Investigating the relationship between ccfDNA concentration,its integrity,and some individual factors in an Iranian population

Abstract

INTRODUCTION:

Circulating cell-free DNA (ccfDNA) increases in some pathologic conditions like cancer. We aimed to investigate the correlation between some individual factors and the ccfDNA level in peripheral blood of Iranian in relation to prostate cancer.

MATERIAL AND METHOD:

30 patients with prostate cancer (PCa), 40 with benign prostate hyperplasia (BPH), and 30 controls were studied. Personal information, ccfDNA concentration, and the integrity index were assessed for the correlation between the disease and different factors. The results were statistically analyzed using SPSS software.

RESULTS:

In PCa group, no association was found between total ccfDNA, BMI, BPH background, non-cancerous diseases, medications, PCa length, and job ( $p$ -value $>$ 0.05). But, total ccfDNA had statistical associations with weight, family history of cancer, and location ( $p$ -value $<$ 0.05). No association was between the integrity of ccfDNA, weight, the background of BPH, and family history of cancer. But, the integrity of ccfDNA was significantly associated with BMI and PCa length ( $p$ -value $<$ 0.05).

In BPH group, no association between total ccfDNA or the integrity of ccfDNA and the assessed factors was obtained ( $p$ -value $>$ 0.05).

In the normal group, neither statistical association was found between total ccfDNA, weight, BMI, and job, nor between the integrity of ccfDNA, weight, BMI, non-cancerous disease, drug, job, and location ( $p$ -value $>$ 0.05). But, a statistical association was found between the integrity of ccfDNA and family history of cancer in the recent group (Based on 95% CI and $P$ -value less than 0.05).

CONCLUSION:

ccfDNA and its integrity as possible prostate cancer biomarkers under the influence of individuals’ physiological status are prone to the pathologic changes toward the disease. Further simultaneous study of the target groups could clarify this matter.

Keywords

ccfDNA correlation individuality individual factors

1. Introduction

Circulating cell-free DNA (ccfDNA) is derived from blood cells by different mechanisms such as cell death, active secretion, and autophagocytosis [3, 20, 22]. CcfDNA sheds blood on all people from all types of cells. But it increases in various pathologic conditions such as cancer, sepsis, autoimmune diseases and in particular, in physiological states such as pregnancy or intense physical exercise [20, 27]. It is shown that the amount of ccfDNA in cancer patients varies by location and progression [12, 27] and in normal subjects, the mean amount of plasma ccfDNA is highly variable from less than 10 ng/mL to more than 1500 ng/mL [11]. These differences can be due to various in-vivo and in-vitro factors. In-vitro factors include different methods for sampling, extraction, detection. In this study in-vitro factors can be ignored using an identical method including specimen type (plasma); the processing of whole blood and ccfDNA isolation [19] and quantification [18].

Differences in ccfDNA level between people by in-vivo factors could be due to extra secretion or apoptosis and necrosis of cells into the blood, elimination processes, physiological distribution, individual conditions and genetics [20, 27]. It may be appropriate to consider these differences for using screening and prognosis in different populations. Our recent study used ccfDNA for the early detection of local and metastatic prostate cancer [18]. Reliable non-invasive ccfDNA testing can be performed as a clinical test if its sensitivity and impact of individual factors are examined to solve non-cancerous increase and decrease (borderline can narrow following omission of false result). Also, ccfDNA can be used as an effective adjuvant tool to complement screening tests to decrease costs and unnecessary precautions following decrease specific tests on the uncertain results of screening.

On the other hand, it seems that in the cancer group, due to the over-effects of cancer, the impact of non-cancerous factors should be neglected. However, in this article, we intend to examine the in-vivo factors related to the individual’s condition in cancer patients, benign hyperplastic, and non-cancerous individuals. Also, we intend to discuss differences in ccfDNA levels that may be due to differences in the factors involved in the production and deletion (nuclease, blood, liver, and kidney) of ccfDNA, based on individual and genetic conditions along with the pathological status. We would like to investigate which factors may affect ccfDNA concentration and its integrity to be able to detect a possible cut-off for PCa screening. Also, ccfDNA values can be achieved through a non-invasive, fast, repeatable and sensitive method as possible biomarkers for molecular detection and prognosis in a variety of diseases like cancers or risk (cancerous/pathologic/physiologic) of starting an event along with increased ccfDNA production or elimination.

2. Materials and methods

2.1 Studied population

100 subjects including 30 PCa, 40 BPH, and 30 normal individuals were studied. The circulating cell-free DNA was estimated by amplification of ALU115 and ALU247 repeat elements using quantitative real-time PCR [18] and individual information was tabulated and investigated. Regardless of overall ccfDNA outcomes and its difference in impact PCa (even local or metastatic), BPH and normal individuals, we studied each three groups independently. The factors we investigated included BMI, weight, job, location, non-cancerous diseases, familial history of cancer, individual history of BPH in PCa group, length of BPH or PCa in both groups, medications, smoking, and addiction.

2.2 Statistical analysis

Statistical analysis was performed by SPSS 13.0. And the $p$ -value $<$ 0.05 was considered as statistically significant. The differences between ccfDNA levels of cancer patients, BPH, and normal individuals’ plasma were analyzed by t-test, chi-square and Post Hoc. Correlations between clinicopathological parameters and plasma ccfDNA levels or the integrity index were performed by a one-way test, as appropriate. We used ROC curve (receiver operating characteristic) data to evaluate the specificity and sensitivity of the determined ccf DNA concentrations and DNA integrity in the studied groups.

3. Results

In PCa group (Table 1), no significant association was found between total ccfDNA and BMI ( $p$ -value $=$ 0.32), background of BPH ( $p$ -value $=$ 0.16), non-cancerous disease ( $p$ -value $=$ 0.08), medications ( $p$ -value $=$ 0.64), PCa length ( $p$ -value $=$ 0.59), and job ( $p$ -value $=$ 0.31).

Table 1
The relation between PCa group characteristics with ccfDNA (ALU115) and integrity (ALU247/ALU115)

	No. of patients	ccfDNA (ALU115) Md. (range) (ng/ $\mu$ l)	$P$ value	Integrity (ALU247/ALU115)	$P$ value
Weight	29		0.009		0.443
$<$ 60	13–14	46.2692		0.3600
60–70	8	31.1875		0.3300
70–80	5	32.2200		0.3600
80–90	2	103.000		0.3750
$>$ 90	1–2	275.000		0.4200
BMI			0.322		0.036
19–25	18	62.9556		0.3711
25–30	10	29.4200		0.3310
30–35	2	51.7500		0.3000
$>$ 40	–	–		–
Affected length			0.597		0.054
$<$ 1 month	2	88.9000		0.3900
$<$ 1 year	23	46.8261		0.3409
1–5 year	5	55.2200		0.3940
$>$ 10 year	–	–		–
Background of BPH			0.163		0.621
Yes	5	94.3200		0.3920
No	25	42.3720		0.3452
Cancer familial history			$<$ 0.001		0.247
Yes	6	95.3833		0.3533
No	24	39.9417		0.3529
Non-cancerous disease
Yes	25	55.5640	0.087	0.3556	0.094
No	5	28.3600		0.3400
Drug
Yes	22	52.3045	0.647	0.3555	0.328
No	8	47.5250		0.3462
Smoking
Yes	10	58.3700	0.850 (yes, no)	0.3456	0.758 (yes, no)
No	18	46.0556	1.000 (yes, quit)	0.3600	0.806 (yes, quit)
Quitted	2	59.1000	0.950 (no, quit)	0.3850	0.565 (no, quit)
Addiction
Yes	1	26.3000		0.3600
No	29	51.8828	–	0.3528	–
Job
Physical	15	49.2933		0.3467
Non physical	15	52.7667	0.313	0.3593	0.188
Location
Healthy (ruralist)	12	59.5083		0.3658
Contaminated (urbanist)	28	45.3778	0.042	0.3444	0.251

PCa ${}^{1}$ : Prostate cancer, Md: Median.

In contrast, statistical associations was found between total ccfDNA and weight ( $p$ -value $=$ 0.009), and familial history of cancer ( $p<$ 0.001), location ( $p$ -value $=$ 0.04).

Furthermore, in PCa group, no significant association was found between the integrity of ccfDNA and weight ( $p$ -value $=$ 0.44), background of BPH ( $p$ -value $=$ 0.62), cancer familial history ( $p$ -value $=$ 0.24), non-cancerous disease ( $p$ -value $=$ 0.09), drug ( $p$ -value $=$ 0.32), job ( $p$ -value $=$ 0.18), location ( $p$ -value $=$ 0.25).

In contrast, statistical associations were found between integrity of ccfDNA and BMI ( $p$ -value $=$ 0.03), PCa length ( $p$ -value $=$ 0.05).

In BPH group (Table 2), no statistical association was observed between total ccfDNA and weight ( $p$ -value $=$ 0.96), BMI ( $p$ -value $=$ 0.43), BPH length ( $p$ -value $=$ 0.18), drug ( $p$ -value $=$ 0.92), location ( $p$ -value $=$ 0.37).

Table 2

Relation between BPH group’s characteristics with ccfDNA (ALU115) and integrity (ALU247/ALU115)

	No. of patients	ccfDNA (ALU115) Md. (range) (ng/ $\mu$ l)	$P$ value	Integrity (ALU247/ALU115)	$P$ value
Weight			0.965		0.972
$<$ 60	13	13.923077		0.150000
60–70	11	13.081818		0.183636
70–80	8	12.462500		0.147500
80–90	6	14.650000		0.170000
$>$ 90	2	13.750000		0.140000
BMI			0.437		0.510
19–25	17	12.1706		0.1494
25–30	19	14.6000		0.1716
30–35	3	13.7333		0.1733
$>$ 40	1	14.5000		0.1300
Affected length			0.180		0.464
$<$ 1 month	2	10.3000		0.1100
$<$ 1 year	9	11.5222		0.1633
1–5 year	23	13.9217		0.1613
$>$ 10 year	6	15.9167		0.1750
Cancer familial history			$<$ 0.001		0.337
Yes	12	16.0083		0.1817
No	28	12.4250		0.1525
Non-cancerous disease			0.010		0.206
Yes	32	13.9812		0.1638
No	8	11.5750		0.1512
Drug			0.921		0.666
Yes	32	13.5406		0.1606
No	8	13.3375		0.1638
Smoking
Yes	10	12.6700	0.887 (yes, no)	0.1520	0.943 (yes, no)
No	13	11.8385	0.285 (yes, quit)	0.1454	0.351 (yes, quit)
Quitted	17	15.2588	0.085 (no, quit)	0.1788	0.156 (no, quit)
Addiction
Yes	1	21.0000	–	0.2300	–
No	39	13.3077		0.1595
Job
Physical	28	14.2929		0.1725
Non physical	12	11.6500	0.018	0.1350	0.142
Location
Healthy (ruralist)	5	14.7200		0.1600
Contaminated (urbanist)	35	13.3257	0.372	0.1614	0.312

BPH ${}^{1}$ : Benign prostate hypertrophy, Md: Median.

In contrast, statistical associations was found between total ccfDNA and cancer familial history ( $p<$ 0.001), non-cancerous disease ( $p$ -value $=$ 0.01), job ( $p$ -value $=$ 0.01).

Furthermore, in BPH group, no statistical association was found between integrity of ccfDNA and weight ( $p$ -value $=$ 0.97), BMI ( $p$ -value $=$ 0.51), cancer familial history ( $p$ -value $=$ 0.33), non-cancerous disease ( $p$ -value $=$ 0.20), drug ( $p$ -value $=$ 0.66), BPH Length ( $p$ -value $=$ 0.46), job ( $p$ -value $=$ 0.14), location ( $p$ -value $=$ 0.31).

In normal group (Table 3), no statistical association were found between total ccfDNA and weight ( $p$ -value $=$ 0.52), BMI ( $p$ -value $=$ 0.19), job ( $p$ -value $=$ 0.09).

In contrast, statistical associations were found between total ccfDNA and cancer familial history ( $p$ -value $=$ 0.007), non-cancerous disease ( $p<$ 0.001), drug ( $p<$ 0.001), location ( $p$ -value $=$ 0.004).

Furthermore, in normal group, no statistical association were found between integrity of ccfDNA and weight ( $p$ -value $=$ 0.33), BMI ( $p$ -value $=$ 0.28), non-cancerous disease ( $p=$ -value 0.92), drug ( $p$ -value $=$ 0.28), job ( $p$ -value $=$ 0.28), location ( $p$ -value $=$ 0.12).

In contrast, statistical associations were found between the integrity of ccfDNA and cancer familial history ( $p$ -value $=$ 0.004).

Table 3

Relation between Normal group’s characteristics with ccfDNA (ALU115) and integrity (ALU247/ALU115)

	No. of patients	ccfDNA (ALU115) Md. (range) (ng/ $\mu$ l)	$P$ value	Integrity (ALU247/ALU115)	$P$ value
Weight	29		0.523		0.333
$<$ 60	5	9.7000		0.1420
60–70	14	9.0142		0.1350
70–80	7	9.3285		0.1357
80–90	2	8.2500		0.1200
$>$ 90	1–2	13.000		0.1200
BMI			0.191		0.282
19–25	16	8.9375		0.1419
25–30	9	10.0556		0.1344
30–35	5	9.0000		0.1200
$>$ 40	–	–		–
Cancer familial history			0.007		0.004
Yes	5	11.3400		0.1400
No	25	8.8720		0.1352
Non-cancerous disease			$<$ 0.001		0.922
Yes	9	9.6667		0.1278
No	21	9.1190		0.1395
Drug			$<$ 0.001		0.284
Yes	5	10.3000		0.1420
No	25	9.0800		0.1348
Smoking
Yes	6	9.2500	0.985 (yes, no)	0.1383	0.946 (yes, no)
No	21	9.1333	0.540 (yes, quit)	0.1343	0.964 (yes, quit)
Quit (left)	3	10.4000	0.380 (no, quit)	0.1433	0.856 (no, quit)
Addiction
Yes	2	7.2500	–	0.1200	–
No	28	9.4286		0.1371
Job
Physical	10	10.1000		0.1370
Non physical	20	8.8750	0.097	0.1355	0.281
Location
Healthy	13	9.5000		0.1292
Contaminated	17	9.1176	0.004	0.1412	0.125

Md: Median.

4. Discussion

In this paper, we examined the possible relationship between BMI, workplace, weight, and family history of other cancers including prostate cancer with DNA integrity and the concentration of ccfDNA fragments which could possibly cause the disease. Any relationship in this regard could be considered in PCa prevention programs. Given that each individual has multiple factors that influence on each other and on ccfDNA concentration at the same time, it is not feasible to study the effect of a single factor. Therefore, in this study, first, we explored each factor as a preliminary idea by ignoring other factors. Some of these factors certainly need to be examined separately in future studies in animal models or in cell culture. In our study, some factors had more frequency in hyperplastic individuals than in the cancerous group with an influence on ccfDNA concentration. The reason of this influence could be due to the nature of the environmental factors as part of people’s lifestyle. Our purpose for examining these factors was to determine the screening range more precisely.

As we found in our previous study, the ccfDNA total and its integrity index in the patients with PCa were significantly higher than those of BPH and healthy groups. High levels of 23.51 ng/ml of ccfDNA and greater than 0.30 of integrity were associated with prostate cancer. Whereas values of 17.9 ng/ml up to 23.51 ng/ml of ccfDNA and 0.30 to 0.20 of integrity were considered as non-overlapping borderline values between benign hyperplasia and prostate cancer. Also, values of 9.1 ng/ml-10.79 ng/ml of ccfDNA and 0.15–0.12 of integrity are borderline values overlapping between benign hyperplasia and healthy controls.

However, the achieved cutoff or a diagnostic range of the measured parameters for healthy, BPH, and PCa subjects need further investigation on bigger population size to be confirmed [18] meaning that the first effective non-cancerous factors have existed in hyperplasia.

As our results reveal, weight had significant association with the ccfDNA level, while BMI showed association with the ccfDNA integrity in PCa the other groups. The effect of weight and BMI on ccfDNA concentration and integrity is an indication of the association between changes in body fat mass and tissue changes in hyperplastic and cancerous conditions. In this regard, increased integrity can be a sign of the final stages of cancer [24, 17, 31]. Concentrations of ccfDNA may be associated with higher post-cancer weight gain, with higher mortality [28]. While low BMI is associated with high integrity, this may be related to weight loss in advanced stages of cancer. Despite the gradual increase and subsequent decrease, the mean of DNA concentration and integrity were significantly different in the cancer patients but did not differ significantly in the hyperplasia group. Therefore, this pilot study reveals that the effect of DNA concentration and its integrity depend on tissue changes, in cancer development. Studies suggest that ccfDNA and integrity can increase and then decrease during the course of treatment and fallow up of cancer, and are important factors for tracking the progress of the disease during treatment [9]. Our study revealed that ccfDNA and integrity show decreasing and then increasing changes during the course of disease before treatment. Since there is no information about the mean of ccfDNA concentration and integrity of cancer patients at the time of hyperplasia, it is not possible to use the ccfDNAs alterations for prevention purposes. Therefore, the ccfDNA evaluation need to be done in BPH and then PCa status for each patient to achieve the molecular differences related to prostate tissue changes and cell free DNA. In some studies, bph is not introduced as a risk for cancer and is likely reported to co-exist with cancer [4]. In some studies, BPH has been associated with an increased risk of prostate and bladder cancer. The risk of prostate cancer is particularly high in Asian BPH patients [7]. We think that ccfDNA is helpful in understanding the association between BPH and prostate cancer. On the other hand, the treatment of hyperplasia and its induced apoptosis with the emergence of pathological clones at the stage of proliferation and stress-induced DNA damage can cause genome instability and initiate tumorigenesis [21].

Our results showed that concentration of ccfDNA was significantly correlated with family history of all types of cancer including prostate cancer in all three groups ( $p$ -value $<$ 0.001). Moreover, the average ccfDNA integrity of those with BPH and family history of cancer was higher than those without a family history of cancer. Also, the average of ccfDNA integrity in normal individuals’ prostate cancer was significantly associated with family history of cancer. Therefore, it can be concluded that there are not much effective factors on integrity in normal people. Understanding the factors affecting integrity can help to manage hyperplasia and cancer at different stages [2]. In prostate cancer screening, familial accumulation of prostate cancer- first-degree relatives (father, brother, child) - is considered a surrogate marker of genetic susceptibility to the disease, although the shared environment cannot be excluded among family members [15]. Perhaps using ccfDNA for family cancer screening will be included in clinical protocols in the future [25].

The history of some diseases such as hypertension, diabetes, coronary heart disease, hyperlipidemia, obesity, prostatitis is associated with prostate cancer [23]. On the other hand, abnormalities associated with abnormal cell death such as cancer, auto-immunity, viral infection, AIDS, sepsis, ischemia, neuro-degeneration, impaired healing and tissue regeneration increase the level of cell death and lead to increased concentrations. ccfDNA [13]. Our results demonstrated that 83.3% of cancer patients and 80% of BPH and 30% of normal people had non-cancerous diseases such as cardiovascular diseases, allergies, diabetes, hypertension, urinary (kidney-bladder) stone or gallstone. These diseases may have no direct effects on ccfDNA but could indirectly be effective. For example, diabetes as a possible risk factor in many epidemiological studies of prostate cancer [30], has been detected in 25% of the BPH, in 16.6% of the cancer, and in 6% of the normal understudy groups. Therefore, non-malignant diseases like diabetes is associated with ccfDNA concentration and integrity. Higher ccfDNA in cancer patients with a history of the disease [14] as well as high numbers in the PCa and BPH groups led us to think that indirectly there may be a cancer risk signaling [23], that is, increased mortality. Cellular can, for whatever reason, weaken the body’s overall health (including disrupting homeostasis and weakening immunity, etc.) and causing cancer.

It should be noted that 73.3% of cancer patients, 80% of hyperplasia, and 16.6% of normal people have consumed medications such as hypertensive drugs like Aspirin (ASA), (Losartan), Amlopres, metformin, and sedative medications from the diazepam family and other drugs. In this study, the concentration of ccfDNA had significant association with medications in normal subjects. It is consistent with previous reports that Aspirin and Metformin have cancer prevention effects [5, 32, 33] and show association with cancer biomarkers [1]. Of course, the association can indirectly depend on the dose, duration of usage, and the interaction of the drug that was not studied in this study. However, the effects of a drug may also be dependent on its ability to affect cell division and growth, or directly induce apoptosis and ccfDNA release [10]. In addition, some drugs can affect the DNA transmission through the cell membranes and some others could affect the blood-brain barrier and the organ capsule to release ccfDNA [20]. Therefore, further studies are needed to identify the molecular and cellular events and the pathways through which drugs affect ccfDNA concentration. On the other hand, ccfDNA is rapidly degraded in blood and is rapidly eliminated by the liver and kidneys nucleases [6, 12, 16]. The increase and consistency of ccfDNA can be a result of the suppression effects by the drugs.

There is a correlation between smoking and aggressive PCa, higher PCa mortality and worse treatment outcome [8]. Regardless of the duration of smoking and smoking cessation, the history of smoking in hyperplasia group (67.5%) was higher than in cancer group (40%) and healthy group (30%), respectively. But regardless of quit smoking, smoking rates in the cancer group (33.3%) were higher than in hyperplasia (25%) and healthy (20%), respectively. In our study, no association was found between smoking and ccfDNA concentration and integrity ( $p$ -value). We found a significant correlation between ccfDNA level and living state in the three understudy groups. Cancerous people of non-industrial areas had a higher ccfDNA average than normal and BPH people, though it was not significant. One could hypothesize that individuals’ physiological adjustments may be different for ccfDNA in different conditions [13, 29].

Air pollution and environmental toxins are linked to cancer risk through epigenetic changes [26]. While in our study living in a non-polluted city with increasing ccfDNA level in cancer played the opposite like some other studies that were irrelevant with cancer risk [26].

Although the high impact of cancer could cover the effects of some factors, we think some factors with synergistic activity on malignancy may be indirectly observed by examining ccfDNA levels. No significant correlation was found between Job and ccfDNA concentration and integrity in the cancer and normal groups. Although the cancer patients had stopped working, interestingly, the ccfDNA and integrity mean in this group with a history of non-physical occupation was higher than those with a history of heavy physical occupation ( $p$ -value $=$ 0.31), showing the effect of physical activity on tissue and organ health. As opposed to the normal group, individuals with heavy jobs had a higher mean

Also, the integrity mean in BPH individuals with a heavy physical job was higher than the rest of the group with no significant difference (ccfDNA $p$ -value $=$ 0.018, integrity $p$ -value $=$ 0.142). In our study, 70% of prostate hyperplasia patients, 50% of prostate cancer patients and 33% of normal individuals had heavy physical activity jobs. We think there is a link between occupation with heavy physical activity and prostate problems especially hyperplasia.

5. Conclusions

A comprehensive assessment of individual factors can provide a better picture of the actual relationship between those factors and ccfDNA characteristics. Finding these links is useful for delineating and determining screening cut-off to eliminate malignancies and other related diseases. The study of the biology of pre-malignancy contributes to a comprehensive description of the molecular and cellular events that lead to the progression of malignancy.

Footnotes

Acknowledgments

We would like to appreciate all participants in this study, particularly the patients and control individuals. Our sincere thanks to Hamid Pouresmaili for the manuscript review and fine edition. The present study was supported by the Research Council and was approved by Shahid Beheshti University of Medical Sciences Ethics Committee.

All participants provided written informed consent, and that this study was conducted in accordance with the Declaration of Helsinki.

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Investigating the relationship between ccfDNA concentration,its integrity,and some individual factors in an Iranian population

Abstract

INTRODUCTION:

MATERIAL AND METHOD:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Studied population

2.2 Statistical analysis

3. Results

Table 1 The relation between PCa group characteristics with ccfDNA (ALU115) and integrity (ALU247/ALU115)

5. Conclusions

Footnotes

Acknowledgments

References

Table 1
The relation between PCa group characteristics with ccfDNA (ALU115) and integrity (ALU247/ALU115)