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Chromosome damage and sister chromatid exchanges in lymphocyte cultures from patients with two primary cancers
Chromosome Damage and Sister Chromatid Exchanges in Lymphocyte Cultures from Patients with Two Primary Cancers Thelma Brown, Audrey A. Dawson, Ian A. McDonald, Irene Bullock, and Jessie L. Watt
ABSTRACT:
Sister chromatid exchanges (SCEs) and chromosome damage were scored in lymphocyte cultures from 11 patients with two or more primary cancers and were compared with normal controls. None of the patients had a constitutional chromosome anomaly, but six showed evidence of chromosome instability, which could not be accounted for by treatment, expressed either as elevated SCE frequency or increased nonspecific chromosome damage and chromosome loss. Chromosome damage included major rearrangements as well as deletions and gaps. The possibility of common mechanisms in chromosome instability leading to susceptibility to a heterogeneous group of primary cancers is discussed.
INTRODUCTION Onset of clinical evidence of cancer is almost always a c c o m p a n i e d by c h r o m o s o m e changes. Specific types of c h r o m o s o m e rearrangements are found in m a n y cancers and m a y show clonal evolution. The P h i l a d e l p h i a c h r o m o s o m e (Ph), for example, is found in the early stages of chronic m y e l o i d leukemia (CML), and clones with trisomy 8 and i s o c h r o m o s o m e 17q may develop later [1-3]. High susceptibility to cancer has been related to constitutional chromosome anomalies that may be specific, such as the elevated incidence of leukemia associated with Down's s y n d r o m e [4], or to general c h r o m o s o m e fragility, as in the chrom o s o m e breakage syndromes, Fanconi's anemia, Bloom's syndrome, ataxia telangiectasia, and xeroderma p i g m e n t o s u m [5-7]. Chromosome fragility can be seen as increased frequency of c h r o m o s o m e breaks and rearrangements or as an increase in the frequency of sister chromatid exchanges (SCEs), either s p o n t a n e o u s l y as in Bloom's s y n d r o m e or as a hypersensitive response to carcinogens [8-10]. Variation in sensitivity to carcinogens, paralleled by differences in SCE frequency, has also been seen in response to smoking. Smoking is k n o w n to cause elevated SCE frequency [11-13], and H o p k i n and Evans [14] found that smokers with lung cancer had more SCEs in their l y m p h o c y t e s than equally heavy smokers without lung cancer.
From the Departments of Genetics and Medicine, University of Aberdeen, Aberdeen, Scotland. Address requests for reprints to Dr. Thelma Brown, Department of Genetics, University of Aberdeen, Medical School Buildings, Foresterhill, Aberdeen, AB9 2ZD, Scotland. Received April 4, 1984; accepted August 24, 1984.
35 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 17, 35~I2(1985) 0165-4608/85/$03.30
36
T. B r o w n et al.
The i n v e s t i g a t i o n p r e s e n t e d h e r e was carried out o n a g r o u p of patients w i t h t w o or m o r e p r i m a r y cancers that w e r e of a t y p e not k n o w n to be a s s o c i a t e d w i t h a h i g h e r i n c i d e n c e of a s e c o n d cancer. Thus, t h e y w e r e c o n s i d e r e d to h a v e an inherent i n c r e a s e in s u s c e p t i b i l i t y to cancer. T h e i n t e n t i o n was to find out if c a n c e r s u s c e p t i b i l i t y is a s s o c i a t e d w i t h e l e v a t e d SCE levels or w i t h an i n c r e a s e in general or specific c h r o m o s o m e breakage and r e a r r a n g e m e n t s .
PATIENTS AND CONTROLS E l e v e n patients (five male, six female, age range 6 0 - 8 1 years) w e r e s e l e c t e d w h o h a d d e v e l o p e d t w o or m o r e n e o p l a s m s that w e r e h i s t o l o g i c a l l y different, so that t h e y w e r e b o t h clearly p r i m a r y cancers, not a single c a n c e r w i t h metastases. The patients can be d i v i d e d into three c l i n i c a l groups as follows: G r o u p 1: T h r e e patients w h o s e first t u m o r was s q u a m o u s c a r c i n o m a of the skin, w i t h o u t m e t a s t a s e s to l y m p h nodes. G r o u p 2: F o u r p a t i e n t s w h o s e t u m o r s w e r e b o t h c a r c i n o m a s of i n t e r n a l organs but w h o h a d r e c e i v e d n e i t h e r r a d i o t h e r a p y nor c h e m o t h e r a p y prior to s a m p l i n g . G r o u p 3: F o u r patients w h o s e t u m o r s w e r e b o t h c a r c i n o m a s of i n t e r n a l organs but w h o had r e c e i v e d r a d i o t h e r a p y or c h e m o t h e r a p y . O n e p a t i e n t (DC8) in this g r o u p had b e e n treated w i t h c o m b i n a t i o n c h e m o t h e r a p y ( c h l o r a m b u c i l , vincristine, and p r e d n i s o l o n e ) o n the 2 days prior to s a m p l i n g . T h e o t h e r three p a t i e n t s had r a d i o t h e r a p y t r e a t m e n t for the first cancer. C l i n i c a l details of the p a t i e n t s are s u m m a r i z e d in Table 1. Six n o r m a l i n d i v i d u a l s , f o u r m a l e a n d two female, age range 2 5 - 5 0 , w h o w e r e all n o n s m o k e r s w e r e u s e d as controls.
METHODS M e t a p h a s e p r e p a r a t i o n s of P H A - s t i m u l a t e d l y m p h o c y t e s w e r e m a d e by r o u t i n e m e t h o d s f r o m s a m p l e s of w h o l e b l o o d [15]. Forty-eight h o u r c u l t u r e s w e r e u s e d to score c h r o m o s o m e breaks and rearrangements. For u n i f o r m q u a n t i t a t i v e assessment, c h r o m o s o m e d a m a g e was s c o r e d in 50 G i e m s a - s t a i n e d m e t a p h a s e s , e a c h w i t h 46 c e n t r o m e r e s , w h i c h w e r e not s e l e c t e d for
Table 1
C l i n i c a l details of d o u b l e c a n c e r patients Time before analysis of
Group 1
Group 2
Group 3
Patient
Sex
First tumor site
DC1 DC2 DC3 DC4 DC5 DC6
M M M F F M
Skin (multiple} Skin (multiple) Skin Colon Colon Bronchus
DC7 DC8
F F
Breast Colon
DC9 DC10 DC11
F M F
Breast Larynx Colon
Second tumor site Larynx Bladder Kidney Bladder Lung Chronic lymphocytic leukemia Rectum Prolymphocytic leukemia Bronchus Prostate Bladder
X-ray
Chemotherapy
Smoker
--2 Months ----
-------
No Yes No Yes No No
---
-" Day
No No
16 Years 12 Years 1 Year, 4 months
----
Yes Yes Yes
37
Cytogenic of Multiple Primary Cancers
quality. A n e u p l o i d and p o l y p l o i d cells seen w h i l e scanning for the 50 complete cells were recorded. Cells with more than 46 c h r o m o s o m e s were not scored as a n e u p l o i d if extra chromosomes were noticeably different and therefore probably "strays" from other broken metaphase cells. Banding (G and C) was used to locate damage. SCEs were scored in 30 fluorescence plus giemsa (FPG)-stained metaphases [16] from 66-hour cultures containing 20 I~g/ml b r o m o d e o x y u r i d i n e (BrdU). Samples from a patient and a control were assayed at the same time under identical conditions. RESULTS
SCE Frequency From Figure 1 and Table 2 it can be seen that one patient in each of groups 1 (DC2) and 2 (DC4) had slightly raised SCE levels that could have been due to smoking, but otherwise, SCE frequencies in patients and controls were similar in these two groups. No SCE score was recorded for one patient in group 1 (DC3) because of lack of metaphases in BrdU cultures. The patients and controls were not m a t c h e d for age, but age has been shown to have little or no effect on SCE frequency [17]. The most significantly raised SCE frequencies occurred in group 3. The high score in DC8 was probably due to recent chemotherapy, but patients DC9 and DCIO
Figure 1 Mean number of SCEs per cell for each patient compared with individual control and with pooled control mean and 95% distribution limits. Pooled control parameters include one mean per individual to avoid bias toward controls providing more than one sample. Significance of difference between patient and individual control: "0.01 < p < 0.05; *'0.001 < p < 0.01; ***p < 0.001. DOUBLE CANCER PATIENTS SCE frequency of patients compared with controls
Group 1
Group 2
Group 3
20 18
OPatient • Control
16 -
14 + 1.96~
12 03
~
Mean of controls
lO -
I
DC1
DC2
DC4
DC5 DC6 DC7 Patients
*1
DC8 DC9 DC10 DC11
1.96~
Significance difference
of
38
T. Brown et al. had similarly high SCE scores that could not be attributed to direct effects of treatment, as both these patients had received only radiotherapy many years previously.
Chromosome Damage and Aneuploidy At 48 hr, metaphase lymphocytes are almost all in first division in culture; therefore, chromosome damage seen at this stage was probably present in vivo. From 48-hr Giemsa-stained preparations (Table 2), 75 major damage events were found in 502 complete (46 centromere) metaphases from these double cancer patients. A complete spectrum of rearrangements was seen, including dicentrics, rings, translocations, pericentric inversions, and deletions (Fig. 2). Most of the damage was of the chromosome type; only two chromatid interchanges (quadriradials) and two chromatid deletions were found. In control samples, only three major damage events (all deletions) were seen in 550 metaphases. Chromosome damage was very unevenly distributed among patients (Table 2). Two patients in group 2 (DC4, DC6) and all four patients in group 3 (DC8 to DC11) had more damage than any of the controls. The frequency of gaps in chromosomes from patients was double that of the control group; nine patients had more than the control mean and two were above the 95% distribution limit. Similarly, aneuploid cells were twice as frequent in the patients as in the controls, with one patient outside the 95% limit. Telomeric stickiness was seen almost exclusively in one patient (DC8), most frequently involving chromosome #16 and G-group chromosomes. With a few exceptions, damage and chromosome loss tended to be distributed as expected [18] among chromosome groups, with damage events being proportional and chromosome loss inversely proportional to the length of the chromosome (Table 3). Chromosome #16 was involved in more events than expected, mainly in one patient (DC8), but G-banding showed no specific breakpoint or type of rearrangement. The only apparently nonrandom location of damage was in chromosome #2, where 12 of the 17 damage events involved the q-arm, including an endoreduplication and 10 events resulting in 2 q - (6 of these occurred in patient DC9). The main difference between distribution of chromosome loss in patients and controls was an excess loss from group C (Table 3), which G-banding showed to be due to selective loss of chromosomes #8, #11, and X. The excess in overall loss of chromosomes in the patients compared to controls could have been mainly mechanical loss during preparation, perhaps due to increased membrane fragility. Selective loss of chromosomes #8, #11, and X, however, probably reflects a true preferential loss of these chromosome in circulating lymphocytes. This selective loss was limited to five of the six female patients and was probably age-related, as preferential loss of C-group chromosomes in aging females is a well-known phenomenon [19, 20].
DISCUSSION The 11 double cancer patients were a heterogeneous group with a probable common susceptibility to cancer. No constitutional changes were detected, although this cannot be considered to be a negative association in view of the small sample size. The patients were divided into groups on a clinical basis (Table 1), and the cytogenetic results show that the first group (with initial skin cancers) was most like the controls, and the third group (with two carcinomas of internal organs, treated with radiotherapy or chemotherapy) deviated most from controls. When all the evidence is taken into account, 6 of the 11 patients showed evi-
DC1 DS2 DC3 DC4 DC5 DC6 DC7 DC8 DC9 DC10 DC11
2
180 30
30
30
10.3
12.26
12.0
1
0.2 1.0
1 0 0 3 0 5 1 38 6 4 17 75
8 30 0 30 30 30 30 27 30 30 30 275
9.9 13.4 0 12.7 11.0 10.0 9.9 17.6 18.6 15.9 9.7 12.9
bFrom all 48-hr preparations, banded and unbanded.
1
0.7
1 0.1
0 0 1 0 O 1 0 0 1 1 0 4
C/;IROMOSOME CHROMATID
Major d a m a g e
10
13
65 6
16 13 6 10 11 22 9 11 11 12 2 123
50
50
550 50
23 50 29 50 50 50 50 50 50 50 50 502
Gaps C e l l s scored
N u m b e r of d a m a g e e v e n t s ° i n 46 c e n t r o m e r e m e t a p h a s e s
°From 48-hr Giemsa-stained preparations only.
Total Pooled c on tr ols Total Mean/ sample Upper limit ( m ea n + 1.96 SD) Maximum in range
3
2
1
M e a n / C el ls cell scored
SCEs
Cytogenetic analysis
Group Patient
Table 2
15
15
68 6
5 6 11 3 10 11 10 44 5 11 12 128
+ (42-45) (47-48)
Aneuploid c e l l s / 50 (46 centromere) cells
1.0
2 0.2
Polyploid
Location
0
1 16
1 20
0
15
18 1
1
1
0
2 13
1 6
1
2
TELOMERIC MULTIPLE CHROMOSOME CHROMOSOME STICKINESS DAMAGE #16 2q 1
Type
Nonrandom damage b
¢.D
_.-
-t--IX..ll”-i^-
.-.
-.=---“..“l”,~““~-_-“_.-..“__~_I
.
;I_ i_._____.__
Examples of damage. (a) Two chromosome SCE score (39) and dicentric (Z C-group chromosomes).
Figure 2
A deletions
and fragments
(chromosomes
#
and #2) and centric
B ring (chromosome
#18). (b) High
41
Cytogenetic of Multiple Primary Cancers
Table 3
Location of damage and missing chromosomes
Damage events
A
B
C
1
2
3
(A) Total (B) Divided by chromosome pairs
20 20
17 17
12 12
14 7
78 10
Missing chromosomes
A
B
C
D
6 1
6 3
86 17
2.8 1.4
4.3 6.5
15.9 9.6
(A) Total Patients Controls (B) Percent of total Patients Controls
D
E 16
17
18
7 2
16 16
4 4
2 2
E
F
G
17 4
42 20
24 6
48 18
8.2 5.8
20.2 29.1
17.3 13.1
31.2 34.6
F
G
2 1
10 4
dence of c h r o m o s o m e instability in different ways (Table 2; patients DC4, 6, 8 through 11). This could only be partially accounted for in two patients; high SCE frequency was related to c h e m o t h e r a p y in patient DC8, and c h r o m o s o m e damage was related to recent r a d i o t h e r a p y in patient DC11. Cytogenetic analysis was carried out on PHA-stimulated lymphocytes, not on cells directly involved in the neoplasia. Even in the cases of chronic l y m p h o c y t i c leukemia (DC6) and p r o l y m p h o c y t i c leukemia (DC8), the leukemic cells were probably B lymphocytes, whereas the cells r e s p o n d i n g to PHA stimulation w o u l d be m a i n l y T l y m p h o c y t e s [21]. The considerable a m o u n t of nonspecific chromosome damage found in cells not directly involved in neoplasia suggests an underlying chromosome instability. Two of the double cancer patients with evidence of c h r o m o s o m e instability (DC9, DC10) had u n a c c o u n t a b l y high SCE scores. Chromosome breakage syndromes are k n o w n to be associated with high SCE frequencies s p o n t a n e o u s l y (Bloom's syndrome) or as an exaggerated reaction to carcinogens. It w o u l d be informative to take blood samples from cancer patients with s u s p i c i o u s l y high SCE frequencies before therapy and expose their l y m p h o c y t e s to a m i l d carcinogen in vitro to see if a hypersensitive response occurs. These results may augment present ideas regarding oncogenes, as cancer-related chromosome rearrangements have been shown to activate oncogenes that i n d u c e cell proliferation [see reference 3 for review]. Chromosome instability, as seen in some patients with double cancers, leads to breaks occurring at m a n y different chromosome locations in different cells. This must increase the chance of damage occurring, w h i c h could activate an oncogene to initiate the b u i l d - u p of a clone with the chromosome rearrangement that triggered the neoplastic transformation. Thus, tissues not involved in the n e o p l a s m may have nonspecific damage, while transformed cells show clonal d e v e l o p m e n t of specific types of damage. Continued instability w o u l d give rise to the possibility of a second p r i m a r y cancer d e v e l o p i n g also by a chance break activating a different oncogene. The types of cancer d e v e l o p e d w o u l d d e p e n d on w h i c h oncogene was activated in w h i c h tissue. A n a p p a r e n t l y heterogenous group of cancers could in this w a y have a c o m m o n initiatory mechanism in c h r o m o s o m e instability. In summary, our results tentatively indicate that a significant p r o p o r t i o n of peo-
42
T. B r o w n et al.
ple with multiple cancers may have an underlying inherent chromosome instability, w h i c h is s i m i l a r to t h e b e t t e r - d e f i n e d c h r o m o s o m e b r e a k a g e s y n d r o m e s . W e h a v e also p r o v i d e d s o m e s u p p o r t for t h e i d e a t h a t u n a c c o u n t a b l y h i g h SCE frequency may indicate cancer susceptibility in some individuals.
This work was carried out with financial support from the Cancer Research Campaign.
REFERENCES 1. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier North Holland, New York. 2. Yunis J] (1983]: The chromosomal basis of h u m a n neoplasia. Science 221:227-236. 3. Mitelman F, Levan G (1981): Clustering of aberrations to specific chromosome in h u m a n neoplasms. IV. A survey of 1,871 cases. Hereditas 95:79-139. 4. Holland WW, Doll R, Carter'CO (1962]: The mortality from leukaemia and other cancers among patients with Down's syndrome (mongols) and among their parents. Br J Cancer 16:177-186. 5. German J (1973): Genetic disorders associated with chromosomal instability and cancer. J Invest Dermatol 60:427-434. 6. Lehmann AR (1981): Cancer-associated h u m a n genetic diseases with defects in DNA repair. J Cancer Res Clin Oncol 100:117-124. 7. Schroeder TM, Kurth R (1971]: Spontaneous chromosomal breakage and high incidence of leukemia in inherited disease. Blood 37:96-112. 8. Chaganti RS, Schonberg S, German J (1974]: A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci USA 71:4508-4512. 9. Auerbach AD, Wolman SR (1979): Carcinogen induced chromosome breakage in chromosome instability syndromes. Cancer Genet Cytogenet 1:21-28. 10. Latt SA, Stetten G, Juargens LA (1975): Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia. Proc Natl Acad Sci USA 72:40664070. 11. Lambert B, Lindblad A, Nordeskjold M, Werelius B (1978): Increased frequency of sister chromatid exchanges in cigarette smokers. Hereditas 88:147-149. 12. Murthy PBK (1979): Frequency of sister chromatid exchanges in cigarette smokers. Hum Genet 52:343-345. 13. Husum B, Wulf HC, Niebuhr E (1982): Increased sister chromatid frequency in lymphocytes in healthy cigarette smokers. Hereditas 96:85-88. 14. Hopkin JM, Evans HJ (1980): Cigarette smoke-induced DNA damage and lung cancer risks. Nature 283:388-390. 15. Moorehead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960): Chromosome preparations of leucocytes cultured from h u m a n peripheral blood. Exp Cell Res 20:613616. 16. Perry P, Wolff S (1974): New Giemsa method for the differential staining of sister chromatids. Nature 251:156-158. 17. Lacassie Y, Fu W, Borgaonkar DS (1975): Chromosome abnormalities and sister chromatid exchanges in senescence. Am J Hum Genet 27:57A. 18. Brown T, Fox DP, Robertson FW, Bullock I (1983): Non-random chromosome loss in PHAstimulated lymphocytes from normal individuals. Mutat Res 122:403-406. 19. Sandberg AA, Cohen WM, Rimm AA, Levin ML (1967): Aneuploidy and age in a population survey. Am J Hum Genet 19:633-643. 20. Jarvik LF, Yen FS, Fu TK, Matsuyama SS (1976): Chromosomes in old age: A six year longitudinal study. Hum Genet 33:17-22. 21. Nowell P, Daniele R, Rowlands D, Finan J (1980): Cytogenetics of chronic B-cell and Tcell leukemia. Cancer Genet Cytogenet 1:273-280.
ABSTRACT:
Sister chromatid exchanges (SCEs) and chromosome damage were scored in lymphocyte cultures from 11 patients with two or more primary cancers and were compared with normal controls. None of the patients had a constitutional chromosome anomaly, but six showed evidence of chromosome instability, which could not be accounted for by treatment, expressed either as elevated SCE frequency or increased nonspecific chromosome damage and chromosome loss. Chromosome damage included major rearrangements as well as deletions and gaps. The possibility of common mechanisms in chromosome instability leading to susceptibility to a heterogeneous group of primary cancers is discussed.
INTRODUCTION Onset of clinical evidence of cancer is almost always a c c o m p a n i e d by c h r o m o s o m e changes. Specific types of c h r o m o s o m e rearrangements are found in m a n y cancers and m a y show clonal evolution. The P h i l a d e l p h i a c h r o m o s o m e (Ph), for example, is found in the early stages of chronic m y e l o i d leukemia (CML), and clones with trisomy 8 and i s o c h r o m o s o m e 17q may develop later [1-3]. High susceptibility to cancer has been related to constitutional chromosome anomalies that may be specific, such as the elevated incidence of leukemia associated with Down's s y n d r o m e [4], or to general c h r o m o s o m e fragility, as in the chrom o s o m e breakage syndromes, Fanconi's anemia, Bloom's syndrome, ataxia telangiectasia, and xeroderma p i g m e n t o s u m [5-7]. Chromosome fragility can be seen as increased frequency of c h r o m o s o m e breaks and rearrangements or as an increase in the frequency of sister chromatid exchanges (SCEs), either s p o n t a n e o u s l y as in Bloom's s y n d r o m e or as a hypersensitive response to carcinogens [8-10]. Variation in sensitivity to carcinogens, paralleled by differences in SCE frequency, has also been seen in response to smoking. Smoking is k n o w n to cause elevated SCE frequency [11-13], and H o p k i n and Evans [14] found that smokers with lung cancer had more SCEs in their l y m p h o c y t e s than equally heavy smokers without lung cancer.
From the Departments of Genetics and Medicine, University of Aberdeen, Aberdeen, Scotland. Address requests for reprints to Dr. Thelma Brown, Department of Genetics, University of Aberdeen, Medical School Buildings, Foresterhill, Aberdeen, AB9 2ZD, Scotland. Received April 4, 1984; accepted August 24, 1984.
35 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 17, 35~I2(1985) 0165-4608/85/$03.30
36
T. B r o w n et al.
The i n v e s t i g a t i o n p r e s e n t e d h e r e was carried out o n a g r o u p of patients w i t h t w o or m o r e p r i m a r y cancers that w e r e of a t y p e not k n o w n to be a s s o c i a t e d w i t h a h i g h e r i n c i d e n c e of a s e c o n d cancer. Thus, t h e y w e r e c o n s i d e r e d to h a v e an inherent i n c r e a s e in s u s c e p t i b i l i t y to cancer. T h e i n t e n t i o n was to find out if c a n c e r s u s c e p t i b i l i t y is a s s o c i a t e d w i t h e l e v a t e d SCE levels or w i t h an i n c r e a s e in general or specific c h r o m o s o m e breakage and r e a r r a n g e m e n t s .
PATIENTS AND CONTROLS E l e v e n patients (five male, six female, age range 6 0 - 8 1 years) w e r e s e l e c t e d w h o h a d d e v e l o p e d t w o or m o r e n e o p l a s m s that w e r e h i s t o l o g i c a l l y different, so that t h e y w e r e b o t h clearly p r i m a r y cancers, not a single c a n c e r w i t h metastases. The patients can be d i v i d e d into three c l i n i c a l groups as follows: G r o u p 1: T h r e e patients w h o s e first t u m o r was s q u a m o u s c a r c i n o m a of the skin, w i t h o u t m e t a s t a s e s to l y m p h nodes. G r o u p 2: F o u r p a t i e n t s w h o s e t u m o r s w e r e b o t h c a r c i n o m a s of i n t e r n a l organs but w h o h a d r e c e i v e d n e i t h e r r a d i o t h e r a p y nor c h e m o t h e r a p y prior to s a m p l i n g . G r o u p 3: F o u r patients w h o s e t u m o r s w e r e b o t h c a r c i n o m a s of i n t e r n a l organs but w h o had r e c e i v e d r a d i o t h e r a p y or c h e m o t h e r a p y . O n e p a t i e n t (DC8) in this g r o u p had b e e n treated w i t h c o m b i n a t i o n c h e m o t h e r a p y ( c h l o r a m b u c i l , vincristine, and p r e d n i s o l o n e ) o n the 2 days prior to s a m p l i n g . T h e o t h e r three p a t i e n t s had r a d i o t h e r a p y t r e a t m e n t for the first cancer. C l i n i c a l details of the p a t i e n t s are s u m m a r i z e d in Table 1. Six n o r m a l i n d i v i d u a l s , f o u r m a l e a n d two female, age range 2 5 - 5 0 , w h o w e r e all n o n s m o k e r s w e r e u s e d as controls.
METHODS M e t a p h a s e p r e p a r a t i o n s of P H A - s t i m u l a t e d l y m p h o c y t e s w e r e m a d e by r o u t i n e m e t h o d s f r o m s a m p l e s of w h o l e b l o o d [15]. Forty-eight h o u r c u l t u r e s w e r e u s e d to score c h r o m o s o m e breaks and rearrangements. For u n i f o r m q u a n t i t a t i v e assessment, c h r o m o s o m e d a m a g e was s c o r e d in 50 G i e m s a - s t a i n e d m e t a p h a s e s , e a c h w i t h 46 c e n t r o m e r e s , w h i c h w e r e not s e l e c t e d for
Table 1
C l i n i c a l details of d o u b l e c a n c e r patients Time before analysis of
Group 1
Group 2
Group 3
Patient
Sex
First tumor site
DC1 DC2 DC3 DC4 DC5 DC6
M M M F F M
Skin (multiple} Skin (multiple) Skin Colon Colon Bronchus
DC7 DC8
F F
Breast Colon
DC9 DC10 DC11
F M F
Breast Larynx Colon
Second tumor site Larynx Bladder Kidney Bladder Lung Chronic lymphocytic leukemia Rectum Prolymphocytic leukemia Bronchus Prostate Bladder
X-ray
Chemotherapy
Smoker
--2 Months ----
-------
No Yes No Yes No No
---
-" Day
No No
16 Years 12 Years 1 Year, 4 months
----
Yes Yes Yes
37
Cytogenic of Multiple Primary Cancers
quality. A n e u p l o i d and p o l y p l o i d cells seen w h i l e scanning for the 50 complete cells were recorded. Cells with more than 46 c h r o m o s o m e s were not scored as a n e u p l o i d if extra chromosomes were noticeably different and therefore probably "strays" from other broken metaphase cells. Banding (G and C) was used to locate damage. SCEs were scored in 30 fluorescence plus giemsa (FPG)-stained metaphases [16] from 66-hour cultures containing 20 I~g/ml b r o m o d e o x y u r i d i n e (BrdU). Samples from a patient and a control were assayed at the same time under identical conditions. RESULTS
SCE Frequency From Figure 1 and Table 2 it can be seen that one patient in each of groups 1 (DC2) and 2 (DC4) had slightly raised SCE levels that could have been due to smoking, but otherwise, SCE frequencies in patients and controls were similar in these two groups. No SCE score was recorded for one patient in group 1 (DC3) because of lack of metaphases in BrdU cultures. The patients and controls were not m a t c h e d for age, but age has been shown to have little or no effect on SCE frequency [17]. The most significantly raised SCE frequencies occurred in group 3. The high score in DC8 was probably due to recent chemotherapy, but patients DC9 and DCIO
Figure 1 Mean number of SCEs per cell for each patient compared with individual control and with pooled control mean and 95% distribution limits. Pooled control parameters include one mean per individual to avoid bias toward controls providing more than one sample. Significance of difference between patient and individual control: "0.01 < p < 0.05; *'0.001 < p < 0.01; ***p < 0.001. DOUBLE CANCER PATIENTS SCE frequency of patients compared with controls
Group 1
Group 2
Group 3
20 18
OPatient • Control
16 -
14 + 1.96~
12 03
~
Mean of controls
lO -
I
DC1
DC2
DC4
DC5 DC6 DC7 Patients
*1
DC8 DC9 DC10 DC11
1.96~
Significance difference
of
38
T. Brown et al. had similarly high SCE scores that could not be attributed to direct effects of treatment, as both these patients had received only radiotherapy many years previously.
Chromosome Damage and Aneuploidy At 48 hr, metaphase lymphocytes are almost all in first division in culture; therefore, chromosome damage seen at this stage was probably present in vivo. From 48-hr Giemsa-stained preparations (Table 2), 75 major damage events were found in 502 complete (46 centromere) metaphases from these double cancer patients. A complete spectrum of rearrangements was seen, including dicentrics, rings, translocations, pericentric inversions, and deletions (Fig. 2). Most of the damage was of the chromosome type; only two chromatid interchanges (quadriradials) and two chromatid deletions were found. In control samples, only three major damage events (all deletions) were seen in 550 metaphases. Chromosome damage was very unevenly distributed among patients (Table 2). Two patients in group 2 (DC4, DC6) and all four patients in group 3 (DC8 to DC11) had more damage than any of the controls. The frequency of gaps in chromosomes from patients was double that of the control group; nine patients had more than the control mean and two were above the 95% distribution limit. Similarly, aneuploid cells were twice as frequent in the patients as in the controls, with one patient outside the 95% limit. Telomeric stickiness was seen almost exclusively in one patient (DC8), most frequently involving chromosome #16 and G-group chromosomes. With a few exceptions, damage and chromosome loss tended to be distributed as expected [18] among chromosome groups, with damage events being proportional and chromosome loss inversely proportional to the length of the chromosome (Table 3). Chromosome #16 was involved in more events than expected, mainly in one patient (DC8), but G-banding showed no specific breakpoint or type of rearrangement. The only apparently nonrandom location of damage was in chromosome #2, where 12 of the 17 damage events involved the q-arm, including an endoreduplication and 10 events resulting in 2 q - (6 of these occurred in patient DC9). The main difference between distribution of chromosome loss in patients and controls was an excess loss from group C (Table 3), which G-banding showed to be due to selective loss of chromosomes #8, #11, and X. The excess in overall loss of chromosomes in the patients compared to controls could have been mainly mechanical loss during preparation, perhaps due to increased membrane fragility. Selective loss of chromosomes #8, #11, and X, however, probably reflects a true preferential loss of these chromosome in circulating lymphocytes. This selective loss was limited to five of the six female patients and was probably age-related, as preferential loss of C-group chromosomes in aging females is a well-known phenomenon [19, 20].
DISCUSSION The 11 double cancer patients were a heterogeneous group with a probable common susceptibility to cancer. No constitutional changes were detected, although this cannot be considered to be a negative association in view of the small sample size. The patients were divided into groups on a clinical basis (Table 1), and the cytogenetic results show that the first group (with initial skin cancers) was most like the controls, and the third group (with two carcinomas of internal organs, treated with radiotherapy or chemotherapy) deviated most from controls. When all the evidence is taken into account, 6 of the 11 patients showed evi-
DC1 DS2 DC3 DC4 DC5 DC6 DC7 DC8 DC9 DC10 DC11
2
180 30
30
30
10.3
12.26
12.0
1
0.2 1.0
1 0 0 3 0 5 1 38 6 4 17 75
8 30 0 30 30 30 30 27 30 30 30 275
9.9 13.4 0 12.7 11.0 10.0 9.9 17.6 18.6 15.9 9.7 12.9
bFrom all 48-hr preparations, banded and unbanded.
1
0.7
1 0.1
0 0 1 0 O 1 0 0 1 1 0 4
C/;IROMOSOME CHROMATID
Major d a m a g e
10
13
65 6
16 13 6 10 11 22 9 11 11 12 2 123
50
50
550 50
23 50 29 50 50 50 50 50 50 50 50 502
Gaps C e l l s scored
N u m b e r of d a m a g e e v e n t s ° i n 46 c e n t r o m e r e m e t a p h a s e s
°From 48-hr Giemsa-stained preparations only.
Total Pooled c on tr ols Total Mean/ sample Upper limit ( m ea n + 1.96 SD) Maximum in range
3
2
1
M e a n / C el ls cell scored
SCEs
Cytogenetic analysis
Group Patient
Table 2
15
15
68 6
5 6 11 3 10 11 10 44 5 11 12 128
+ (42-45) (47-48)
Aneuploid c e l l s / 50 (46 centromere) cells
1.0
2 0.2
Polyploid
Location
0
1 16
1 20
0
15
18 1
1
1
0
2 13
1 6
1
2
TELOMERIC MULTIPLE CHROMOSOME CHROMOSOME STICKINESS DAMAGE #16 2q 1
Type
Nonrandom damage b
¢.D
_.-
-t--IX..ll”-i^-
.-.
-.=---“..“l”,~““~-_-“_.-..“__~_I
.
;I_ i_._____.__
Examples of damage. (a) Two chromosome SCE score (39) and dicentric (Z C-group chromosomes).
Figure 2
A deletions
and fragments
(chromosomes
#
and #2) and centric
B ring (chromosome
#18). (b) High
41
Cytogenetic of Multiple Primary Cancers
Table 3
Location of damage and missing chromosomes
Damage events
A
B
C
1
2
3
(A) Total (B) Divided by chromosome pairs
20 20
17 17
12 12
14 7
78 10
Missing chromosomes
A
B
C
D
6 1
6 3
86 17
2.8 1.4
4.3 6.5
15.9 9.6
(A) Total Patients Controls (B) Percent of total Patients Controls
D
E 16
17
18
7 2
16 16
4 4
2 2
E
F
G
17 4
42 20
24 6
48 18
8.2 5.8
20.2 29.1
17.3 13.1
31.2 34.6
F
G
2 1
10 4
dence of c h r o m o s o m e instability in different ways (Table 2; patients DC4, 6, 8 through 11). This could only be partially accounted for in two patients; high SCE frequency was related to c h e m o t h e r a p y in patient DC8, and c h r o m o s o m e damage was related to recent r a d i o t h e r a p y in patient DC11. Cytogenetic analysis was carried out on PHA-stimulated lymphocytes, not on cells directly involved in the neoplasia. Even in the cases of chronic l y m p h o c y t i c leukemia (DC6) and p r o l y m p h o c y t i c leukemia (DC8), the leukemic cells were probably B lymphocytes, whereas the cells r e s p o n d i n g to PHA stimulation w o u l d be m a i n l y T l y m p h o c y t e s [21]. The considerable a m o u n t of nonspecific chromosome damage found in cells not directly involved in neoplasia suggests an underlying chromosome instability. Two of the double cancer patients with evidence of c h r o m o s o m e instability (DC9, DC10) had u n a c c o u n t a b l y high SCE scores. Chromosome breakage syndromes are k n o w n to be associated with high SCE frequencies s p o n t a n e o u s l y (Bloom's syndrome) or as an exaggerated reaction to carcinogens. It w o u l d be informative to take blood samples from cancer patients with s u s p i c i o u s l y high SCE frequencies before therapy and expose their l y m p h o c y t e s to a m i l d carcinogen in vitro to see if a hypersensitive response occurs. These results may augment present ideas regarding oncogenes, as cancer-related chromosome rearrangements have been shown to activate oncogenes that i n d u c e cell proliferation [see reference 3 for review]. Chromosome instability, as seen in some patients with double cancers, leads to breaks occurring at m a n y different chromosome locations in different cells. This must increase the chance of damage occurring, w h i c h could activate an oncogene to initiate the b u i l d - u p of a clone with the chromosome rearrangement that triggered the neoplastic transformation. Thus, tissues not involved in the n e o p l a s m may have nonspecific damage, while transformed cells show clonal d e v e l o p m e n t of specific types of damage. Continued instability w o u l d give rise to the possibility of a second p r i m a r y cancer d e v e l o p i n g also by a chance break activating a different oncogene. The types of cancer d e v e l o p e d w o u l d d e p e n d on w h i c h oncogene was activated in w h i c h tissue. A n a p p a r e n t l y heterogenous group of cancers could in this w a y have a c o m m o n initiatory mechanism in c h r o m o s o m e instability. In summary, our results tentatively indicate that a significant p r o p o r t i o n of peo-
42
T. B r o w n et al.
ple with multiple cancers may have an underlying inherent chromosome instability, w h i c h is s i m i l a r to t h e b e t t e r - d e f i n e d c h r o m o s o m e b r e a k a g e s y n d r o m e s . W e h a v e also p r o v i d e d s o m e s u p p o r t for t h e i d e a t h a t u n a c c o u n t a b l y h i g h SCE frequency may indicate cancer susceptibility in some individuals.
This work was carried out with financial support from the Cancer Research Campaign.
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