words:

Papillary thyroid carcinoma (PTC) is the most common type of differentiated thyroid cancer, accounting for about 80-85% of all cases of thyroid cancer (1). Based on Global cancer statistics 2020, 586.202 new cases of thyroid cancer worldwide were diagnosed (2). For the majority of patients with PTC, the prognosis is good. However, PTC can be aggressive; up to 35% of patients have local tumor renewal or systemic spread and about 10% of cases may present with metastases at initial presentation. Around 60-70% of this subset of patients will become radioiodine refractory (1, 3-5). Non-invasive diagnostic methods such as ultrasound are primarily used to differentiate thyroid nodules. However, if thyroid cancer is suspected, invasive procedures such as fine-needle biopsy (FNB) must be performed. However, even the FNB is often unable to make a definitive diagnosis and we must undertake an even more aggressive invasion-surgery. FNB cytology by ultrasonography is still the main diagnostic method of PTC, but there is a technical limitation and 15-30% of cases are cytological indeterminate (6, 7).

A minimally invasive diagnostic test that can accurately diagnose the onset of the disease remains the subject of research. Plasma sampling is the easiest way to investigate the presence of malignancies. It has very recently been recognized as the sample of choice over serum and whole blood for investigating levels of cfDNA (8).

Circulating cell-free DNA (cfDNA) is different length nucleic acid fragments released to the bloodstream from body cells. The length of cfDNA fragments depends on the mechanism of cell death. Short cfDNA fragments, less than 180 bp, are usually produced to the bloodstream from normal cells after apoptosis, while longer cfDNA fragments occur due to necrosis in tumor cells (9, 10).

The cfDNA in blood plasma was first reported by Mandel and Metais in 1948, but only very recently has been recognized as the potential diagnostic and prognostic biomarker in a variety of tumors (11, 12). Current analysis shows potential for diagnostic use in colorectal, liver, kidney, breast, prostate and lung cancer, and hepatocellular carcinoma (13-19). cfDNA can also be measured at higher levels in trauma, stroke, myocardial infarction and sepsis (20, 21). Unlike benign cells, tumor cells release longer DNA fragments in the blood because of necrosis or autophagy.

Recent studies demonstrate that longer DNA strands circulating in plasma can be considered a biomarker for thyroid cancer. However, the structural features of cfDNA for diagnosis and prognosis of differentiated thyroid cancer are poorly characterized. GAPDH as well as β-actin is the most commonly used endogenous housekeeping genes that have a stable expression pattern and other characteristics in cancer. Low amplification and deletion probability of GAPDH gene is detected in different types of cancer. Therefore, GAPDH is a useful reference gene in measuring cfDNA concentration released to the bloodstream from various types of cancer (21, 22).

Strong correlation between β-actin and a multitude of cancers was shown, but also controversial results were found. Some studies found that β-actin is deregulated in melanoma, liver, kidney, gastric, colorectal, oesophageal, lymphoma, prostate, pancreatic, lung, ovarian, breast, and leukaemia (15, 23). However, β-actin is upregulated in most cancer tissues. It was shown that β-actin polymerization and expression changes may be consistent with metastasis and invasive cancer Thus, we chose GAPDH and β-actin genes to identify the role of cfDNA status for early non-invasive diagnosis and prognosis of papillary thyroid cancer. We investigated whether the occurrence and development of PTC might be predicted using levels of circulating cfDNA.

Materials and Methods

Sample collection. PTC patients, patients with nodular goiter (NG) and healthy controls (HC) were involved in this study. Plasma samples were obtained from patients with PTC 24 h before and 4-6 weeks after surgery at the Hospital of Lithuanian University of Health Sciences Kaunas Clinics between 2020 and 2021. PTC patients underwent thyroidectomy, and the diagnosis of PTC was confirmed histopathologically after surgery. Classification of patients with PTC was performed according to the 8th edition of the AJCC/UICC staging system (24). Histological PTC subtypes were divided into two groups: aggressive (diffuse sclerosing variant and tall cell carcinoma) and non-aggressive subtypes (classical and follicular variant). The PTC group had no benign nodes of thyroid, severe or critical condition and no previous history of any other cancer.

Plasma samples from patients with NG were obtained 24 h before surgery. NG patients underwent thyroidectomy, and the diagnosis of multiple hyperplastic nodes was confirmed histopathologically. The HC group had no thyroid disease, autoimmune illness or previous history of any cancer and their family history for thyroid diseases and cancer was negative.

Thyroid ultrasound, hormones (TSH and fT4) and anti-thyroid peroxidase (anti-TPO) antibodies were performed on all prospective subjects before inclusion to the study. Plasma thyroglobulin (Tg) was performed on all PTC patients three month after surgery, before I131 therapy. Tg <0.1 ng/ml was considered suppressed.

Venous blood was drawn from PTC, NG and HC patients. All peripheral venous blood samples (10 ml) were collected in EDTA (BD Vacutainer PPT™ Plasma Preparation Tube; 13 x 100 mm/5 ml) tubes and separated by the centrifugation 1,900 × g for 10 min at 4°C. Supernatant then was transferred to a new 15 ml tube and centrifuged in conical tubes at 16,000 × g for 10 min at 4°C. Purified plasma was transferred to 1.5 ml aliquots and stored at –80°C until nucleic acid purification. Plasma cfDNA was extracted from 5 ml blood plasma using QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany) according to the manufacturer’s protocol. Eluted cfDNA was transferred into 0.2 ml Eppendorf tubes and stored at –80°C. Total cfDNA was quantified by the qPCR assay targeting the 67 bp amplicon on the APP gene.

The study was approved by the Kaunas Regional Committee of Biomedical Research (Lithuania, approval No. BE-2-64; 2020-02-07). Written informed consent was obtained from each participant of the study after a full explanation of the purpose and nature of all procedures used. This study was conducted in accordance with the Declaration of Helsinki.

Measurement of cfDNA integrity and cfDNA concentration. Plasma cfDNA concentration and cfDNA integrity index were analysed in our study. Plasma cfDNA was quantified by the qPCR assay targeting the 99 bp amplicon on the β-actin gene and 108 bp amplicon on the GAPDH gene. β-actin394 primers were used to amplify the long DNA fragments, released mainly from non-apoptotic cells. cfDNA integrity index was calculated as the ratio between the 394 and 99 bp amplicons of the β-actin gene as previously described. The concentration of cfDNA was measured by qPCR using three primer sets β-actin99 (F: 5’-CCACACTGTGCCCATCTACG-3’; R: 5’-AGGATCTTCATGA GGAGTCAGTCAG-3’), β-actin394 (F: 5’-CCACACTGTGCCCATC TACG-3’; R: 5’-TTAGCTTCCACAGCACAGCC-3’) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F: 5’-ATGTTCGTC ATGGGTGTGAA-3’; R: 5’-GGTGCTAAGCAGTTGGTGGT-3’) as previously described.

qPCR was performed in 20 μl reaction volume with 0.4 μM of forward and reverse primers, 2 μl of cfDNA, 6.4 μl of nuclease free water and 10 μl PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA, USA). qPCR was performed in Applied Biosystems 7500 Fast Real-Time PCR System thermal cycler (Applied Biosystems, USA) using the following cycling profile: 50°C for 2 min, 95°C for 2 min, 40 cycles at 9°C for 15 s, 60°C for 15 s, and 72°C for 1 minute, and ended with a melt curve from 65 to 95°C for 5 s. All reactions were performed in triplicate. The amount of DNA fragments for each gene was determined using a standard curve created by performing qPCR with serial dilutions (from 2.5 to 25,000 ng/ml) of human genomic DNA.

Statistical analysis. The normality of data distribution was tested using Kolmogorov–Smirnov criteria. Student’s t test and analysis of variance (ANOVA - for normal distribution) or Mann-Whitney U-test and Kruskal – Wallis H-test criterion - for abnormal distribution were used to determine the differences in quantitative traits between the comparison groups. The association between qualitative values in comparative groups was assessed by the Chi-square (χ2) test. The relationships between the amount of cfDNA in the plasma and clinic - pathological parameters were determined by Pearson correlation. The predictive capability (diagnostic performance) of each biomarker was investigated by means of the area under the ROC (Receiver-Operating Characteristics) curve (AUC). Statistical analyses were performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). The results were considered statistically significant at p<0.05.

Results

Study population. One hundred and eighty five patients were included in the study: 68 patients with a histologically confirmed diagnosis of PTC after surgical treatment, 31 patients with NG and 86 healthy controls. In the PTC group, there were 8 male patients (11.8%) and 60 female patients (88.2%), while in the NG group, 5 patients (16.1%) were male and 26 patients (83.9%) were female. In the HC group, there were 65 (75.6%) female and 21 (24.4%) male patients. The mean age [standard deviation (SD)] of PTC patients was 48.19 (14.9), 49.61 (17.16) of NG patients and 45.30 (12.07) of HC patients. Clinicopathological characteristics of the study population with PTC are shown in Table I.

View this table:
Table I.

Clinicopathological characteristics of the population with papillary thyroid cancer (PTC).

cfDNA concentration in PTC, NG and HC groups. Plasma cfDNA concentration was quantified according to β-actin99 and GAPDH concentrations. The concentration of β-actin99 was significantly higher in the PTC patients compared to HC (1,005.97 vs. 623.12 ng/ml, p<0.05), while there was no significant difference of GAPDH between these groups (Figure 1 and Figure 2). The concentration of GAPDH was significantly higher in the NG group compared to the PTC and HC (1,167.50 vs. 879.76 and vs. 751.34 ng/ml, p<0.05 in both cases). The concentration of β-actin99 in the NG group was significantly higher compared to the PTC and HC (1,336.89 vs. 1,005.97 and vs. 623.12 ng/ml, p<0.001 and p<0.05, respectively). The concentration of β-actin394 in the PTC group was significantly higher than in the HC (456.35 vs. 157.27 ng/ml, p<0.001). The NG group also had a higher concentration of long fragments than the control group (474.04 vs. 157.27 ng/ml, p<0.001) (Figure 3).

Figure 1.
Figure 1.

The comparison of GAPDH concentration in papillary thyroid cancer (PTC), nodular goiter (NG) and healthy controls (HC) patients. Analysis of Kruskal – Wallis H-test criterion was used to determine the differences in quantitative traits between the comparison groups. *p<0.05.

Figure 2.
Figure 2.

The comparison of β-actin99 concentration in papillary thyroid cancer (PTC), nodular goiter (NG) and healthy controls (HC) patients. Analysis of Kruskal – Wallis H-test criterion was used to determine the differences in quantitative traits between the comparison groups. *p<0.05, **p<0.001.

Figure 3.
Figure 3.

The comparison of β-actin394 concentration in papillary thyroid cancer (PTC), nodular goiter (NG) and healthy controls (HC) patients. Analysis of Kruskal – Wallis H-test criterion was used to determine the differences in quantitative traits between the comparison groups. *p<0.001.

The cfDNA integrity index was significantly higher in the PTC patients compared to HC and NG (0.475 vs. 0.134 and vs. 0.359, p<0.001 and p<0.05, respectively), While the index of NG patients was significantly higher compared to the HC group (0.359 vs. 0.134, p<0.001) (Figure 4).

Figure 4.
Figure 4.

The comparison of cfDNA integrity index in papillary thyroid cancer (PTC), nodular goiter (NG) and healthy controls (HC) patients. Analysis of ANOVA - for normal distribution, was used to determine the differences in quantitative traits between the comparison groups. *p<0.05, **p<0.001.

cfDNA concentrations in PTC patients before and after surgery. The specificity of the assay was demonstrated considering the variation of plasma cfDNA concentrations from GAPDH and β-actin99 genes and cfDNA integrity index comparing before and after surgery. After surgery, samples showed a significantly lower concentration of β-actin394 and cfDNA integrity index as compared to before. Moreover, concentration of β-actin394 and cfDNA integrity index after the surgery significantly decreased in PTC patients with suppressed Tg concentration (p<0.05 and p<0.001, respectively) (Figure 5 and Figure 6). However, after surgery concentrations of GAPDH and β-actin99 cfDNA decrease was not significantly lower (Table II).

Figure 5.
Figure 5.

The comparison of β-actin394 dynamic after surgery in papillary thyroid cancer (PTC) patients with suppressed and not suppressed thyroglobulin. *p<0.05.

Figure 6.
Figure 6.

The comparison of cfDNA integrity index dynamic after surgery in papillary thyroid cancer (PTC) patients with suppressed and not suppressed thyroglobulin. *p<0.001.

View this table:
Table II.

Plasma GAPDH, β-actin99, β-actin394 concentration and cfDNA integrity index in papillary thyroid cancer (PTC) patients before and after thyroidectomy.

Association of cfDNA concentration with clinicopathological features of PTC. A trend towards higher cfDNA concentrations has been observed between many clinicopathological features in the presence of more advanced PTC or in the presence of adverse disease features, although no significant differences were identified (Table III). We observed that GAPDH and β-actin99 concentrations were significantly higher in PTC with greater tumor size (>2 cm) compared to lower (≤2 cm) tumor size (p<0.05 in both cases). Similarly, PTC patients with a larger tumor size (>2 cm) were more likely to have a higher cfDNA integrity index compared to lower (≤2 cm) tumor size (p<0.05). The concentration of GAPDH, β-actin99, β-actin394 and cfDNA integrity index in aggressive histology variants subtypes of PTC to other, non-aggressive subtypes of PTC was compared, but no statistically significant relation was found (Table III).

View this table:
Table III.

Correlation between clinicopathological features of papillary thyroid cancer (PTC) and relative concentration of GAPDH, β-actin99, β-actin394 and cfDNA integrity index in plasma samples.

The total tumor size was calculated as the sum of the diameters of all tumors in PTC multifocal cases. A weak positive correlation between the concentration of GAPDH and β-actin99 with the total size of PTC tumors was found (p<0.05, r=0.245 and p<0.05, r=0.304, respectively) (Figure 7). The analysis showed a weak positive correlation between the concentration of β-actin394 and the total size of PTC tumors (p<0.05, r=0.239) (Figure 8).

Figure 7.
Figure 7.

The correlation between GAPDH (A) and β-actin99 (B) and total tumor size.

Figure 8.
Figure 8.

The correlation between β-actin394 concentration and the total tumor size.

The diagnostic value of cfDNA. To evaluate the possible diagnostic value of cfDNA, ROC analysis was performed. Concentration of β-actin99, β-actin394 and cfDNA integrity index had statistically significant satisfactory or good diagnostic values to differentiate PTC patients from HC (Figure 9C, 9E, 9G; Table IV). cfDNA integrity index had the highest AUC of 0.901 (95% CI=0.855-0.948), with 98.5% sensitivity and 64.0% specificity at the cutoff value of 0.065 (PTC vs. HC, Figure 9G). The concentration of GAPDH did not show statistically significant differences between PTC and HC (Figure 9A; Table IV). However, GAPDH and β-actin99 had statistically significant differences between PTC and NG. GAPDH had an AUC of 0.703 (95% CI=0.597-0.809), β-actin99 - 0.644 (95% CI=0.520-0.767) with a low sensitivity and specificity (Figure 9B, 9D; Table V). In addition, the comparison between PTC patients and those with benign nodules showed that the cfDNA integrity index had an AUC of 0.629 (95% CI=0.513-0.746), with 69.1% sensitivity and 67.6% specificity at the cutoff value of 0.279 (p<0.05). This might be useful for differentiating PTC from NG (Figure 9H; Table V).

Figure 9.
Figure 9.
Figure 9.

Diagnostic value of circulating cfDNA (GAPDH, β-actin99, β-actin394) and cfDNA integrity index in discriminating papillary thyroid cancer (PTC) from nodular goiter (NG) and healthy controls (HC). Receiver operating characteristic (ROC) curves were used to distinguish the groups. (A) Plasma GAPDH in PTC vs. HC and (B) NG. (C) Plasma β-actin99 in PTC vs. HC and (D) NG. (E) Plasma β-actin394 in PTC vs. HC and (F) NG. (G) Plasma cfDNA integrity index in PTC vs. HC and (H) NG. AUC, Area under the curve.

View this table:
Table IV.

ROC curves analysis for plasma concentration of GAPDH, β-actin99, β-actin394 and cfDNA integrity index to discriminate papillary thyroid cancer patients from a healthy control.

View this table:
Table V.

ROC curves analysis for plasma concentration of GAPDH, β-actin99, β-actin394 and cfDNA integrity index to discriminate papillary thyroid cancer patients from nodular goiter.

Discusion

Currently, there are no non-invasive tests that can specifically identify the presence of early-stage PTC and give high-confidence results. cfDNA has been the subject of significant interest in the field of cancer researchers, but little has been published with regards to PTC. In order to expand our knowledge, we choose GAPDH gene, β-actin99 and β-actin394 genes to identify the role of cfDNA status for early non-invasive diagnosis and prognosis of PTC.

The annealing sites of β-actin99 are within the β-actin394 ones. The β-actin99 primers could amplify both shorter fragments, truncated by apoptosis (β-actin99), and longer DNA fragments (β-actin394), so the results of β-actin99 quantitation represent the total amount of cfDNA. However, β-actin394 primers amplify only longer DNA fragments. Therefore, the results of β-actin394 quantitation more closely represent the amounts of DNA released from necrotic cell death. The main origin of short cfDNA fragments in healthy individuals has been attributed to apoptotic cells, while a majority of longer DNA fragments could represent a diagnostic biomarker for malignant tumors.

In this study, we compared cfDNA concentration determined by GAPDH, β-actin99 and β-actin394 and cfDNA integrity index in plasma samples of PTC, NG and HC. We observed that β-actin99 and β-actin394 concentrations and cfDNA integrity index in the PTC group were significantly higher compared to HC. Meanwhile, the concentration of GAPDH and β-actin99 in the NG group was significantly higher compared to the PTC and HC. Only the cfDNA integrity index was significantly higher between the PTC and NG groups. After surgery, PTC plasma samples showed a lower β-actin394 concentration and cfDNA integrity index, compared to the levels before. cfDNA (GAPDH and β-actin99) concentration, β-actin394 concentration and cfDNA integrity index was higher in tumors with greater tumor size (>2 cm) compared to lower (≤2 cm) tumor size. The concentration of GAPDH, β-actin99 and β-actin394 correlated with total tumor size. ROC analysis revealed that cfDNA integrity index provides higher accuracy in distinguishing PTC from NG and may be used as a potential tumor marker.

A higher concentration of circulating β-actin has previously been observed in hepatocellular and renal cell carcinomas, when compared to healthy controls (15, 23). Zane et al. found a significantly higher concentration of cfDNA in thyroid cancer patients compared with healthy individuals (25). Similarly, Jianga et al. found a significantly higher concentration of plasma GAPDH cfDNA in the thyroid cancer patients, than that of healthy controls, while in nodular goiter, they were not significantly different from the other two groups (26). Our study showed that the concentration of plasma β-actin was higher in the PTC group compared to HC, while the concentration of GAPDH did not differ significantly.

The concentration of cfDNA (GAPDH and β-actin99) and β-actin394 in the NG group was significantly higher compared to the PTC and HC. Given the fact that the large-sized nodes (>2 cm - 61.5% in the NG group) were diagnosed for the majority of NG group patients, concentration differences could be related to the size of the lesions. Thakur et al. observed that concentration of cfDNA was significantly higher in the plasma samples of patients with benign nodules compared to thyroid cancer patients (27). This is consistent with a few studies focused on other cancers that documented the cfDNA concentration correspondence to tumor size (28, 29). Tissue necrosis may develop at the center of large benign thyroid nodules due to circulatory and homeostatic disorders. Therefore, we hypothesize that the increase in cfDNA concentration in the NG group could be related to necrosis of the thyroid nodule cells.

Previous studies suggested that cfDNA integrity index was significantly higher in patients with gastrointestinal and hepatocellular tumors, melanoma, urologic tumor and breast cancer, as compared to healthy controls. Most studies have used GAPDH, ALU and β-actin qPCR to calculate the cfDNA integrity index (23, 30-33). It was observed, that the integrity index was most specific parameter indicating tumor status (9). Our results also confirm that cfDNA integrity index is a more specific parameter in differentiating PTC from NG and HC than measurement of β-actin99 and β-actin394 concentrations separately. Salvianti et al. found a higher cfDNA integrity index in thyroid cancer patients compared to healthy individuals and patients with NG (21). In accordance, our findings also suggest that cfDNA integrity index is significantly higher in PTC compared with NG and HC groups. This finding may support the cfDNA integrity index as a promising biomarker in differentiating PTC patients from thyroid nodules and healthy individuals.

Moreover, in most PTC patients with suppressed Tg, the β-actin394 and cfDNA integrity index was significantly decreased after surgery. The decrease in Tg levels may take up to six or twelve months. In our study, we obtained significant differences only three months after surgery. Our findings suggest that the plasma concentration of β-actin394 and cfDNA integrity index changed significantly after surgery and it therefore might be an indication of successful removal of the tumor and a useful prognostic marker after thyroidectomy. However, it would be useful to assess the changes after six or twelve months.

The findings by Salvianti et al. also suggest that cfDNA integrity index was lower after surgery than that taken before surgery in patients with thyroid cancer (21). Gang et al. also found the relation between cfDNA integrity index and the presence of thyroid tumor (32). Further studies of plasma cfDNA concentration measurements before and after surgery with more participants and a longer follow-up period would disclose more information about cfDNA as a prognostic biomarker of PTC patients.

It has been shown that cfDNA levels correlate with disease stage and primary tumor size (28). Umetani et al. found that cfDNA integrity index positively correlated with the size of invasive cancers and was significantly higher in the presence of lymphovascular invasion and lymph node metastasis. Our findings suggest that plasma concentration of cfDNA and β-actin394 and cfDNA integrity index were significantly higher in patients with greater tumor size. The concentration of GAPDH, β-actin99 and β-actin394 positively correlated with total tumor size. However, there was no correlation between lymphovascular invasion, lymph node metastasis and other clinico - pathological features. It is known that thyroid cancer releases lower amounts of cfDNA to the bloodstream compared with other more aggressive cancer types. Moreover, about half of the patients in our study were diagnosed with non-aggressive PTC subtype and pT1 stage. Therefore, we can assume that we do not have cancer invasion or metastasis for these reasons.

Previous studies suggest the significant diagnostic value of cfDNA concentration and integrity index in breast cancer patients. In our study, ROC curves were used to evaluate the diagnostic value of GAPDH, β-actin99, β-actin394 concentration and cfDNA integrity index in PTC, HC, and NG groups. Our findings indicated that β-actin99, β-actin394 concentrations and cfDNA integrity index changes may be used as parameters in differentiating PTC patients from HC, but the diagnostic value of GAPDH, β-actin99 and β-actin394 concentrations does not allow to differentiate NG from PTC. Meanwhile, ROC curve analysis confirmed that only plasma cfDNA integrity index might be a reliable marker in discriminating PTC from NG [AUC of 0.629 (95% CI=0.513-0.746)]. cfDNA has several limitations that restrict its use as a marker for PTC detection. Firstly, cfDNA is not specific and increase in its level could be observed in other malignancies and conditions such as in infections, severe diseases or inflammations; therefore, it must be evaluated with other findings. Secondly, due to a short patient follow-up period after the surgery it is difficult to determine prognostic value of cfDNA in PTC. Another limitation of this study is the relatively small sample size and lack of advanced disease cases. Finally, further large-scale and longer follow-up prospective investigations are needed to confirm our results in the diagnosis and prognostic prediction of PTC. It would also be useful to include more genes in the study that may be relevant to the pathogenesis of PTC.

In conclusion, the changes of cfDNA integrity index are expected to be a sensitive and specific parameter for the diagnosis of PTC and differentiating PTC from NG. The concentration of β-actin99 and β-actin394 are superior to GAPDH in differentiating PTC from HC, but further studies with larger sample size and longer follow-up period are necessary to confirm circulating cfDNA relevance as a biomarker for non-invasive prognostic tool of PTC.

Acknowledgements

This study was supported by the Lithuanian Research Council (Grant No. S-SEN-20-14 “The role of epigenetic markers for early detection of papillary thyroid carcinoma and prognostic significance in long-term outcome in elderly patients”).

Footnotes

  • Authors’ Contributions

    Conceptualization, A.D. and B.Z.; methodology, M.K., D.D., A.D.; validation, R.K., A.D., R.V.; formal analysis, R.K., A.K., M.K.; investigation, A.K., R.K., M.K, A.D.; resources, B.Z., A.D., R.V.; data curation, A.K., M.K., R.K., A.D., D.D.; writing – original draft preparation, R.K., M.K.; supervision, D.D., A.D., B.Z.; writing – review and editing, A.D., D.D., B.Z., R.V.; visualization, R.K., M.K., D.D., A.D.; project administration, A.D., B.Z.; funding acquisition, B.Z, A.D. All authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    The Authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.

  • Received February 14, 2022.
  • Revision received March 3, 2022.
  • Accepted March 14, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Limaiem F,
    2. Rehman A and
    3. Mazzoni T
    : Papillary thyroid carcinoma. 2021. PMID: 30725628.
  2. ↵
    1. Sung H,
    2. Ferlay J,
    3. Siegel RL,
    4. Laversanne M,
    5. Soerjomataram I,
    6. Jemal A and
    7. Bray F
    : Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71(3): 209-249, 2021. PMID: 33538338. DOI: 10.3322/caac.21660
    OpenUrlCrossRefPubMed
  3. ↵
    1. Kowalska A,
    2. Walczyk A,
    3. Kowalik A,
    4. Pałyga I,
    5. Trybek T,
    6. Kopczyński J,
    7. Kajor M,
    8. Chrapek M,
    9. Pięciak L,
    10. Chłopek M,
    11. Góźdź S and
    12. Kamiński G
    : Increase in papillary thyroid cancer incidence is accompanied by changes in the frequency of the BRAF V600E mutation: a single-institution study. Thyroid 26(4): 543-551, 2016. PMID: 26889698. DOI: 10.1089/thy.2015.0352
    OpenUrlCrossRefPubMed
    1. Medas F,
    2. Canu GL,
    3. Boi F,
    4. Lai ML,
    5. Erdas E and
    6. Calò PG
    : Predictive factors of recurrence in patients with differentiated thyroid carcinoma: a retrospective analysis on 579 patients. Cancers (Basel) 11(9): 1230, 2019. PMID: 31443531. DOI: 10.3390/cancers11091230
    OpenUrlCrossRefPubMed
  5. ↵
    1. Fugazzola L,
    2. Elisei R,
    3. Fuhrer D,
    4. Jarzab B,
    5. Leboulleux S,
    6. Newbold K and
    7. Smit J
    : 2019 European Thyroid Association guidelines for the treatment and follow-up of advanced radioiodine-refractory thyroid cancer. Eur Thyroid J 8(5): 227-245, 2019. PMID: 31768334. DOI: 10.1159/000502229
    OpenUrlCrossRefPubMed
  6. ↵
    1. Lewiński A and
    2. Adamczewski Z
    : Papillary thyroid carcinoma: a cancer with an extremely diverse genetic background and prognosis. Pol Arch Intern Med 127(6): 388-389, 2017. PMID: 28680025. DOI: 10.20452/pamw.4058
    OpenUrlCrossRefPubMed
  7. ↵
    1. Ward LS and
    2. Kloos RT
    : Molecular markers in the diagnosis of thyroid nodules. Arq Bras Endocrinol Metabol 57(2): 89-97, 2013. PMID: 23525286. DOI: 10.1590/s0004-27302013000200001
    OpenUrlCrossRefPubMed
  8. ↵
    1. Nuzzo PV,
    2. Berchuck JE,
    3. Korthauer K,
    4. Spisak S,
    5. Nassar AH,
    6. Alaiwi SA,
    7. Chakravarthy A,
    8. Shen SY,
    9. Bakouny Z,
    10. Boccardo F,
    11. Steinharter J,
    12. Bouchard G,
    13. Curran CR,
    14. Pan W,
    15. Baca SC,
    16. Seo JH,
    17. Lee GM,
    18. Michaelson MD,
    19. Chang SL,
    20. Waikar SS,
    21. Sonpavde G,
    22. Irizarry RA,
    23. Pomerantz M,
    24. De Carvalho DD,
    25. Choueiri TK and
    26. Freedman ML
    : Author Correction: Detection of renal cell carcinoma using plasma and urine cell-free DNA methylomes. Nat Med 26(10): 1663, 2020. PMID: 32895574. DOI: 10.1038/s41591-020-1078-y
    OpenUrlCrossRefPubMed
  9. ↵
    1. Salem R,
    2. Ahmed R,
    3. Shaheen K,
    4. Abdalmegeed M and
    5. Hassan H
    : DNA integrity index as a potential molecular biomarker in colorectal cancer. Egyptian Journal of Medical Human Genetics 21(1): 38, 2021. DOI: 10.1186/s43042-020-00082-4
    OpenUrlCrossRef
  10. ↵
    1. Diefenbach RJ,
    2. Lee JH,
    3. Kefford RF and
    4. Rizos H
    : Evaluation of commercial kits for purification of circulating free DNA. Cancer Genet 228-229: 21-27, 2018. PMID: 30553469. DOI: 10.1016/j.cancergen.2018.08.005
    OpenUrlCrossRefPubMed
  11. ↵
    1. Mandel P and
    2. Metais P
    : Nuclear acids in human blood plasma. C R Seances Soc Biol Fil 142(3-4): 241-243, 1948. PMID: 18875018.
    OpenUrlPubMed
  12. ↵
    1. Sanchez C,
    2. Snyder MW,
    3. Tanos R,
    4. Shendure J and
    5. Thierry AR
    : New insights into structural features and optimal detection of circulating tumor DNA determined by single-strand DNA analysis. NPJ Genom Med 3: 31, 2018. PMID: 30479833. DOI: 10.1038/s41525-018-0069-0
    OpenUrlCrossRefPubMed
  13. ↵
    1. Hao TB,
    2. Shi W,
    3. Shen XJ,
    4. Qi J,
    5. Wu XH,
    6. Wu Y,
    7. Tang YY and
    8. Ju SQ
    : Circulating cell-free DNA in serum as a biomarker for diagnosis and prognostic prediction of colorectal cancer. Br J Cancer 111(8): 1482-1489, 2014. PMID: 25157833. DOI: 10.1038/bjc.2014.470
    OpenUrlCrossRefPubMed
    1. Sobhani N,
    2. Generali D,
    3. Zanconati F,
    4. Bortul M and
    5. Scaggiante B
    : Cell-free DNA integrity for the monitoring of breast cancer: Future perspectives? World J Clin Oncol 9(2): 26-32, 2018. PMID: 29651384. DOI: 10.5306/wjco.v9.i2.26
    OpenUrlCrossRefPubMed
  15. ↵
    1. Yamamoto Y,
    2. Uemura M,
    3. Nakano K,
    4. Hayashi Y,
    5. Wang C,
    6. Ishizuya Y,
    7. Kinouchi T,
    8. Hayashi T,
    9. Matsuzaki K,
    10. Jingushi K,
    11. Kato T,
    12. Kawashima A,
    13. Ujike T,
    14. Nagahara A,
    15. Fujita K,
    16. Imamura R and
    17. Nonomura N
    : Increased level and fragmentation of plasma circulating cell-free DNA are diagnostic and prognostic markers for renal cell carcinoma. Oncotarget 9(29): 20467-20475, 2018. PMID: 29755665. DOI: 10.18632/oncotarget.24943
    OpenUrlCrossRefPubMed
    1. Qu C,
    2. Jiao Y and
    3. Wang Y
    : A liquid biopsy assay for identifying early-stage hepatocellular carcinoma in asymptomatic HBsAg-seropositive individuals. Mol Cell Oncol 6(5): e1614419, 2019. PMID: 31528693. DOI: 10.1080/23723556.2019.1614419
    OpenUrlCrossRefPubMed
    1. Vymetalkova V,
    2. Cervena K,
    3. Bartu L and
    4. Vodicka P
    : Circulating cell-free DNA and colorectal cancer: a systematic review. Int J Mol Sci 19(11): 3356, 2018. PMID: 30373199. DOI: 10.3390/ijms19113356
    OpenUrlCrossRefPubMed
    1. Fawzy A,
    2. Sweify KM,
    3. El-Fayoumy HM and
    4. Nofal N
    : Quantitative analysis of plasma cell-free DNA and its DNA integrity in patients with metastatic prostate cancer using ALU sequence. J Egypt Natl Canc Inst 28(4): 235-242, 2016. PMID: 27634416. DOI: 10.1016/j.jnci.2016.08.003
    OpenUrlCrossRefPubMed
  19. ↵
    1. Szpechcinski A,
    2. Rudzinski P,
    3. Kupis W,
    4. Langfort R,
    5. Orlowski T and
    6. Chorostowska-Wynimko J
    : Plasma cell-free DNA levels and integrity in patients with chest radiological findings: NSCLC versus benign lung nodules. Cancer Lett 374(2): 202-207, 2016. PMID: 26854716. DOI: 10.1016/j.canlet.2016.02.002
    OpenUrlCrossRefPubMed
  20. ↵
    1. Caglar O,
    2. Cilgin B,
    3. Eroglu M and
    4. Cayir A
    : Evaluation of circulating cell free DNA in plasma as a biomarker of different thyroid diseases. Braz J Otorhinolaryngol 86(3): 321-326, 2020. PMID: 30826312. DOI: 10.1016/j.bjorl.2018.12.008
    OpenUrlCrossRefPubMed
  21. ↵
    1. Salvianti F,
    2. Giuliani C,
    3. Petrone L,
    4. Mancini I,
    5. Vezzosi V,
    6. Pupilli C and
    7. Pinzani P
    : Integrity and quantity of total cell-free DNA in the diagnosis of thyroid cancer: correlation with cytological classification. Int J Mol Sci 18(7): 1350, 2017. PMID: 28672797. DOI: 10.3390/ijms18071350
    OpenUrlCrossRefPubMed
  22. ↵
    1. Qin Y,
    2. Wang JR,
    3. Wang Y,
    4. Iyer P,
    5. Cote GJ,
    6. Busaidy NL,
    7. Dadu R,
    8. Zafereo M,
    9. Williams MD,
    10. Ferrarotto R,
    11. Gunn GB,
    12. Wei P,
    13. Patel K,
    14. Hofmann MC and
    15. Cabanillas ME
    : Clinical utility of circulating cell-free DNA mutations in anaplastic thyroid carcinoma. Thyroid 31(8): 1235-1243, 2021. PMID: 33599171. DOI: 10.1089/thy.2020.0296
    OpenUrlCrossRefPubMed
  23. ↵
    1. Chen H,
    2. Sun LY,
    3. Zheng HQ,
    4. Zhang QF and
    5. Jin XM
    : Total serum DNA and DNA integrity: diagnostic value in patients with hepatitis B virus-related hepatocellular carcinoma. Pathology 44(4): 318-324, 2012. PMID: 28193336. DOI: 10.1097/PAT.0b013e328353a24c
    OpenUrlCrossRefPubMed
  24. ↵
    1. Tuttle RM,
    2. Haugen B and
    3. Perrier ND
    : Updated American Joint Committee on cancer/tumor-node-metastasis staging system for differentiated and anaplastic thyroid cancer (eighth edition): what changed and why? Thyroid 27(6): 751-756, 2017. PMID: 28463585. DOI: 10.1089/thy.2017.0102
    OpenUrlCrossRefPubMed
  25. ↵
    1. Zane M,
    2. Agostini M,
    3. Enzo MV,
    4. Casal Ide E,
    5. Del Bianco P,
    6. Torresan F,
    7. Merante Boschin I,
    8. Pennelli G,
    9. Saccani A,
    10. Rubello D,
    11. Nitti D and
    12. Pelizzo MR
    : Circulating cell-free DNA, SLC5A8 and SLC26A4 hypermethylation, BRAF(V600E): A non-invasive tool panel for early detection of thyroid cancer. Biomed Pharmacother 67(8): 723-730, 2013. PMID: 23931930. DOI: 10.1016/j.biopha.2013.06.007
    OpenUrlCrossRefPubMed
  26. ↵
    1. Jiang Z,
    2. Bahr T,
    3. Zhou C,
    4. Jin T,
    5. Chen H,
    6. Song S,
    7. Ikeno Y,
    8. Tian H and
    9. Bai Y
    : Diagnostic value of circulating cell-free mtDNA in patients with suspected thyroid cancer: ND4/ND1 ratio as a new potential plasma marker. Mitochondrion 55: 145-153, 2020. PMID: 33035689. DOI: 10.1016/j.mito.2020.09.007
    OpenUrlCrossRefPubMed
  27. ↵
    1. Thakur S,
    2. Tobey A,
    3. Daley B,
    4. Auh S,
    5. Walter M,
    6. Patel D,
    7. Nilubol N,
    8. Kebebew E,
    9. Patel A,
    10. Jensen K,
    11. Vasko V and
    12. Klubo-Gwiezdzinska J
    : Limited utility of circulating cell-free DNA integrity as a diagnostic tool for differentiating between malignant and benign thyroid nodules with indeterminate cytology (Bethesda category III). Front Oncol 9: 905, 2019. PMID: 31620364. DOI: 10.3389/fonc.2019.00905
    OpenUrlCrossRefPubMed
  28. ↵
    1. Bettegowda C,
    2. Sausen M,
    3. Leary RJ,
    4. Kinde I,
    5. Wang Y,
    6. Agrawal N,
    7. Bartlett BR,
    8. Wang H,
    9. Luber B,
    10. Alani RM,
    11. Antonarakis ES,
    12. Azad NS,
    13. Bardelli A,
    14. Brem H,
    15. Cameron JL,
    16. Lee CC,
    17. Fecher LA,
    18. Gallia GL,
    19. Gibbs P,
    20. Le D,
    21. Giuntoli RL,
    22. Goggins M,
    23. Hogarty MD,
    24. Holdhoff M,
    25. Hong SM,
    26. Jiao Y,
    27. Juhl HH,
    28. Kim JJ,
    29. Siravegna G,
    30. Laheru DA,
    31. Lauricella C,
    32. Lim M,
    33. Lipson EJ,
    34. Marie SK,
    35. Netto GJ,
    36. Oliner KS,
    37. Olivi A,
    38. Olsson L,
    39. Riggins GJ,
    40. Sartore-Bianchi A,
    41. Schmidt K,
    42. Shih lM, Oba-Shinjo SM,
    43. Siena S,
    44. Theodorescu D,
    45. Tie J,
    46. Harkins TT,
    47. Veronese S,
    48. Wang TL,
    49. Weingart JD,
    50. Wolfgang CL,
    51. Wood LD,
    52. Xing D,
    53. Hruban RH,
    54. Wu J,
    55. Allen PJ,
    56. Schmidt CM,
    57. Choti MA,
    58. Velculescu VE,
    59. Kinzler KW,
    60. Vogelstein B,
    61. Papadopoulos N and
    62. Diaz LA Jr.
    : Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6(224): 224ra24, 2014. PMID: 24553385. DOI: 10.1126/scitranslmed.3007094
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Catarino R,
    2. Ferreira MM,
    3. Rodrigues H,
    4. Coelho A,
    5. Nogal A,
    6. Sousa A and
    7. Medeiros R
    : Quantification of free circulating tumor DNA as a diagnostic marker for breast cancer. DNA Cell Biol 27(8): 415-421, 2008. PMID: 18694299. DOI: 10.1089/dna.2008.0744
    OpenUrlCrossRefPubMed
  30. ↵
    1. Mead R,
    2. Duku M,
    3. Bhandari P and
    4. Cree IA
    : Circulating tumour markers can define patients with normal colons, benign polyps, and cancers. Br J Cancer 105(2): 239-245, 2011. PMID: 21712823. DOI: 10.1038/bjc.2011.230
    OpenUrlCrossRefPubMed
    1. Pinzani P,
    2. Salvianti F,
    3. Zaccara S,
    4. Massi D,
    5. De Giorgi V,
    6. Pazzagli M and
    7. Orlando C
    : Circulating cell-free DNA in plasma of melanoma patients: qualitative and quantitative considerations. Clin Chim Acta 412(23-24): 2141-2145, 2011. PMID: 21839068. DOI: 10.1016/j.cca.2011.07.027
    OpenUrlCrossRefPubMed
  32. ↵
    1. Gang F,
    2. Guorong L,
    3. An Z,
    4. Anne GP,
    5. Christian G and
    6. Jacques T
    : Prediction of clear cell renal cell carcinoma by integrity of cell-free DNA in serum. Urology 75(2): 262-265, 2010. PMID: 19962739. DOI: 10.1016/j.urology.2009.06.048
    OpenUrlCrossRefPubMed
  33. ↵
    1. Zaher ER,
    2. Anwar MM,
    3. Kohail HM,
    4. El-Zoghby SM and
    5. Abo-El-Eneen MS
    : Cell-free DNA concentration and integrity as a screening tool for cancer. Indian J Cancer 50(3): 175-183, 2013. PMID: 24061455. DOI: 10.4103/0019-509X.118721
    OpenUrlCrossRefPubMed
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Diagnostic Value of Circulating Cell-free DNA in Patients With Papillary Thyroid Cancer
RAIMONDA KLIMAITE, MINTAUTE KAZOKAITE, AISTE KONDROTIENE, DALIA DAUKSIENE, RASA VERKAUSKIENE, BIRUTE ZILAITIENE, ALBERTAS DAUKSA
Anticancer Research May 2022, 42 (5) 2289-2299; DOI: 10.21873/anticanres.15708
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