(CANCER RESEARCH 52. 6348-6352. November 15. 1992]

Intrinsic Radiosensitivity of Normal Human Fibroblasts and Lymphocytes afterHigh- and Low-Dose-Rate Irradiation1

Fady B. Geara,2 Lester J. Peters, K. Kian Ang, Jennifer L. Wike, Susan S. Sivon, Roland Guttenberger,

David L. Callender, Edmond P. Malaise, and William A. Brock

Departments of Experimental Radiotherapy [F. B. G., J. L. W., S. S. S., W. A. B.]. Clinical Radiotherapy [F. B. G., L. J. P., K. K. A.], Head and Neck Surgery[D. L. C.], and Biomathematics ¡R.G.], The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030, and Laboratoire de RadiobiologieCellulaire, UnitéINSERM 247, Institut Gustave Roussy, 94805 Villejuif, France ¡E.P. M.]

ABSTRACT

The existence of heritable radiosensitivity syndromes and clinicalobservations in radiotherapy patients suggests that human cellular radiosensitivity differs among individuals. We report here an in vitrostudy of radiosensitivity in 30 fÃ-broblast and 29 lymphocyte culturesobtained from cancer patients and controls. In 25 cases, both fibroblastsand lymphocytes were obtained from the same donors. Fibroblasts werecultured from skin biopsy samples, and peripheral T-cell lymphocytes

were cultured from blood. Clonogenic survival assays were performed byusing high- and low-dose-rate irradiation; lymphocytes were in ( >,,phase

and fibroblasts in confluent plateau phase. Various end points werecalculated and compared (i.e., surviving fraction at 2 Gy, initial slope ofthe survival curve, and doses resulting in 10 and 1% survival, respectively). Depending on the end point, the coefficient of variation of thesurvival parameters ranged from 31 to 68% for lymphocytes and 21 to41% for fibroblasts following high-dose-rate irradiation. Similar ranges»ere obtained after low-dose-rate irradiation. Variance analysis per

formed on replicate assays in cultures derived from the same patientshowed that variation due to technical or sampling errors was significantly lower than variation between individuals (/' = 0.00034 and 0.014

for fibroblasts and lymphocytes, respectively). No correlation was observed between the radiosensitivity of lymphocyte and fibroblast cultures derived from the same donors. We conclude that there is significant variation in normal cell radiosensitivity among individuals. On theother hand, comparisons of lymphocyte and fibroblast radiosensitivitiessuggest that tissue-specific characteristics, such as differentiation sta

tus, may variably modulate radiosensitivity.

INTRODUCTION

As several previous reports have argued, the existence of avariety of heritable syndromes expressing a radiosensitive phe-notype is evidence for a genetic influence upon radiosensitivity(1-4). The most striking example is AT,3 an autosomal reces

sive disorder that causes immunological dysfunction, pronenessto cancer, and an unusual susceptibility to radiation injury(5-10). This increased sensitivity is found in all cells and tissues, both in vivo (5, 9) and in vitro (6-8).

Clinical observations in radiotherapy patients with no knowngenetic syndromes have revealed large individual differences intissue reactions after treatment. This has been quantified byTuresson (11), who studied normal tissue reactions in breast

Received 6/18/92; accepted 9/3/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This investigation was supported by research grants from the National CancerInstitute (CA-50192, CA-06294, and CA-16672), the Association pour la Recherche sur le Cancer, the University Cancer Foundation, and the Katharine M. Un-sworth Charitable Annuity Lead Trust.

2 To whom requests for reprints should be addressed, at Department of Radiotherapy, Box 97, The University of Texas M. D. Anderson Cancer Center, 1515Holcombe Boulevard, Houston, TX 77030.

1The abbreviations used are: AT, ataxia telangiectasia: LDR. low-dose rate;HDR, high-dose rate; SF2, survival at 2 Gy; «,initial slope of the survival curve;DIO, DI, dose resulting in 10 and 1% survival, respectively; DO,dose resulting in 1log cell kill; CV, coefficient of variation.

cancer patients following a given physical dose of radiationadministered over a given period of time to the same tissues.Long-term follow-up of these patients revealed wide differencesin individual normal tissue responses, with some patients experiencing minimal skin reactions and others experiencing severe fibrosis and telangiectasia. These findings further supportthe existence of a significant genetic component in the determination of cellular and tissue radiosensitivity.

The ability to detect differences in radiosensitivity could havean important impact upon clinical radiotherapy, since dose limitations are based on acute and late toxicity, and the severity ofearly and late effects cannot be predicted ahead of time. Latetoxicity remains a major concern in radiotherapy planning,where dose limits have been set based on the estimated probability of the risk of injury for a given physical absorbed dosewithout regard to individual variability of response. Therefore,identifying patients who have a greater or lesser than averagerisk for complications could allow for an adjustment of radiation dose. Such individual adjustments could, theoretically, prevent complications in "sensitive" patients and increase theprobability of cure in "resistant" patients (4, 12).

Although the variability in radiosensitivity among normalindividuals is not clearly documented, several studies have compared the in vitro radiosensitivity of cultured cells derived fromnormal individuals (3, 13-20; summarized in Table 1). Most

reports conclude that heterogeneity between individuals doesexist, but the degree of variability reported is generally small. Ithas been suggested that the use of LDR irradiation in measuring cellular radiosensitivity may provide better discrimination(13, 15, 21-23). The rationale is that, during LDR irradiation,damage and repair take place at the same time, thus amplifyingthe influence of different repair rates between cultures. Anotheradvantage of LDR irradiation is that it results in simple exponential survival curves that can be mathematically fit by using alinear model, resulting in less error related to curve fitting thanwith nonlinear data.

If individual heterogeneity in radiosensitivity exists for one ormore cell types (e.g., fibroblasts or lymphocytes), and if thesedifferences are due to genetic differences, then one may expectthat individuals from whom, for example, sensitive fibroblastcultures are derived, would also demonstrate sensitivity in cultures from other normal cell types. This is indeed the case forindividuals with AT, who show extraordinary radiosensitivity invirtually all cell types tested. However, two studies in apparently normal individuals found no correlation between the radiosensitivity of cycling fibroblasts and resting peripheral T-cell

lymphocytes (17, 18). This may be partly accounted for by thefact that, in both studies, radiosensitivity was measured in different phases of the cell cycle for each cell type. Also, one ofthese studies (18), showed no significant differences in D,0values between individuals for either fibroblasts or lymphocytes, thus precluding any tests for correlation.

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l.\ IITKO NORMA! CKLL RADIOSKNSITIVITY

Table 1 Variation among individuals in normal cell radiosensitivity, reportedin the literature, for fibroblasts (F) and lymphocytes (L) in terms of coefficient

of variation of different radiosensitivity parametersItalic numbers are calculated from the published data or graphs.

CVi

Authors (Ref.) Cell type n" SF2 010

Cox and Masson(16)Deschavanneet al.(14)Little

et al.(1)Littleétal.(19)Kushiro

et al. (18,27)Weeks

étal.(13)Banétal.(15)FFFFFLFFF»34702473222262151541202415.51811.65.92010914191114

" Number of cell strains studied.* Fibroblast cultures derived from breast cancer patients.

In this study we investigated the degree of radiosensitivityvariation in normal peripheral T-cell lymphocyte and skin fi-broblast cultures derived from individuals with no known radiosensitivity syndromes. This report focuses on variation between individuals, the technical aspects of lymphocyte andfibroblast survival measurement, comparison of HDR andLDR irradiation, and comparison of the radiosensitivity of lymphocytes and fibroblasts from the same individual.

MATERIALS AND METHODS

Patients. For peripheral T-cell lymphocyte cultures, a total of 33blood samples were obtained from 23 patients with head and neckcancers, 2 patients with breast cancer, and 8 normal healthy volunteers.For fibroblast cultures, 31 skin samples were obtained: 25 by punchbiopsy from the inside of the forearm (23 head and neck cancer, and 2breast cancer patients), and 6 were skin samples from mastectomyspecimens (Table 2). Repeat measurements were performed on fourfibroblast cell strains (one derived from a patient and three from controls) after HDR irradiation, and on two cell strains after LDR irradiation (both derived from controls). For lymphocytes, repeat measurements were performed on six blood samples derived from the samedonor (Table 3).

Fibroblast Cultures and Radiosensitivity Measurements. After obtaining informed consent, 6-mm punch biopsies of the skin were takenunder local anesthesia. All chemical products were purchased fromSigma Chemical Co. (St. Louis, MO) unless otherwise noted. Subcutaneous fat was removed from the sample, and the remaining tissue wasminced with scissors into 1- to 2-mm pieces, which were disaggregatedfor 30 min by magnetically stirring in an enzyme cocktail containing1% collagenase type III (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 1% trypsin (GIBCO, Grand Island, NY) in calcium/magnesium-free Hanks' balanced salt solution. Cells were spun down,

resuspended and counted, then inoculated into minimum essentialmedium, supplemented with 15% fetal bovine serum, antibiotics(100 Mg/ml penicillin, 50 Mg/ml streptomycin), and 5 mg/ml ampho-tericin B. All cultures were fed twice weekly with the same medium, butwithout amphotericin B. After 2-3 weeks of incubation, cells weretrypsinized (0.25% trypsin, 0.04% EDTA) and subcultured.

Radiosensitivity was measured by clonogenic assay. Contact-inhibited confluent fibroblast cultures (third passage) were irradiated asmonolayers at different doses with the use of both HDR and LDRirradiations. Cultures were trypsinized as described above, counted, andthen inoculated into 100-mm culture dishes, using five inoculum sizelevels for each dose point.

Lymphocyte Culture and Radiosensitivity Measurements. Peripheral T-cell lymphocytes along with other mononuclear cells were separated from blood samples by centrifugation on a Ficoll/Hypaque density gradient (Becton Dickinson and Co., Lincoln Park, NJ). Themononuclear cell layer was then removed, rinsed twice with phosphatebuffered saline, and the lymphocytes were counted by using a hemacy-

tometer. The lymphoblastoid cell line BL 3590 was used as a source offeeder cells; it was maintained in RPMI media (JRH Biosciences, Len-exa, KS) supplemented with 15% fetal calf serum, 200 Mg/mlglutamine,100 Mg/ml penicillin, and 50 mg/ml streptomycin.

The limiting dilution assay for primary lymphocytes was used tomeasure clonogenic survival of lymphocytes. It was a modification ofthe method published by Trainor and Morley (24) and others (8, 17, 18,20, 25). After cell counts had been adjusted for the expected platingefficiencies and surviving fractions, cells were irradiated and then inoculated into 96-well, round-bottomed plates. The media used was RPMI(GIBCO, Grand Island, NY) supplemented with 15% fetal calf serum,0.5% phytohemagglutinin (Wellcome Diagnostic, United Kingdom),10 lU/ml recombinant human interleukin 2 (Boehringer Mannheim),100 Mg/ml penicillin, and 50 Mg/ml gentamicin. One thousand lethallyirradiated feeder cells (50 Gy) were added to each well to give a finalvolume of 100 Mi-

After incubation for 15 days, 28 n\ of a 0.5-mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dissolved inphosphate-buffered saline was added to each well. The 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which is converted toan insoluble dark blue formazan salt in metabolically active cells, wasused to visualize lymphocyte colonies.

Irradiations. HDR irradiations were performed at room temperature by using a li7Cs source with a dose rate of 455 cGy/min. Fibro

blasts were irradiated as confluent monolayers and lymphocytes wereirradiated in suspension. LDR irradiations, for both lymphocytes andfibroblasts, were carried out at 37°Cin a 5% CO2, humidified chamber,using a 137Cssource with a dose rate of 2.85 cGy/min.

Data Analysis. Fibroblast survival cunes generated with HDR irradiations were fitted by using a linear-quadratic model (S = e~aD~"D2).

The following parameters were calculated: «,SF2, O10, and D¡.Radiosensitivity parameters for lymphocytes were obtained by a si

multaneous fit of the data from all 12 dilutions. Our analysis usedmaximum-likelihood principles that were adapted from a publishedmethod of direct analysis (26). The parameters «,SF2, Dw, and D¡weredetermined by the same methods as used for fibroblasts.

Analyses of variance were performed for all data from different individuals and from repeated measurements from the same individuals.The variances for repeated measurements were pooled and considered

Table 2 Sources of skin and blood samples used to generate ßhroblastandlymphocyte cultures and measure radiosensitivity

LymphocytesRadiotherapy

patients beforetreatmentRadiotherapypatients with severereactionsSkin

specimens frommastectomyNormalvolunteersTotalSuccessful

primaryculturesCompletedassaysSuccess

rate214833332988%Fibroblasts214631303097%

Table 3 Variance analysis for fibrohlast and lymphocyte SF2 after high- andlow-dose-rate irradiation for all cell strains and repeated measurements

All P values showed the difference in variance to be highly significant."

CelltypeFibroblastFibroblast''FibroblastFibroblast''DoserateHDRHDRLDRLDRn30272724m22»2215'15Varianceratio7.35.49.74.9P0.0000410.000340.0000580.0024OriginalCV(%)31.1625.7031.0722.34AdjustedCV(%)28.9523.2129.4218.14

Lymphocyte HDR 29 6 8.1 0.014 32.70 30.61" The adjusted CV is derived from the original CV of all cell strains after

adjusting for the variance due to technical error in repeated measurements, n.number of cell strains analyzed: m, number of repeated measurements.

* Twenty-two repeat measurements performed in four cell strains.' Analysis performed excluding three cell strains that exhibited in vitro senes

cence.rfFifteen repeat measurements performed in two cell strains.

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IN VITRO NORMAL CELL RADIOSENSITIVITY

to represent the reproducibility or experimental variation. The ratio ofthe variance of all individuals to the pooled variance would be 1 if allvariations between individuals were due only to experimental variation.An F statistic was used to refute the null hypothesis of equal variance.The adjusted CV was derived from the variance that resulted aftersubtracting the pooled variance from that obtained in all individuals.

RESULTS

Data Collection. From the 31 skin specimens collected, 30primary fibroblast cultures were generated. Clonogenic survivalassays were performed for all 30 cultures, using HDR and for27 cultures using LDR irradiation.

Of the 33 lymphocyte cultures set up, 29 generated évaluablelimiting dilution assay survival data after HDR irradiation; theother 4 were not analyzable because of low growth.

Fibroblast Radiosensitivity. Different sources of experimental variability with regard to the culture and assay of fibroblastswere investigated. While standardizing the fibroblast survivalassay, we found that the normally linear relationship betweenthe number of cells inoculated into a culture and the number ofresulting colonies changed whenever the number of colonies ina culture exceeded 50-80, above which fewer than the expectednumber of colonies appear. Fig. 1 illustrates this point from oneexperiment. This problem was overcome by plating cells atseveral different densities, as shown in Fig. 1, and using onlydata showing a linear relationship between cell number andcolony formation. There was no systematic relationship between control plating efficiencies and radiosensitivity.

To estimate assay error, the variance in SF2 values frommultiple assays performed in four cell strains was determined.The assay variation was found to be significantly lower thanthat obtained in all individuals (P = 0.000041). Table 3 showsthat the CV for SF2 for all individuals was 31.16%. After adjusting for technical variation, the CV for differences amongindividuals was found to be 28.95%. The same analysis was alsoperformed by using LDR irradiation in two fibroblast strains.The SF2 variance resulting from repeat assays was also significantly lower than that obtained in all individuals (P =0.000058). The adjusted CV was 29.42%, whereas the originalCV was 31.07% (Table 3). Therefore, for both HDR and LDRirradiations, differences among individuals are greater than canbe accounted for by technical variation.

3000 6000 9000

Number of cells inoculated

Fig. 1. Comparison of the number of cells inoculated into a 100-mm culturedish with the number of fibroblast colonies formed. Each dose point is representedby five different cell inoculum sizes. As illustrated, the number of colonies decreased from that expected when the number of cells increased, particularly withlarger inoculum sizes. Only data that were linear with the origin were selected forsurvival calculations.

flI os-

CV= 41%

0.5-,

CV= 21% CV= 22%

Fig. 2. Fibroblast radiosensitivity parameters (a, SF2, DIO, and D,). The darkest columns represent three strains that exhibited cell senescence. Diagonallystriped columns represent measurements performed in fibroblast cultures derivedfrom surgical skin specimens. Note that the SF2 distribution panel contains threemore diagonally striped columns, which represent cultures in which we measuredSF2 only.

Fig. 2 shows the survival results for all fibroblast strains, aswell as the distributions for the low-dose (a, SF2) and high-doseparameters (DÃŒO,DI) in rank order. The CVs were 41, 31, 21,and 22% for a, SF2, D10, and Z),, respectively (Table 4), thusconfirming that the parameters associated with the low-doseregion of the survival curve are the most useful for identifyingindividual differences in radiosensitivity.

In our study, the use of LDR irradiation did not increasethe observed radiosensitivity differences among individuals(Table 4).

Lymphocyte Radiosensitivity. Potential sources of experimental variability were also examined for lymphocyte assays.The variance due to technical and sampling errors in measuringSF2 was found to be significantly lower than that observed in allindividuals (P = 0.014). The adjusted CV was 30.61%, whereasthe nonadjusted CV was 32.70% (Table 3). There was no systematic relationship between control plating efficiencies andradiosensitivity.

a, SF2, DIO, and D¡all showed notable variation amongindividuals (Fig. 3). As with the fibroblast assays, comparisonbetween LDR and HDR assays in our study showed no advantage for LDR in amplifying the differences among individuals(Table 4).

Fibroblast versus Lymphocyte Radiosensitivity. As mentioned above, previously published reports (17, 18) have shownno correlation between the radiosensitivity of resting lymphocytes and cycling fibroblasts derived from the same individuals.In the present study, we compared the radiosensitivity of bothlymphocytes and fibroblasts, in the G0 phase of the cell cycle.Of the 25 lymphocyte and fibroblast cultures, 16 were usable forthis comparison. Comparison of the two cell types was made byusing both SF2 and a. Fig. 4 shows a random association between the radiosensitivity of the two cell types (r = 0.37,P = 0.152, and r = 0.30, P = 0.258 for SF2 and a, respectively).

DISCUSSION

The main purpose of this study was to investigate the degreeto which normal cell radiosensitivity varies among individuals.The existence of normal cell radiosensitivity variation from

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Table 4 Mean, SD, and CV for four radiosensitivity parameters(a, SF2, BIO, and D¡,respectively)

LymphocytesParameter"aSF2Dio0iHDR

LDRHDR

LDRHDR

LDRHDR

LDRMean

±SD0.373

±0.2410.388 ±0.2240.374

±0.1230.401±0.1113.965

±1.0164.684 ±1.3076.786±

1.9448.780 ±3.131CV

(%)68

58332731283527FibroblastsMean

±SD0.573

±0.2260.507 +0.1360.291

±0.1020.350 ±0.1133.551

±0.9124.707 ±1.1286.125±

1.4299.392 ±2.428CV

(%)412731

3121

2422

23" All parameters were calculated by using a linear-quadratic fit for both lympho

cytes and fibroblasts after HDR and LDR irradiations.

individual to individual may indicate that genetic diversity playsa role in determining cellular radiosensitivity. In addition, itwas our goal to compare the radiosensitivity of two normal celltypes (e.g., fibroblasts and lymphocytes) in the same individual.

The most important result of this study is the statistical evidence for interindividual variation in lymphocyte and fibro-blast radiosensitivity. The actual differences among individualswere found to be significantly greater than variation due totechnical or sampling errors. This is reflected by the adjustedcoefficients of variation which were calculated after analyzingthe variance of the whole group and the variance of the repeatedmeasurements in several cell strains (Table 3). These observations are in general agreement with those of most of the published reports in which CVs ranged from 14 to 41% for differentradiosensitivity end points (D0, D10, SF2) (Table 1). On theother hand, Kushiro et al. (18) reported that normal fibroblastand lymphocyte radiosensitivity showed no significant interindividual variation in D10, D50, or D90. However, the CV of theSF2 values listed in one of their publications (27) is 20%, whichis comparable to what was found by others. Another significantdifference between our study and others is that the majority ofour study population was composed of cancer patients, a groupthat may not represent the general population in this regard.

A surprising observation in this study was that LDR irradiation did not amplify the differences in the low-dose parametersof the survival curves, as reported by others (13, 21). A possibleexplanation may be that our LDR (2.85 cGy/min) is higherthan what was used by others. Therefore, it is likely that complete repair was not occurring during our LDR treatments. Thiswas confirmed by allowing 24 h for the recovery of any remaining potentially lethal damage after LDR irradiations, or bytreating cells with dose rates of less than 1 cGy/min. In thesecases, ßwas near zero and the «value was lower.4- 5

A lack of correlation between the radiosensitivity of G0-phaseresting lymphocytes and plateau-phase fibroblasts from thesame individuals underscores the importance of tissue-specificeffects on the expression of radiosensitivity. Even though wemeasured the radiosensitivity of cells in the same phase of thecell cycle, our results are similar to those obtained by others, inwhich resting lymphocytes were compared with cycling fibroblasts (17, 18). This could be explained by the assumption thata large number of genes influence radiosensitivity (28) and thatsome of them exhibit differential expression in different celltypes. Some of these genes may have an essential role in the celland may therefore be expressed in all cell types. In this case, any

4 F. B. Geara el al., unpublished observations.5 E. P. Malaise et al., unpublished observations.

dysfunction in these genes would result in increased radiosensitivity in virtually all cell types and tissues such as that observed in individuals with homozygous AT genes. The role thatdifferentiation plays in determining radiosensitivity, however,will not be clearly understood until a number of such genes havebeen identified and their expression studied in different celltypes.

Cell-specific differences in radiosensitivity may also be attributed to the difference in ratios of lymphocyte subtypes in individuals. Blood contains a heterogeneous cell population, especially with regard to the proportions of lymphocyte subtypes.The notion of subset influence on radiosensitivity is supportedby a recent report by Uckun et al. (29), showing that CD3surface antigen expression is associated with cellular radiore-sistance in some types of malignant T-lineage lymphoblasticcells. Yet another possible reason could be the potentially variable expression of programmed cell death by different subsetsof T-lymphocytes. However, Nakamura et al. (30) reported thatlymphocytes expressing the surface antigens CD4 or CDSshowed no difference in their radiosensitivities. Their studygroup, however, was composed of only normal individuals; ingroups like ours, made up of cancer patients, large shifts inlymphocyte-subtype ratios are possible.

Another potential source of variability in fibroblast measurement is the role of cell senescence in radiosensitivity. All cultures that became senescent exhibited increased radiosensitivity. The development of in vitro senescence is highly variable.

CV= 68% 0.5-

ÕÕra

B

3-

O

CV= 35%

Fig. 3. Lymphocyte radiosensitivity parameter (

/A VITRO NORMAL CELL RADIOSENSITIVITY

unpredictable, and independent of plating efficiency, and variesfrom one cell strain to another (31). Morphologically senescentcells become larger with increasing filopodia density. Theirgrowth rate decreases and plateau phase is reached at lower celldensities, with open spaces between cells. Because of this association between in vitro senescence and increased radiosensitiv-ity, we performed an analysis excluding data derived from thethree fibroblast cell strains that exhibited in vitro senescence(Table 3). Little et al. reported no relationship between fibroblast culture passage number and radiosensitivity (19), and ourresults agree with their findings only until morphologically senescent cells begin to appear.

In conclusion, it appears from this study that the radiosensitivity of normal cells varies among individuals and that thesedifferences are greater than can be accounted for by samplingand methodology errors. These differences are likely due tovariation in one or more genes that affect radiosensitivity. However, differences among individuals in the sensitivity of one celltype do not predict what will be found in other cell types. Weassume that this means that differential gene expression resultsin cell and tissue types with different arrays of active genes thatinfluence radiosensitivity.

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1992;52:6348-6352. Cancer Res Fady B. Geara, Lester J. Peters, K. Kian Ang, et al. Lymphocytes after High- and Low-Dose-Rate IrradiationIntrinsic Radiosensitivity of Normal Human Fibroblasts and

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