Malignant melanoma: Sister chromatid exchange analysis in three families

Malignant Melanoma: Sister Chromatid Exchange Analysis in Three Families Achille Ghidoni, Enrica Privitera, Elena Raimondi, Dario Rovini, Maria Teresa Illeni, and Natale Cascinelli

ABSTRACT: Sister chromatid exchange (SCE) was analyzed in stimulated lymphacytes and skin fibroblasts in members of three families with cutaneous malignant melanoma (CMM). Two of these families were characterized by familial CMM; the other family had one patient affected by CMM and two others with other cutaneous melanocytic lesions. All the patients had undergone surgery but no chemotherapy. Higher and differing SCE rates were found in lymphocytes and in fibroblasts of all patients. A wide range of SCE distribution was found in patients with high SCE rate. A few healthy close relatives also showed relatively high SCE rates and wide range distributions. These subjects may be regarded as a subset of family members at high risk for developing cancer. The variability of SCE rates and distribution may reflect genetic heterogeneity of CMM. INTRODUCTION Sister chromatid exchange (SCE) has been used as an indicator of possible alterations in the genetic material of cancer patients [1-6], although the exact nature of the lesions giving rise to SCE is still u n k n o w n [7-11]. In the neoplastic cells of a few tumors, such as leukemias [12-13] and lymphomas [14], the SCE rate has been found to be higher than the control rate. Similar results were obtained in cultured malignant m e l a n o m a cells [15]. Higher SCE rates have also been found in nonneoplastic cells (peripheral lymphocytes) of subjects affected by tumors such as multiple nevoid carcinoma [14], retinoblastoma [16], and in patients with cutaneous cancer after chronic exposure to arsenic [14]. These observations accord with the finding of high SCE frequency in some genetic diseases as Bloom syndrome [17], Xeroderma p i g m e n t o s u m after UV irradiation [18], or familial polyposis after mutagenic challenge in vitro [19], which are k n o w n to favor cancer development. In other investigations i n c l u d i n g sporadic cutaneous malignant m e l a n o m a (CMM) patients [20, 21], the possibility that SCE was influenced by the malignant disorder was excluded. However, the data reported in one of these studies [20] suggest that at least in some cases SCE rates may be influenced by malignant melanoma; another investigation on 36 sporadic m e l a n o m a patients examined in our laboratory [22] seems to confirm this view.

From the Istituto di Genetica, Universit~di Milano (A. G., E. P., E. R.), and the Istituto Nazionaleper lo Studio e la Cura dei Tumeri, Milano (D. R., M, T. I., N. C.), Italy. Address requests for reprints to Prof. A. Ghidoni, Istituto di Genetica, Via Celoria, 26, 20133 Milano, Italy. Received October 13, 1982; accepted December 30, 1982. 347 © ElsevierSciencePublishingCo., Inc., 1983 52 VanderbiltAve., New York, NY 10017

Cancer Geneticsand Cytogenetics 0165-4608/63/$03.00

348

A. Ghidoni et al. In the present work, an analysis was made of SCE frequency in three families with members affected by CMM or other cutaneous melanocytic lesions. The purpose of this study was to obtain a better knowledge of genetic factors that may favor this neoplasm for which various genetic mechanisms have been proposed [23-28].

MATERIAL ANDMETHODS Three families with two or three subjects affected by cutaneous malignant melanoma (CMM) or by a precancerous lesion were analyzed. In each family peripheral blood was obtained from all living patients and some of their close relatives (see pedigrees in Fig. 1). All the patients and one of the unaffected subjects also donated a skin fragment.

Family 1. CMM was diagnosed in the three patients, and subject 2 died from this neoplasm before the beginning of the study. Subject 3 was affected by multiple primary CMM. Subjects 3 and 4 had undergone radical surgery. Family 2. The diagnoses of the patients were as follows: CMM (subject 2), juvenile melanoma (subject 3), and junctional melanotic nevus (subject 4). All the patients had undergone surgery.

Figure 1 Pedigrees of families 1, 2, and 3 analyzed: malignant melanoma (hatchmarks), juvenile melanoma (broken lines), and junctional nevus (dots).

FAMILY 1

FAMILY

q

2

2

5 O1

FAMILY

3

$3&

D

~6

~ -~2

D ~5

349

Malignant Melanoma

Family 3. CMM was diagnosed in the two patients (3 and 4) and both had undergone radical surgery. None of the patients underwent chemical treatment with cytostatic drugs, immunological treatment, or radiotherapy. Sex, age, smoking habits, and other data of all subjects analyzed are reported in Table 1.

Whole blood cultures. Whole blood 10-ml cultures were set up with 0.7 ml blood in RPMI 1640 medium (Labtek) supplemented with 15% newborn calf serum, 100 IU/ml penicillin, 100 ~g/ml streptomycin, 0.2 mg/ml phytohemagglutinin (M form, Difco}, and 10 ~g/ml BuDR. Cultures were kept in the dark for 72 hr at 37°C. Two hours before arresting the cultures, colcemid was added at a final concentration of 0.05 ~g/ml. Blood cultures could not be repeated.

Skin fibroblasts cultures. Fibroblast cultures were established in 5% CO2 incubator from skin biopsies in Eagle's Minimum Essential Medium (Labtek) supplemented with 18% fetal calf serum and antibiotics as described above. Slides were prepared after a hypotonic treatment with 0.075 M KC1 for 10 rain at room temperature and fixation in the methanol-acetic acid mixture, by the air

Table 1

Clinical data of members

of the three families analyzed

Family

Subject a

Sex

Age

S m o k e r or nonsmoker

Clinical status b

Cell t y p e examined c

1

1 2 3 4 5 6 7 8 1 2 3 4 5 1 2 3 4 5 6 7

F M F M F M F F F M M F M F M F F F F M

67 69 d 35 43 37 42 12 7 44 48 21 19 / 16 72 78 41 48 46 17 13

NS NS NS NS NS S (15) e NS NS NS S (20) NS NS NS NS NS S (7-8) S (20) S (20) NS NS

healthy (s) C M M (s) C M M (s) C M M healthy healthy healthy healthy healthy (s) C M M (s) JM (s) JN healthy healthy healthy (s) C M M (s} C M M healthy healthy healthy

none none L,F L,F L none L L L,F L,F L,F L,F L L L L,F L,F L L L

2

3

~Numbered as in Figure 1. bCMM = cutaneous malignant melanoma; JM = juvenile melanoma; JN = junctional nevus; (s) = had undergone surgery for the neoplasm (8 months to 10 yr prior to the beginning of this investigation}. CL = lymphocytes; F = cutaneous fibroblasts. ~Deceased. eCigarettes/day in parentheses. rThis subject only was taking hormone contraceptives.

350

A. G h i d o n i et al. drying technique. Sister c h r o m a t i d differentiation was obtained by the FPG technique [29]. SCEs were observed and counted in cells w h i c h had replicated twice in presence of BuDR.

RESULTS

The pedigrees and all the available data of three families with recurrent malignant m e l a n o m a are reported in Figure 1 and in Tables 1 and 2.

Family 1. Two family members (3 and 4), sister and brother, affected by CMM were analyzed. Their ally high mean 8.8 ± 0.4. All case of subject patient 3).

father had died of the same neoplasm. Patient 3 revealed an u n u s u SCE rate (28.5 ± 1.5), whereas patient 4 h a d a m e a n SCE rate of unaffected relatives had lower mean SCE rates (6.4 +_ 0.3, in the 5, sister of the patients; 5.8 ± 0.3, in the case of the daughter of

Family 2. Father, son, and daughter were patients 2, 3, and 4, respectively, of this family. Their respective m e a n SCE rates were 8.1 ± 0.5, 9.2 ± 0.5, and 8.9 ± 0.4. The mean SCE rate values of the unaffected relatives were 5.2 ± 0.4 and 5.6 ± 0.4.

Family 3. Patients 3 and 4 are sisters. The study was e x t e n d e d to five close relatives, i n d i c a t e d as subjects 1, 2, 5, 6, and 7. Patient 3 had an SCE rate of 8.9 +- 0.6 and her sister, patient 4, an u n u s u a l l y high SCE rate of 28.0 + 1.7. Two unaffected relatives (subjects 1 and 2) h a d SCE rates w i t h i n normal range--i.e., 6.9 ± 0.4 and 6.4 ± 0.5, respectively. On the contrary, other unaffected relatives (subjects 5, 6, and 7) had high SCE rates (28.5 -+ 1.6, 18.7 -+ 1.2, and 25.7 ± 3.8, respectively). SCEs were also analyzed in low passage skin fibroblasts from seven affected subjects and one healthy donor. The SCE rate per cell was found to differ from that found in lymphocytes. In most cases (five out of seven patients) the fibroblasts showed higher values; in other cases the fibroblasts revealed lower values than the stimulated lymphocytes. The average SCE rate in 20 unrelated subjects was 7.05 ± 0.14. Single data of each of these subjects are not reported, since the s a m p l e was rather h o m o g e n e o u s concerning the SCE rates. The SCE rate in fibroblasts of the unique healthy donor e x a m i n e d was 9.5 ± 0.4, whereas the rate in l y m p h o c y t e s was 5.2 ± 0.4. As shown by standard errors reported in Table 2, the higher mean SCE rates are often a c c o m p a n i e d by a greater variability in the distribution of SCE per cell. If the patients of the three families are considered together, the majority (five out of seven) had a m o d e r a t e l y high SCE rate, the r e m a i n d e r (two out of seven) an u n u s u a l l y high SCE rate. The opposite was found with regard to the unaffected relatives: the majority (seven out of ten had normal SCE rates, the r e m a i n d e r (three out of ten) relatively high SCE rates. DISCUSSION

The higher SCE rates found in l y m p h o c y t e s of malignant m e l a n o m a patients agree with similar findings reported for only a few other n e o p l a s m s [14, 16]. Other authors [21] suggested that spontaneous SCE is not a marker of cancer risk in general. However, besides the w e l l - k n o w n SCE patterns in the Bloom s y n d r o m e [17, 30] and Xeroderma p i g m e n t o s u m [18], increased SCE rates have also been found in lymphocytes of untreated cancer patients after exposure to cigarette smoke condensate [31] and in l y m p h o c y t e s of cancer-prone subjects after treatment w i t h m i t o m y c i n C

2 3 4 3 4

2

35 35 25 30 45

35 50

NO. OF CELLS

8.1 9.2 8.9 8.9 28.0

28.4 8.8

SCE/ CELL

0.5 0.5 0.4 0.6 1.7

1.5 0.4

SE

Patients

lymphocytes

Lymphocytes

in stimulated

85 50 45 30 32

14 31

NO. OF CELLS

and

11.7 11.4 15.8 12.4 6.4

21.7 14.0

sC~_J CELL

0.3 0.4 0.5 0.7 0.3

1.6 0.7

SE

fibroblasts

Fibroblasts

in skin

1 2 5 6 7

8 1 5

5 7

Subject

of subjects

aThe mean SCE rate per cell was 7.05 (SE = 0.14) in a g r o u p of 20 u n r e l a t e d subjects (mean age: 39).

3

3 4

1

SCE analysis

Subject

Z

Family

Table

30 30 19 45 17

35 35 35 35 36

NO. OF CELLS

described

1.a

6.9 6.4 28.5 18.7 25.7

6.4 5.8 5.5 5.2 5.6

SCE/ CELL

Lymphocytes

0.4 0.5 1.6 1.2 3.8

0.3 0.2 0.5 0.4 0.4

SE

m

m

---67 --

NO. OF CELLS

Unaffected Relatives

in table

m

m

_

_

---9.5 --

SCF_d CELL

Fibroblasts

D

m

m

---0.4 --

SE

352

A. Ghidoni et al. [19]. These observations are in keeping with the widely assumed view that genetic lesion represents an important step in the development of cancer [32-34]. Different genetic mechanisms are known to underlie syndromes which favor cancer [11], and are thought to exist for tumors with identical diagnosis [24, 26]. Our results on CMM patients and their close relatives further support the concept of the existence of genetic lesions leading to the development of CMM and bring further evidence of its genetic heterogeneity [28, 35]. The findings of the present study acquire greater significance from the fact that the differences were observed within families. In each family higher and differing SCE rates were found in melanoma patients, while lower and more homogeneous SCE rates were found in the unaffected relatives, with the exception of family 3, where three subjects with high SCE rates had not developed tumors by the age of 46, 17, and 13 years, respectively. It appears that at least two levels of relatively high SCE rates are common to all CMM patients examined by us in this study. In families 1 and 3 both levels are apparently present, whereas in family 2 only the lower level is present in all patients. In family 2, however, only one of the patients was diagnosed with CMM, the others having other melanocytic lesions. A variety of causes, including the genetic makeup, could account for the variation in the SCE patterns observed between families. No conclusion could be drawn, in this regard, on the basis of the available data. Differences between SCE rates in lymphocytes and fibroblasts, respectively, also deserve further investigation, with particular attention to cases with an unusual discrepancy, such as that of 28.0 +- 1.7 (lymphocytes) vs 6.4 -+ 0.3 (fibroblasts) found in patient 4 of family 3. The greater variability observed in the distribution per cell of SCE frequencies in lymphocytes of two patients (patient 3 of family 1; patient 4 of family 3) and in three unaffected relatives (patients 5, 6, and 7 of family 3) cannot readily be explained. A similar observation was made in fibroblasts of patient 3 of family 1 and in patient 4 of family 2. This variability appears to largely exceed any expectation based upon the increase of the SCE rate, although cells with increased SCE rate have been observed to contribute to the skewness of the distribution significantly deviating from normal [36]. An interpretation of this phenomenon is made difficult by our poor knowledge of the mechanism for SCE formation. However, this observation, together with that of altered SCE in cultivated hamster cells treated with DNA damaging agents [37], suggests the existence of genetic damage in some of the subjects examined by us. The smoking habit of the subjects analyzed in the present study is apparently of little significance owing to the small number of smoking subjects, even though an effect of cigarette smoking on the SCE rate in cultured lymphocytes has been shown [38]. No effect of age on SCE could be observed in this small sample. Only patient 4 of family 2 was taking hormonal contraceptives. If a relationship exists between the SCE rate (and/or distribution) and the development of CMM or other precancerous lesions, as these observations seem to suggest, the SCE data could possibly serve as an indicator, in families with recurrent melanoma, of the risk of developing the neoplasm. In this case, the unaffected relatives in family 3 with high SCE rates and variability could be considered as subjects at high risk. This hypothesis can be tested only by observing the clinical status of all members of these families in the course of time. The existence of different levels of SCE rates and variability in different families may indicate, perhaps, different degrees of risk, in accordance with the probable existence of different genetic mechanisms for the development of melanomas [24]. This is not incompatible with

Malignant Melanoma the finding of differences between SCE rates of familial m e l a n o m a sporadic m e l a n o m a patients [22, 39]. With regard to inheritance of SCE rates, the observations made inconclusive; the SCE rate patterns observed in families 2 and 3 with d o m i n a n t inheritance of higher SCE rates and the possible c o m p l e m e n t a r y factors as was suggested for familial CMM [28].

353 patients and of in family 1 are are compatible i n v o l v e m e n t of

Supported by the G. Ambrosoli Fund. The authors are grateful to Prof. M. Sari for guidance in the statistical elaboration of data.

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