Chromosomal Analysis of Colonic Adenomatous Polyps Michel Longy, Robert Saura, Laurent Schouler, Cyril Mauhin, Jean-Franqois Goussot, Olivier Grison, and Patrice Couzigou

ABSTRACT: Chromosomal analysis of 25 colonic adenomatous polyps was performed by o direct method similarto that used in prenatal diagnosis of chramosomal aberration on charionic villi. Fourteen lesions showed an abnormal km'yotype. Two changes were recurrent: trisomy 7 /observed in eight cases) and trisomy 13 (abser~ed in seven cases). No monosomv of the sh()rt arm o) chromosome 17 was observed even at the level of two polyps with in situ carcinoma lesions.

INTRODUCTION K a r y o t y p e a n a l y s e s on c o l o n i c a d e n o c a r c i n o m a s h a v e s h o w n the e x i s t e n c e of recurring c h r o m o s o m a l r e a r r a n g e m e n t s in these lesions. A certain c h r o n o l o g y was prop o s e d c o n c e r n i n g the o c c u r r e n c e of the different aberrations o b s e r v e d [1, 21. This c h r o n o l o g y does not, h o w e v e r , take into a c c o u n t the c h r o m o s o m a l e v e n t s that can o c c u r before the m a l i g n a n t t r a n s f o r m a t i o n . A n u m b e r of t e c h n i c a l difficulties h a v e p r e v e n t e d c h r o m o s o m a l analysis of a d e n e m a t o u s p o l y p s from being t h o r o u g h l y d o c u m e n t e d . H o w e v e r , the r e l a t i o n s h i p b e t w e e n both lesions is u n i v e r s a l l y a d m i t t e d [31, and the p r e s e n c e of c h r o m o s o m a l aberrations at the level of these t u m o r s was o b s e r v e d as early as 1963 15]. A n original t e c h n i q u e [4] based on the m e t h o d s of c h r o m o s o m a l analysis u s e d for p r e n a t a l d i a g n o s i s on c h o r i o n i c villi [6, 7] a l l o w e d us to establish the k a r y o t y p t e of 25 c o l o n i c a d e n o m a t o u s polyps.

MATERIALS A N D METHODS T h e 25 p o l y p s c o r r e s p o n d to 23 different patients (average age, 66 years; range, 3 2 - 7 8 years; 68% male}. T h r e e a m o n g then: (cases 17, 18, and 19) w e r e from the s a m e patient, w h o had G a r d n e r s y n d r o m e . T h e first six cases h a v e been p u b l i s h e d p r e v i o u s l y [4, 8]. Each l e s i o n was o b t a i n e d t h r o u g h e n d o s c o p i c excision; a b i o p s y was p e r f o r m e d on the surface of the polyp, and the s a m p l e was i m m e d i a t e l y p l a c e d it: a cell c u l t u r e m e d i u m (RPMI + 10% fetal calf serum), p r e h e a t e d at 37°C, to w h i c h antibiotics had been added. From the Laboratoire de Cytogf3n6.tique,Centre H6pitalier Pellegrin, Bordeaux (M. L., R. S., C), G.), Servi(:e de. Gastro-ent6rologie, U,S.N. Haut-L6v6que. Pessac {L. S., C. M., P. C.I, and Laboratoire d'Anatomo-pat hologie, H6pita[ Saint-Andr6, Bordeaux (J.-E. G.I, France. Address reprint requests to: Michel Longy, M.D., Laboratoire de Cytogenetique, Maternit6, C. H. Pellegrin, Place Am61ie Raba L6on, 33076 Bordeaux Cedex, France. Received December 5, 1989; accepted March 7, 1990.

249 ~3 1990 Elsevier Science Publishing Co., Inc. 655 Avenue o{ the Americas, New York. NY 1001{I

CancerGenet Cytogenet 49:249 257 (1990} 0165-4608/90/$03,50

250

M. Longy et al.

i

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I q¢

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Figure 1

(;T(;-banded metat)hase with the karyotype 49,XY, + 7, + 9, + ]3 (case 6).

The remainder of the lesion was fixed for histopathological examination according to the usual protocol. This examination was performed by the same pathologist for all samples. The lesions were graded in five stages: mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ, and invasive carcinoma. The method used to establish the karyotype has been described previously [4]. The sample was dissociated with the aid of binocular lenses to separate the villous digitations at the surface of the lesion. This material was incubated overnight in the m e d i u m described above. A hypotonic preshock with a citric trisodic solution (1%) was administered before three methanol : acetic acid fixations were performed. Nuclear ejection was performed with a solution of acetic acid diluted at 50%: mechanical spreading was performed on a slide heated at 40°C, followed by standard staining (Giemsa) and chromosomal banding (RHG or GTG) (Figs. 1 and 2).

RESULTS

Results are summarized in Table 1: 56% of the lesions showed an abnormal karyotype. Numerical abnormalities were by far the most frequent, and two chrmnosomes (chromosomes 7 and 13), were particularly involved; trisomy 7 was observed in eight cases {57% of rearranged karyotypes), and trisomy 13 was observed in seven cases (50% of rearranged karyotypes). The other trisomies observed with at least one recurrence were; trisomy 20 (four times), trisomy 8 (twice), and trisomy 12 (twice). Two cases {cases 12 and 13) were characterized by a high percentage of near-tetraploidy. A precise chromosomal analysis could be performed on certain cases. Details of these results are shown in Table 2. Structural rearrangements (Fig. 3) were observed in only three lesions (cases 1, 8 and 24); these i n c l u d e d a deletion of the short arm of a chromosome 3, a rearrangement of the short arm of a chromosome 1 with deletion of part of this segment, a rearrangement of chromosome 22 that could correspond to a dic (22), and a marker chromosome resulting from a t(20;?)(pter;?). No monosomy of the short arm of chroinosome 17 was

29 38 21 3"1 30 20 24 18

15 11 12 28 28 57 20 47 22

9 10 11 12 13 14 15 16

17 18 19 2(I 21 22 23 24 25

" i d e m : XX,

8

30 65

15 17 36 3 5 8

2 5

7

4 27 14 16 2 4 2

27 7

2 9 18 13 2

16

4t5

2 13 4

3

3 12 4 2

2

9

13 22

16 2

2

12

20

5

1

9

11

48

8

47

19

17

49

3

50

1

2

51

number

l.+13,-18.+der[1)t{l:?){p12:?),+?dic{22),

1 4

3 3 2 6 4 12

3

1 1

5 3 5 7 7 9 2 5 6

2 10 4 4

1 1

2 3 2 5 1

2

45

2 1 1 2

2 3

1 4 10 6 1

26 16 3O 24 27

7

4

22

1 2 3 4 5 6

45

counted

Case

Chromosome

Summary of results

No-, of cells

Table 1

3

52

9

53

•1 {92)

1 (gz?)

I [1021

11 { 7 0 - 8 4 ) 23 ( 7 0 - 9 2 1

1

Near-teh'aploidy

4 4 3 8 4

3 2 3 3

5

5

6 6

7 3 6

3

7 9 4 6 5 7

kar?,'olvped

No. t~f (:ells

5 . - 1:}

12 4 8 , X Y . -

1 2 , - 20

46,XX 4 6 , X X / 4 7 , X X , -- 20 46,XX 46,XY 46,XY 46,XY 4 8 . X Y , - 7 , - 13 48,XY,-7,-8. 2(},- d e r { 2 O l t ( 2 0 : ? ) ( p t e r : ? ] 46,XY, 47.XY,- 7

46,XY/47.XY,- 7 46,XY 4 7 , X X . + 1 3 . 4 8 . X X . - 7 . * 13 4 7 , X Y , + 20 4 8 , X Y . - - 7 , ± 13 5 3 , X X X , ~ 7 . - 9 . - 1 0 , ~ 1 2 , - 13, ~ 16

46,XY/47,XY,46,XY

47,XX.-1,*13.-18, *der(1)t(l:?)(p12:?). 4- ? d i c ( 2 2 ) ' 4 8 , i d e m " , 8 4 9 , i d e m C ~ 8. 8 / 5 0 . i d e m ' ; , ÷ 8. - 8. - der{l)

46.XY

4 6 , X X 46.XX,(lel{3}{p 12) 4 7 . X Y . - 13 4 8 . X Y . - 3. ~ 1 3 / 4 8 . X Y . 46,XY 46.XY 46.XX 4 9 . X Y , ~ 7 , ~ 9 , + 13

Karyotype

Mild Mild Mihl Moderate Mild Sex e r e Mild

Moderate Moderale

Mild Moderate Mild Mild Moderate Modcrale Mild havasive (~ilFCilIOIll(I

Moderate In s i l u carcii]onla

Moderate Mo(lerale Mild Moderate Sever{.' in situ (iarcinonl~l

l)ysplasia

bO W1

Metaphase2

1

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1

1

+ '2

4

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-

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Metaphase3

-1

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1

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1

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2

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1

- 2

-2

3

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4

1

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1

1

1

1

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1

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-2

8

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9

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11

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1

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1{3

Detailed Karyotypes of the near-tetraploid mitoses of cases 12 and 13

Metaphase2

Metaphase

Case

Table2

-'2

-1

-

-1

-

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17

1

I

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18

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1

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1

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77

73

{~}

84

85

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253 f

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Figure 2 h:ase 16l.

RH(;-banded metaphase with the, karyotype, 53,XXX,+ 7 , ÷ 9 . + 1 0 , + 12, + 13,+ l{i

e

II II

°-°-I

a

c

b

d

Figure 3 Marker chromosomes: (a) pair 1 with der(llt(1:?)(p12:?) (case 8l: (hi pair 22 with supenmmerary ?die(22) (case ~); (c) pair 3 with del(3)(p12] (case 1); and (d) pair 20 with der(20]t(20:?)(pter;?] (case 24].

254

M. Longy et al.

observed, nor was a rearrangement of the short arm of chromosome 5 that could have corresponded to the lap locus observed [4, 101. For each lesion, the cytogenetic analysis data were compared with the anatomoclinical characteristics (Table 3). No clear correlation was observed between the size of the polyps and the existence of a chromosomal rearrangeinent. Similarly, there was a correlation between the histopathological grading with chromosomal anomalies only for evolved lesions. The karyotypes showing the most marked rearrangements corresponded to the polyps with in situ carcinoma (cases 6 and 8) and invasive carcinoma (case 16). DISCUSSION

Karyotype normality of certain adenomatous polyps was reported after the first attempts to analyze the chromosomes in these tumors [11]. Mainly they were lesions with minor atypia only. This correlation did not appear to be evident in our study, in which half of the cases of mild dysplasia had rearranged karyotypes and one case of severe dysplasia had a normal karyotype. The occurrence of chromosomal aberrations was also reported as early as the first attempts [5], but the published series were few [12-15], so that the estimation of a n e u p l o i d y was made mainly by static 1161 or flux [17-19] cytometry. The rate of a n e u p l o i d y varied then between 6 and 27% of the lesions according to the series. This rate is inferior to the one obtained by cytogenetic analysis; i.e., in the 25 studied lesions we observed 14 (56%) with (:hromosomal aberrations. Recurrence of trisomy 7 and 13 has been reported previously [201, but its frequency was not as high as that which we observed. These two changes are usual at the level of colonic adenocarcinoma where they have been reported in 30 to 50% of the cases [1, 21, 22]. In the same manner, several rearrangements particularly frequent at the level of colonic adenocarcinoma were observed in at least one of the polyps of this series: they were m o n o s o m y 18, monosomy lp, trisomy 20, and duplication of the X chromosome. Adenomas and adenocarcinomas appear to have in c o m m o n a certain "chromosomal adaptation to the proliferation." For this reason, the study of peritetraploid mitosis is revealing. The transition to tetraploidy appears to be associated with a certain instability with chromosomal losses. These losses occur specifically on chromosomes "lost" at the level of adenocarcinoinas and usually spare the chromosomes that usually r e d u n d a n t (Table 3). Thus, chromosomes 18 were rarely present in four sets and most of the time were observed in a diploid state, which is equivalent to a relative monosomy, whereas the 7.13, and 20 chromosome pairs were frequently observed in four sets, sometimes even in five or six sets. Similar conclusions can be drawn from two observations reported earlier concerning two different polyps. In one, the modal n u m b e r was 79 ]20], and in the other, the n u m b e r of chromosomes ranged from 18 to 87 [23]. Both showed the same chroinosomal pattern we noticed in our observations. Monosomy of the short arm of chromosome 17 is considered a fundamental occurrence in the genesis of colonic carcinoma [24]. This rearrangement was observed in 90% of infiltrated adenocarcinomas, and allelic losses for genie sequences on the short arm of 17 were observed in 80% of these tumors [25-27]. That this type of rearrangement has not been observed in in situ carcinoma lesions leads us to believe that loss of the short arm of chromosome 17 may play an important role in the transition toward the invasive stage. It is also tempting to try to establish a correlation with the i17q frequently associated with the blastic phase of chronical myeloid leukemia 128]. The correlation between this rearrangement and the iuvasive nature of the proliferation does not appear to be complete, however, because of the existence of allelic losses involving the short arm of chromosome 17, which was reported in

3-40 3-20

14.07

11.18

With chromosomal aberrations Without chromosomal aberrations

Range

Average

Size (mm)

5

6

Mild

5

4

Moderate

C o r r e l a t i o n s b e t w e e n c l i n i c a l , p a t h o l o g i c a l , a n d c y t o g e n e t i c data

Condition

Table 3

Severe

Dysplasia

2

carcinoma

In situ

Invasive

1

carcinoma

Yes

Previous No

t'O ¢J1

256

M. Longy et al.

6% of c o l o n i c a d e n o m a s w i t h o u t areas of i n v a s i v e c a r c i n o m a o b t a i n e d by a c r y o s t a t s e c t i o n i n g t e c h n i q u e [29]. In a d d i t i o n , the s a m e t e a m w a s able to extract DNA f r o m a d e n o m a t o u s a n d a d e n o c a r c i n o m a t o u s areas of d e g e n e r a t e d p o l y p s . O n e p o l y p s h o w e d t h e s a m e loss of s e q u e n c e s in the s h o r t arm of c h r o m o s o m e 17 in t h e s e t w o z o n e s [30]. M o r e o v e r , o n e of the l e s i o n s w e r e p o r t c o r r e s p o n d s to a d e g e n e r a t e d i n v a s i v e c a r c i n o m a t o u s p o l y p . T h e k a r y o t y p e is h o m o g e n o u s w i t h 53 c h r o m o s o m e s a n d d o e s not s h o w m o n o s o m y 17p or m o n o s o m y 18. This relative d i s c r e p a n c y probably r e s u l t s f r o m t h e focal n a t u r e of the d e g e n e r a t i o n . It is u n f o r t u n a t e that the karyot y p e d z o n e , w h i c h d o e s not n e c e s s a r i l y c o r r e s p o n d to the z o n e of i n v a s i v e {:arcinoma, has not b e e n i d e n t i f i e d in this s a m p l e . Still, the r e s u l t s t e n d to c o n f i r m that loss of m a t e r i a l o n t h e s h o r t a r m of c h r o m o s o m e 17 c o n s t i t u t e s a f u n d a m e n t a l d i f f e r e n c e b e t w e e n c o l o n i c a d e n o m a a n d a d e n o { : a r c i n o m a . C o n v e r s e l y , several a b e r r a t i o n s , in p a r t i c u l a r t r i s o m i e s 7 an{1 13, are re(:urrent both in c o l o n i c a d e u o m a s a n d a d e n o c a r c i n o m a s . T h e p r e s e n t data are i n a d e q u a t e , h o w e v e r , to t)rovi(te p r e d i c t i v e v a l u e to the p r e s e n c e or a b s e n c e of s u c h a b n o r m a l i t i e s at the level of coloni(" a d e n o m a t o u s p o l y p s .

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