The role of dna repair processes in the response of human tumors to fractionated radiotherapy

In1 J Rad~armn Oncolo~ RIOI Phvs Vol Pnmcd in the U.S.A. All rights mrvcd

10, pp

I 127-1 134

036%3016/84

103.00 + .oO Press Ltd.

copyright & 1984 Pcrgamon

0 ASTR Special Feature THE ROLE OF DNA REPAIR PROCESSES IN THE RESPONSE OF HUMAN TUMORS TO FRACTIONATED RADIOTHERAPY RALPH

R. WEICHSELBAUM,

M.D.

Joint Center for Radiation Therapy, Department of Radiation Therapy, Harvard Medical School, and Department of Cancer Biology, Harvard School of Pubiic Health The relationship of inherent radiosensitivity, sublethal and potentially lethal damage repair to radiation therapy is not yet fully explrrined. I will examine how these varlous repair processes might be relevant to radiotherapy, based on laboratory investigation of human tumor cells in vitro. Although radiocurability is a complex function, recovery processes manifested in the post-radiation period may be a determinant of radiocurability. DNA Repair, Radiobiology, Radiotherapy.

The importance of radiotherapy in modem cancer treatment has focused attention on the determinants of radiocurability. Clinical analysis shows that small tumors are generally more radiocurable than large tumors. Histologic subtype may also be an important determinant of radiocurability. For example, properly treated breast carcinoma or seminoma is more highly radiocurable than glioblastoma, osteosarcoma, or chondrosarcoma. However, the complex relationship between these clinical observations and the underlying biological data is not yet well understood. Inherent cellular factors may determine tumor cell survival after radiation Quantitatively, these factors are expressed in terms of survival curve parameters; Do (radiosensitivity), the inverse of the slope of the exponential portion of the survival curve, and ii or extrapolation number, which represents the ability of a cell to accumulate and repair sublethal X ray damage.‘,” Major DNA lesions produced by X rays are double and single stranded DNA breaks, as well as base damage. A wide variety of enzymes is known to be involved in the repair of these lesions and thus it has been suggested that mammalian radiation survival curves represent molecular repair processes.’ However, the specific mechanisms of repair of radiation-induced DNA lesions have not yet been precisely defined, possibly because of the biochemical complexity of studying DNA repair in mammalian cells.‘~** Two human autosomal recessive diseases have enabled investigators to correlate the response to physical agents in vivo with the response of cells in vim. Xeroderma

pigmentosum (XP) is a disease characterized by unusual sensitivity to ultraviolet light in vivo as well as a high incidence of skin cancer. *’When fibroblasts from patients with this disease are examined in vitro, they are unusually sensitive to the lethal effects of ultraviolet light; in some forms of this disease, cells from patients are deficient in the excision repair process. 6*27The second disease, ataxia telangiectasia (AT), is characterized by occulocutaneous telangiectasia, severe immune deficiency, cerebellar ataxia, and high incidence of leukemias and lymphomas. Patients with AT treated by radiotherapy for this malignancy exhibit severe normal tissue responses.‘3q20 AT fibroblasts are extremely sensitive to ionizing radiation when studied in vitro, although the precise mechanism(s) of radiationinduced lethality in AT cells is unknownm These human diseases provide models which ‘aid in the study of DNA repair and lend validity to the fact that the study of human tissue in vitro may reflect processes that occur in vivo. Human tumors show a wide range of response in clinical radiotherapy; however, the radiosensitivity of cells derived from human tumors tested under aerobic conditions lie within a fairly narrow range.33,35*”Exceptions have been reported however. Gerweck et al. and Nilsson et al. have reported several radioresistant glioblastoma lines.‘2*23Weichselbaum et al. reported an inherently radioresistant melanoma line and Richie et al. reported two radioresistant human tumor bladder cancer lines.26*47Our laboratory has investigated approximately 35 clonogenic human tumor lines in vitro and approximately 5 have Do% greater than 190 rad and 7 DO’s greater than 170

Reprint requests to: Ralph R. Weichselbaum, M.D., Joint Center for Radiation Therapy, 50 Binney St., Boston, MA 02 115.

Accepted for publication

11627

2 1 March 1984.

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Radiation Oncology 0 Biology 0 Physics

Table 1. Calculated cumulative survival fraction Survival fraction

x=

x=

lo-” IO-‘0 IO+ 10-8 IO-’ 10-6

0.45 0.49 0.52 0.56 0.60 0.65

0.28 0.32 0.35 0.40 0.45 0.50

x3?

X20

Note: Calculated cumulative survival fraction for either 32 or 20 equal fractions when the fractional survival is varied. Adapted from Hellman.16

rad. Except for one radiosensitive leukemia cell line (Do = 76 rad), most tumor Do’s lie between 100 and 160 rad.34.44 The enhanced survival that occurs when a radiation dose is split with an interval of several hours between fractions has been interpreted to be a result of the repair of sublethal damage induced by the first dose in cells that survive this dose.” The capacity of cells to accumulate and repair sublethal damage (SLDR) is reflected by the width of the shoulder region of the survival curve. In fractionated radiation schemes, the shoulder region may be recapitulated many times. Thus, the surviving fraction after a single dose may be magnified to an exponent equal to the number of fractions and therefore the shoulder region has a profound influence on the final surviving fraction at the end of treatment (Table 1).9.‘o.‘6Thus the ability of cells in a tumor to accumulate and repair sublethal injury has been postulated to be a major cellular determinant of radiocurability.9,‘0*‘6 Large extrapolation numbers have been reported in some human tumor cell lines, especially melanoma. For example, Barranco et al. reported several melanoma lines with large shoulders.’ Selby and Courtenay2* investigated two xenografied human melanomas and also reported

July 1984. Volume 10. Number 7

large extrapolation numbers. Camey et ~1.~studied two large cell lung carcinoma lines with relatively large extrapolation numbers although much smaller than those reported by Barranco et al. Experiments performed in our laboratory to examine sublethal damage repair in exponentially growing cultures of various human tumor cell lines demonstrated no significant differences in their capacity for split-dose recovery and confirmed that the amount of SLDR may be predicted from the extrapolation number.46,47 Extrapolation numbers of cells studied in our laboratory are relatively small compared to those found for human melanoma, although they are consistent with findings of other investigators for many melanoma as well as nonmelanoma cell lines.23.29.46.47 It should be noted that with the exception of the report of Camey et al., most survival curves performed on human cells which show a large shoulder were performed on human melanomas. ‘,3.7,28 Since most cells in human tumors cycle slowly or are noncycling, the investigation of DNA repair processes in these cells may be important for cancer therapy. Little and Hahn suggested the use of density inhibited plateau phase cultures of mammalian cells to study the repair of potentially lethal damage since the constant feeding of cells in a crowded density inhibited state is a physiological condition that may resemble one that exists among some populations of tumor cells in vim. 15.” When density inhibited plateau phase cultures are treated with X rays and subculture of the cells at low density is delayed, an enhancement in survival occurs. This has been referred to as reflecting recovery from potentially lethal X ray damage and is analogous to liquid-holding recovery in bacteria and yeast. “,14PLDR has been described in animal ascities and solid tumors as well as established human tumor cell lines.g,‘8.25.47 To establish a molecular basis of PLDR in human cells we studied PLDR in fibroblasts from patients with xeroderma pigmentosum and ataxia telangiectasia.42 Fi-

Table 2. Radiosensitivity and repair of potentially lethal damage in human diploid fibroblast strains

Cell strain and source Li 106 (primary culture) CRL 1343 (ATCC) XPl2BE (ATCC CRL 1223) XP8BE (IMR-GM 671) XP4BE (ATCC CRL I 162)

Clinical classification Normal skin fibroblasts Skin fibroblasts from ataxia telangiectasia Skin fibroblasts

from xeroderma pigmentosum (XP) (group A)

XP skin libroblasts (group C) XP skin fibroblasts (variant)

Enhancement after X ray

Survival l&I (ultraviolet light)

7

4.13 + 0.03

31 t6

4.33 + 0.34

3

1.78 + 0.13

29 2 3

3.10 r 0.55

160 ? 17

4.63 t 1.04

Cloning efficiency

0, (X ray)

1.O-6.3%

1492

0.7 f 3.5%

462

7.9-23.0s

Survival enhancement after ultraviolet light

6tl

0.96 it 0.15

8.3-16.9%

-

-

821

0.91 ? 0.21

0.7-1.2s

-

-

20 t_ 2

2.30 2 0.40

Role of DNA

1129

in response of tumors to radiotherapy 0 R. R. WEICHSELBAUM

broblasts from XP complementation groups A & C, were defective in UVPLD: however, fibroblasts from patients with the xeroderma variant were proficient in UVPLD. This suggests that UVPLDR reflects activity in the excision repair pathway since fibroblasts from patients in complementation groups A & C are known to be defective in excision repair whereas fibroblast from the XP variant are excision repair proficient. Fibroblasts from patients with ataxia telangiectasia are almost completely defective in X ray PLDR (Table 2). Thus, we concluded that X

al

5ot

ray PLDR most likely reflected the activity of a molecular repair process although we could not directly prove this

because of the complexity of X ray repair of mammalian cells.42 We studied the repair of X ray induced potentially lethal damage in 9 established human tumor lines derived from tumors of classically different radiocurability. Cells lines derived from 3 tumors considered non-radiocurable ( 1 osteosarcoma and 2 melanomas) repaired more PLD than cell lines derived from 3 tumors considered radiocurable (2 breast carcinoma lines and 1 neuroblastoma line) (Fig. 1). The remaining lines (glioblastoma. melanoma, and hypemephroma) were intermediate in their ability for PLDR. We suggested that the repair of X ray induced PLD might be one important cellular determinant of clinical radiocurability in some tumors since the Do and ii of all the cell lines were similar with the exception of a radioresistant human melanoma line C-32.47 Other laboratories have simultaneously investigated PLDR in human tumor cells.5.8 For example, Couxtenay et ~1.’ were the first to demonstrate PLDR in a xenografied human pancreatic carcinoma. We extended our investigations to measure potentially lethal damage repair after fractionated radiation in plateau phase cultures of a human melanoma cell line and a human breast carcinoma cell line, to study whether PLDR occurs at doses employed in clinical radiotherapy.38 Although the radiation survival curve parameters in exponential growth of these cell lines is similar, the repair of potentially lethal X ray damage after fractionated radiation in plateau phase cultures conferred significant radioresistance on the human melanoma cells but not on human breast cancer cells. Therefore, we concluded that in a multifractionated regimen, potentially lethal damage repair might confer radioresistance on some human tumor cells and occurs at doses employed in clinical radiotherapy (Figs. 2a and 2b). The repair of potentially lethal damage may have implications for fractionation. For example, in clinical radiotherapy large-dose fractions have been employed in an attempt to control unusually radioincurable tumors, especially melanoma. I4 Results of experiments designed to study PLDR in a human melanoma line in vitro following single radiation doses from 2- 1200 rad, are shown in Figure 3. These data indicate that significant PLDR occursfollowing large fraction sizes. Thus, although more radiation damage is seen initially (0 hour subculture),

TIME OF POTENTIALLY LE?-HAL DAMAGE REFJIR fhl 50

I-

b

I

I

I

I

I

J

8 24 0 2 4 6 TIME OF R77iWTfALL Y LETHAL DAMAGE REPA/R fN

Fig. 1a. PLDR following X-irradiation in 3 human tumour lines 0; melanoma cell line C-32. (Dose 7 Gy; PE 4.6-6.92). A: melanoma cell line C-143. (Dose was 7 Gy; PE 4.8-29.41). 0: osteosarcoma cell line TX-4. (Dose 7 Gy; PE 3.1-9.8%). Fig. 1b. PLDR following X-irradiation in 3 human tumour lines MCF-7 (0) breast carcinoma. (Dose 5 Gy.) PE 2.0-20.4%. MDAMB 231 (A)breast carcinoma. (Dose 5 Gy.) PE 51.0-85.48,

LAN-l (0) neuroblastoma. (Dose 5 Gy.) PE 2.4-36.8%. much of this damage may be repaired (24 hour subculture).4’ These data indicate that the use of large fractions may not be the optimal method to permanently control radioincurable tumors, since such tumors may contain many noncycling cells proficient in PLDR, which is possibly analogous to those observed in plateau phase cultures.37*4’Our data on human tumors in cell culture thus far suggest that PLDR is dose modifying.39q40Therefore, large fractions may lower the therapeutic ratio especially if the total overall dose is decreased because the amount of tumor cell kill may be underestimated and also because damage to normal tissues is generally increased by large fraction sizes.37

1130

Radiation Oncology 0 Biology 0 Physics

C- 143-

July 1984. Volume IO, Number 7

MELANOMA

1

I

1

1

0

2

4

6

w

HOURS BETWEEN TOTAL DOSE AND EXPLANT

Fig. 2a. Effects of fractionated X rays on plateau cultures of human melanoma line C- 143. Dashed line in the figure represents control experiments of a single dose (7 Gy) performed at the same time as fractionated experiments. Adapted from Weichselbaum et al.3n

Few clinical investigations demonstrate an advantage for large fraction irradiation; some demonstrate an actual disadvantage when permanent local control and not regression or palliation is the end point assessed. For example, Byhardt et al.* examined local control in 132 patients with squamous carcinoma of the oral cavity and oropharynx, treated by either 3 or 5 fractions per week.2 For patients treated three times a week, larger fraction sizes were employed and the NSD’s for both groups were approximately equal. All patients were distributed equally in each fractionation scheme by stage. Local control of the primary was significantly better in patients treated 5 times per week. Eichom’ studied various fractionation schemes in patients with operable and inoperable carcinpma of the lung. Patients with operable lung cancer were treated preoperatively with treatment schedules which included 250 rad given 22 times, a second group was treated with 400 rad given 4 times and 600 rad delivered 6 times, and a third group treated with 1000 rad given twice and 600 rad delivered 6 times. Patients with inoperable lung tumors were treated with analogous fractionation schemes (although not exactly the same). In patients with operable lung cancer, tumor destruction was assessed by gross and histopathologic examination after thoracotomy. In pa-

tients with inoperable lung cancer, autopsy results were employed to examine local control. Survival was not considered a direct measure of local control, and multiple biopsies were taken from all regions which appeared still viable macroscopically. The percentage of patients with operable lung cancer showing complete tumor destruction was significantly increased in the group treated with smaller multiple fractions. In patients with inoperable lung cancer, the groups that received large fraction radiotherapy had approximately half the number of negative tumor specimens compared to groups that treated by multifractionation, in spite of the fact that the NSD’s were higher in all cases in patients receiving larger fractionation schemes (asses& by autopsy). Eichom’ suggests that kinetics of the individual tumors may have contributed to the effects of fractionation and that the larger fraction sizes may not have been effective in patients who had a large component of nonproliferating tumor cells. Thus large fractions may induce more repairable potentially lethal damage and may have accounted for decreased radiocurability in these clinical examples. In an attempt to assess more directly the clinical contribution of X ray repair parameters to local control of cancer, we studied 10 early passage tumor cell lines derived from patients with head and neck squamous cell carci-

Role

of DNA in response of tumors to radiotherapy e R. R.

FRACTIONATION

EXPOSURE

C-143

+

WEICHSELBAUM

24 HOUR

1131

PLDr

MELANOMA

MCF BREAST

Fig. 2b. Comparison of 24 hr repair time survival points after fractionated radiation in plateau phase human melanoma line C-143 and human breast cancer line MCF-7. Significant differences in radiosensitivity are seen between the cell lines (Do = 860 vs Do = 176). Adapted from Weichselbaum et al.”

nomao3* Five biopsies were obtained from tumors before the initiation of therapy (Table 3) and five biopsies from tumors after the patients suffered radiation failures (Table 4). Our study is unique in that cell populations from each tumor were serially cultivated under identical conditions and were studied between 10 and 15 passages after initial explant. We determined X ray survival curve parameters including ii and Do as well as the repair of potentially lethal X ray damage for each cell line. Also our study was the first to examine well-characterized early passage clonogenic tumor cells for which clinical outcome (local control) is known Among cell lines derived from tumors that failed radiotherapy, SCC-35 was radioresistant (Do = 184 rad) and lines SCC-4 and SK-49 (Do = 169 t-ad/ 170 tad) were well above the mean for the entire group (Do = 143 tad). Two cell lines, SCC-25 and SCC-13, derived from patients who failed radiotherapy, were intermediate in

radiosensitivity (Table 4). Three cell lines, SCC-4, SCC25 and SCC-49, derived from patients who failed radiation were intermediate in the ability to perform PLDR and two lines, SCC-35 and SCC-13, were relatively deficient in this ability. Among cell lines established from patients before treatment with radiotherapy, two lines, SCC-61 and SCC-73, were radiosensitive (Do = 107 t-ad/ 108 tad) and two lines, SCC-9 and SCC-66, were intermediate in radiosensitivity (Do = 134 and 129 rad) (Table 3). Three lines, SCC-9, SCC-61 and SCC-73, were extremely proficient in PLDR, and two lines were relatively deficient in the repair process. Thus, although inherent radioresistance was associated with therapeutic failure in 4 of 8 patients, other factors such as the repair of sublethal and potentially lethal X my damage may have been important. For example, line SCC-6 1 was the most radiosensitive in our group, but also the most proficient in PLDR. This line was derived from a patient who failed radiotherapy.

1132

Radiation Oncology 0 Biology 0 Physics C- 143 MELANOMA

200 rad

+

0

‘2

‘4

‘6

HOURS BElIVEN

‘8

/!

‘24

EXPLANT

Fig. 3. Enhancement in survival following subculture as a function ofdose in human melanoma line C-143. Twenty-four hour survival levels may be more analogous to the in viva situation in clinical radiotherapy than are survival levels obtained from exponentially growing cells or 0 hour subculture survival points. Note increase in repair with increasing doses of X ray similar to observations in bacteria and yeast. Adapted from Weichselbaum et al.”

July 1984. Volume 10, Number 7

This tumor was unusual in that it grew through standard fractionation Similarly, the tumor that yielded SCC-25 grew through standard fractionation requiring alteration of treatment regimens, and SCC-25 was the most proficient in PLDR of cell lines derived from tumors that failed radiotherapy. The cell lines with the largest extrapolation numbers, SCC-66 and SCC-13, were associated with treatment failures; as previously stated a small enhancement in surviving fraction is exponentially magnified during fractionation. It is not known whether the slightly larger extrapolation numbers seen here are biologically significant or whether these tumors failed on a stochastic basis. Cell lines SCC-9 and SCC-13 were proficient in PLDR but the tumors were successfully treated by radiation therapy. Interpretation is clouded by the fact that one patient had an excellent response to chemotherapy (SCC-73) prior to radiotherapy and another patient had surgical excision of the primary lesion after preoperative radiotherapy, which was followed by postoperative radiotherapy (SCC-9). In these cases, cell pop ulations proficient in PLDR may have been removed and/or PLDR may not have been expressed in the tumors of these individuals.32 Conditions that influence the repair of potentially lethal damage in vitro may differ from those in vivo. For example, treatment of some tumors with X rays or other cytotoxic agents is known to stimulate cell proliferation and repopulation. Ifthis proliferation occurs at early time periods after radiation, much potentially lethal damage repair may not be expressed since proliferation may fix damage and thus potentially lethal damage converted to lethal damage, perhaps analogous to early subculture points (O-2 hours) in vitro. If proliferation begins at later

Table 3. Cell lines derived from tumors before initiation Line

Stage

Comments

N

of radiotherapy 4

(rad)

24HR R/R, (PLDR)

see-9

T2N, oral tongue

No tumor in surgical specimen, died of distant disease, local control, no tumor at autopsy

1.39

134

7.1

SCC-6 I

Tc4rJ%~x,oral tongue

Unusually aggressive tumor, enlarged on standard fractionation treatment. Changed to 100 rad three times a day

I.83

107

20.3

see-73

T.,No retromolar trigone

Good partial response to chemotherapy prior to radiation therapy, local and distant control, died result of myocardial infarction, no tumor at autopsy

I.17

108

9.3

see-7 I

T,N, soft pallate

Persistent disease

1.45

160

2.3

see-66

T4,No floor of mouth

Persistent disease

2.14

129

2.7

1133

Role of DNA in response of tumors to radiotherapy 0 R. R. WEICHSELBAUM

Table 4. Cell lines derived from tumors that failed fractionated Line

N

Comments

Stage

radiotherapy DO

(rad)

24HR R/R, (PLDR)

see-4

T~No floor of mouth

In-field persistence

1.49

169

4.4

see-25

TIN, oral tongue

Unusually aggressive tumor, patient treated with 200 rad twice per day, no effect on rapidly increasing tumor size

1.53

142

6.2

see-35

TIN0 pyriform sinus

In-field recurrence 2 years later

1.63

184

1.4

see- 13

TzNo skin of face

Recurrence

2.1 I

128

2.2

see-49

TzNo tonsil

In-field recurrence later

1.55

170

4.9

5 months later

time periods ( 1-3 weeks) fixation of damage may not occur and PLD may proceed in cells genetically competent to do so. Thus differing amounts of PLDR may occur early and late during a multifractionated treatment course, depending on the amount of proliferation stimulated by initial doses of radiation and/or pretreatment chemotherapy. If radiation induced proliferation increases fixation of lethal radiation lesions, a decrease in PLDR at the end of fractionated treatment might be observed since more cells might be expected to be cycling. McNalley and deRonde” found a decrease in the experimentally obtained surviving fraction when compared to predicted survival at the end of a fractionation scheme when plateau phase cultures of V79 cells were treated with 1.5 or 2.0 Gy every 6 hours. The cells’inherent capacity for recovery may have been reduced, or radiation induced proliferation may have prevented PLDR. Cells may have a genetically determined maximum recovery capacity following treatment with X rays. This may be expressed “instantaneously” as Do or as recovery

14 months

over a period of hours (PLDR). For example, cell lines SCC-35 and SCC-61 have markedly different Do’s and PLDR capacities. However, the surviving fraction after 24 hours repair time is the same for both cell lines. Therefore, in tumors that contain cells analogous to the SCC35 line that express recovery “‘instantaneously” as an increase in Do, a “true” radiosensitizer such as BUDR might be an effective mode of investigation. In cells which express their maximal recovery over a period of hours, such as (SCC-6 1) compounds such as Ara-C, Ara-A, and 3-aminobenzamide which have been shown to inhibit PLDR in vitro and in vivo might be effective in reducing cellular recovery between fractions of radiation.*’ Radiocurability is undoubtedly a highly complex function and no single explanation or avenue of investigation is likely to provide all insights into the radiobiology of clinical radiotherapy. However, the study of DNA repair processes has implications not only for radiotherapy and cytotoxic treatment in general but implications for the prevention of malignancy as we11.3’.36,43,45

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Radiation Oncology 0 Biology 0 Physics

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July 1984, Volume 10, Number 7 hancement

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295-297, 1977. 41. Weichselbaum, R.R., Malcolm,

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