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

  • Jürgen Dunst
  • Susanne Neubauer
  • Axel Becker
  • Erich Gebhart
Originalarbeit

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.

Key Words

Radiotherapy Radiosensitivity Normal tissue reaction FISH Ataxia telangiectasia 

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

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.

Schlüsselwörter

Radiotherapie Radiosensibilität Normalgewebsreaktion FISH Ataxia telangiectasia 

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Copyright information

© Urban & Vogel 1998

Authors and Affiliations

  • Jürgen Dunst
    • 1
    • 2
  • Susanne Neubauer
    • 1
  • Axel Becker
    • 2
  • Erich Gebhart
    • 3
  1. 1.Department of RadiooncoloyUniversity of ErlangenErlangenGermany
  2. 2.Department of RadiooncologyMartin-Luther-University HalleHalleGermany
  3. 3.Department of Human GeneticsUniversity of ErlangenGermany

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