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Chromosomal in-vitro radiosensitivity of lymphocytes in radiotherapy patients and AT-homozygotes

Chromosomale In-vitro-Radiosensitivität von Lymphozyten von Bestrahlungspatienten und AT-Homozygoten

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Abstract

Background

We investigated the in-vitro radiosensitivity of peripheral blood lymphocytes with a special FISH/CISS-technique.

Patients and Methods

From October 1993 through April 1996, a total number of 52 cancer patients was enrolled in the study. The tumor sites in these patients were: breast (n=41), lung (n=4), head and neck (n=3) as well as prostate, bladder, rectal cancer and Hodgkin’s disease (each n=1). Twenty-six of them were examined prior to planned radiotherapy (prospective group) and 26 after radiotherapy (retrospective group). Three additional individuals (without cancer or radiotherapy) with proven ataxia telangiectasia (Louis-Bar syndrome, AT-homozygotes) were also investigated and their blood samples served as positive control for radiosensitivity. The clinical radiation response of normal tissue in radiotherapy patients was scored according to the WHO grading system for acute and according to the RTOG grading system for late effects. For to estimate the intrinsic radiosensitivity, blood samples were taken and irradiated in vitro with 0 (control) or 0.7 or 2 Gy with a 6 MV-linear accelerator, standard 48-hour lymphocyte cultures were prepared, chromosomes #1, #2 and #4 were simultaneously labeled with a FISH/CISS-technique and 200 to 1 000 metaphase spreads were scored for chromosomal aberrations. The radiation sensitivity of lymphocytes was expressed as the number of radiation-induced chromosomal breaks per mitosis after 0.7 Gy or 2 Gy corrected for the 0-Gy control value.

Results

The frequency of chromosomal breaks/mitosis in the unirradiated control lymphocytes was 0.020±0.015 in prospective patients who had not yet received radiotherapy. It was significantly higher in retrospective patients (0.264±0.164 breaks/mitosis) as a result of the previous radiation exposure. The 3 At-homozygotes showed also an increased number of spontaneous chromosomal breaks (0.084±0.016 breaks/mitosis), probably resulting from the chromosomal instability in this disease. This figure, however, was significantly lower than in retrospective patients. — The number of radiation-induced breaks after in-vitro irradiation was comparable in lymphocytes of patients who showed no normal tissue reaction (n=11) as compared to those with mild to moderate radiation reaction (n=32, acute reactions Grade 1 to 2, late reactions Grade 0 to 2). In 9 patients with unexpected severe plus late Grade 3 to 4 reactions, however, a significantly higher number of radiation-induced chromosomal breaks was measured; the highest number was observed in a patient with a radiation myelitis. The 3 AT-homozygotes showed, as expected, an extreme radiosensitivity of their lymphocytes. The number of breaks/mitosis after 0.7 Gy in vitro irradiation of lymphocytes was 0.103±0.059 in patients with no normal tissue radiation reaction (n=11), 0.122±0.146 in the group with mild to moderate radiation reactions Grade 1 to 2 (n=32), 0.359+0.226 in patients with unexpected Grade 3 to 4 normal tissue reactions (n=9) and 0.550±0.243 in the 3 AT-homozygotes (p < 0.01, t-test). The difference in lymphocyte radiosensitivity between these 4 groups was also detected after in-vitro irradiation with 2 Gy (0.484±0.132 vs. 0.535±0.228 vs 0.926±0.349 vs. 1.423±0.072).

Conclusion s

We found a significantly higher number of chromosomal breaks in lymphocytes of patients with severe or extreme radiation reaction of normal tissues as compared to patients with no or only mild to moderate radiation reactions. The radiosensitivity of lymphocytes in these radiosensitive patients was in the range between normal radiosensitivity and the radiosensitivity of AT-homozygotes. Detection of patients with severely enhanced intrinsic radiosensitivity might be possible with this method.

Zusammenfassung

Hintergrund

Es wurden Lymphozyten von Radiotherapiepatienten in vitro bestrahlt und überprüft, ob ein Zusammenhang zwischen der gemessenen In-vitro-Strahlenempfindlichkeit und der klinischen Strahlenreaktion des Normalgewebes besteht.

Patienten und Methodik

Von Oktober 1993 bis April 1996 haben wir 62 Radiotherapiepatienten auf Strahlenempfindlichkeit untersucht. Zehn Patienten wurden nicht bestrahlt bzw. erschienen nicht zu Nachuntersuchungen, so daß 52 ausgewertet werden konnten. Die Tumorentitäten waren Mammakarzinom (n=41), Bronchialkarzinome (n=4), Kopf-Hals-Tumoren (n=3) sowie jeweils ein Prostata-, Blasen- und Rektumkarzinom und ein Patient mit Morbus Hodgkin. 26 Patienten wurden vor der Radiotherapie (prospektiv) und 26 nach der Radiotherapie (retrospektiv) untersucht. Ferner standen uns Blutproben von drei AT-Homozygoten zur Analyse zur Verfügung; sie dienten als positive Kontrolle für erhöhte Strahlenempfindlichkeit. — Zur Bestimmung der In-vitro-Strahlenempfindlichkeit wurden Blutproben aus peripherem Venenblut entnommen und an einem 6-MV-Linearbeschleuniger mit 0,7 Gy und 2 Gy bestrahlt (eine Blutprobe wurde nicht bestrahlt und diente als Kontrolle). Danach wurden klassische 48-Stunden-Lymphozytenkulturen angesetzt, die Chromosomen 1, 2 und 4 mittels einer FISH-Technik simultan gefärbt und die Bruchrate dieser Chromosomen in 200 bis 1 000 Mitosen unter einem Fluoreszenzmikroskop ausgezählt. Als Maß für die Strahlenempfindlichkeit galt die Zahl der Brüche pro Mitose nach In-vitro-Bestrahlung, korrigert um die Bruchrate der nicht bestrahlten Kontrolle.

Ergebnisse

Die Frequenz von Chromosomenbrüchen (in Bruchereignissen pro Mitose) betrug in nichtbestrahlten Lymphozyten der 0-Gy-Kontrolle 0,020±0,015 bei prospektiven Patienten, die noch nicht bestrahlt waren. Retrospektive Patienten nach Strahlentherapie hatten signifikant höhere Bruchraten in der 0-Gy-Kontrolle (0,264±0,164) als Folge der vorausgegangenen therapeutischen Strahlenexposition. AT-Homozygote zeigten ebenfalls als Ausdruck der für diese Erkrankung charakteristischen genomischen Instabilität eine erhöhte Basisbruchrate von 0,084±0,016. Die Häufigkeit von Chromosomenbrüchen nach In-vitro-Bestrahlung war bei Patienten mit unerwartet schweren Nebenwirkungen am Normalgewebe höher als bei Patienten, die keine oder nur geringe Normalgewebsreaktionen gezeigt hatten. Die Zahl von Chromosomenbrüchen pro Mitose, nach 0,7 Gy In-vitro-Bestrahlung betrug 0,103±0,059 bei elf Patienten ohne Strahlenreaktion am Normalgewebe, 0,122±0,146 bei 32 Patienten mit moderater Normalgewebsreaktion (nicht signifikant gegenüber der erstgenannten Gruppe), 0,359±0,226 bei neun Patienten mit extremer/unerwarteter Normalgewebsreaktion (p=0,009 bzw. 0,014 gegenüber Patienten ohne bzw. mit moderater Strahlenreaktion) und 0,550±0,243 bei AT-Homozygoten (p < 0,01 gegenüber den anderen Gruppen). Vergleichbare Unterschiede bestanden nach In-vitro-Bestrahlung mit 2 Gy.

Schlußfolgerung en

Ausgewählte Patienten mit unerwartet starker klinischer Strahlenreaktion am Normalgewebe hatten in dieser Untersuchung signifikant höhere chromosomale Bruchraten ihrer Lymphozyten nach In-vitro-Bestrahlung mit 0,7 bzw. 2 Gy. Ihre Strahlenempfindlichkeit lag zwischen derjenigen einer Normalpopulation und derjenigen von AT-Homozygoten. Die verwendete Methode könnte sich deshalb als prädiktiver Test zur Detektion strahlenempfindlicher Individuen eignen.

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References

  1. Arlett CF, Harcourt SA. Survey of radiosensitivity in a variety of human cell strains. Cancer Res 1980;40:926–32.

    CAS  PubMed  Google Scholar 

  2. Bauchinger M, Schmid E, Zitzelsberger H, et al. Radiation-induced chromosome aberrations analysed by two-colour fluorescence in situ hybridization with composite whole chromosome-specific DNA-probes and a pancentrometric DNA probe. Int J Radiat Biol 1993;64:179–84.

    Article  CAS  PubMed  Google Scholar 

  3. Begg AC, Russell NS, Knaken H, et al. Lack of correlation of human fibroblast radiosensitivity in vitro with early skin reaction in patients undergoing radiotherapy. Int J Radiat Biol 1993;64:393–405.

    Article  CAS  PubMed  Google Scholar 

  4. Bentzen SM.; Overgaard J. Clinical correlations between late normal tissue endpoints after radiotherapy: implication s for predictive assays of radiosensitivity. Eur J Cancer 1993;29A:1373–6.

    Article  CAS  PubMed  Google Scholar 

  5. Brock WA, Tucker SL, Geara FB, et al. Fibroblast radiosensitivity versus acute and late normal skin responses in patients treated for breast cancer. Int J Radiat Oncol Biol Phys 1995;32:1371–9.

    Article  CAS  PubMed  Google Scholar 

  6. Brown JM, Evans J, Kovacs MS. The prediction of human tumor radiosensitivity in situ: an approach using chromosome aberrations detected by fluorescence in situ hybridization. Int J Radiat Oncol Biol Phys 1992;24:279–86.

    Article  CAS  PubMed  Google Scholar 

  7. Budach W, Hartfort A, Gioioso D, et al. Tumors arising in SCID-mice share enhanced radiation sensitivity of SCID normal tissues. Cancer Res 1991;52:6292–6.

    Google Scholar 

  8. Budach W. Genetische Prädisposition und Strahelnempfindlichkeit von Tumoren. Strahlenther Onkol 1997;173:469–79.

    Article  CAS  PubMed  Google Scholar 

  9. Burnet NG, Nyman J, Turesson I, et al. The relationship between cellular radiation sensitivity and tissue response may provide the basis for individualising radiotherapy schedules. Radiother Oncol 1994;33:228–38.

    Article  CAS  PubMed  Google Scholar 

  10. Cole J, Arlett CF, Green MHL, et al. Comparative cellular radiosensitivity II: The survival following gamma-irradiation T-lymphocytes, T-lymphocyte cell lines, lymphoblastoid cell lines and fibroblasts from normal donors, from ataxia telangiectasia patients, and ataxia telangiectasia heterozygotes. Int J Radiat Biol 1988;54:929–43.

    Article  CAS  PubMed  Google Scholar 

  11. Cunliffe PN, Mann JR, Cameron AH, et al. Radiosensitivity in ataxia teleangiectasia. Br J Radiol 1975;48:374–6.

    Article  Google Scholar 

  12. Dahl, O, Horn A, Mella O. Do acute side effects during radiotherapy predict tumor response in rectal carcinoma? Acta Oncol 1994;33:409–13.

    Article  CAS  PubMed  Google Scholar 

  13. Dunst J, Gebhart E, Neubauer S. Can extremely enhanced radiosensitivity in radiotherapy patients be detected by in-vitro testing of lymphocytes? Strahlenther Onkol 1995;171:581–6.

    CAS  PubMed  Google Scholar 

  14. Geara FB, Peters LJ, Ang KK, et al. Prospective comparision of in vitro normal cell radiosensitivity and normal tissue reactions in radiotherapy patients. Int J Radiat Oncol Biol Phys 1993;27:1173–9.

    Article  CAS  PubMed  Google Scholar 

  15. Gebhart, E, Neubauer S, Schmitt G, et al. Use of a three-colour chromosome-in-situ-suppression technique for the detection of past radiation exposure. Radiat Res 1996;145:47–52.

    Article  CAS  PubMed  Google Scholar 

  16. Gray JW, Pinkel D, Brown JM. Fluorescence in situ hybridization in cancer and radiation biology. Radiat Res 1994;137:275–89.

    Article  CAS  PubMed  Google Scholar 

  17. Hart RN, Kikler BF, Evans RG, et al. Radiotherapeutic management of medulloblastoma in a pediatric patient with ataxia telangiectasia. Int J Radiat Oncol Biol Phys 1987;13:1237–40.

    Article  CAS  PubMed  Google Scholar 

  18. Humphreys MW, Nevin NC, Woolridge MAW. Cytogenetic investigations in a family with ataxia telangiectasia. Hum Genet 1989;83:79–82.

    Article  CAS  PubMed  Google Scholar 

  19. Johansen J, Bentzen SM, Overgaard J, et al. Evidence for a positive correlation between in vitro radiosensitivity of normal human skin fibroblasts and the occurrence of subcutaneous fibrosis after radiotherapy. Int J Radiat Biol 1994;66:407–12.

    Article  CAS  PubMed  Google Scholar 

  20. Johansen J, Bentzen SM, Overgaard J, Overgaard M. Relationship between the in vitro radiosensitivity of skin fibroblasts and the expression of subcutaneous fibrosis, telangiectasia, and skin erythema after radiotherapy. Radiother Oncol 1996;40:101–9.

    Article  CAS  PubMed  Google Scholar 

  21. Kuhnt T, Richter C, Enke E, et al. Acute radiation reaction and local control in breast cancer patients receiving postmastectomy radiotherapy. Strahlenther Onkol 1998;174:257–61.

    Article  CAS  PubMed  Google Scholar 

  22. Little JB, Nove J, Strong LC, et al. Survival of human diploid skin fibroblasts from normal individuals after X-irradiation. Int J Radiat Biol 1988;54:899–910.

    Article  CAS  PubMed  Google Scholar 

  23. Loeffler JS, Harris JR, Dahlberg MS, et al. In vitro radiosensitivity of human diploid fibroblasts derived from women with unusually sensitive clinical responses to definitive radiation therapy for breast cancer. Radiat Res 1990;121:227–31.

    Article  CAS  PubMed  Google Scholar 

  24. Lucas J, Awa A, Straume T, et al. Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation. Int J Radiat Biol 1992;62:53–63.

    Article  CAS  PubMed  Google Scholar 

  25. Morell D, Cromartie E, Swift M. Mortality and cancer incidence in 263 patients with ataxia teleangiectatica. J Natl Cancer Inst 1986;77:89–92.

    Google Scholar 

  26. Natarajan AT, Vyas RC, Darroudi F, et al. Frequency of X-ray induced chromosome translocations in human peripheral lymphocytes as detected by in situ hybridization using chromosome-specific DNA-libraries. Int J Radiat Biol 1992;61:199–203.

    Article  CAS  PubMed  Google Scholar 

  27. Neubauer S, Gebhart E, Schmitt G, et al. Is chromosome in situ suppression (CISS) hybridization suited as a predictive test for intrinsic radiosensitivity in cancer patients? Int J Oncol 1996;8:707–12.

    CAS  PubMed  Google Scholar 

  28. Norman A, Iwamoto KS, Kagan AR, et al. Radiation sensitive breast cancer patients. Radiother Oncol 1992;23:196–7.

    Article  CAS  PubMed  Google Scholar 

  29. Parshad R, Sanford KK, Jones, GM, et al. G2 chromosomal radiosensitivity of ataxia telangiectasia heterozygotes. Cancer Genet Cytogenet 1985;14:163–8.

    Article  CAS  PubMed  Google Scholar 

  30. Peters LJ. Inherent radiosensitivity of tumor and normal tissue cells as a predictor of human tumor response. Radiother Oncol 1990;17:177–90.

    Article  CAS  PubMed  Google Scholar 

  31. Peters LJ. Radiation therapy tolerance limits. For one or for all? Cancer 1996;77:2379–85.

    Article  CAS  PubMed  Google Scholar 

  32. Richter C, Kuhnt T, Becker A, et al. Acute normal tissue reaction and local control in breast cancer patients with postmastectomy radiotherapy. Int J Radiat Oncol Biol Phys 1996;36:Suppl 1:285.

    Article  Google Scholar 

  33. Schmid E, Zitzelsberger H, Braselmann H, et al. Radiation induced chromosome aberrations analyzed by fluorescence in situ hybridization with a triple combination of composite whole chromosome-specific DNA probes. Int J Radiat Biol 1992;62:673–8.

    Article  CAS  PubMed  Google Scholar 

  34. Streffer C. Genetische Prädisposition und Srahlenempfindlichkeit bei normalen Geweben. Strahlenther Onkol 1997;173:462–8.

    Article  CAS  PubMed  Google Scholar 

  35. Swift M, Reitnauer PJ, Morrel D, et al. Breast and other cancers in families with ataxia teleangiectatica. N Engl J Med 1987;316:1289–94.

    Article  CAS  PubMed  Google Scholar 

  36. Tucker SL, Turesson I, Thames HD. Evidence for individual differences in the radiosensitivity of human skin. Eur J Cancer 1992;A:1783–91.

    Article  Google Scholar 

  37. Tucker SL, Geara FB, Peters LJ, et al. How much could the radotherapy dose be altered for individual patients based on a predeictive assay of normal-tissue radiosensitivity? Radiother Oncol 1996;38:103–13.

    Article  CAS  PubMed  Google Scholar 

  38. Turesson I. Individual variation ad dose dependency in the progression rate of skin telangiectasia. Int J Radiat Oncol Biol Phys 1990;19:1569–74.

    Article  CAS  PubMed  Google Scholar 

  39. Virsik-Peuckert P, Rave-Fränk M, Schmidberger H. Further studies on the possible relationship between radiation-induced reciprocal translocations and intrinsic radiosensitivity of human tumor cells. Radiother Oncol 1996;40:111–19.

    Article  CAS  PubMed  Google Scholar 

  40. Virsik-Peuckert P, Rave-Fränk M, Schmidberger H, et al. Prädiktive Bedeutung von strahleninduzierten Translokationen gemessen an primären Tumorzellen. Strahlenther Onkol 1996;172:34.

    Google Scholar 

  41. Weichselbaum RR, Epstein J, Little JB. In vitro radiosensitivity of human diploid fibroblasts derived from patients with unusual clinical responses to radiation. Radiology 1976;121:479–82.

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Jürgen Dunst.

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Dunst, J., Neubauer, S., Becker, A. et al. Chromosomal in-vitro radiosensitivity of lymphocytes in radiotherapy patients and AT-homozygotes. Strahlenther Onkol 174, 510–516 (1998). https://doi.org/10.1007/BF03038983

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  • DOI: https://doi.org/10.1007/BF03038983

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