Increased chromosomal radiosensitivity in patients undergoing radioimmunoglobulin therapy

Mutation Research, 227 (1989) 39-45 Elsevier

39

MUTLET 0247

Increased chromosomal radiosensitivity in patients undergoing radioimmunoglobulin therapy S h u q i n X i a o a, D a v i d J a c o b s o n - K r a m a, Steven P i a n t a d o s i b a n d J e r r y R. W i l l i a m s a "Radiobiology Laboratory and bOncology Biostatistics, Johns Hopkins University Oncology Center, Baltimore, MD 21205 (U.S.A.) (Accepted 1 May 1989)

Keywords." Radioimmunoglobulin therapy; Chromosomal radiosensitivity; Immunoglobulin therapy, lymphocytes

Summary Lymphocytes from individual patients undergoing radiolabeled immonoglobulin therapy have been examined both for chromosome aberrations expressed immediately upon explant, or for chromosome aberrations induced by a subsequent challenge of -/-rays after PHA-stimulated proliferation. Despite interpatient variation, there is strong correlation between levels of chromosome aberrations observed in the initial mitosis after mitogenic stimulation and levels induced by a challenge dose of radiation in replicate cultures after several cell cycles of growth. These data indicated that even after proliferation, human lymphocytes retain a memory of in vivo exposure to ionizing radiation that can be observed by challenge with a clastogenic agent. This persistent hypersensitivity occurs at high frequency, suggesting that it may be related to initial steps in multistage carcinogenesis.

Persistent memory of exposure to carcinogens in somatic cells is clearly established through the observed latency for cancer in human populations. This memory may be retained by changes in the primary structure of the DNA, e.g., mutation, as has been shown for certain oncogenes. The mechanism of cellular memory to exposure from nongenotoxic carcinogens is less clearly understood, but this memory of exposure may be manifested as mutational hypersensitivity to subseCorrespondence: Dr. Jerry R. Williams, Johns Hopkins University Oncology Center, 600 North Wolfe Street, Room 2-121, Baltimore, MD 21205 (U.S.A.).

quent exogenous challenge or endogenous processes. Cellular exposure to ionizing radiation, both in vitro and in vivo, has been reported to alter cytogenetic responses to subsequent in vitro radiation challenges (Wiencke et al., 1986; Shadley et al., 1987). The nature of the responses to the second dose appears dependent on the magnitude and timing of the initial radiation exposure. Exposure of human peripheral blood lymphocytes to very low doses of X-rays (0.01 and 0.05 Gy) or radiation from incorporated tritiated thymidine has been reported to induce an adaptive response. This response is characterized by a 25% reduction in the

0165-7992/89/$ 03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

40 number of chromatid and isochromatid breaks seen in cells given 1.5 Gy during mid to late Sphase. The adaptive response can be induced in lymphocytes as early as 4 h after mitogenic stimulation and once induced, appears to persist for up to 66 h after the initial radiation exposure. Higher doses of radiation, e.g. 0.5 Gy are not effective for inducing the response. In contrast to the protective effects of low radiation doses, accidental irradiation of human beings and experimental irradiation of monkeys with relatively high doses (2-10 Gy, total dose) result in an induced sensitivity to the clastogenic effects of a challenge dose of radiation delivered in vitro (Guedeney et al. 1986). This effect appeared to persist for at least one year post exposure. In order to better characterize the long term effects of repeated exposures to radiation, we are studying a group of patients undergoing radioimmunoglobulin therapy for treatment of primary hepatoma. These patients are infused with immunoglobulins conjugated with suitable radioisotopes, in this case 131-iodine. Injected isotope levels vary from 30 to 50 mCi per therapy cycle, and patients receive multiple cycles, depending on tumor response. The circulating radiolabeled immunoglobulins produce a relatively homogeneous irradiation of blood elements. Patients have received up to 11 cycles of therapy, treatment that produces total radiation doses over 50 Gy to the liver, and probably over 10 Gy to bone marrow, which is over 2 x the lethal dose if given as a single acute exposure. These individuals represent a unique opportunity, even if less than experimentally ideal, to study hematopoietic cells that have received, or whose progenitors have received, higher radiation doses than ever before possible. The studies presented here represent initial data on this important patient population. Materials and methods

Patients Primary hepatoma patients who are candidates for radioimmunoglobulin therapy (RIT) are generally given induction therapy consisting of 21 Gy of

external beam radiation to the tumor and are then cyclically injected at approx. 8-week intervals with 30 mCi of 13~I-labeled antiferritin. Most patients also receive low dose chemotherapy consisting of 15 mg adriamycin and 500 mg 5-fluorouracil (Order et al., 1985). Although the exact whole body radiation dose experienced by a particular patient during each cycle of RIT depends on factors such as the extent of targeting of the antibody to the tumor as well as normal tissue exposure, body mass of the patient, and the half-life of the antibody in vivo, whole body doses are on the order of 0.3 Gy per cycle (Leichner et al., 1983). Cell cultures Peripheral blood lymphocytes were cultured using 0.5 ml of whole blood in a total volume of 4.5 ml. Culture medium consisted of RPMI 1640 supplemented with 10070 fetal bovine serum (Hazleton), l°70 L-glutamine (Gibco), l°70 penicillinstreptomycin (Gibco) and 0.4°7o phytohemagglutinin (Gibco). For studies of the background frequencies of chromosomal aberrations, cultures additionally contained 10 /xg/ml of 5-bromo2-deoxyuridine (BrdUrd) and were incubated for a total of 48 h. Parallel cultures from the same individuals to be challenged with radiation in vitro were incubated for 72 h in the absence of BrdUrd. In each case a parallel 72-h control culture was also analyzed for background aberration levels since aberration frequencies are known to decline between 48 and 72 h of culture (Bender and Brewen, 1969). Cultures to be challenged with radiation received 0.25 Gy of 7-rays from a Gammacel140 irradiator and were incubated for l h prior to addition of colcemid (Gibco). All cultures were treated with a final concentration of 0.2/~g/ml of colcemid for 2 h. Cell cultures were pelleted by centrifugation at 1000 rpm for l0 min, resuspended in 0.075 M KC1 and fixed in 3 changes of methanol:acetic acid (3:1). Cells cultured in the presence of BrdUrd were stained by the fluorescence plus Giemsa technique (Goto et al., 1978) while cultures grown in the absence of BrdUrd were stained with Giemsa alone. All slides were scored with an Olympus standard microscope at a final magnification of

41

1250×. The frequency of aberrations was determined in 100 cells per point and were quantified both as percent aberrant cells and as aberrations per cell.

Statistical analysis The major statistical endpoint of this investigation was the estimation of a quantitative relationship between chromosome-type aberrations induced in vivo by RIT and chromatid-type aberrations induced by a challenge dose of radiation delivered in vitro during G2 prophase. Two methods for quantifying these effects were used: bivariate Pearson correlation, and simple linear regression. All computations were done using the statistical analysis system (SAS) (SAS Institute, 1985). For linear regressions, the dependent variable was G2-induced chromatid aberrations. Regression coefficients were estimated using least squares, and individual data points were studied for their influence on parameter estimates using standard influence diagnostics (Draper and Smith, 1981). All p values reported are two-sided.

Results Ideally, it would be desirable to examine the effects o f cumulative in vivo radiation dose on in vitro cellular susceptibility to mutagenic challenges. As discussed above, it is not possible to convert quantities of injected isotopes to whole body doses directly due to variations in tumor and normal tissue targeting, and variations in the in vivo half-life of the radiolabeled antibody. Because symmetrical chromosome-type aberrations (e.g. dicentrics, rings) increase as a function of wholebody radiation dose (Evans et al., 1979), the frequencies of these events are often used as a reflection o f cumulative in vivo exposure. Chromosometype aberrations seen in the first metaphase after mitogenic stimulation result from damage induced in the mitotically arrested Go lymphocytes and presumably reflect DNA damage which occurred in vivo. Frequencies of chromatid-type aberrations after cellular exposure in G2 prophase were used as an indicator of in vitro radiation sensitivity.

Because BrdUrd greatly sensitizes cells to the clastogenic effects of radiation, it could not be included in the culture medium of ceils which were challenged with radiation in vitro. The lack of BrdUrd in the medium precluded direct identification of the number of cell cycles traversed by individual cells analyzed for chromatid aberrations. However, previous studies indicate that the majority of cells are in the second, third and fourth replication cycle cells after 72 h of culture (Schneider et al., 1978). Parallel control cultures indicated that the vast majority of ceils had traversed more than a single cell cycle after 72 h and that only relatively low levels of background chromatid aberrations were present. The results in Fig. I indicate a positive correlation between frequencies of chromosome aberrations quantified in first replication cycle cells and frequencies of chromatid aberrations induced by .,/-radiation in cells cultured for 72 h. The Pearson correlation between G0-induced chromosome aberrations and G2-induced chromatid aberrations was 0.445 which was statistically significant from 0 (p = 0.002). The slope of the estimated linear regression was 0.657 and the estimated intercept was significantly different from 0 (p -- 0.0001). Using influence diagnostics, no individual observations were observed to be particularly influential on the estimate of this slope. The clastogenic responses to in vitro G2 radiation challenge were measured in a group of normal control individuals as a comparison for the RIT patient group (Table 1). The chromatid-aberration frequency seen in cells after a 0.25 Gy challenge varied from 0.12 to 0.19 aberrations per cell. While some patient responses also fell into or below this range, even after one or more cycles of RIT, most showed higher levels of sensitivity. The positive correlation between levels of chromosome damage sustained in vivo and in vitro susceptibility to a challenge dose o f radiation may have at least two possible explanations. First, the patient population examined may display a spectrum of intrinsic susceptibilities to the clastogenic effects of ionizing radiation. This spectrum of sensitivity would be evident as range of aberration

42 0.6

0.5

0.4

G2-induced chromatid o.a breaks

Y

C)

0.2

~~"i0.1

0

I) () )
~L)

c) (~

()

(_)

i

__L

0.2

0.4

±

i

0.6

0.8

1

1.2

Go-induced chromosome aberrations Fig. 1. C h r o m o s o m a l radiosensitivity of cells from RIT patients displayed as a function of levels of chromosome damage sustained in vivo. Symmetrical chromosome-type aberrations were scored in first-division metaphases. Parallel cultures from the same individuals to be challenged with radiation in vitro were incubated for 72 h.

levels induced both in vivo and in vitro. Thus, an intrinsically radiosensitive patient might be expected to demonstrate relatively high aberration levels in response to radiation delivered as part of therapy as well as in response to a challenge dose in vitro. A second explanation might be that patients become increasingly radiosensitive with increasing cycles of RIT. Data derived from two patients at different times during RIT suggest that lymphocytes become increasingly radiosensitive with increased numbers of RIT cycles (Table 2).

Discussion

The present study indicates that a group of patients undergoing RIT for primary hepatoma display increasing sensitivity to the clastogenic effects of "r-rays delivered in vitro. This increased sensitivity to chromosome damage seen in RIT patients may have several explanations. First, the major side-effect which limits the use of RIT is hematopoietic toxicity. Destruction and repopulation of large numbers of cells in the hematopoietic

TABLE 1 B A S E L I N E A N D R A D I A T I O N - I N D U C E D C H R O M O S O M E A B E R R A T I O N S IN N O R M A L C O N T R O L S Volunteer No,

Baseline frequency of chromosome-type aberrations a

Radiation-induced chromatid aberrations b

Baseline frequency of chromatid aberrations

Net induced aberrations

1

0.01 0.00 0.00 0.00 0.01 0.01 0.01 0.01

0.12 0.17 0.19 0.11 0.20 0.15 0.16 0.19

0.00 0.02 0.02 0.00 0.01 0.01 0,00 0.00

0.12 0.15 0.17 0.11 0.19 0.14 0.16 0.19

2 3 4 5 6 7 8

a Dicentrics and rings per cell. b Excluding gaps.

43 TABLE 2 CHROMOSOMAL RADIOSENSITIVITY IN RIT PATIENTS Patient

Date sampled Cumulative quantity

of 1 3 1 1

Net induced chromatid aberrations per cell a

J.T. J.T.

9/08/87 1/13/88

ll0mCi 188mCi

0.02 0.22

R.B. R.B. R.B.

9/14/87 1/19/88 8/08/88

30mCi 90mCi 180mCi

0.11 0.37 0.26

a Excluding gaps. Fig. 1. Chromosomal radiosensitivity of cells from RIT patients displayed as a function of levels chromosome damage sustained in vivo. Symmetrical chromosome-type aberrations were scored in first-division metaphases. Parallel cultures from the same individuals to be challenged with radiation in vitro were incubated for 72 h.

system might result in generation of new populations o f peripheral lymphocytes with different sensitivities to mutagenic challenges. Although this explanation cannot be ruled out, the observation by Guedeney et al. (1986) that sensitivity to in vitro challenge can persist for many years after in vivo radiation exposure argues against transient shifts in cellular populations. The pattern of induced hypersensitivity seen in RIT patients is similar to other manifestations of radiation-induced sensitivities seen in other cellular systems, both in vitro and in vivo. We have previously reported that bone marrow cells of mice acutely exposed to 1-3 Gy of "y-radiation show increased sensitivity to induced chromatid aberrations after exposure to a challenge dose of radiation in G2 prophase (Jacobson-Kram and Williams, 1985). More recently we have found that pretreatment doses as low as 0.125 Gy are also capable of inducing this radiosensitivity (JacobsonKram and Williams, 1988). Analysis of the distributions of cells with aberrations suggests that the induced sensitivity is a characteristic of the entire population and not a small subset of outliers. We have observed a similar manifestation o f radiationinduced sensitivity in cultured V79 cells (Frank and

Williams, 1982). Cells exposed to 9 Gy of X-ray, delivered acutely or by protracted exposures, and subcultured for up to 108 days (270 mean population doublings) were highly susceptible to PUVA (8-methoxypsoralen followed by exposure to ultraviolet light, 265 nm)-induced mutation. Mutations were induced at doses of PUVA which did not elicit mutations above background in unirradiated cells. Subsequent studies have shown that cultured cells pretreated with UVC (254 nm) are also rendered hypersensitive to mutations induced by P U V A at the H G P R T locus (D'Arpa et al., 1989). However, hypersensitivity was not seen when the challenging agent was UVC instead of PUVA or when the locus assayed was the sodium-potassium ATPase locus. Although the mechanism(s) of this hypersensitivity is unknown, mutation at any specific locus seems remote as a causal mechanism, since specific-locus mutations are rare. A more likely explanation might entail heritable alterations in chromatin structure or responses to toxic exposures that induce heritable changes in gene expression. For example, abnormal DNA methylation patterns have been found both in polyps of the colon and in frank malignancies (Goelz et al., 1985). Mutagen-induced hypersensitivity has been reported for inducing agents other than X-ray and for endpoints other than clastogenesis and gene mutations. Ikebuchi et al. (1988) have recently reported that V79 cells exposed to 80 fractions of mid-UV light were more sensitive to UV-induced mutations at the H G P R T locus than control cells which were not preirradiated. V79 cells similarly exposed to the same number of far-UV fractions showed a sensitivity to mutation equal to control unirradiated cells. The authors hypothesized that increased sensitivity to mutation was the result of a compromised DNA repair capacity or induction of an error-prone repair pathway. Another expression of induced heritable hypersensitivity has been reported for the induction of sister-chromatid exchanges in cells pretreated with UVC and challenged with mitomycin C (Kim et al., 1985). Cells preirradiated with 5 J / m s showed an approx. 2-fold increased level of SCE in response to mitomycin C challenges. This sensitivity was seen even when the

44 p r e t r e a t m e n t a n d the c h a ll e n g e were s e p a r a t e d by 25 cellular g e n e r a t i o n s . O u r results suggest a possible m e c h a n i s m f o r a causal r e l a t i o n s h i p b e t w e e n the i n d u c t i o n o f hypersensitivity an d

the

process

of

multistage

car-

cinogenesis. H y p e r s e n s i t i v e cells w o u l d be at higher risk f o r i n c u r r i n g s u b s e q u e n t m u t a t i o n s a n d m i g h t t h e r e f o r e be m o r e a d a p t a b l e to a c h a n g i n g e n v i r o n m e n t . I n c r e a s e d a d a p t a b i l i t y c o u l d give such cells a g r o w t h a d v a n t a g e o v e r n o r m a l cells. This h y p o t h e sis is s u p p o r t e d by the o b s e r v a t i o n s that m a n y t u m o r s are k a r y o t y p i c a l l y a b n o r m a l a n d carry b o t h random

an d

tumor-specific chromosomal

rear-

r a n g e m e n t s (Yunis, 1985). A d d i t i o n a l l y , evidence is increasing t h a t t u m o r cell lines a n d cell lines derived

from

markedly

cancer-prone

increased

i n d iv id u a ls

susceptibility to

induced chromatid aberrations

show

a

radiation-

(Parshad

et al.,

1983; P a r s h a d et al., 1984, 1985). T h e s e o b s e r v a t i o n s t a k e n t o g e t h e r with o u r o w n f r o m the present i n v e s t i g a t i o n m a y help to e x p l a i n high rates o f treatment

associated

leukemias

associated

with

radiation therapy.

Acknowledgements This

work

CA43791

an d

was

supported

CA39543.

The

by

NIH

expert

grants

technical

assistance o f Y a d i n W a n g is g r a t e f u l l y a c k n o w l edged.

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Goelz, S.E., B. Vogelstein, S.R. Hamilton and A.P. Feinberg (1985) Hypomethylation of DNA from benign and malignant human colon neoplasms, Science, 228, 187-190. Goto, K., S. Maeda, Y. Kano and T. Sugiyama (1978) Factors involved in differential Giemsa-staining of sister chromatids, Chromosoma, 66, 351-356. Guedeney, G., M. Harou-Kouka, M.T. Doloy and R. Masse (1986) Modification of individual chromosomal radiosensitivity after total-body irradiation in man and monkey, Br. J. Cancer, 53(7), 167-168. Ikebuchi, M., M. Osmak, A. Han and C.K. Hill (1988) Multiple, small exposures of far-ultraviolet or mid-ultraviolet light change the sensitivity of acute ultraviolet exposures measured by cell lethality and mutagenesis in V79 Chinese hamster cells, Radiation Res., 114, 248-267. Jacobson-Kram, D., and J.R. Williams (1985) In vivo exposure to X-irradiation induces a hypersensitivity to subsequent induction of G1 aberrations by X-rays in mouse bone marrow, Fourth International Conference on Environmental Mutagens, p. 169. Jacobson-Kram, D., and J.R. Williams (1988) Failure to observe adaptive response to ionizing radiation in mouse bone marrow cells in vivo, Environ. Mol. Mutagen., 11, 49-50. Kim, J.P., P. D'Arpa, D. Jacobson-Kram and J.R. Williams (1985) Ultraviolet-light exposure induces a heritable sensitivity to the induction of SCE by mitomycin C, Mutation Res., 149, 437-442. Leichner, P.K., J.L. Klein, S.S. Siegelman, D.S. Ettinger and S.E. Order (1983) Dosimetry of ~3q-labeled antiferritin in hepatoma: specific activities in the tumor and liver, Cancer Treatment Reports, Vol. 67, No. 7-8, pp. 647-658. Order, S.E., G.B. Stillwagon, J.L. Klein, P.K. Leichner, S. Siegelman, E.K. Fishman, D.S. Ettinger, T. Haulk, K. Kopher and S.A. Leibel (1985a) 1-131 antiferritin: a new treatment modality in hepatoma, A ROTG study, J. Clinical Oncol., 3, 1573-1582. Order, S.E., J.L. Klein, P.K. Leichner, D.S. Ettinger, K. Kopher, K. Finney, M. Surdyke and S.A. Leibel (1985b) Radiolabeled antibody in the treatment of primary and metastatic liver malignancies, in: Recent Results in Cancer Research, Therapeutic strategies in primary and metastatic liver cancer, Springer, Heidelberg, Vol. 100, pp. 307-314. Parshad, R., K.K. Sanford and G.M. Jones (1983) Chromatid damage after G2 phase X-irradiation of cells from cancerprone individuals implicates deficiency in DNA repair, Proc. Natl. Acad. Sci. (U.S.A.), 80, 5612-5616. Parshad, R., R. Gantt, K.K. Sanford and G.M. Jones (1984) Chromosomal radiosensitivity of human tumor cells during the G: cell cycle period, Cancer Res., 44, 5577-5578. Parshad, R., K.K. Sanford and G.M. Jones (1985) Chromosomal radiosensitivity during the G2 cell cycle period of skin fibroblasts from individuals with familial cancer, Proc. Natl. Acad. Sci. (U.S.A.), 82, 5400-5403.

45 SAS Institute Inc. (1985) SAS User's Guide: Statistics, version 5th edn., Cary, NC. Schneider, E.L., R.R. Tice and D. Kram (1978) Bromodeoxyuridine differential chromatid staining technique: A new approach to examining sister chromatid exchange and cell replication kinetics, in: D.M. Prescott (Ed.), Methods in Cell Biology, Academic Press, New York, Vol. 10, pp. 379-409. Shadley, J.O., V. Afzal and S. Wolff (1987) Characterization of the adaptive response to ionizing radiation induced by low dose of X-rays to human lymphocytes, Radiation Res., 111, 511-517.

Wiencke, J.K., V. Zfzal, G. Olivvieri and S. Wolff (1986) Evidence that [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism, Mutagenesis, 1, 375-380. Yunis, J.J. (1985) The chromosomal basis of human neoplasia, Science, 221,227-236. Communicated by F.H. Sobels