50
Bas.J.Vet.Res.Vol.19, No.2, 2020.
ASSOCIATION BETWEEN SOME RISK FACTORS AND PROSTATE
CANCER PROGRESSIONIN BASRAH, IRAQ
Rasha A. Al-Badran *, Adnan Issa Al-Badran *, Majed Azkar **,
Loma Al-Mansouri ***
*Department of Biology, College of Science, University of Basrah,Basrah,Iraq
**Department of surgery, College of Medicine, University of Basrah, Iraq
***Department of Internal Medicine and Oncology, College of Medicine, University of Basrah,
Iraq
(Received14 May 2020 ,Accepted 6 June 2020)
Keywords: Prostate Cancer; Risk Factors, Basrah.
*Corresponding Author: Adnan.albadran@uobasrah.edu.iq
ABSTRACT
Prostate cancer is a complex disease in which both genetic and environmental influences
led to the development and growth of tumors. at the recent time, is considered the second most
common malignancy after lung cancer in men and the sixth leading cause of cancer – related
death worldwide.Sixty-seven blood samples of prostate cancer patients collected from Basrah
oncology and Hematology center aging between (45-90). On the other hand, seventy blood
samples of men without cancer were collected as control group aging between (45-90). Two ml
of peripheral blood were drawn by sterilized syringe from the two groups than kept in the EDTA
tube for DNA extraction for molecular study (not included). Both study groups have answered
questions about personal information such as age, address, cigarette smoking, occupation, and
family history.The present study found evidence that the aging was a significant risk factor for
developing prostate cancer, as well as family history increased the risk of disease about
eighteenfold (OR=18.8).Type of work people do related to the development of prostate cancer,
free business increase the risk about eightfold (OR=8.18), and military increase the risk for fold
(OR=4.09) while men deal with chemical material increase the risk about threefold (OR=2.5).
In our study, the place of living was divided into three regions (South, North, and the center of
Basra) the risk of disease increases about threefold for both South and North of Basra compare
with the Centre of Basra. Cigarette smoking also increases the risk of twofold compare with nonsmokers.
51
Bas.J.Vet.Res.Vol.19, No.2, 2020.
INTRODUCTION
Prostate cancer was the most common cancer among men, there are three non-modifiable
risk factors for prostate cancer: age, race, and a positive family history of prostate
cancer. Furthermore, it was found that a variety of modifiable or behavioral causes are associated
with the risk of prostate cancer. Although not firmly established, protective behavioral factors
can include physical activity and frequent tomato consumption, cruciferous vegetables, and soy
foods. In contrast, high dietary intake of dairy products and meat may increase prostate cancer
risk. On the other hand, factors such as smoking and obesity were weakly associated with
prostate cancer incidents and mortality (1). Prostate cancer is an example of the age –depending
disease. This affects predominantly men older than 40 years. The average age is about (65-70)
years. It is rare before 40 years old, but the rate increases with age in constant to other types of
cancer. Tumor at this site occurs with insignificant frequency in men that are less than 55 years
old, the vast majority of the cases (86%) occur over the age of 66 (2).
The disease is most common among Australian, New Zealand, and African American
men, followed by Western and Northern Europeans, in these countries, the high incidence is
partly due to the high detection rate resulting from routine screening and diagnostics. Prostate
cancer is also relatively common in the Caribbean, Southern Africa, and South America. In
contrast, Prostate cancer is less incidence in Eastern and South-Central Asia (3). Prostate cancer
as many common cancers tend to cluster in families, 5-10% of prostate cancer cases describe as
familial cancer which is considered to result from heritable risk genetic factors. The risk of
prostate cancer increases with the increasing number of affected relatives, especially those
diagnosed at a young age. Men with an inherited predisposition are more likely to be affected at
a younger age than 55 years (4). Athletes and men with high physical activity are less likely to
develop prostate cancer and urinary tumor (5). Physical activity can reduce fatigue, elevate
mood, reduce physical limitations, decrease falls, attenuate bone loss, and promote weight
(6).The purpose of this work was to determine the association between some risk factors and
prostate cancer progression.
52
Bas.J.Vet.Res.Vol.19, No.2, 2020.
MATERIALS AND METHODS
In the present study sixty-seven of blood samples were collected from prostate cancer
patients from Al-Sadder teaching Hospital – Basra center for Oncology and Hematological
disease their ages range between (45-90) years old. On the other hand, seventy blood samples of
males without cancer were collected as a control group their ages range between (45-90) years
old. Two ml of peripheral blood was drawn by sterilized syringe from the two groups than they
have been kept in sterilized EDTA tubes for DNA extraction using Genomic DNA Mini Kit
(Geneaid, Taiwan) and molecular study (not included) was done to amplifying segment of the
gene HOXB13 by PCR technique using pair of specific primers.
Questions were asked to both patients and healthy groups about age, place of residence,
family history of cancer, type of work, and smoking. Descriptive statistics were applied to
describe patients and controls characteristic according to use percentage. ORs and 95%CLs were
calculated by using the SPSS program. OR were considered significant if OR≥ 1.5.
RESULTS
Demographic Variables
All study subjects, prostate cancer patients (n=67) and control group (n=70) have
answered questions in the questionnaire form as in appendix.Different parameters from both
groups were obtained from questionnaire form; some of them are shown in table (1)
Table (1): Distribution of demographic variables between patients and control group.
Variable Control (n=70) Patient (n=67)
Age (years)
Mean age(+SD)
Range
60 (±9)
45-90
71(±8)
45-90
Obesity
Normal
Thin
Obesity
5 (7%)
34 (49%)
31 (44%)
4 (6%)
37 (55%)
26 (39%)
53
Bas.J.Vet.Res.Vol.19, No.2, 2020.
Family history
Table (2) shows the family history as a strong significant factor in development of
prostate cancer for eighteen folds comparing with no family history.
Table (2): distribution of patients and controls according to family history
Family history control Patient OR 95/CI P value
No 69(98.57%) 52(78%) 1.0 ------ -------
Yes 1(1.43%) 15(22%) 18.8 1.101-1.423 0.0002
Age
The age distribution of patients and control groups showed in table (3) which explain that
the maximum number of patients were in the age group of 66-75 year ( 49.3%) followed by the
age group of 56-65 year (21%), followed byb76-85 year( 22.3%), 45-55year (4.4%), and the
minimum number was in age group 86-95 year (3%). In other hand the age group 56-65year
(41%) from the control group was the highest, followed by the age group of 45-55year (39%),
followed by 66-75year (14%), 3% for both age groups (76-85), and (86-95) respectively. The
present study showed that the age was a significant risk factor for developing the disease with
age progress
Table (3): Distribution of age among patients and control groups.
Age (years) control patient OR 95% CI P Value
45-55 27 (39%) 3 (4.4%) ------- ------- ----
56-65 29 (41%) 14 (21%) 4.34 1.123-16.804 0.02
66-75 10 (14%) 33 (49.3%) 29.7 7.421-118.865 0.65
76-85 2 (3%) 15(22.3%) 67.5 10.124- 450.064 0.77
86-95 2 (3%) 2 (3%) 9 0.907- 89.266 0.03
54
Bas.J.Vet.Res.Vol.19, No.2, 2020.
Address
The majority of patients with prostate cancer were from south of Basrah16.3%, followed
by north of Basrah 14.2% then by center of Basrah 15.6% as shown in table (4). The risks of
prostate cancer increased about three folds in south of Basrah compared with center of Basrah
OR=2.73 (95%CI=1.3-60925) and P value= 0.008 as shown in Table (4-4).
Table (4): Distribution of study subjects according to their address
Address control patient OR 95% CI P value
Center of Basra 42 (٦٠%) 23 (٣4%) 1.0 ----- ------
North of Basra 14 (٢٠%) 20 (٣٠%) 2.73 1.175-6.383 0.012
South of Basra 14 (٢٠%) 24 (36%) 3 1.3-6.925 0.008
OR= Odds ratio, 95%CI=95% confidence interval
Occupation
Table (5) shows that patient’s exposure to chemical material in their job were large
number than other group, these group had odds ratio about 2.5, military group formed 4.09 odds
ratio, and free job group had highest odds ratio 8.18. In other hand large group of control work in
business office followed by chemical group then by military group, finally group of free
business.It has been noticed that significant risk factor increased development of prostate cancer
in patients who work in free business to about eight folds OR=8.18 than group of business office,
followed by military group, then by chemical works group.
Table (5): Distribution of occupation among patients and control groups
Occupation control Patient OR 95% CI P value
Business office 30(٤٣%) 11(١٦.4%) 1.0 ----- -----
Chemical works 25(٣6%) 23(٣٤.3%) 2.5 1.027-6.13 0.041
Free business 7(١٠%) 21(٣1.3%) 8.18 2.725-24.568 8.1E-5
Military 8(١١%) 12(١8%) 4.09 1.321-12.668 0.012
OR= Odds ratio, 95%CI=95% confidence interval
55
Bas.J.Vet.Res.Vol.19, No.2, 2020.
Cigarette Smoking
As shown in table (6), heavy smoking was significantly associated with risk of prostate cancer
OR=1.69(95%CI=0.387-2.507).
Table (6): Association of cigarette smoking with prostate cancer
Type of smoking control patient OR 95% CI P value
Non 55 (79%) 51 (76.1%) 1.0 ----- -----
Light smoking 12 (17%) 11 (16.4%) 0.93 0.38-2.297 0.881
Heavy smoking 3 (4%) 5 (7.5%) 1.69 0.387-2.507 0.479
OR= Odds ratio, 95%CI=95% confidence interval
DISCUSSION
The most common disease among men is prostate cancer, the incidence and mortality of
prostate cancer vary widely. While some of these differences can be attributed to variation in
diagnostic intensity, the considerable heterogeneity in prostate cancer mortality across countries
indicates that behavioral factors play an important role. The sizeable rise in the incidence and
mortality of prostate cancer following migration from low-risk to high-risk countries, as well as
changing lifestyle like changing dietary habits, some behaviors such as smoking and direct
contact with chemical substances are major contributors to the risk factors. (1)
In our study, it was found that the mean age for prostate cancer is 71 with standard
deviation (± 8) as shown in table (1), table (3) cleared that incident of prostate cancer increased
with age and the age group of (66-75) recorded high percentage 48% which agreed with previous
research which consider age As a risk factor for prostate cancer (7).Aging exhibits an increase to
various autoimmune, led to a decline in the immune response, and triggers the inflammatory
pathways causing the development of prostate cancer (8). on the other hand, high levels of blood
cholesterol in advanced age, progressing incident of prostate cancer because the metabolism of
this fatty acid affected cellular activity including cell division (9). Prostate cancer in high age
may increase due to an irregular level of testosterone (10).
On the other hand, the Body Mass Index (BMI) and prostate cancer relationship result
was not clear, a study by (11) detected that high BMI may be related to an increased risk of
56
Bas.J.Vet.Res.Vol.19, No.2, 2020.
aggressive prostate cancer. while the inverse relationship was observed between high BMI and
nonaggressive prostate cancer. Another study by (12) suggests that BMI was strongly correlated
with prostate cancer mortality and that men with high BMI (overweight or obese) who had
aggressive prostate cancer would have markedly greater odds of death. Body Mass Index (BMI)
was not significant in our study. Moreover in our study, the family history as shown in table (2)
was associated with a significant increase in the risk of prostate cancer about eighteen –fold.
Family history is widely recognized as a risk factor for prostate cancer with an estimated
heritability of 58%, while the other 42% explained by other common causes. low penetrance
variants, a study by Mancuso estimates that as much as 42% of ‘missing’ heritability is likely to
be explained by rare variants (13).
Ewing and colleagues observed that the case carrier frequency was higher in a familial
cohort (14). Several studies have since replicated this finding in the Caucasian family and casecontrol
populations and estimated that the variant is associated with a 4- to 8-fold increase in the
risk of prostate cancer and early disease (15) We agreed with the study by (16) which explained
the role of hereditary of family history with prostate cancer.
Table (4) shows the distribution of patient and control group at different parts of Basrah,
the risk of prostate cancer increased in south and north of Basrah (OR=3,59, 2.7) respectively
comparing to the center of Basrah, this may due to accumulation of high level of carcinogens in
these reigns such as depleted uranium and other chemical materials which used in previous wars.
In addition, these areas lay near oil fields which throws many kinds of pollutants especially
hydrocarbons, also the pollution from fumes and wastes of many industrial factories present in or
near south and north of Basrah. The annual report of the Iraqi Cancer Registry (2015) confirms
this fact. this result also agreed with other researches consider chemicals and radiation material
as carcinogens for cancer incidents and mortality (17,18).
In addition to working around dust, the fibers, chemicals, and fumes can also increase the
risk for developing an occupational disease such as cancer. Our study found that the risk of
prostate cancer increases the risk of cancer eight-fold (OR=8, 95%CI= 2.7-24.5) in free business
included workers in construction, drivers, shop owners, farmers, and Oil stations. Military and
chemical workers were also at high risk to develop prostate cancer (OR=4,59, 2.5) respectively.
Previous studies have reported elevated risks associated with employment in construction and
transportation, with some evidence linked to whole􀀀body vibrations, diesel exhaust, and
57
Bas.J.Vet.Res.Vol.19, No.2, 2020.
polycyclic aromatic hydrocarbons (PAH). (19). On the other hand, men who deal with pesticides
and diesel engine exhaust from farm equipment or have a store of agriculture material and
machinery are suspected of contributing risk factors. Exposure to pesticides may affect hormone
levels and function by disrupting endocrine activity and increasing estrogen levels leading to
tumor promotion (20).
Military men have elevated risk for prostate cancer may be the reason is exposure to
diesel exhaust, dust and particulate matter, chemical agents, radiation, and other mixed agents,
and they can be under constant psychological stress which may impact biological processes
leading to the development of cancer(21). Of course, deal with chemical induce forming a tumor,
our result agreed with other research (22). In addition to above factor it is clearly that cigarette
smoking harms nearly every organ of the body, causes many diseases including cancer, and
reduces the health of smokers in general. Heavy smoking increases the risk of prostate cancer
two-fold comparing with non-smoking as shown in table (6). Smoking may cause a genetic
mutation by affected hormones that exhibit tumor suppressor genes lead to tumor formation. (23)
Smoking was associated with a higher risk of dying from prostate cancer as well as other causes
of death. In contrast, past smoking was not associated with prostate cancer-specific mortality
suggesting that smoking may be a modifiable risk factor (24), study of (25) found an association
between tobacco smoking and incident of prostate cancer.
العلاقة بین بعض عوامل الخطورة وزیادة الاصابة بسرطان المثانة فی مدینھ البصرة – العراق
رشا عبدالامام عبدالرزاق*، عدنان عیسى البدران*، ماجد اصغر**، لمى احمد المنصوری***
* قسم علم الحیاة، کلیة العلوم ،جامعة البصرة ، البصره ،العراق.
**قسم الجراحة، کلیة الطب، جامعة البصرة ، البصره ،العراق.
***قسم الباطنیة والاورام، کلیة الطب، جامعة البصرة ، البصره ،العراق.
الخلاصة
یعد سرطان البروستات مرضًا معقدًا یحدث نتیجة التأثیرات الوراثیة والبیئیة معا حیث تسبب تکون الأورام
ونموھا. فی الآونة الأخیرة ، یعتبر ثانی أکثر الأورام الخبیثة شیوعًا بعد سرطان الرئة لدى الرجال والسبب الرئیسی السادس
للوفاة المرتبطة بالسرطان فی جمیع أنحاء العالم. سبعة وستون عینة دم لمرضى سرطان البروستات تم جمعھا من مستشفى
58
Bas.J.Vet.Res.Vol.19, No.2, 2020.
٩٠ ). و جمع سبعین عینة دم من الرجال - البصره التعلیمی و مرکز البصرة للأورام وأمراض الدم تتراوح اعمارھم بین ( ٤٥
٩٠ ). تم سحب ٢ مل من الدم الوریدی عن طریق حقنة معقمھ من - الاصحاء کمجموعة ضابطة اعمارھم تتراوح بین ( ٤٥
لاستخلاص الحمض النووی. تمت الاجابة على بعض الأسئلة حول EDTA المجموعتین ، تم الاحتفاظ بھا فی أنبوب
المعلومات الشخصیة مثل العمر والعنوان وتدخین السجائر والوظیفة وتاریخ الأسرة من قبل کلا المجموعتین وجدت الدراسة
الحالیة ان العمر عامل خطر کبیر لتطور سرطان البروستات ، کما تبین ان تاریخ الأسرة یزید من خطر المرض حوالی ثمانیة
کما ان نوع العمل الذی یقوم بھ الافراد المشمولین بالدراسة الحالیة لھ علاقة بتطور سرطان . (OR = عشر ضعفا ( 18.8
والعسکریة تزید من (OR= البروستات ، فالأعمال التجاریة الحرة زادت من خطر الإصابة بحوالی ثمانیة أضعاف ( ٨.١٨
بینما الرجال الذین یتعاملون مع المواد الکیمیائیة ازداد خطر الإصابة لدیھم بحوالی ثلاثة أضعاف (OR= خطر الإصابة ( ٤.٠٩
تم تقسیم مدینة البصره الى ثلاث مناطق (جنوب وشمال ووسط البصرة) وجد ان خطر الإصابة یزید بحوالی .(OR = 2.5)
ثلاثة أضعاف لکل من جنوب وشمال البصرة مقارنة بمرکز البصرة. کما وجد ان تدخین السجائر یزید من خطر الإصابة
بعشرین ضعفًا مقارنةً بعدم التدخین.
REFERENCES
1-Leitzmann, M. F., & Rohrmann, S. (2012). Risk factors for the onset of prostatic cancer: Age,
location, and behavioral correlates. Clinical Epidemiology, 4(1), 1–11.
https://doi.org/10.2147/CLEP.S16747
2-Tao, Z. Q., Shi, A. M., Wang, K. X., & Zhang, W. D. (2015). Epidemiology of prostate cancer:
Current status. European Review for Medical and Pharmacological Sciences, 19(5), 805–
812.
3-Center, M. M., Jemal, A., Lortet-Tieulent, J., Ward, E., Ferlay, J., Brawley, O., & Bray, F.
(2012). International variation in prostate cancer incidence and mortality rates. European
urology, 61(6), 1079-1092.
4-Carter, B. S., Bova, G. S., Beaty, T. H., Steinberg, G. D., Childs, B., Isaacs, W. B., & Walsh, P.
C. (1993). Hereditary prostate cancer: Epidemiologic and clinical features. Journal of
Urology, 150(3), 797–802. https://doi.org/10.1016/S0022-5347(17)35617-3
5-Sommer, F., Klotz, T., & Schmitz-Dräger, B. J. (2004). Lifestyle issues and genitourinary tumors.
World journal of urology, 21(6), 402-413.
59
Bas.J.Vet.Res.Vol.19, No.2, 2020.
6-Bostwick, D. G., Burke, H. B., Djakiew, D., Euling, S., Ho, S. M., Landolph, J., … Timms, B.
(2004). Human prostate cancer risk factors. Cancer, 101(10 SUPPL.), 2371–2490.
https://doi.org/10.1002/cncr.20408
7-Vaidyanathan, V., Karunasinghe, N., Jabed, A., Pallati, R., Kao, C. H. J., Wang, A., ... &
Ferguson, L. R. (2016). Prostate Cancer: Is It a Battle Lost to Age?. Geriatrics, 1(4), 27.
8-Solana, R., Tarazona, R., Gayoso, I., Lesur, O., Dupuis, G., & Fulop, T. (2012, October). Innate
immunosenescence: effect of aging on cells and receptors of the innate immune system in
humans. In Seminars in immunology (Vol. 24, No. 5, pp. 331-341). Academic Press.
9-Krycer, J. R., & Brown, A. J. (2013). Cholesterol accumulation in prostate cancer: a classic
observation from a modern perspective. Biochimica et Biophysica Acta (BBA)-Reviews on
Cancer, 1835(2), 219-229.
10-Jones, H. (2008). Testosterone for the aging male; current evidence and recommended practice.
Clinical Interventions in Aging, Volume 3, 25–44. doi:10.2147/cia.s190.
11-Xie, B., Zhang,G., Wang, X.& Xu, X.( 2017). Body mass index and incidence of nonaggressive
and aggressive prostate cancer: a dose-response meta-analysis of cohort studies :
Oncotraget,57(8),97584-97592
12-Haque, R., Van Den Eeden, S. K., Wallner, L. P., Richert-Boe, K., Kallakury, B., Wang, R., &
Weinmann, S. (2014). Association of body mass index and prostate cancer mortality.
Obesity Research and Clinical Practice, 8(4), 1–15.
https://doi.org/10.1016/j.orcp.2013.06.002
13-Mancuso, N., Rohland, N., Rand, K. A., Tandon, A., Allen, A., Quinque, D., Mallick, S., Li, H.,
Stram, A., Sheng, X. & Kote-Jarai, Z. (2016). The contribution of rare variation to
prostate cancer heritability. Nature genetics, 48(1), 30.
14-Ewing, C. M., Ray, A. M., Lange, E. M., Zuhlke, K. A., Robbins, C. M., Tembe, W. D., Wiley,
K.E., Isaacs, S.D., Johng, D., Wang, Y., Yan, G., Gielzak, M., Partin, A.W. & Bizon, C.
(2012). Germline mutations in HOXB13 and prostate-cancer risk. New England Journal of
Medicine, 366(2), 141-149.
60
Bas.J.Vet.Res.Vol.19, No.2, 2020.
15-Xu, J., Lange, E. M., Lu, L., Zheng, S. L., Wang, Z., Thibodeau, S. N., … Isaacs, W. B. (2013).
HOXB13 is a susceptibility gene for prostate cancer: Results from the International
Consortium for Prostate Cancer Genetics (ICPCG). Human Genetics, 132(1), 5–14.
https://doi.org/10.1007/s00439-012-1229-
16-Pierce, B. L., Friedrichsen􀀀Karyadi, D. M., McIntosh, L., Deutsch, K., Hood, L., Ostrander, E.
A., Melissa, A.A. & Stanford, J. L. (2007). Genomic scan of 12 hereditary prostate cancer
families having an occurrence of pancreas cancer. The Prostate, 67(4), 410-415.
17-Epstein, S. S. (1990). Losing the war against cancer: Who’s to blame and what to do about it.
International Journal of Health Services, 20(1), 53–71. https://doi.org/10.2190/W318-FRD5-
KVX8-9QJJ
18-Boström, C. E., Gerde, P., Hanberg, A., Jernström, B., Johansson, C., Kyrklund, T., Rannug,
A., Törnqvist, M., Victorin, K. & Westerholm, R. (2002). Cancer risk assessment,
indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air.
Environmental health perspectives, 110(suppl 3), 451-488.
19-Sass􀀀Kortsak, A. M., Purdham, J. T., Kreiger, N., Darlington, G., & Lightfoot, N. E. (2007).
Occupational risk factors for prostate cancer. American journal of industrial medicine, 50(8),
568-576.
20-Barry, K. H., Koutros, S., Lubin, J. H., Coble, J. B., Barone-Adesi, F., Freeman, L. E. B.,
Sandler, D.P., Hoppin, J.A., Ma, X., Zheng,T. & Alavanja, M. C. (2012). Methyl
bromide exposure and cancer risk in the Agricultural Health Study. Cancer Causes &
Control, 23(6), 807-818.
21-Zhu, K., Devesa, S. S., Wu, H., Zahm, S. H., Jatoi, I., Anderson, W. F., ... & McGlynn, K. A.
(2009). Cancer incidence in the US military population: comparison with rates from the
SEER program. Cancer Epidemiology and Prevention Biomarkers, 18(6), 1740-1745.
22-Sritharan, J., MacLeod, J., Harris, S., Cole, D. C., Harris, A., Tjepkema, M., Peters, P.A. &
Demers, P. A. (2018). Prostate cancer surveillance by occupation and industry: the
61
Bas.J.Vet.Res.Vol.19, No.2, 2020.
Canadian Census Health and Environment Cohort (CanCHEC). Cancer medicine, 7(4),
1468-1478.
23-Hickey, K., Do, K. A., & Green, A. (2001). Smoking and prostate cancer. Epidemiologic reviews,
23(1), 115-125.
24-Riviere, P., Kumar, A., Luterstein, E., Vitzthum, L. K., Nalawade, V., Sarkar, R. R., … Rose,
B. S. (2019). Tobacco smoking and death from prostate cancer in US veterans. Prostate
Cancer and Prostatic Diseases. https://doi.org/10.1038/s41391-019-0178-6
25-Jiménez-Mendoza, E., Vázquez-Salas, R. A., Barrientos-Gutierrez, T., Reynales-Shigematsu, L.
M., Labra-Salgado, I. R., Manzanilla-García, H. A., & Torres-Sánchez, L. E. (2018).
Smoking and prostate cancer: A life course analysis. BMC Cancer, 18(1), 1–11.
https://doi.org/10.1186/s12885-018-4065-7