Jump to ContentJump to Main Navigation
Show Summary Details
More options …


Emerging Science

Open Access
See all formats and pricing
More options …

Biomarkers used to assess radio- and chemotherapy-induced lymphocyte genome instability in a case of cerebral infarction during relapse of a testicular seminoma

Marija Gamulin / Ana Pastuhović / Fedor Šantek / Mislav Grgić / Nevenka Kopjar
Published Online: 2014-06-09 | DOI: https://doi.org/10.2478/bimo-2014-0003


We report a case of a testicular seminoma patient with relapse who was irradiated after acute cerebral infarction induced by cisplatin-based chemotherapy. Lymphocytic genome instability was studied using an alkaline comet assay, analysis of structural chromosome aberrations, and cytokinesis-block micronucleus assay in blood samples collected before and after PET CT scanning that preceded radiotherapy, as well as before the administration of the first and after the administration of the last fraction of 3D conformal radiation. A challengetest with hydrogen peroxide (H2O2) was performed on isolated peripheral blood lymphocytes in order to establish to what extent earlier therapies had modified the response of the patient’s DNA to external stimuli with a genotoxic chemical. Levels of primary DNA damage in lymphocytes increased after diagnostic exposure, lowered prior to administration of a conformal 3D radiotherapy, and were the highest at the end of radiotherapy. Ex vivo exposure to H2O2 caused additional lymphocyte DNA damage, which gradually increased 15 and 30 minutes after treatment. Diagnostic and therapeutic exposure to radiation caused measurable cytogenetic damage that was subjected to extensive repair. All of the obtained results point to increased genomic instability in the patient which should be taken into account in his future medical surveillance.

Keywords : Cisplatin-based chemotherapy; chromosome aberrations; comet assay; irradiation; lymphocyte; micronucleus; testicular cancer


  • [1] Schmoll H.J., Jordan K., Huddart R., Pes M.P., Horwich A., Fizazi K., et al., ESMO Guidelines Working Group, Testicular seminoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol., 2010, 21(Suppl 5): 140-146. CrossrefGoogle Scholar

  • [2] Cancer Research UK, Testicular cancer incidence statistics, http://www.cancerresearchuk.org/cancer-info/cancerstats/ types/testis/incidence/uk-testicular-cancer-incidencestatistics (2013, accessed 30 June 2013) Google Scholar

  • [3] National Comprehensive Cancer Network, Clinical Practice Guidelines in Oncology, Testicular Cancer, http://www.nccn.org (2013, accessed 30 June 2013) Google Scholar

  • [4] Jones D.A., Ester E.C., Leavitt D., Sweet R., Konety B., Jha G., et al., Adjuvant radiotherapy for synchronous bilateral testicular seminoma: a case report and a review of the pertinent literature. Case Reports Urol., 2013, http://dx.doi. org/10.1155/2013/241073. CrossrefGoogle Scholar

  • [5] Choo R., Quevedo F., Choo C.S., Blute M., Can radiotherapy be a viable salvage treatment option for the relapsed seminoma confined to the infra-diaphragm region recurring after primary chemotherapy for bulky stage II seminoma? Can. Urol. Assoc. J., 2010, 4, E137-140. Google Scholar

  • [6] Singh N.P., McCoy M.T., Tice R.R., Schneider L.L., A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res., 1988, 175, 184-191. Google Scholar

  • [7] Gamulin M., Kopjar N., Grgić M., Ramić S., Viculin T., Petković M., Garaj-Vrhovac V., Cytogenetic follow-up in testicular seminoma patients exposed to adjuvant radiotherapy. Coll. Antropol., 2010, 34, 455-465. Google Scholar

  • [8] Sigma-Aldrich Inc. Histopaque®-1077 (Procedure No. 1077), http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/ General_Information/1/1077.Par.0001.File.tmp/1077.pdf (2010, accessed 30 June 2013) Google Scholar

  • [9] Duke R.C., Cohen J.J., Morphological and biochemical assays of apoptosis. In: Coligan J.E., Kruisbeal A.M. (Eds.), Current Protocols in Immunology, John Willey and Sons, New York, 1992 Google Scholar

  • [10] Sham A.S.K., Szeto Y.T., Benzie I.F.F., Tan-Un K.C., Preliminary study of DNA damage in peripheral lymphocytes from lung cancer patients and healthy subjects, Turk. J. Med. Sci., 2003, 33, 149-154. Google Scholar

  • [11] IAEA, PAHO, WHO, Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies, http://www-pub.iaea.org/MTCD/publications/PDF/ EPR-Biodosimetry%202011_web.pdf (2011, accessed 30 June 2013) Google Scholar

  • [12] Fenech M., Morley A.A., Measurement of micronuclei in lymphocytes, Mutat. Res., 1985, 147, 29-36. Google Scholar

  • [13] Fenech M., Chang W.P., Kirsch-Volders M., Holland N., Bonassi S., Zeiger E., HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures, Mutat. Res., 2003, 534, 65-75. Google Scholar

  • [14] Eastmond D.A., Tucker J.D., Identification of aneuploidyinducing agents using cytokinesis-blocked human lymphocytes and antikinetochore antibody, Environ. Mol. Mutagen., 1989, 13, 34-43. CrossrefGoogle Scholar

  • [15] Bosl G.J., Patil S., Carboplatin in Clinical Stage I Seminoma: Too much and too little at the same time, J. Clin. Oncol., 2011, 29, 949-956. CrossrefGoogle Scholar

  • [16] Gamulin M., Grgić M., Stage I testicular seminoma: Results of adjuvant irradiation, detection of patients with relapsed disease and results of relapse therapy, J. Clin. Oncol. (Meeting Abstracts), 2013, 31(No. 15_suppl), e15509. Google Scholar

  • [17] Classen J., Schmidberger H., Meisner C., Winkler C., Dunst J., Souchon R., et al., Para-aortic irradiation for stage I testicular seminoma: Results of a prospective study in 675 patients. A trial of the German testicular cancer study group (GTCSG), Br. J. Cancer, 2004, 90, 2305-2311. Google Scholar

  • [18] Martin J.M., Joon D.L., Ng N., Grace M., Gelderen D.V., Lawlor M., et al., Towards individualised radiotherapy for stage I seminoma, Radiother. Oncol., 2005, 76, 251-256. CrossrefGoogle Scholar

  • [19] Gerl A., Vascular toxicity associated with chemotherapy for testicular cancer, Anticancer Drugs, 1994, 5, 607-614. Google Scholar

  • [20] Doehn C., Büttner H., Fornara P., Jocham D, Fatal basilar artery thrombosis after chemotherapy for testicular cancer, Urol. Int., 2000, 65, 43-45. CrossrefGoogle Scholar

  • [21] Weijl N.I., Rutten M.F., Zwinderman A.H., Keizer H.J., Nooy M.A., Rosendaal F.R., et al., Thromboembolic events during chemotherapy for germ cell cancer: A cohort study and review of the literature, J. Clin. Oncol. 2000, 18, 2169-2178. Google Scholar

  • [22] Russmann S., Winkler A., Lövblad K.O., Stanga Z., Bassetti C., Lethal ischemic stroke after cisplatin-based chemotherapy for testicular carcinoma and cannabis inhalation, Eur. Neurol., 2002, 48, 178-180. CrossrefGoogle Scholar

  • [23] Nuver J., Smit A.J., van der Meer J., van den Berg M.P., van der Graaf W.T.A., Meinardi M.T., et al., Acute chemotherapyinduced cardiovascular changes in patients with testicular cancer, J. Clin. Oncol., 2005, 23, 9130-9137. CrossrefGoogle Scholar

  • [24] Shahab N., Haider S., Doll D.C., Vascular toxicity of antineoplastic agents, Semin. Oncol., 2006, 33, 121-138. CrossrefGoogle Scholar

  • [25] Azak A., Oksüzoğlu B., Deren T., Oneç B.M., Zengin N., Cerebrovascular accident during cisplatin-based combination chemotherapy of testicular germ cell tumor: an unusual case report, Anti-Cancer Drugs, 2008, 19, 97-98. Google Scholar

  • [26] Batra R., Davies J.N., Wheatley D., Extensive arterial and venous thrombo-embolism with chemotherapy for testicular cancer: a case report, Cases J., 2009, http://www.casesjournal.com/ content/pdf/1757-1626-2-9082.pdf. Google Scholar

  • [27] Meattini I., Scotti V., Pescini F., Livi L., Sulprizio S., Palumbo V., et al., Ischemic stroke during cisplatin-based chemotherapy for testicular germ cell tumor: case report and review of the literature, J. Chemother., 2010, 22, 134-136. Google Scholar

  • [28] Moore R.A., Adel N., Riedel E., Bhutani M., Feldman D.R., Tabbara N.E., et al, High incidence of thromboembolic events in patients treated with cisplatin-based chemotherapy: a large retrospective analysis, J. Clin. Oncol., 2011,; 29, 3466-3473. CrossrefGoogle Scholar

  • [29] Khadjooi K., Adab N., Kenton A., Acute stroke secondary to carotid artery dissection in a patient with germ cell tumour: did cisplatin play a role?, Onkologie, 2013, 36, 46-48. CrossrefGoogle Scholar

  • [30] Dietrich J., Marienhagen J., Schalke B., Bogdahn U., Schlachetzki F., Vascular neurotoxicity following chemotherapy with cisplatin, ifosfamide, and etoposide, Ann. Pharmacother., 2004, 38, 242-246. Google Scholar

  • [31] Abouassaly R., Fossa S.D., Giwercman A., Kollmannsberger C., Motzer R.J., Schmoll H.J., Sternberg C.N., Sequelae of treatment in long-term survivors of testis cancer, Eur. Urol., 2011, 60, 516-526. CrossrefGoogle Scholar

  • [32] Hall E.J., Wuu C.S., Radiation-induced second cancers: The impact of 3D-CRT and IMRT, Int. J. Radiat. Oncol. Biol. Phys., 2003, 56, 83-88. Google Scholar

  • [33] Bokemeyer C., Schmoll H.J., Treatment of testicular cancer and the development of secondary malignancies, J. Clin. Oncol., 1995, 13, 283-292. Google Scholar

  • [34] Norppa H., Bonassi S., Hansteen I.L., Hagmar L., Strömberg U., Rössner P., et al., Chromosomal aberrations and SCEs as biomarkers of cancer risk, Mutat. Res., 2006, 600, 37-45. Google Scholar

  • [35] Bonassi S., Znaor A., Ceppi M., Lando C., Chang W.P., Holland N., et al., An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans, Carcinogenesis, 2007, 28, 625-631. Google Scholar

  • [36] Boffetta P., van der Hel O., Norppa H., Fabianova E., Fucic A., Gundy S., et al., Chromosomal aberrations and cancer risk: results of a cohort study from Central Europe, Am. J. Epidemiol., 2007, 165, 36-43. Google Scholar

  • [37] Murgia E., Ballardin M., Bonassi S., Rossi A.M., Barale R., Validation of micronuclei frequency in peripheral blood lymphocytes as early cancer risk biomarker in a nested case-control study, Mutat. Res., 2008, 639, 27-34. Google Scholar

  • [38] Bonassi S., El-Zein R., Bolognesi C., Fenech M., Micronuclei frequency in peripheral blood lymphocytes and cancer risk: evidence from human studies, Mutagenesis, 2011, 26, 93-100. CrossrefGoogle Scholar

  • [39] Mohrenweiser H.W., Jones I.M., Variation in DNA repair is a factor in cancer susceptibility: a paradigm for the promises and perils of individual and population risk estimation?, Mutat. Res., 1998, 400, 15-24. Google Scholar

  • [40] Jagetia G.C., Jayakrishnan A., Fernandes D., Vidyasagar M.S., Evaluation of micronuclei frequency in the cultured peripheral blood lymphocytes of cancer patients before and after radiation treatment, Mutat. Res., 2001, 491, 9-16. Google Scholar

  • [41] Faust F., Kassie F., Knasmüller S., Boedecker R.H., Mann M., Mersch-Sundermann V., The use of the alkaline comet assay with lymphocytes in human biomonitoring studies, Mutat. Res., 2004, 566, 209-229. Google Scholar

  • [42] Moller P, The alkaline Comet assay: towards validation in biomonitoring of DNA damaging exposures, Basic. Clin. Pharmacol. Toxicol., 2006, 98, 336-345. CrossrefGoogle Scholar

  • [43] Wasson G.R., McKelvey-Martin V.J., Downes C.S., The use of the comet assay in the study of human nutrition and cancer, Mutagenesis, 2008, 23, 153-162. CrossrefGoogle Scholar

  • [44] McKenna D.J., McKeown S.R., McKelvey-Martin V.J., Potential use of the comet assay in the clinical management of cancer, Mutagenesis, 2008, 23,183-90. CrossrefGoogle Scholar

  • [45] Baciuchka-Palmaro M., Orsière T., Duffaud F., Sari-Minodier I., Pompili J., Bellon L., et al., Acentromeric micronuclei are increased in peripheral blood lymphocytes of untreated cancer patients, Mutat. Res., 2002, 520, 189-198. Google Scholar

  • [46] M’Kacher R., Girinsky T., Koscielny S., Dossou J., Violot D., Béron-Gaillard N., et al., Baseline and treatment-induced chromosomal abnormalities in peripheral blood lymphocytes of Hodgkin’s lymphoma patients, Int. J. Radiat. Oncol. Biol. Phys., 2003, 57, 321-326. CrossrefGoogle Scholar

  • [47] Palyvoda O., Polańska J., Wygoda A., Rzeszowska-Wolny J., DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests, Acta Biochim. Pol., 2003, 50, 181-190. Google Scholar

  • [48] Mothersill C., Seymour C.B., Mechanisms and implications of genomic instability and other delayed effects of ionizing radiation exposure, Mutagenesis, 1998, 13, 421-426. CrossrefGoogle Scholar

  • [49] Hendry J.H., Genomic instability: potential contributions to tumour and normal tissue response, and second tumours, after radiotherapy, Radiother. Oncol., 2001, 59, 117-126. CrossrefGoogle Scholar

  • [50] Kopjar N., Želježić D., Garaj-Vrhovac V., Evaluation of DNA damage in the white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test, Acta Biochim. Pol., 2006, 53, 321-336. Google Scholar

  • [51] Garaj-Vrhovac V., Đurinec M., Kopjar N., Oreščanin V., A survey on the cytogenetic status of the Croatian general population by use of the cytokinesis-block micronucleus assay, Mutat. Res., 2008, 649, 91-100. Google Scholar

  • [52] Kopjar N., Kašuba V., Milić M., Rozgaj R., Želježić D., Gajski G., et al., Normalne i granične vrijednosti mikronukleus-testa na limfocitima periferne krvi ispitanika opće populacije Republike Hrvatske, Arh. Hig. Rada Toksikol., 2010, 61, 219-234. Google Scholar

  • [53] Gamulin M., Katić J., Milić M., Grgić M., Fučić A., Long-term follow-up study of genome damage elimination in patients with testicular seminoma exposed to ionising radiation during radiotherapy. Arh. Hig. Rada Toksikol., 2011, 62, 51-55. Google Scholar

  • [54] Gamulin M., Grgić M., Ramić S., Garaj-Vrhovac V., Kopjar N., Rani učinci radioterapije na razini oštećenja genoma u bolesnika liječenih od raka prostate i seminoma testis / Early effects of radiotherapy on genome damage in patients with prostatic cancer and testicular seminoma, In: Knežević Ž., Majer M., Krajcar Bronić I. (Eds.), Proceedings of the Ninth Symposium of the Croatian Radiation Protection Association (10-12 April 2013, Krk, Croatia), Croatian Radiation Protection Association Zagreb, 2013, 327-333. Google Scholar

  • [55] Wei Z., Lifen J., Jiliang H., Jianlin L., Baohong W., Hongping D., Detecting DNA repair capacity of peripheral lymphocytes from cancer patients with UVC challenge test and bleomycin challenge test, Mutagenesis, 2005, 20, 271-277. CrossrefGoogle Scholar

  • [56] Harrison L., Malyarchuk S., Can DNA repair cause enhanced cell killing following treatment with ionizing radiation?, Pathophysiology, 2002, 8, 149-159. CrossrefGoogle Scholar

  • [57] Migliore L., Guidotti P., Favre C., Nardi M., Sessa M.R., Brunori E., Micronuclei in lymphocytes of young patients under antileukemic therapy, Mutat. Res., 1991, 263, 243-248. Google Scholar

  • [58] Tates A.D., van Dam F.J., Natarajan A.T., Zwinderman A.H., Osanto S., Frequencies of HPRT mutants and micronuclei in lymphocytes of cancer patients under chemotherapy: a prospective study. Mutat. Res., 1994, 307, 293-306. Google Scholar

  • [59] Carbonell E., Demopoulos N.A., Stefanou G., Psaraki K., Parry K.M., Marcos R., Cytogenetic analysis in peripheral lymphocytes of cancer patients treated with cytostatic drugs: results from an EC Collaborative Study, Anticancer Drugs, 1996, 7, 514-519. Google Scholar

  • [60] Elsendoorn, T.J., Weijl N.I., Mithoe S., Zwinderman A.H., Van Dam F., De Zwart F.A., et al., Chemotherapy-induced chromosomal damage in peripheral blood lymphocytes of cancer patients supplemented with antioxidants or placebo, Mutat. Res. 2001, 498, 145-158. Google Scholar

  • [61] Kopjar N., Garaj-Vrhovac V., Milas I., Assessment of chemotherapy-induced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline comet assay, Teratogen. Carcinogen. Mutagen., 2002, 22, 13-30. CrossrefGoogle Scholar

  • [62] Kopjar N., Garaj-Vrhovac V., Milas I., Acute cytogenetic effects of antineoplastic drugs on peripheral blood lymphocytes in cancer patients: chromosome aberrations and micronuclei, Tumori, 2002, 88, 300-312. Google Scholar

  • [63] Kopjar N., Milas I., Garaj-Vrhovac V., Gamulin M., Cytogenetic outcomes of adjuvant chemotherapy in non-target cells of breast cancer patients, Hum. Exp. Toxicol. 2007, 26, 391-399. CrossrefGoogle Scholar

  • [64] Liu X., Zhao J., Zheng R., Protection against damaged DNA in single cell by polyphenols, Die Pharmazie, 2002, 57, 852-854. Google Scholar

  • [65] Liu X., Zhao J., Zheng R., DNA damage of tumor-associated lymphocytes and total antioxidant capacity in cancerous patients, Mutat. Res., 2003, 539, 1-8. Google Scholar

  • [66] Slupphaug G., Kavli B., Krokan H.E., The interacting pathways for prevention and repair of oxidative DNA damage, Mutat. Res., 2003, 531, 231-251. Google Scholar

  • [67] Anand S.S., Singh H., Dash A.K., Clinical applications of PET and PET-CT, Med. J. Arm. For. India, 2009, 65, 353-358. Google Scholar

  • [68] Huang B., Law M.W., Khong P.L., Whole-body PET/CT scanning: estimation of radiation dose and cancer risk, Radiology, 2009, 251,166-174. Google Scholar

  • [69] Téoule R., Radiation-induced DNA damage and its repair, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med., 1987, 51, 573-89. CrossrefGoogle Scholar

  • [70] Li L., Story M., Legerski R.J., Cellular responses to ionizing radiation damage, Int. J. Radiat. Oncol. Biol. Phys., 2001, 49, 1157-1162. CrossrefGoogle Scholar

  • [71] McMillan T.J., Tobi S., Mateos S., Lemon C., The use of DNA double-strand break quantification in radiotherapy, Int. J. Radiat. Oncol. Biol. Phys. 2001, 49, 373-377. CrossrefGoogle Scholar

  • [72] Weidner Maluf S., Monitoring DNA damage following radiation exposure using cytokinesis-block micronucleus method and alkaline single-cell gel electrophoresis, Clin. Chim. Acta, 2004, 347, 15-24. Google Scholar

  • [73] Jianlin L., Jiliang H., Lifen J., Wei Z., Baohong W., Hongping D., Measuring the genetic damage in cancer patients during radiotherapy with three genetic end-points, Mutagenesis, 2004, 19, 457-464. CrossrefGoogle Scholar

  • [74] Cheong N., Zeng Z.C., Wang Y., Iliakis G., Evidence for factros modulating radiation-induced G2-delay: potential application as radioprotectors, Phys. Med., 2001, Suppl 1, 205-209. Google Scholar

  • [75] Tice R.R., Agurell E., Anderson D., Burlinson B., Hartmann A., Kobayashi H., et al., Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen., 2000, 35, 206-221. CrossrefGoogle Scholar

  • [76] Leprat F., Alapetite C., Rosselli F., Ridet A., Schlumberger M., Sarasin A., et al., Impaired DNA repair as assessed by the “comet” assay in patients with thyroid tumors after a history of radiation therapy: a preliminary study, Int. J. Radiat. Oncol. Biol. Phys., 1998, 40, 1019-1026. CrossrefGoogle Scholar

  • [77] Müller W.U., Bauch T., Stüben G., Sack H., Streffer C., Radiation sensitivity of lymphocytes from healthy individuals and cancer patients as measured by the comet assay, Radiat. Environ. Biophys., 2001, 40, 83-89. Google Scholar

About the article

Received: 2014-04-03

Accepted: 2014-05-09

Published Online: 2014-06-09

Citation Information: Biomonitoring, Volume 1, Issue 1, ISSN (Online) 2300-4606, DOI: https://doi.org/10.2478/bimo-2014-0003.

Export Citation

© 2014 Marija Gamulin et al. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

Comments (0)

Please log in or register to comment.
Log in