Variation of SCE Frequency in Lymphocyte Cultures from Patients with Hodgkin's Disease Before, During, and After Treatment

Thelma Brown, Audrey A. Dawson, Derek J. King, Irene Bullock, and Jessie L. Watt

ABSTRACT: Seven patients with newly-diagnosed Hodgkin's disease, having standard mustine-vin- blastine-prednisolone-procarbazine therapy, had a large and regular pattern of rise in SCE frequency in their peripheral lymphocytes, which apparently started to fall before the end of the course (32 wk). In contrast, SCE frequency fell below normal in the lymphocytes of nine patients having mantle radiotherapy. Twenty-eight patients studied 2-13 yr after initial che- motherapy or radiotherapy for Hodgkin's disease had normal or near normal SCE frequencies.

INTRODUCTION

Combinat ion chemotherapy of widespread or systemic Hodgkin 's disease (HD) has resul ted in dramatic improvement in remiss ion and survival rates. In recent years, however, a disquiet ing number of pat ients have deve loped secondary malignancies, especial ly acute nonlymphoblas t ic leukemia (ANLL) [1]; a l though prior to the ad- vent of combinat ion chemotherapy this was rare in HD [2].

Sister chromat id exchange (SCE) frequency in per iphera l b lood lymphocytes provides a sensit ive method of assaying exposure to chemical mutagens and carcin- ogens [3]. The technique was used in this s tudy to moni tor the effect of first-line chemotherapy or radiotherapy during and after t reatment in young adul t pat ients newly diagnosed as having HD. Monitoring the effects of therapy at the level of cel lular DNA damage and repair might a l low recommendat ion on modif icat ion of t reatment to min imise r isk of leukemia in suscept ible patients.

Long-term effects of t reatment on SCE frequency were invest igated in patients with Hodgkin 's disease diagnosed at least 2 yr previously.

From the Department of Genetics, and Department of Medicine. University of Aberdeen; Aberdeen, Scotland.

Address requests for reprints to Dr. Thelma Brown, Department of Medical Genetics, Uni- versity of Aberdeen, Medical School Building, Foresterhill, Aberdeen, Scotland.

Received October 22, 1984; accepted March 11, 1985.

5 3

© 1986 Elsevier Science Publishing Co., Inc. Cancer Genet Cytogenet 20:53~61 (1986) 52 Vanderbilt Ave., New York, NY 10017 0165-4608/86/$03.50

54 T. B r o w n et al.

P A T I E N T S

Group 1

G r o u p 1 c o n s i s t e d of 16 p a t i e n t s w i t h n e w l y d i a g n o s e d HD (Table 1). B lood s a m p l e s we re t a k e n before a n y t r e a t m e n t , a n d f o l l o w - u p s a m p l e s we re

t a k e n at i n t e r v a l s d u r i n g t r e a t m e n t , i m m e d i a t e l y before a p u l s e in c h e m o t h e r a p y p a t i e n t s a n d ove r seve ra l m o n t h s f o l l o w i n g t r e a t m e n t in b o t h c h e m o t h e r a p y a n d r a d i o t h e r a p y pa t i en t s .

Group 2

G r o u p 2 c o n s i s t e d of 28 p a t i e n t s (age range , 2 4 - 6 9 yr) w i t h HD d i a g n o s e d b e t w e e n 2 a n d 13 yr p r ev i ous l y . T h e s e p a t i e n t s we re i n v e s t i g a t e d to see if SCE f r e q u e n c y c o r r e l a t e d w i t h t h e t ype of p r e v i o u s t r e a t m e n t , or t he t i m e i n t e rva l s ince s u c h t reat- men t . Al l p a t i e n t s we re in r e m i s s i o n at t he t i m e of s a m p l i n g (Table 2).

T a b l e 1 N e w H o d g k i n ' s d i s ease p a t i e n t s

Splenectomy ° Smoker Total number

Therapy of patients Sex Yes No Age range (yr) Yes No

Chemotherapy b 7 5M 3 2 14-47 3 2 2F 2 0 18-21 1 1

Mantle radiotherapy 9 6M 6 0 15-49 5 1 3F 3 0 32-41 3 0

aStaging laparotomy with splenectomy.

bMVPP chemotherapy was given as a course of six pulses, each lasting 2 wk, with 4 wk between pulses.

T a b l e 2 P a t i e n t s w i t h H o d g k i n ' s d i sease d i a g n o s e d 2 - 1 3 yr p r e v i o u s l y (all i n r e m i s s i o n )

Time since treatment (mo)

Start End Clinical Initial Number of history treatment patients Mean Range Mean Range

In remission since initial therapy

Initial therapy + Chemotherapy after relapse b

Mantle 2 33 32-33 32 31-32 radiotherapy

MVPP chemotherapy

6 pulses 4 116 80-132 104 77-122 9 pulses 11 89 25-136 67 5-106 Combined 4 62 36-92 45 15-65

modality a

Mantle 3 109 54-165 51 1-113 radiotherapy

MVPP 4 94 51-135 37 1-75

aCombined modality treatment consisting of MVPP + mantle radiotherapy.

bChemotherapy including salvage MVPP for relapse after initial radiotherapy or more than one form of chemotherapy (e.g., CCNU-vinblastine-bleomycin (CVB), Goldman and Dawson [17]) for relapse after initial MVPP.

SCE in HD Patients 5 5

Patient in Relapse

One patient was s tudied in relapse and on chemotherapy at the t ime of sampling. He had had mul t ip le courses of combinat ion chemotherapy, wi th pal l ia t ive radio- therapy over the 12 yr from init ial diagnosis, having had numerous previous re- lapses. He deve loped overt acute myelo id leukemia (AML) 4 mo after sampling.

NORMAL CONTROLS

Eight laboratory controls were tested, at least one on each occasion on which an assay was set up. All controls were non-smokers.

TREATMENT

Chemotherapy

Patients with widespread HD (stages IIIB and IV) were given a course of m u s t i n e - v inb la s t ine -p redn i so lone -p roca rbaz ine (MVPP) fol lowing a highly s tandardized regimen [4]. The treatment was given in six pulses, each lasting 2 wk, with a 4-wk interval between pulses.

Radiotherapy

Patients wi th local ized disease (stages I and II) above the d iaphragm were treated with mant le rad io therapy (4000 cGy tumor dose) given in 20 fractions over a 4-5 wk period.

METHODS

Metaphase preparat ions from PHA-st imulated lymphocytes were made by s tandard procedures [5] wi th the addi t ion of BrdU (20 ~g/ml) to differentiate sister chroma- tids. Sl ides were FPG-stained [6], coded, randomized, and scored blind. SCE were counted in 20-30 second divis ion metaphases where possible. Fewer than 20 score- able metaphases were found in some of the samples taken during or immedia te ly following a course of treatment. The mean number of SCE per cell (SCE frequency) and the dis t r ibut ion of SCE between cells were recorded for each sample. Since smoking is known to cause an increase in SCE frequency, the smoking habits of the patients were recorded.

A sample from one of the eight laboratory controls was assayed with each batch of samples from patients to moni tor exper imenta l and normal ind iv idua l variation. Statistical parameters for the normal popula t ion were der ived from the first sample from each control.

RESULTS

SCE in HD Patients Before Treatment

The samples taken from patients before chemotherapy and before radio therapy could be treated as separate statist ical populat ions , since pat ients were treated with radio therapy if the disease was localized, and with chemotherapy if the disease was widespread.

Populat ions of SCE frequencies can be expected to have a normal or near normal

56 T. Brown et al.

distr ibution, because they are popula t ions of means. Variance ratio- and t-tests showed that there was no significant difference between variances or means of SCE frequencies in prechemotherapy patients (mean, 9.2 -+ 0.7) preradio therapy pa- tients (mean, 10.4 +- 0.7) or the control group (mean, 10.1 + 0.4). HD itself, whether local ized or systemic, therefore, does not appear to cause any change in SCE frequency. The popula t ions were too small to register a significant difference in SCE frequency between smokers and non-smokers.

Effect of Treatment on SCE Frequency

Since the same patients were moni tored throughout, the pretreatment samples pro- v ided controls for the effects of t reatment on SCE frequencies. SCE frequencies in sequential samples from patients treated with chemotherapy or rad io therapy are shown in Figure 1, where they are compared with the mean and 95% popula t ion l imits of the normal control group. Since the courses of t reatment were highly stan- dardized, sequential samples from all the pat ients wi th in a group were at compa- rable stages of treatment.

With mant le rad io therapy there was a fall in SCE frequency, which cont inued for at least 2 yr fol lowing treatment (regression coefficient, - 0 . 0 2 9 ; p < 0.05), and the later samples were outside the normal range (p ~ 0.05) al though not signifi- cant ly different from the mean of patients before treatment.

Figure 1 demonstrates the dramatic effect of MVPP on SCE frequency. There was a sharp rise in mean SCE per cell, typica l ly appearing wi thin I wk of chemotherapy init iation. The rise cont inued throughout approximate ly the first 20 wk of therapy when it peaked, and apparent ly started to fall before the end of treatment. Succes- sive results from each pat ient fol lowed this pat tern but wi th ind iv idua l variat ion in rate and extent of response. Immedia te ly after comple t ion of the whole course of chemotherapy, the frequency of SCE fell to the lower l imits of normal. The mean of samples after this fall (8.9 _+ 0.3) was not significantly below the mean of pretreat- ment samples (9.2 -+ 0.7). The fall in SCE frequency during the late stages of che- motherapy and fol lowing radio therapy could not be at tr ibuted to possible changes in smoking habits, since it occurred to the same extent in both smokers and non- smokers.

Three patients re lapsed wi th in 3 mo of finishing MVPP therapy. Their SCE fre- quencies were slower to rise or reached a lower max imum than those of patients with good cl inical response (Fig. 1). This a typical response in SCE score was not reflected in the leukocyte counts recorded before each pulse of chemotherapy. Ex- cept for an init ial rise immedia te ly fol lowing splenectomy, no regular pat tern of change in leukocyte counts was observed, nor was there a significant difference between the counts of patients who re lapsed and those who did not.

The dis t r ibut ion of SCE between cells was skewed in untreated patients. During chemotherapy this effect was exaggerated due to a few cells having very high SCE numbers. Figure 2 demonstrates the dis t r ibut ion of SCE between cells related to the t ime from the start of chemotherapy. Results from all the chemotherapy pat ients were pooled to show the dis t r ibut ion before treatment (column 1) and over 10-wk per iods from the start of treatment. Most of the pretreatment cell popula t ion had between 5 and 15 SCE with a cut-off at 20. During chemotherapy the rise in mean SCE frequency appeared to be part ly due to a general rise in SCE scores and also to a few cells wi th except ional ly high SCE scores outs ide the range of the main cell populat ion. The number of "high SCE" cells tended to rise and fall wi th the mean of the main cell popula t ion and they did not persist long after the end of treatment. The origin and significance of these high frequency cells is difficult to surmise, especial ly since a s imilar but less marked effect has been shown to cause a skewed intercell SCE dis t r ibut ion in a popula t ion of normal ind iv idua ls [7].

SCE in HD Patients 5 7

HODGKIN'S DISEASE PATIENTS

(SCE/Time from start of treatment)

35 -

30 -

25

LIJ o c/) "S ,. 20

E t -

t -

15

:" .;!:

m'nl

5 - I

- 5 0

End of End of XRT Chemotherapy

(10) (~)

7 ~3(9)

v ~z E~(IO) (9) |

• Radiotherapy

[] Chemotherapy

v Chemotherapy--I~ relapse

V

0

Io

w i w w ==(9) •

I I I I I i 20 40 60 80 100 120

Weeks from start of treatment

~ : + 1.96 b

iiiiiiiii!iiii!!iiii!iiiiiii Mean of

control

~ - 1.96 b °OOOOoo

Figure I Composite graph of SCE frequencies from HD patients compared with the mean and 95% distribution limits of the pooled controls. Continuous line, chemotherapy, mean of individual trends; broken line, radiotherapy, regression line r = -0.029. Numbers in paren- theses indicate number of cells scored if fewer than 20.

Long-Term Effects of Treatment

In Figure 3 SCE frequencies in samples cultured from the 28 patients diagnosed more than 2 yr previously are compared schematical ly with normal controls as- sayed at the same time. The SCE frequencies were all normal or nearly normal, although some of the patients had received more than one course of chemotherapy, in some cases completed only recently (e.g., one patient at the end of a third course of chemotherapy had a mean SCE frequency of 8.1, compared with the mean of the control populat ion of 10.8).

Three chemotherapy patients had slightly elevated SCE frequencies. In one case fmean, 17.2) this was due to persistence of a few high SCE cells 5 mo after the end

Number

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SCE in HD Patients 59

20

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UJ ~3 03

t ' -

10

HODGKIN'S DISEASE PATIENTS

(SCE/Years from start of treatment)

© Chemotherapy

• Combined XRT and Chemotherapy

• X-ray therapy

• Run control

R

Mean of control population

I I I I I I I I I I I I I I 0 1 2 3 4 5 6 7 8 9 10 11 12 13

Years from start of treatment

Figure 3 SCE frequencies in HD patients at least 2 yr from start of treatment, compared with individual controls and with pooled control mean and 95% distribution limits.

of a n ine-pulse course of chemotherapy, and a sample taken 1 yr later from the same pat ient had a normal SCE frequency (mean, 8.6) with no high SCE cells. This was the only pat ient from this group with more than one high SCE cell (in 20-30 cells). The other two patients wi th elevated SCE frequencies (mean, 15.5 and 17.3) had a general increase in SCE scores. It is poss ible that smoking contr ibuted to the high SCE count in one of these patients, al though other smokers were wi th in 95% dis t r ibut ion l imits of the control populat ion.

The two patients who had received mantle radiotherapy approximate ly 3 yr pre- viously main ta ined the low SCE frequencies seen in the more recent ly treated pa- tients.

A mean SCE score of 14.5 was found in the patient who developed overt AML 16 wk later. This was wi th in the normal range, al though he was assessed during a course of chemotherapy.

DISCUSSION

The present invest igat ion was carried out on a sizeable group of HD patients un- dergoing highly s tandardized ini t ial treatment, either mantle rad io therapy or com- binat ion chemotherapy, and the same group of patients was compared before, dur- ing, and after treatment.

The results showed that HD, itself, caused no increase in SCE frequency in any of the 16 patients in this investigation. However, Kurvink et al. [8] found that the mean SCE frequency of a group of 13 untreated lymphoma patients ( including five HD patients) was significantly greater than the mean of their control group.

60 T. Brown et al.

Ionizing radiat ion has been shown to have a min imal influence on SCE fre- quency, compared with its clastogenic effects [4]. The steady decl ine in SCE fre- quency seen in the nine pat ients during 2 yr fol lowing mantle radiotherapy, and a similar low SCE frequency after rad io therapy reported by Kurvink et al. [8], may have been due to change in the propor t ions of different cell types rather than a direct effect on SCE frequency. Posner et al. [9] noted a decl ine in the relative propor t ion of T- lymphocytes to B-lymphocytes for at least 1 yr following mantle and paraaort ic i r radi t ion in HD patients, and a higher frequency of SCE in T-lym- phocytes than in B-lymphocytes has been reported by Santesson et al. [10] in PHA- s t imulated cultures.

Many of the cytotoxic drugs used in chemotherapy are known to cause an in- crease in SCE frequency. In part icular, b i funct ional alkylat ing agents have been shown to have this effect in vivo [11] and a direct dose response has been demon- strated in vitro [12]. Increase in SCE frequency is an indica t ion of exposure to chemical carcinogens [4] and is direct ly correlated with mutageneci ty [13]. MVPP therapy involves intensive combinat ion chemotherapy given in a l imited number of pulses. In response to this t reatment SCE frequency rose rapidly, at tained a peak after 2-3 pulses, began to decl ine before the end of treatment, and finally leveled out close to the mean of the pretreatment samples. This pat tern of changes in SCE can be compared with other publ i shed reports of changes associated with different regimens using other cytotoxic drugs. Drugs that cause an increase in SCE fre- quency start to do so rapidly , wi th in hours, wi th the subsequent pat tern of response depending on the dose and part icular drug used. Gebhart et al. [14] found that a plateau level of SCE frequency occured after the 7th pulse when a combinat ion of methyl-CCNU vincris t ine and fluorouracil was given at 5-wk intervals (with methyl- CCNU given on alternate pulses). A plateau effect was also noted by Stetka et al. [15], who adminis te red mitomycin-C via int raper i toneal inject ion in weekly doses to rabbits. He observed that SCE frequency rose and fell after the first three injec- tions but remained high after subsequent doses, largely due to the persis tence of a few high SCE frequency cells.

On the other hand, Palmer et al. [16] observed a cont inuous rise in SCE fre- quency in patients wi th Behcet 's syndrome treated with chlorambuci l in a long- term low-dose manner. The pat ient wi th the highest cumulat ive dose had the high- est mean SCE frequency (partly due to high SCE frequency cells) and developed AML. In the present study, the pat ient who deve loped AML had received the most t reatment (both chemotherapy and radiotherapy) over 12 yr but his SCE level was normal (mean, 14.6) wi th only one high SCE cell in a sample of 20 cells. More information on the long-term effects fol lowing MVPP therapy wil l be gained after a suitable t ime lapse in the present carefully moni tored group.

In HD patients treated with s tandard MVPP therapy, SCE frequency provides a method of moni tor ing ini t ial response to treatment. Most patients are in cl inical remission after two pulses of MVPP, which is when the max imum SCE frequency was observed. Patients who re lapsed had a delayed or low increase in SCE fre- quency. This abnormal ly low SCE frequency after two pulses of t reatment may be a means of early identif icat ion of pat ients who are l ikely to need further treatment.

The significance of the high SCE score several years after therapy in a few pa- tients, and whether or not a normal SCE score is typical prior to developing a sec- ond mal ignancy require more observation on pat ients who have re lapsed and have received mul t ip le courses of treatment.

Supported by a grant from the Cancer Research Campaign. The authors thank Prof. F. W. Robertson, Department of Genetics, and Prof. A. S. Douglas, Department of Medicine, Aber- deen University for their interest and encouragment.

SCE in HD Pat ients 61

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