Cytogenetic and Interphase FISH Analyses of 73 Basal Cell and Three Squamous Cell Carcinomas

Cytogenetic and Interphase FISH Analyses of 73 Basal Cell and Three Squamous Cell Carcinomas: Different Findings in Direct Preparations and Short-Term Cell Cultures Rosario Casalone, Daniela Mazzola, Rossana Righi, Paola Granata, Elio Minelli, Maurizio Salvadore, Maurizio Lombardo, and Egidio Bertani

ABSTRACT: Cytogenetic analysis performed on 73 sporadic basal cell carcinomas (BCCs) and three squamous cell carcinomas (SCCs) showed different findings in direct preparations (24 hours) and in short-term cell cultures. Except for loss of the Y chromosome, not one of the other clonal (⫹6, ⫹16, add(2)(q37), del(3)(q13), add(1)(p31), and near triploidy) or sporadic changes found in direct preparations was found in cell cultures and vice versa. Clonal trisomy 6 found in two BCC direct preparations and demonstrated by interphase fluorescence in situ hybridization in 8 other cases seems to be a nonrandom change in basal cell carcinoma. Immunohistochemistry showed that the cell type investigated was different in the two methods of analysis used: epithelial in direct preparations and fibroblastic in cell cultures. Thus, the results obtained in direct preparations indicate the BCC or SCC epithelial karyotype, whereas the aberrations found in cell cultures indicate the presence of chromosome instability in the fibroblastic stroma. The apparent lack of correspondence between direct and indirect preparations and the presence of clonal chromosome changes in both epithelial and stromal cells suggest tumor cell heterogeneity of BCC. The fibroblastic stroma seems to be implicated in the neoplastic process. This is not evident in SCC, in which clonal changes are present only in direct preparations. The chromosomal distribution of the breakpoints involved in structural changes in direct and cell culture preparations is random; together with those reported in the literature, the breakpoints found in BCC cultures show, however, a cluster to 1p36, 3q13, 9q22, 14p11, 15p11, and Xp11 bands. We did not find any significant correlations between BCC cytogenetic results and the clinical data (site, age, sex, recurrence). The incidence of cases of BCC (38%) and of SCC (100%) showing clonal chromosome changes agree with their benign and malignant nature, respectively. Finally, a significantly high incidence of constitutional inv(9) and dup(9)(q11q21) was found in the group of patients with BCC. © Elsevier Science Inc., 2000. All rights reserved.

INTRODUCTION The results of cytogenetic analysis performed on basal cell carcinoma (BCC) of the skin show the presence of numerical or structural clonal chromosome changes or both in 42 of the 79 cases reported in the literature [1–9]. Except for a

From the Laboratorio di Citogenetica (R. C., D. M., R. R., P. G., E. M.), the Instituto Anatomia Patologica (M. S.) and the Clinica Dermatologica (M. L.), Ospedale di Circolo e Università, Varese, Italy; and the Clinica Dermatologica, Ospedale di Gallarate (E. B.), Gallarate, Italy. Address reprint requests to: Dr. R. Casalone, Laboratorio di Citogenetica, Ospedale di Circolo e Università, Viale Borri 57, 21100 Varese, Italy. Received August 10, 1998; accepted September 3, 1999. Cancer Genet Cytogenet 118:136–143 (2000)  Elsevier Science Inc., 2000. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

reciprocal t(9;16)(q22;p13), found in three BCC short term cell cultures [8], no other recurrent structural chromosome abnormality has been identified as yet. Simple clonal numerical changes, most commonly ⫹18, ⫹9, ⫹20, ⫹7, and ⫹5 were recently described [9]. The data reported in the literature were, however, obtained from histologically undefined cell culture populations or from a mixture of fibroblastic and epithelial cells; thus we cannot exclude the possibility that the chromosome changes described in BCC are restricted to the nonneoplastic stroma. To prevent fibroblastic contamination, we have performed cytogenetic analysis on direct preparations from 73 BCCs. Fluorescence in situ hybridization (FISH) analysis with the use of a centromeric probe specific for chromosome 6 was also performed in 16 cases to test the pres-

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Cytogenetic Analyses of BCC and SCC Table 1 Clinical data and cytogenetic findings in 73 basal cell carcinomas Direct preparations Case Age/ no. sex

Tumor site

Total no. of cells

1 2 3 4 5 6 7

74/M 59/F 34/M 46/M 71/F 58/F 78/F

Abdomen Head Head Head Head Head Thorax

0 4 12 22 5 0 0

8 9 10 11 12 13 14 15 16 17 18 19 20

67/M 62/F 49/F 81/M 65/F 64/M 80/F 76/M 69/M 52/F 51/M 80/F 79/F

Neck Back Back Head Neck Head Abdomen Back Head Head Head Head Head

11 0 9 10 11 5 5 0 11 10 5 5 0

21 22

70/M Head 82/M Head

23 24 25 26 27 28

62/F 60/F 77/M 69/F 65/F 56/M

29 30

65/F Head 58/M Head

1 4

31

59/F

Head

32

Clonal changes

Cell culture preparations Sporadic changes

Days Total no. in vitro of cells

Clonal changes t(6;19)(q12;q14)[5]

⫹7[1] ⫺X[1]

90ⵑ92,XXX,⫺Y[24]

del(7)(q22)[1]

17 15 13 18 12 16 13

7 16 20 48 1 16 3

14 17 10 16 15 13 10 13 14 16 18 13 15

0 26 0 0 0 10 13 10 18 13 19 21 42

18 14

15 11

13 13 14 17 13 13

18 27 22 15 19 37

15 13

6 28

0

13

49

83/M Head

0

15

40

⫺Y[15]

33

69/F

Head

7

i(18)(q11)[1]

14

14

inv(9)(p11q13)c

34 35 36 37 38

75/F 69/M 80/F 64/M 59/M

Head Head Head Head Head

0 14 16 15 6

⫹8[1]

18 23 28 13 13

21 10 0 16 10

Head Thorax Back Thorax Head Head

⫹22[1] add(2)(q37)[2] ⫺Y[1]

⫹22[1]

add(15)(q25)[1] inv(9)(p11q13)c

0 4

0 0 10 0 11 0

⫺Y[1]

⫹6[10]

⫺Y[4]

inv(9)(p11q13)c

(see FISH results) ⫹6[2] ⫹16[9]

Sporadic changes

t(13;15)(q14;q15)[10] t(1;11)(q32;q11) [1]

⫹Y[18] ⫺Y[18] ⫹i(X)(p11)[2] der(9)t(9;3) (p21;p13)[6]

del(X)(p11)[1] t(1;3)(q43;q11)[1] ⫺Y,⫹16[1] add(4)(q32)[1] ⫺Y[1] ⫹Y[1]

inv(9)(p11q13)c ⫹20[1] ⫺3,⫹3mar[1] der(15) t(Y;15)(q21.2;p11)c ⫹15[1] inv(5)(p15q12), t(1;12)(p32;q24)[4] del(9)(p12), t(1;12)(p12;q24)[4] add(5)(q35)[1] i(7)(p11)[1] add(13)(q34)[1] t(1;3)(q42;q11)[1] ⫹3[1] add(7)(p22)[1] del(7)(q22)[1] ⫺Y,⫹11[1] ⫹mar1,⫹mar2[1] del(1)(q41)[1] del(3)(q23)[1] add(3)(p27), del(5)(p15), t(7;12) (p22;q23)[1]

(continued)

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Table 1 Continued Direct preparations Case Age/ no. sex

Tumor site

Total no. of cells

39 40 41 42 43 44 45 46 47 48 49

91/F 68/M 66/M 53/M 83/M 70/M 34/F 51/F 60/M 57/F 57/M

Head Thorax Head Head Head Head Groin Back Head Head Head

19 0 0 20 4 5 4 0 5 0 21

50 51 52 53 54 55 56 57 58 59

53/M 84/F 75/F 46/M 85/F 61/M 75/F 61/F 70/F 67/M

Back Head Abdomen Thorax Neck Head Back Head Head Thigh

ND ND 2 1 0 0 21 0 4 4

60 61

60/F Head 74/M Head

10 4

62

80/M Head

12

63

70/M Head

16

64 65 66 67 68 69 70 71 72 73

54/M 76/M 64/M 62/M 74/M 67/M 58/F 70/M 56/F 62/M

7 20 20 4 2 24 21 25 24 24

Head Head Thorax Thorax Head Thorax Head Head Head Thorax

Clonal changes

Cell culture preparations Sporadic changes

(see FISH results) (see FISH results) (see FISH results) (see FISH results) inv(9)(p11q13)c (see FISH results) (see FISH results) t(1;15)(q25;q23) [1]

(see FISH results) (see FISH results) (see FISH results) ⫹6[1] (see FISH results) (see FISH results)

inv dup(9) (q11;q21)c ⫺Y[3] (see FISH 58ⵑ60,XY[4] results) 70ⵑ75,XXYY,del (3)(q13),add(1) (p31)[6] ⫺Y[2] (see FISH results) ⫹6[1] (see FISH results) ⫺Y[1] ⫹6[1]

Days Total no. in vitro of cells 18 ND ND 10 15 16 13 13 12 10 12

15 18 20 40 20 20 18 25

15 13 15 13 15 13 12 12 10 15

40 25 25 20 20 26 20 20 20 42

19 13

20 26

16

44

18

20

15 13 15 ND 22 19 22 20 25 18

0 20 0

Clonal changes

Sporadic changes

10

30 32 40 56 50

⫺Y[9] t(2;14)(q24;q24)[9] inv(9)(p11q13)c inv(9)(p11q13)c ⫹mar[3]

⫺Y[5]

⫹14[1]

⫺Y[3] t(1;3)(p32;q23),del (3)(q13)[7] ⫺Y[11] inv dup(9)(q11;q21)c ⫺Y[9]

del(1)(q21)[3] ⫺Y[1]

Abbreviations: c, constitutional; ND, not done.

ence of trisomy 6 that was observed by conventional cytogenetics. The results obtained by the direct method are compared with those obtained from short-term cell cultures, and the differences are discussed. Three squamous cell carcinomas (SCCs) also were analyzed, and the results are compared with those found in BCC. MATERIALS AND METHODS Basal cell carcinoma samples were surgically obtained from 73 patients (39 men and 34 women) 34–91 years of age. Age, sex, and lesion site are reported in Table 1. Dur-

ing this work, three SCCs were analyzed; the results are reported in Table 2. Cytogenetics Each sample was divided into two parts, one of which was directly processed (24 hours), and the other of which was submitted to short-term cell culture (10–28 days). Cytogenetic analysis was performed on direct preparations by using a standard method utilized for chorionic villi [10]. Briefly, small pieces of tissue were transferred in 3-mm Petri dishes containing RPMI medium. After 24 hours of incubation, colcemid (0.04 ␮g/mL) was added to

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Cytogenetic Analyses of BCC and SCC Table 2 Clinical and cytogenetic findings in three squamous cell carcinomas of the skin Direct preparations Case no.

Age /sex

Tumor site

Total no. of cells

1

77/F

Head

20

2

85/F

Head

14

3

67/M

Head

8

Cell culture preparations Sporadic changes

Clonal changes ⫺1, ⫹2der(1)t(1;17)(q36;q21),dup(7)(q12qter) ⫹8,⫹9,⫹16,⫹21,⫺14,⫹i(14q) ⫽ 20 ⫹8[6] ⫹6, ⫹11[3] ⫹6, ⫹8, ⫹11[2] 67⬃82,XY[8]

the medium for 1 hour. Then the medium was removed and replaced by a hypotonic solution (1% sodium citrate). Ten minutes later, the hypotonic solution was removed and replaced by methanol:acetic acid (3:1) fixative (20 min). Treatment with 60% aqueous acetic acid solution (10 min) led to cell dissociation; the cell suspension was distributed on warmed slides. Cell culture methods and the cytogenetic analysis technique performed on cultured cells were standard and previously described [11]. QFQ banding technique was applied. Clonal chromosome changes were defined as follows: at least two metaphases with the same structural change or trisomy and at least three metaphases with the same chromosome loss. Cases in which direct or culture preparations provided fewer than four metaphases are reported in Table 1 but are not taken into account. We also performed cytogenetic analysis (direct preparations and short-term cultures) on 12 samples of normal skin from 10 non-BCC patients (6 men, 4 women, mean age 55 years, sun-unexposed body sites: abdomen, back) and from 2 BCC-affected patients (cases 56 and 63) sampled in site close to the tumor. Constitutional karyotypes were defined on PHA-stimulated lymphocytes and were normal in all but 7 cases: variant inv(9)(p11q13) was found in 5 cases (nos. 13, 23, 33, 45, and 46); the rare variant dup(9)(qhq21) was found in case 61; and a t(Y;15)(q21.2;p11) in case 28. Fluorescence In Situ Hybridization Interphase FISH analysis with the use of a centromeric biotinylated probe specific for chromosome 6 (D6Z1, biotin, Oncor) was performed on nuclei from BCC direct preparations in 10 cases and on both direct and short-term cell culture preparations in 11 cases. FISH analysis was also performed on a direct preparation from one normal skin sample. FISH with the use of a centromeric probe specific for chromosome 8 (D8Z1, biotin, Oncor) was also performed as a control in 6 cases. Slides were treated with RNase 1⫻ for 1 hour at 37⬚C. After having been rinsed in 2 ⫻ SSC solution (70⬚C), slides were hybridized with a mixture containing 10 ng/␮L of biotin-labeled probe. After an overnight incubation at 37⬚C, the slides were rinsed in postwashing solution (high stringency 65% formamide/ 2 ⫻ SSC) at 43⬚C and then in 2⫻ SSC (pH 7) 1⫻ at 37⬚C. Slides were placed in a PBD solution before proceeding to detection and amplification with fluorescein-avidinlabeled and anti-avidin Ab. Interphase nuclei were stained

⫹2[1] ⫹mar[1]

Days in vitro

Total no. of cells

11

15

15

20

17

16

Clonal changes

Sporadic changes

by propidium iodide antifade. A percentage of three spotted nuclei in direct preparations was considered significant if higher than 10% and higher than the mean ⫹ two SD of three spotted nuclei in fibroblast from BCC or direct preparation of normal skin. Cell Type Characterization The nature of the cytogenetically analyzed cells was investigated by observations of their morphological aspect at the inverted microscope and by immunohistochemical analysis: cells from cultures, resuspended in Hanks’, and directly dissociated cells were centrifuged onto slides and fixed by Merckofix (Merck, Darmstadt, Germany); antikeratin (cocktail CK22, Biomeda) and anti-vimentin (DAKO-Vimentin, V9) antibodies were tested by the avidin-biotin peroxidase immunohistochemical technique [12]. Controls included use of nonimmune globulins and sera and antigen-adsorbed antibodies. RESULTS Cytogenetics BCC. Cytogenetic results (at least four metaphases analyzed) were obtained in both direct preparations and cell cultures in 34 cases; in 12 cases, only in direct preparations; and, in 27 cases, only in cell cultures. The results are reported in Table 1. In direct preparations, more than three metaphases were obtained in 46 of the 73 cases. Acquired chromosome abnormalities were found in 18 cases (39% of total cases), whereas 28 cases showed normal metaphases only. Clonal chromosome changes were found in 7 cases (38.8% of the abnormal cases, 15% of total) and they were: ⫹6 (cases 25 and 36; Fig. 1A), ⫺Y (case 30), ⫹16 (case 37), add(2q)(q37) (case 3), 58ⵑ60,XY and ⫺Y (case 62), 70ⵑ75, XXYY, del(3)(q13),add(1)(p31) (case 63). Sporadic changes, in one case associated with clonal changes, were found in 13 cases (68.4% of the abnormal cases) and were: ⫺Y (cases 4, 22, 64, and 70), and ⫹6 (cases 56, 67, and 69), ⫹22 (cases 2 and 8), add (15)(q25) (case 12), i(18)(q11) (case 33), ⫹8 (case 36), t(1;15)(q25;q23) (case 49). In one case (no. 62), two unrelated abnormal clones were found. In short-term cell cultures, cytogenetic results (at least four metaphases analyzed), were obtained in 65 cases. In 37 cases, only normal metaphases were found. In 27 cases (41.5% of total), different structural and numerical acquired chromosome changes were found, together with

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normal metaphases. Clonal changes were found in 17 cases (62.9% of the abnormal cases) and were: ⫹Y (case 16), ⫺Y (cases 18, 32, 43, 55, 61, and 62), t(6;19)(q12;q14) (case 1), 90ⵑ92,XXX,⫺Y (case 4), t(13;15)(q14;q15) (case 6), i(X)(p11) (case 19), der(9)t(3;9)(p13;p21) (case 20), inv(5)(p15q12), t(1;12)(p32;q24), and del(9)(p12),t(1;12) (q12;q24) (case 30), t(2;14)(q24;q24) (case 44), ⫹mar (case 48), t(1;3)(p32;q23),del(3)(q13),⫺Y (case 59), and del(1) (q21) (case 72). In two cases (30 and 59), two unrelated cytogenetically abnormal clones were present. Different numerical (⫺Y,⫹Y,⫺X,⫹3,⫹7,⫹8,⫹11,⫹14, ⫹15,⫹16,⫹20) and structural [del(7)(q22), t(1;11)(q32;q11), del(X)(p11),t(1;3)(q42;q11),add(4)(q32),add(5)(q35),i(7)(p11),

add(13)(q34),t(1;3)(q42;q11),add(7)(p22),del(7)(p22), different markers, del(1)(q41),del(3)(q23),add(3)(p27),del(5)(p15), t(7;12)(p22;q23)] sporadic changes were also present in addition to clonal changes or as a single abnormality in 16 cases (59% of the abnormal cases). The karyotypes (10 metaphases analyzed in direct preparations and 20 in cell cultures for each case) were normal in 11 skin samples, whereas, in one case (56), cell culture showed the presence, in three metaphases, of loss of the Y chromosome. SCC. Clonal chromosome changes [der(1)t(1;7)(q36;q21); dup(7)(q12ⵑqter);i(14q);⫹6,⫹8,⫹9,⫹11,⫹16,⫹21, and near

Figure 1 (A) Trisomy 6 (arrows) in a metaphase from BCC case 25. (B) Specific positive immunoreactivity (ABC peroxidase) for cytokeratin in a direct preparation from a case of BCC (case 4). (C) Nuclei isolated from a BCC (case 34), displaying three and two signals per nucleus, after hybridization with a probe specific for chromosome 6 centromere (Oncor).

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Cytogenetic Analyses of BCC and SCC triploid karyotype] were present in direct preparations in all three cases analyzed, whereas corresponding cell cultures showed normal metaphases only. The results are reported in detail in Table 2. Cell Type Characterization Morphological investigation at the inverted microscope showed fibroblastic-like cells only in all the cytogenetically analyzed cell cultures. Immunohistochemical investigation of uncultured mechanically dissociated cells from one BCC sample (case 4) showed the epithelial nature of the 99% of the cells that were cytokeratin positive (Fig. 1B) and vimentin negative. In contrast, the cultured cells were exclusively fibroblasts (vimentin positive, cytokeratin negative) in the two cases investigated (32 and 33). Interphase In Situ Hybridization The results of interphase FISH analysis with the use of an alpha centromeric probe specific for chromosome 6 (D6Z1, biotin, Oncor) are reported in Table 3. A cell population with trisomy 6 was present in 6 of 16 cases of BCC epithelial cells from direct preparations. In fact, three spotted nuclei (Fig. 1C) were present in 32, 19.8, 19.8, 19.5, 35.3, 11.5, and 16.8%, respectively in cases 34, 44, 54, 56, 59, 67, and 68. Each value exceeds the mean ⫹ two SD (0.1 ⫾ 1.6) of the number of three spotted nuclei found in shortterm cell cultures and in direct preparation from normal skin. Fisher exact test showed significant differences (P ⬍ 0.05) between direct preparations and short-term cultures when cells from both methods were investigated. FISH analysis performed on fibroblastic nuclei from 6 BCC short-term cell cultures (cases 34, 40, 41, 44, 47, and 68) and on nuclei from direct preparations of 1 normal skin sample did not show the presence of trisomy 6. BCC case 59, which showed trisomy 6 in 35.3%, and case 71, which showed trisomy 6 in 14% of the nuclei, failed to show the presence of trisomy 8, performed as a control, by alpha centromeric probe specific for this chromosome (D8Z1, biotin, Oncor). Interphase FISH analysis with the use of the same probe, D8Z1, was performed in four other cases (69, 70, 72, and 73) and failed to show the presence of trisomy 8. DISCUSSION The main result of this work is the demonstration of clonal numerical and structural chromosome changes in epithelial cells from BCC direct preparations. To our knowledge, these are the first cytogenetic and FISH data from direct preparations of BCC, whereas the literature data are based on cytogenetic analysis performed in short-term cell cultures in which evidence of pure epithelial cell population is lacking. Although the percentages of cytogenetically abnormal cells and of metaphases with clonal changes do not significantly differ in direct and cell culture methods, differences were found in the type of chromosome abnormalities. Except for the loss of the Y chromosome, not one of the clonal or sporadic changes found in direct preparations was found in the corresponding short-term cell cultures

Table 3 Percentage of nuclei with one to four spots resulting from interphase hybridization with centromeric probe for chromosome 6 in epithelial fibroblastic cells or both from 21 basal cell carcinomas Case Cell Total no. type nuclei 34 40 41 42 44 46 47 53 54 56 58 59 62 66 67 68 69 70 71 72 73

E F E F E F E E F E E F E E E E E E E E E F E E E E E

313 342 265 330 117 155 210 279 247 209 326 135 80 253 77 194 255 104 320 260 214 200 200 200 200 200 200

1

2

3

4

Cytogenetic evidence of trisomy 6

4% 5% 3% 3.60% 5% 8.4% 0% 20.1% 22.1% 2.9% 18.70% 23.7% 28.7% 1.2% 1.3% 0% 1.6% 10.6% 6.20% 11.5% 4.7% 4% 7.5% 6.5% 9.0% 3.0% 1%

64% 95% 96.6% 96.4% 95% 91.6% 98.1% 79.9% 76.9% 95.7% 81.3% 76.9% 71.3% 79% 77.9% 99.5% 62.7% 89.4% 93.8% 77% 78% 95% 91% 93% 72% 95% 97.5%

32% 0% 0% 0% 0% 0% 0.5% 19.8% 0% 0.9% 0% 0% 0% 19.8% 19.5% 0.5% 35.3% 0% 0% 11.5% 16.8% 0.5% 0.5% 0% 14% 1.5% 0.5%

0% 0% 0.4% 0% 0% 0% 1.4% 0% 0.4% 0.5% 0% 0% 0% 0% 1.3% 0% 0.4% 0% 0% 0% 0.5% 0.5% 1.0% 0.5% 5% 0.5% 1.0%

n.i. No n.i. n.i. n.i. n.i. No No No n.i. No No n.i. n.i. Sporadic No No No No Sporadic n.i. No No No Sporadic No No

Number of spots

Abbreviations: E, Epithelial cells; F, Fibroblastic cells; n.i., not informative.

and vice versa; the same results were obtained when our data from direct preparations were compared with the literature data obtained in short-term cell cultures. In particular, in our cases, clonal trisomy of chromosome 6 was present in direct preparations from 10 patients. Of them, two showed cytogenetic evidence of clonal trisomy 6; in 8, a cell population with trisomy 6 was demonstrated by interphase FISH analysis, in 3 of which sporadic trisomy 6 also was cytogenetically found. Trisomy 6 was absent in BCC and normal cell cultures investigated by cytogenetics and FISH, and no evidence of clonal trisomy 6 was present in the literature data from BCC or normal skin cell cultures. These data suggest that ⫹6 may be a nonrandom change in BCC. The fact that this clonal change was also found in a case of squamous cell carcinoma (see Table 2) and in three cases of cutaneous Merkel cell carcinomas [13–15] analyzed by a direct method suggests that this anomaly may be common to different skin tumors. Although control FISH with the use of centromeric probe specific for chromosome 8 performed in 6 cases failed to show trisomy 8 in direct preparations, we cannot exclude the possibility that, in cells in which tri-

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somy 6 was identified by FISH, other trisomies may be present. Loss of the Y chromosome in direct preparations is of uncertain significance because it may be associated with several types of neoplasia [16] and has also been described in nonneoplastic cells [11, 17, 18], trisomy 16, add (2)(q37);del(3)(q13), and add(1)(p31) were not recurrent changes. It should be noted that two cases showed neartriploid karyotypes. According to the literature, in BCC cell cultures, chromosome instability (41% of the cases showed chromosome abnormalities) is present and clonal numerical (⫺Y, ⫹Y, and near tetraploidy) and structural chromosome changes were found. They were, however, different from case to case. No statistically significant difference exists regarding the incidence of balanced or unbalanced structural or numerical chromosome changes between direct and culture preparations. The different cytogenetic changes found in direct preparations and in short-term cell cultures could be explained by the different cell types analyzed. In fact, the observation at the inverted microscope and the immunohistochemical studies showed that only fibroblasts are present in the cytogenetically analyzed short-term cell cultures, whereas epithelial cells are preponderant in direct preparations. The cytogenetic results obtained by a direct method reflect the epithelial BCC karyotype, whereas the chromosome changes found in the short-term cell cultures are confined to the fibroblastic stroma. Alternatively, the apparent lack of the correspondence between cytogenetic changes found

in direct and indirect preparations could be attributed to the small number of metaphases obtainable in the same case from direct preparations. The finding of clonal chromosome changes both in epithelial and fibroblastic cells suggests the cell heterogeneity of BCC in which the stroma may also take part in the neoplastic process. The involvement of stroma is not evident in the three SCC cases analyzed, in which the cell cultures showed abnormal karyotype, whereas clonal and sporadic changes were present only in direct preparations. Correlations between cytogenetic findings obtained in BCC and clinical data (sex, site, mean age, recurrence after 1 year) did not show a statistically significant difference between cytogenetically normal and abnormal cases. The relatively low percentage (38%) of the BCC cases showing clonal changes (identified in direct preparations and in cell cultures, or by FISH) agree with their essentially benign nature, whereas the high percentage of clonal abnormalities (3/3 in our cases) found in SCC correlates with their malignancy. The chromosomal distribution of the breakpoints in structural rearrangements is reported in Fig. 2. The statistical analysis with the use of the formula proposed by Brogger [19] showed a random distribution in our cases and a clustering to bands 1p36, 3q13, 9q22, 14p11, 15p11Xp11 if literature data are also considered. We have not found clonal rearrangement of the 9q22–q31 region, where genes involved in BCC are mapped [20] and loss of heterozygosity occurs in sporadic BCC [21]. The incidence

Figure 2 Location of breakpoints in clonal and sporadic chromosome rearrangements found in direct preparations (open circles), in short-term cell cultures (solid circles), and reported in the literature (solid triangles).

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Cytogenetic Analyses of BCC and SCC of constitutional inv(9)(p11q13) and of the rare variant dup(9)(q11q21) is significantly higher in BCC patients (6.8% and 1.3%) than in our control population (0.95% and 0.01%) (P ⬍ 0.05, Fisher exact test). The relevance of this finding remains to be established.

10.

Daniela Mazzola is an Anna Villa Rusconi Foundation Scholar.

11.

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