Cytogenetics in hereditary malignant melanoma and dysplastic nevus syndrome: Is dysplastic nevus syndrome a chromosome instability disorder?

Cytogenetics in Hereditary Malignant Melanoma and Dysplastic Nevus Syndrome: Is Dysplastic Nevus Syndrome a Chromosome Instability Disorder? Nell Caporaso, Mark H. Greene, Shein Tsai, Linda Williams Pickle, and John J. Mulvihill

ABSTRACT: Analysis of peripheral blood lymphocyte Giemsa-banded karyotypes was performed on 163 family members from 13 melanoma-prone families. Patients were classified regarding the presence of cutaneous melanoma and dysplastic nevi (a well characterized melanoma precursar), and each karyotype was scared far the n u m b e r of cells containing the following: major structural, minor structural, and numerical abnormalities. No clonal cytogenetic abnormalities were observed. Cutaneoas malignant melanoma and dysplastic nevi syndrome patients each had increased abnormalities of all types combined, compared with paoled controis (i.e., normal family members, and spouses; respectively, X2 = 6.02, p = 0.01; X2 = 5.29, p = 0.02). There was a statistically significant p-value for major structural abnormalities far melanoma patients and numerical abnormalities for the dysplastic nevi patients. Minor structural abnormalities did not differ in any of the groups. In addition, studies of ultraviolet induced sister chromatid exchange, in vitro tetraploidy, and extended prophase banding were performed on a limited number of patients. No significant differences between cases and controls were observed in these tests. Our data suggest that a chromosome instability abnormality may contribute to the pathogenesis of hereditary melanoma.

INTRODUCTION F a m i l i a l c u t a n e o u s m a l i g n a n t m e l a n o m a (CMM) w a s first d e s c r i b e d i n 1820 b y Sir W i l l i a m Norris, w h o r e p o r t e d a n a f f e c t e d f a t h e r a n d s o n [1]. O n e h u n d r e d fifty y e a r s later, E. P. C a w l e y r e p o r t e d C M M i n t h r e e m e m b e r s of a s i n g l e f a m i l y [2]. O v e r t h e n e x t t h r e e d e c a d e s s i m i l a r k i n d r e d s w e r e r e p o r t e d , b u t t h e b a s i s for f a m i l i a l s u s c e p t i b i l i t y to C M M r e m a i n e d o b s c u r e . In 1978, t h e c l i n i c a l a n d h i s t o l o g i c f e a t u r e s of a m e l a n o c y t i c p r e c u r s o r to C M M w e r e d e s c r i b e d i n s e v e n f a m i l i e s . T h e d i s o r d e r w a s d e s i g n a t e d t h e B - K m o l e s y n d r o m e ( n a m e d after f a m i l i e s B a n d K, t h e first t w o k i n d r e d s s t u d i e d ) [3, 4]. A f f e c t e d i n d i v i d u a l s h a d m o r p h o l o g i c a l l y a t y p i c a l m o l e s

From the Family Studies Section (N. C, M. H. G.) and Population Studies Section (L. W. P.), Environmental Epidemiology Branch, and Clinical Epidemiology Branch (J. J. M./, National Cancer Institute, Bethesda, and Biotech Research Laboratories (S. T.), Rockville, MD.

Address requests for reprints to Dr. Nell Caparaso, Family Studies Section, Environmental Epidemiology Branch, National Cancer Institute, Landou Bldg., Room 3C-29, Bethesda, MD 20892. Received June 26, 1985; accepted March 13, 1986.

299 This paper is U.S. government work, cannot be copyrighted, and lies in the public domain.

Cancer Genet Cytogenet 24:299 314(1987) 0165-4608/87/$0.00

300

N. Caporaso et al. that were larger and more irregular in outline, and more numerous than c o m m o n acquired nevi, variably pigmented, and tended to occur on sunshielded skin (e.g., the scalp and bathing trunk area), unusual sites for ordinary moles [5-9]. Histologically, these lesions were characterized by nuclear atypia and a disorderly growth pattern of melanocytes, leading to their current designation as "dysplastic nevi" (DN) [10]. The clinical features of familial dysplastic nevus syndrome (DNS) and the importance of its recognition in the identification of high risk patients and early diagnosis and cure of CMM, have recently been described [9-11]. Furthermore, such high-risk families provide a h u m a n model of the evolution of CMM from directly observable precursor lesions (dysplastic nevi); biological studies in this context may clarify disease mechanisms. For example, we and others have demonstrated that normal cells derived from patients with familial CMM and DN are unusually sensitive to the cytotoxic and mutagenic effect of ultraviolet (UV) light and UV-mimetic chemical carcinogens [12, 13, 34, 35]. Formal genetic analysis of our data has indicated that familial CMM/DN is an autosomal dominant disorder, and suggested that a CMM susceptibility gene might be located on the short arm of chromosome #1, near the Rh blood group locus [15-18]. Here we report the results of cytogenetic studies in members of CMM-prone families, in an effort to determine if chromosome abnormalities contributed to the development of CMM.

MATERIALS AND METHODS Study Design and Specimen Collection The total study cohort included 401 members of 14 melanoma-prone families. Details of the overall study design have been reported previously [11, 18, 19]. Briefly, all surviving first-degree relatives of family members with CMM (and the CMM patients themselves) were examined and classified in one of five groups: (a) melanoma (with or without DN); (b) dysplastic nevi (only); (c) normal blood relative; (d) spouse; and (e) status indeterminate (usually becuase of young age) [6]. With rare exceptions, the diagnoses of CMM and DN were established histologically. With informed consent, peripheral blood lymphocytes were obtained by venipuncture, and fibroblasts for tissue culture were derived from 4-mm punch biopsies of normal skin.

Peripheral Blood Karyotypes Coded, heparinized samples of peripheral blood from 163 subjects from 13 of the 14 melanoma-prone families were karyotyped. Buffy-coat cells were cultured for 72 hours at 37 ° C in RPMI 1640 medium supplemented with heat-inactivated 10% fetal calf serum, 100 U/ml penicillin, 100 p.g/ml streptomycin, and 2% phytohemagglutinin (PHA). Cells were harvested following 1-2 hours incubation in the presence of 0.05 ~g/ml colcemid. Chromosome preparations were made from fixed (methylacetic acid) cells exposed to hypotonic solution (0.075 M KC1) for 15 minutes. Chromosomes were stained using a standard Giemsa-trypsin technique [20]. From 20 to 57 metaphases (mode, 30) were examined for each individual. Each karyotype was classified as normal or demonstrating one or more of three classes of abnormalities: (a) major structural abnormalities, such as rings, transtocations, fragmentation (three or more chromosomes), dicentrics, insertions, or deletions; (b) minor structural abnormalities, such as breaks or gaps; (c) numerical abnormalities, such as scored in cells with aneuploidy including polyploidy, trisomy, pseudodiploidy, etc., but excluding isolated monosomies unless the same chromosome or chromosome groups was involved in two or more cells [21, 22]. The percentage of cells

Hereditary CMM and NS

301

with each type of abnormality was recorded. Certain patients were excluded from the final analysis: eight patients whose DN status was indeterminate, and five CMM patients who had received chemotherapy prior to phlebotomy. Melanoma patients who were treated with surgery, Bacillus-Calmette-Guerin (BCG), or local radiation were included. Extended prophase banding was performed on five CMM/DN patients. The high resolution banding technique used is described in detail elsewhere [23].

In Vitro Tetraploidy Fibroblast cultures were established from 4-mm punch skin biopsies (Meloy Laboratory, Springfield, VA; Flow General Laboratory, McLean, VA). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum, 100 U/ml penicillin, and 100 ~.g/ml streptomycin. To make chromosome preparations, colcemid (0.1 ~g/ml, final concentration) was added to a flask of actively growing fibroblast culture for 2-4 hours before harvesting. Slides were made using standard techniques, treated with Giemsa stain, and examined for tetraploidy. Between 450 and 500 metaphases were examined for each of 23 patients (HCMM/DNS) and 24 controls (spouses). Disease status, age, sex, passage number (representing the length of time that cells were carried in tissue culture), and the percentage of cells exhibiting tetraploidy were recorded. Specimens in which tetraploidy was observed in more than 7% of metaphases were classified as abnormally elevated, in accord with previous studies [24, 25].

Induced Sister Chromatid Exchange Heparinized blood samples were obtained from nine patients (CMM/DNS) and nine controls. The blood was allowed to settle for 1-2 hours, at which time 1 ml of lymphocyte rich plasma was added to 9 ml of Ham's F-12 medium (without thymidine) supplemented with 20% fetal calf serum and 0.1% penicillin and streptomycin. Irradiation was performed with a germicidal lamp emitting predominantly 254 nm UV. A total UV dose of 15 ergs/mm 2 was delivered to the cell suspensions in Petri dishes (100 × 20 mm) at an incident flux of 2.5 ergs/mm2/sec. The suspensions were transferred to plastic flasks (25 cm2), PHA and BrdU (final concentration, 25 Win) added, and the cultures grown in the dark for 72 hours at 37°C. Cells were harvested following a 1-hour treatment with colchicine (0.2 ~g/ml, final concentration) and chromosome preparations were made in a standard manner. Differential staining for the detection of SCEs was accomplished using the method of Goto et al. [26]. Slides were coded, and 30 second-division metaphase cells were analyzed for sister chromatid exchanges (SCE) for each individual, both baseline and following UV treatment.

Statistical Methods Means of assay results from affected patients and controls for the continuous variables were compared using the t-test [27]. A chi-square statistic was used to test for the independent effects of the categorical variables [28J, and a linear regression procedure was used initially to test for the effects of interactions among the variables [29]. In the karyotyped patients, the presence or absence of each type of chromosome aberration was initially treated as a dichotomous variable. A Wilcoxon ranking statistic, using the SAS NPARlWAY procedure [30], was employed to compare the case and control groups after having ranked each patient by percentage of cells with each of the three types of chromosome abnormalities.

302

N. Caporaso et al.

RESULTS Peripheral Blood Karyotypes Of 150 patients eligible for analysis, 33 had CMM (of w h o m 31 also had DN), 32 had DN only, 50 were clinically normal blood relatives and 35 were spouses. These patients were d r a w n from 13 i n d e p e n d e n t m e l a n o m a - p r o n e families. No consistent clonal abnormalities were observed in any patient. Overall, 21% (31/150) of subjects' karyotypes i n c l u d e d at least one cell with a major structural abnormality, 25% (38/150) had minor structural abnormalities, and 33% (50/150) had n u m e r i c a l abnormalities. There were 85 w o m e n and 65 men in the study; in the aggregate the w o m e n had slightly more major structural abnormalities and numerical aberrations, but the m e n had slightly more abnormalities of all type combined. 1 In no instance was the difference statistically significant. Normal family members and spouses did not differ significantly in any of the three categories (Table 1), although spouses had somewhat more numerical abnormalities than normal family members (×2 = 2.94, p = 0.09), whereas, normal family members had slightly more structural abnormalities than spouses (×2 = 1.51, p = .22). Normal family members (mean age, 30.1) were considerably younger than spouse controls (mean age, 46.7). The age difference accounted for the different rates of n u m e r i c a l abnormalities observed in these two control groups. This was expected because the frequency of sex chromosome abnormalities increases with age [31]. Age was not related to structural abnormalities. Accordingly, w h e n considering the presence of any of the three types of aberrations, normal family members and spouses were pooled to simplify comparisons. Pooling the control groups resulted a nearly identical age distributions in cases (mean, 37.8 years) and controls (mean, 36.9 years). The relationship between age and chromosome abnormalities is d e p i c t e d in Figures 1-4. We analyzed the data by family of origin, in order to explore the possibility that a specific family, or subgroup of families, possessed an exceptionally high proportion of i n d i v i d u a l s with abnormal karyotypes, and was thus " d r i v i n g " the association attributed to the group as a whole. We ranked families according to the percentage of i n d i v i d u a l s per family with chromosome abnormalities and on this basis grouped families into three categories of high, intermediate, and low levels. Then we calculated the percentage of both affected and normal patients in each group, for each type of chromosome abnormality. In every category, the percentage of affected i n d i v i d u a l s possessing karyotype abnormalities was greater than the percentage of normals (Table 2). Although the numbers in each subgroup are too small to demonstrate statistical significance, these data suggest that the prevalence of c h r o m o s o m e aberrations in affected individuals is i n d e p e n d e n t of family source. CMM patients and DN patients each showed significant excesses of " c o m b i n e d " chromosomal abnormalities (×2 = 6.02, p = 0.01 and ×2 = 5.29, p -- 0.02, respectively) (Table 3). Major structural abnormalities were increased in both groups, significantly so in the CMM group. Numerical abnormalities were also increased in both groups, but statistical significance was achieved only in the DN group w h e n c o m p a r e d with pooled normals (Table 3). Patients with both m e l a n o m a and DNS did not differ significantly from those with DNS alone. Minor structural abnormalities did not differ significantly among any of the subgroups. Patients were ranked 1Combined abnormalities defined as subjects with cells having either (or both) major structural or numerical abnormalities. This was considered a nominal (dichotomous) variable. Minor structural abnormalities were not included.

85

Pooled normals 37.3

36.9 31

12

11" 8 19 3 9

No.

Major

21

14

33 25 29 9 18

%

38

19

8 11 19 9 10

No.

Structural Minor

25

22

24 34 30 26 20

%

50

21

29 b 12 9

175

12

No.

25

36 49 45 34 19

%

33

Numerical

65

28

19 ° 18 375 13 15

No.

TotaF

43

33

58 56 57 37 30

%

once.~T°talpersons with either major structural, numerical, or both types of cytogenetic abnormalities. Patients with more than one class of abnormality are counted only

bWhen compared with pooled normals, p < 0.01 (chi-square).

aWhen compared with pooled uormals, p < 0.05 (chi-square}.

150

33 32 65 35 50

Melanoma Dysplastic uevi Pooled disease Spouse Normal relation

Total

(yr}

Number

status 40.6 35.0 37.8 46.7 30,1

Mean age

Chromosome abnormalities by type and diagnosis

Subject

Table 1

09

z

C~

t.,0 uJ

o

tr o z <

.50

I I-

.40

NUMERICAL

_J

< D a

.30

t~ z

---4

.20

.10

0

MAJOR STRUCTURAL

I

I

II

III

IV

AGE QUARTILES

Figure 1

The percentage of individuals w i t h a b n o r m a l cells by age quartiles. A trend tow a r d increased numerical abnormalities w i t h age is seen, whereas, no s u c h trend is a p p a r e n t w i t h major structural abnormalities. The age qnartiles b r e a k d o w n is as follows: I, n = 37, range 8-25, m e a n 18.5; II, n = 38, range 26-35, m e a n 29.8; III, n = 36, range 36-49, m e a n 42.6; IV, n = 39, range 50-79, m e a n 57.5. Vertical bars over data points define standard error of the mean. F i g u r e 2 The m e a n percentage of abnormal cells per individual by age quartiles. As in Figure 1, numerical abnormalities increase w i t h age, whereas, major structural abnormalities s h o w no particular trend. The age distribution is the same as in Figure 1. NUMERICAL ' ABNORMALITIES

D

Z

,q, (3

rr"

O Z

,< z<

MAJOR STRUCTURAL ABNORMALITIES

1

L

IV AGE QUARTILES

305

Hereditary CMM and NS

6

POOLED DISEASE

> 5 Z

-~-

5

~

4

o z m <

3

z

POOLED NORMALS

1

0

I

I

I

I

I

II

Ill

IV

AGE QUARTILES F i g u r e 3 The relation between age and n u m e r i c a l abnormalities by breaking d o w n the subjects into p o o l e d controls (spouses and n o r m a l relations) and p o o l e d disease (DNS and C M M / DNS). A trend t o w a r d increasing n u m e r i c a l abnormalities in the highest age quartJles is seen. In a d d i t i o n , the significance of the difference in n u m e r i c a l abnormalities between diseased and n o r m a l is apparent in three of the f o u r quartiles. The age quartiles b r e a k d o w n is as follows: POOLEDDISEASE: I, n = 15, range 10--25, mean 19.6; H, n = 17, range 26-35, mean 30.4; III, n = 16, range 38-49, mean 43.6; IV, n = 17, range 50-75, mean 55.8. POOLEDCONTROLS: I, n = 22, range 8--23, mean 17.8; II, n = 21, range 26 34, mean 29.3, IlI, n = 20, range 36-49, mean 41.9. IV, n = 22, range = 52-79, mean = 58.8.

by the percentage of cells exhibiting each of the three classes of c h r o m o s o m e abnormalities; case and control groups were compared using a Wilcoxon statistic. Results were similar to those found using dichotomous variables, i.e., both the melanoma and DN groups had significantly more abnormalities than pooled controls; n u m eri c a l abnormalities were significantly increased in the DN group (Table 4), whereas, structural abnormalities were significantly increased in the m e l a n o m a group. W h e n all DN patients were analyzed as a group (combining patients with

Table 2

Affected versus controls in high, intermediate, and low c h r o m o s o m e abnormality families Major structural

High Intermediate Low Overall

Numerical

Combined

Normal (%)

Affected (%)

Normal (%)

Affected (%)

Normal (%)

Affected (%)

29 14 5 14

40 50 8 29

47 22 13 25

59 29 21 45

55 36 16 33

81 53 29 57

306

N. C a p o r a s o et el.

D

3

SPOUSES

Z g3 uJ

"~ .<

2

rr

o z rn <

RELATIVES

o~ Z

< ILl

1

J

I

II

J

I

ill

IV

AGE QUARTILES

F i g u r e 4 The control group is divided into spouses and normal relations, illustrating their variation with age. Both groups show increased numerical abnormalities with age. Note that there are no spouses in the youngest age quartile. This figure supports the contention that the difference in mean numerical abnormalities between spouses and normal relations is accounted for by the difference in their respective age distributions. The age quartile breakdown is as follows: NORMALRELATIONS: I, n = 22, range 8-23, mean 17.8; II, n = 12, range 26-33, mean 29.1; III, n = 9, range 36-46, mean 39.4; IV, n = 7, range 52-67, mean 58.4. SPOUSES:I, n = 0; II, n = 9, range 26-34, mean 29.6; III, n = 11, range 36-49, mean 43.8; IV, n - 15, range 52-79, mean = 59.

DN o n l y a n d DN p l u s m e l a n o m a ) , b o t h s t r u c t u r a l a n d n u m e r i c a l a b n o r m a l i t i e s s h o w e d h i g h l y s i g n i f i c a n t i n c r e a s e s c o m p a r e d w i t h c o n t r o l s (Table 4/.

Prophase Banding P r o p h a s e b a n d i n g w a s p e r f o r m e d o n five p a t i e n t s ( r e p r e s e n t i n g five s e p a r a t e f a m i lies) w i t h CMM/DN. All five h a d n o r m a l k a r y o t y p e s .

Tetraploidy F i b r o b l a s t s f r o m 23 f a m i l y m e m b e r s ( r e p r e s e n t i n g 14 s e p a r a t e families) w i t h C M M / DN a n d 24 s p o u s e c o n t r o l s w e r e s t u d i e d . Overall, 13 of t h e 47 f i b r o b l a s t l i n e s rev e a l e d s i g n i f i c a n t t e t r a p l o i d y (greater t h a n 7%). F i v e of 24 (21%) n o r m a l s e x h i b i t e d s i g n i f i c a n t t e t r a p l o i d y , w h e r e a s , e i g h t of 23 C M M / D N (35%) w e r e a b n o r m a l , a diff e r e n c e t h a t w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t (Table 5).

307

Hereditary CMM and NS

Table 3

Summary

of s t a t i s t i c a l c o m p a r i s o n s

various subgroups

made between

of a f f e c t e d f a m i l y m e m b e r s

and

pooled normal study subjects

Pooled normals (n = 85) Major s t r u c t u r a l Xz= p= Numerical X ~= p= Combined b

13% ° --25% --33%

Melanoma (n = 33)

DNS without melanoma (n = 32)

Pooled affecteds (n = 65)

33%

25%

29%

5.59 0.02 36%

1.94 0.16 53%

1.60 0.20 58%

5.13 0.02 45%

8.56 0.003 56%

6.57 0.01 57%

X z=

--

6.02

5.29

8.63

p

--

0.01

0.02

0.003

=

n = Number of individuals in each study group. Percentage of individuals with at least one of the indicated abnormalities. b Combined abnormalities, either major structural or numerical abnormalities.

Table 4

A n a l y s i s of d i s e a s e g r o u p s u s i n g W i l c o x o n ranking statistic

Diagnosis

Major structural

Numerical

z = 2.39 p = 0.02

1.77 0.08

2.80 0.005

z = 1.45 p = 0.16

3.25 0.001

2.81 0.005

z = 2.31 p = 0.02

3.05 0.002

3.44 0.0006

Combined °

Melanoma

Dysplastic nevi

DN p l u s m e | a n o m a b

°Combined abnormalities here refer to the sum of the percentage of cells counted having either major structural, or numerical abnormalities, or both. bIncludes all subjects with either melanoma, or dysplastic nevi, or both.

Table 5

Prevalence

of i n v i t r o

tetraploidy among melanoma/dysplastic

nevus

patients and controls Gro up

N u m b e r > 7%

n

%

Co ntro ls CMM/DN

5 8

24 23

21 35 °

aXZ = 1.14;p = 0.29.

308

N. Caporaso et al.

The percentage of cells in each line exhibiting tetraploidy was then e x a m i n e d as a continuous variable and linear stepwise regression was performed to determine if any of the d e p e n d e n t variables influenced tetraploidy levels significantly. Neither age, sex, passage number, nor disease status were related to tetraploidy. Of all the variables examined, passage n u m b e r was most closely correlated with increased t e t r a p l o i d y (R 2 = 0.059; F = 1.65; p = 0.21), but none of the variables was highly predictive. We also c o m p a r e d mean percent tetraploidy, treating the data as continuous rather than ordinal, using both a t-test, and a Wilcoxon ranking procedure (ordering i n d i v i d u a l s in terms of increasing percentage of cells with tetraploidy); both techniques detected no significant difference between affected and unaffected study subjects. Induced Sister C h r o m a t i d Exchange

The nine CMM/DN patients and nine controls had similar rates of SCE, both on baseline determination and following exposure to UV radiation (Table 6). DISCUSSION

We a p p l i e d various cytogenetic assays to fresh l y m p h o c y t e s and fibroblast cell lines obtained from carefully characterized members of m e l a n o m a - p r o n e families, in order to clarify the pathogenesis of this autosomal d o m i n a n t cancer syndrome. The most striking finding was a significant excess of a p p a r e n t l y r a n d o m c h r o m o s o m e abnormalities in subjects with CMM/DN or DN alone, c o m p a r e d with normal family members and spouses studied at the same time in the same laboratory. Neither age, sex, nor family of origin could account for the abnormalities found. Because significant abnormalities were seen in both the disease groups (CMM and DNS) and because most m e l a n o m a patients had DNS as well, linear stepwise analysis of the data was performed to separate the relative contributions of m e l a n o m a and DN to the abnormalities recorded. The overlap between these two groups was so extensive (i.e., 31 of 33 m e l a n o m a patients also had DN) that the regression technique could not identify a relationship between CMM and the cytogenetic abnormalities that was i n d e p e n d e n t of that identified for DN. The similarity between dysplastic nevi patients who have or have not d e v e l o p e d m e l a n o m a provides further support for the strong relationship between these two conditions [11, 15]. We identified no specific, consistent, clonal cytogenetic abnormality in persons with or at high risk of hereditary melanoma. These data suggest that a chromosomal instability disorder resembling that seen in Bloom's s y n d r o m e [32] or F a n c o n i ' s anemia [33] may be present in high-risk i n d i v i d u a l s from m e l a n o m a - p r o n e families.

Table 6

Baseline and post-UV sister c h r o m a t i d exchange in m e l a n o m a / d y s p l a s t i c nevus patients and controls SCE/cell (_+ SD)

Diagnosis

n

Baseline

Post-UV challenge

Normal CMM/DNS

8 9

14.3 (+ 2.66) 13.0 (+ 0.87)

17.0 (+ 1.79) 16.5 (+ 1.92)

°t-Test, p > 0.05.

Hereditary CMM and NS

309

Such an abnormality might contribute to a mutagenic or carcinogenic event if the chromosomes in CMM/DN patients were unusually susceptible to injury by environmental carcinogens. Considerable data suggest that normal fibroblasts and Ebstein-Barr virus transformed peripheral blood lymphocytes from CMM/DNS patients are unusually sensitive to the cytotoxic and mutagenic effects of both UV radiation [13, 34-36] and the UV-mimetic chemical carcinogen 4-nitroquinoline-1oxide [12, 36]. The possibility that these cytogenetic abnormalities reported here represent indirect evidence of a chromosomal instability syndrome is also consistent with the recent observation that fibroblasts from CMM/DN patients show a significant excess of chromosome gaps and breaks following exposure to ionizing radiation during G2 of the cell cycle [14]. It must be emphasized that the standard karyotype analyses performed in the current survey provide no direct link between environmental carcinogens and the cytogenetic abnormalities detected. Such a hypothesis will require further study employing methods designed specifically to test such a model. Danes et al. have reported that cells cultured from dermal skin biopsies from family members with or at risk of certain heritable cancer syndromes reveal in vitro alterations in chromosome number, which apparently distinguish affected from unaffected members of the same kindreds [24]. Tetraploidy in cultured skin fibroblasts was reported to be increased in affected patients with Gardners' syndrome but not in familial polyposis coli [37-40], and in normal persons with a family history of cancer [41]. They proposed that increased tetraploidy occurred in those epithelial tissues that are at increased risk of malignant transformation. More recently, these investigators have reported an increase in chromosome number, "in vitro hyperdiploidy," in familial breast cancer patients and their high-risk female relatives [25], and in patients with familial malignant melanoma [42]. In the melanoma study, increased hyperdiploidy occurred in dermal monolayer cultures from four of four affecteds, two of 14 high-risk family members, and none of four spouses. It is not clear to what extent tetraploidy accounted for the hyperdiploidy observed in this series. We determined tetraploidy levels on 47 patients and controls, and found no significant correlation with sex, age, number of passages in cell culture, or disease status. Using standard fibroblast cell lines, and considering age, sex, passage number, and disease status, we found no difference in the prevalence of tetraploidy among the cells of CMM/DN patients compared with simultaneously tested controls. Overall, the levels of tetraploidy were high compared with those reported elsewhere [43, 44]. This may be due to differences in technique or other nonspecific or unknown factors [45]. An analysis of the 14 kindreds from which these subjects were chosen showed no excess of nonmelanoma cancers, thus, family cancer history is not a likely explanation [46]. Slides from 34 subjects were also examined to determine if hyperdiploidy exclusive of tetraploidy contributed to observed polyploidy. Of the 218 polyploid ceils counted, only six (3%) had a chromosome number less than 86 (Table 7). Thus nontetraploid hyperdiploidy did not contribute significantly to polyploidy in fibroblasts in either cases or controls in our series. SCE is the reciprocal interchange of DNA between chromatids, which is visualized after differential staining in chromatids substituted with BrdU [47]. It is considered a sensitive indicator of chromosome damage and the clastogenicity of various physical and chemical agents [48]. Consistently elevated levels of SCE have been demonstrated in lymphocytes and fibroblasts from Bloom's syndrome patients [49, 50]. Normal frequencies of SCE are found in cells heterozygous for the Bloom's syndrome gene, and also in cells either homozygous or heterozygous for ataxia telangiectasia [51], Fanconi's anemia [52], and xeroderma pigmentosum [53], other

•. Caporaso et al.

310

Table 7 Distribution of chromosome number in polyploid cells in skin fibroblast cell cultures Chromosome number

<86

86

87

88

89

90

91

92

93

94

Total

N o r m a l s (n ~ 15) C M M / D N S (n - 19) T o t a l c e l l s (n - 34)

4 2 6

2 0 2

1 7 8

4 2 6

5 14 19

10 27 37

17 20 37

31 51 82

7 11 18

1 2 3

82 136 218

n

n u m b e r af individuals in each group.

disorders characterized by chromosomal instability. Thus, the chromosome instability syndromes [54-56], with the notable exception of Bloom's syndrome [57, 58], demonstrate normal SCE levels. Overall, there is not a consistent correlation of SCE levels and chromosome aberrations in human malignant disease [59]. Studies of SCE and melanoma have not revealed consistent abnormalities [6062]. Ramsay reported that spontaneous and UV light-induced SCE frequencies were similar in high-risk members of two melanoma families and controls [34]. In our study we found no indication of baseline or UV-induced SCE abnormalities in HCMM/DN. We have also recently failed to find significant differences in either baseline or stimulated (4-NQO, mitomycin-C, MNNG) SCE in six additional CMM/ DN patients compared with six controls [63]. The lack of SCE abnormalities may not be surprising because the other chromosome instability syndromes with the exception noted above, have normal SCE levels. In addition, it is apparent that SCE show wide variability in sensitivity to different environmental insults, disease states, therapies employed, and tissues tested (e.g., fibroblasts versus lymphocytes). Various changes in experimental design can be envisioned that might reveal SCE abnormalities, e.g., employing fibroblasts instead of peripheral blood lymphocytes, using UV-B rather than UV-C irradiation, testing nevus or tumor tissue, controlling for smoking, or using greater numbers of samples to increase the power of the test. The range of chromosome abnormalities linked with specific tumors and increased risk of tumors is quite broad. At one extreme are highly specific abnormalities generally involving malignant cells only (i.e., Philadelphia chromosome in CML), which contrasts with seemingly random, constitutional abnormalities of the chromosome instability disorders. The constitutional abnormalities described here in melanoma-prone family members resemble the latter type. However, direct studies of melanoma tissue have suggested that nonrandom chromosome abnormalities occur [64], although no uniform, precisely delineated specific lesion has been identified as a characteristic of all melanoma tumor cells [65]. One mechanism of chromosome damage that might encompass both general types of chromosome injury is breakage, which preferentially involves fragile sites. An association between the human fragile sites and the specific sites of chromosome rearrangement characteristic of a number of different tumors has been postulated [66, 67]. One possible explanation for a "chromosome instability" pattern of nonspecific defects such as that seen in our families may be an abnormality of chromosome fragile sites. Such a mechanism could explain the seeming paradox of the occurrence of site-specific cancer excesses in chromosomal instability disorders, which have been considered conditions in which chromosome breaks are random [69, 70]. That is, the apparent random nature of these cytogenetic lesions may have as its basis multiple specific sites of damage (i.e., the fragile sites). Direct cytogenetic studies of melanoma tissue have revealed various nonrandom changes, particularly of chromosomes #1, #2, #3, #6, and #7 [64, 71,721. Linkage analysis of the same families whose cytogenetic studies are summarized above has suggested that a familial melanoma susceptibility gene may be located on the short

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arm of chromosome #1, near the Rh blood group locus [18]. The gene controlling expression of the m e l a n o m a tumor antigen p97 has been tentatively mapped to chromosome #3, near the transferrin receptor [73]. Some investigators have suggested linkage between the familial m e l a n o m a gene and the major histocompatibility complex on chromosome #6, although evidence from a recent linkage analysis study appears to refute this hypothesis [74]. In spite of these clues regarding specific chromosome segments that might be involved in the origin of melanoma, neither standard nor extended cytogenetic studies have revealed consistent or clonal defects in persons with or at high risk of melanoma. Rather, our data and those of others [35] suggest a complex interplay of multiple genetic, cellular (? metabolic), cytogenetic and e n v i r o n m e n t a l factors in the pathogenesis of familial melanoma. The "chromosomal instability" pattern described herein is compatible with either enhanced susceptibility to DNA damage from e n v i r o n m e n t a l carcinogens or abnormal repair of such injury. Previous studies of these families provide precedent for both hypotheses. Additional research is required to clarify our u n d e r s t a n d i n g of the neoplastic process in hereditary CMM, and to determine if this pattern of cytogenetic abnormalities is specific for hereditary CMM, DNS, or m e l a n o m a patients in general. Supported by NIH contracts N01-CP-21021, N01-CP-21037, and N01-CP-21031. The authors thank Dr. Uta Francke for conducting the extended chromosome studies; Drs. Kenneth H. Kraemer and Peter Kohn for performing the sister chromatid exchange assays; and Drs. Jacqueline Whang-Peng, Gloria Balaban, and Sherri Bale for helpful comments on this manuscript.

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