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Tubulovillous adenoma of the colon with hyperdiploidy, double-minute chromosomes, and inversion of chromosome 1
Tubulovillous Adenoma of the Colon with Hyperdiploidy, Double-Minute Chromosomes, and Inversion of Chromosome 1 Marie-H~lene Couturier-Turpin, Albert Louvel, Daniel Couturier, Catherine Esnous, Yvonne Poirier, and Paul Nepveux
A s e s s i l e adenoma of the, left .f/exare of the colon wus studied after st~rgical colectamy. Specimens w e r e obtained ]or complete histalogic evaluation. The t u m o r consisted of glandular tubes with decreased m a t i n production and a papilla U, s t r u c t u r e on the, luminal aspect. The mllscularis F/It/CO,q(l l/cOs not involved: there was no (:ar(:inomat(~us fu(:us. Cytogenetic study was carried o u t on 56 (:el/s; n o n e w a s n o r m a l . 77% were byperdiploid (52-87 c h r o m o s a m o s ) , 16% w e r e hypodiploid 118-39 chrornosonles), a n d 7% w e r e paradiploid. The s u p e r n u m e r a r y chromosomes w e r e chromosomes #3. #6, #13, #19, a n d #20; chromosome # 1 8 w a s m i s s i n g in 80% of the ceils. A marker .for chromosome #1 restdting from a (t21.1-q21.2 brook with inversion of the centromere-bearing segment (pter-q21) was observed in 58'!4, of the cells. Twenty-five. p e r c e n t of the cells had double m i n u t e chromosomes. D e s p i t e the histologically benign nature of the t u m o r , all the cells showed significanl cytogenetic a b e r r o lions, some of which are considered to be m m ' k e r s of neoplastic transformation (polyploidy, double minutes, (:hromosome #1 markerl.
ABSTRA(~:
INTRODUCTION Cytogenetic results published to date on colonic adenomas concern a limited nulnber of cases because of the low mitotic index of these lesions I1. 21. Chromosomal number is normal or only slightly different from 46. Numerical anolnalies (trisomy} seem to preferentially involve chromosolnes -#7, #8. and -#13, whereas, structural aberrations most often involve groups A, C, and F chromosomes. Chromosomal rearrangements appear to be frequent in villous adenomas and adenomas with sev e r e dysplasia. Cytogenetic analysis of villous adenomas, which have a higher potential for neoplastic transforrnation than do other histologic forms, furnish an opportunity to test for cancer risk associated vvith these markers and to deterlnine the significance of chromosomal rearrangements in colonic cancer. From the Laboratoire d'Histoloy, ic-Biologie Ct~llulaire-Cytogl~netique. UEI?. X. Bichat (M-H (]-'1'.. Y. P.}, (;roupe fie Recherche en Patho/ogie Digestive, tl6pital Co(:hin {Pavilion G. Roussy) (D. C.. C. E., F. N.). and Service (l'Anatomie l~atholo~ique. H6pilal Co[:hin (A, L.). Paris, Frant:e.
Address r e q u e s t s for reprints to M-H. Couturier-Turpin. Luboratoire d'ttistologie-Biologie cellulaire-Cytog6.n(~.tique, UER X. Bichat, 16 r u e H e n r i tluchard, 75018 Paris. France. Iteceived July 15. 1987: accepted December 17. 1987.
253 ~(') 1988 Elsevier Scierlt:e Publishing Co., Inc. 52 Vanderhilt Ave., New York. NY 10017
C~mt:er (;enet Cytogenet 32:253-262 (1988) 9165-4608:'88;S03.50
254
M-H. Couturier-Turpin et al. In the case reported here, a complete pathologic study was available (because the tumor required surgical resection) as well as a detailed cytogenetic study because of the high quality of the cell cultures.
CASE REPORT The case concerned a 70-year-old female with a noninvasive sessile polypoid tumor, 3.5 cm in its largest diameter, on the descending colon (50-70 cm from the anal margin), discovered by colonoscopy. Endoscopic resection was attempted three times, several days apart (10/20/85, 10/30/85, 11/13/85), but total resection was not possible because the tumor had a broad base; the macroscopic appearance of the tumor suggested a villous tumor. Surgical resection was performed on 11/26/85 and consisted of left colectomy. The p o l y p o i d tumor, 2.5 cm in diameter, was located 15 cm from the distal site of the surgical resection. HISTOLOGIC STUDIES
Endoscopic Specimens On October 24, 1985, a forceps biopsy was performed. Six of the ten specimens consisted of a proliferation of glandular tubes b u d d i n g into a villous structure on the luminal aspect; mucin production was decreased. The nuclei, some of which were globular, were arranged in a pseudostratified manner. There were numerous mitoses. The other specimens consisted of edematous colonic mucosa. On October 30, 1985, three specimens were obtained by diathermal biopsy. The results were similar to the preceding and led to the conclusion that the tumor was a tubulovillous a d e n o m a with moderate dysplasia. The proliferation did not involve the axis of the polyp. The section passed through the tumor (incomplete resection). On November 15, 1985, one specimen was obtained by diathermal biopsy. The results were the same as in the two previous biopsies.
Colectomy Specimen A sessile polyp measuring 2.5 x I cm was obtained. The proliferation consisted of glandular tubules lined with cells exhibiting decreased mucin production. The cells were basophilic with oblong or round nuclei. The glandular tubes had a papillary structure oil the a d l u m i n a l aspect. There were a few erosions s u r r o u n d e d by inflammatory interstitial tissue. The tumor confined to the mucosa and submucosa was not extended beyond the muscularis mucosae. CYTOGENETIC STUDY
Materials and Methods Ten specimens were obtained a p p r o x i m a t e l y 15 minutes after surgical resection and opening of the surgical specimen, at the tip and periphery of the tumor. The specimens were treated according to a previously described technique 131. Briefly, the technique consists of washing the specimen, mincing it with scissors into small fragments for culture in McCoy's m e d i u m with gentamycin for 18 hours, incubation with colcemid (0.5 i~g/ml for 3 hours), filtration of the tissue through gauze, hypotonic shock with 75 mM KCI, fixation (methanol/glacial acetic acid), and preparation of slides. The Seabright G-banding technique was used.
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The results are expressed as r e c o m m e n d e d by the ISCN 1978 [32]. A cell clone was defined if at least two cells carried the same aberration. A single normal cell indicated the presence of a normal clone. The presence of at least two clones indicated a mosaic. Results Fifty-eight cells were examined. The modal number was established for 42 cells: complete results were obtained for 13 cells (Table 1). None of the cells studied were normal. Thirty-two cells (77%) had more than the normal number of chromosomes (52-81 chromosomes), three cells (7%) were pseud o d i p l o i d (Fig. 1), and seven (16%) were h y p o d i p l o i d (18-39 chromosomes). The supernumeraries observed in over 50% of the cells were chromosomes #3, #6, #13, #15, #19, and #20. Chromosome #18 was missing in 80% of the cells. A chromosome #1 marker resulting from a q21.1-q21.2 break with inversion of the centromere-bearing segment (pter-q21) was observed in 58% of the cells (Fig. 2). Twenty-five percent of the studied cells showed double minutes (dlnin) {Fig. 3). Despite the intercellular diversity of cytngenetic aberrations, a principal cell clone could be distinguished, wbich was characterized by a modal number of 67 (54-81 chromosomes): all the (:ells with the c h r o m o s o m e 1 marker and one or more supernumerary chromosomes #13: seven of eight cells showed a supernumerary chromosome #14, five of eight cells showed one or more DM, one or more supernulnerary chromosome #20, and c h r o m o s o m e #18 was missing in four of eight cells. DISCUSSION Until the present, evaluation of the neoplastic transformation potential of colonic adenomas rested oil indirect evidence: tumor size, histologic structure (tubular, tubulovillous, villous), and degree of dysplasia [4]. Ill the case reported here, the histologically benign character of the tumor demonstrated on three different histologic specimens and studies of the surgical resection specimen, contrast with tile demonstration of significant cytogenetic aberralions in the 56 metaphases studied. According to the theory of clonal evolution of cancer as a p p l i e d to the colonic a d e n o m a - a d e n o c a r c i n o m a sequence, adenomas should only show limited chromosomal aberrations, and in fact, most of the cytogenetic analyses of adenomas do show preservation of normal cell clones with only a few numerical (trisomy 7, 8, and 13) and structural aberrations (chromosomes #1, #7, #8} [5-71. In one case of colonic villous adenoma, aberrations included h y p e r d i p l o i d y {48-52 chromosomes), trisomy 8, loss of the Y chromosome, and presence of a chromosome #1 marker (8). in view of the histologically benign nature of the tumor that we studied, three types of aberrations draw particular attention: the p o l y p l o i d y of the majority of the cells, the presence of a chromosome #1 marker, and dmin. In certain neoplastic processes (histiocytosis), a n e u p l o i d y and more specifically p o l y p l o i d y are considered to be signs of malignant transformation 191. Generally speaking, a n e u p l o i d y is considered to be related to the severity of prognosis in puhnonary and ovarian tumors [101 and various solid tumors [111. The principal clone of colonic a d e n o c a r c i n o m a s is p o l y p l o i d in a p p r o x i m a t e l y 50% of cases: the noninvasive forms are more often p a r a d i p l o i d [3, 12]. Most colonic adenomas studled by cytogenetics show a near normal number of chromosomes. It has been observed previously that villous a d e n o m a s frequently have more than 48 chromosomes. The villous a d e n o m a studied in detail by Reichmann et al. [8] showed 4 8 -
256
257
15
14
13
dms
21
16
10
Karyotype of a pseudodiploid cell [cell 18) with particular numerical aberrations chromosome 1 marker): 48,XX,+X,+X,+2,+3,+7,-8,-12,+13,-14,-17,-17,-18,-18,+19,+19,+20,-21,-22,-22, + mar (X rearranged), + dmin.
Figure 1
8
7
6
and
17
11
two dmin
X
Xou (absence
18
12
M of
259
r u b u l o v i l l o u s Adenoma of the Colon
q
Cell No.57
-/
Cell No. 25
Cell No.43 .o
Normal Chr I
Chr =1 Marker
Figure 2 Rearrangements of chromosome #1 in four different ceils. One or two normal chromosomes #1 are often found, 52 chromosomes; in the villous adenoma that we studied. 75% of the cells were polyploid, with some cells having mare than 69 chromosomes. The most common numerical aberrations were a supernumerary chromosome #13 and the loss of chromosome #18. The former was previously reported in a villous tumor [8J, the latter was reported to be one of the most frequent aberrations in colonic adenocarcinoma [13]. As concerns structural aberrations, a chromosome #1 marker was observed in ten of the 13 cells for which the karyotype was established. Rearrangements of chromosome #1 have been reported in a n u m b e r of different hematologic disorders and solid tumors [14, 15], the sites of the breaks and types of rearrangements vary [16]. Aberrations of chromosome #1 are observed in approximately 40% of colonic adenocarcinomas; the type varies from one case to another, but certain regions seem to be involved more often (i.e., q25--)q32; q24; q41--,q44) [17]. In the case reported here, the break was on q21, which is a site of chromosomal break often involved in neoplastic diseases [18]. The chromosome # 5 that bears the familial adenomatous polyposis gene near band 5q21-q22 is also implicated in 20% of the common colorectal cancers [19]. In the case reported here, one chromosome #5 was missing in only two of 13 cells. In some cytogenetic studies, a frequent deletion of the short arm of chromosome #17 has been reported, and a loss of one allele has been demonstrated using poly-
260
M-H. Couturier-Turpin et al.
.r'., ",.t.
Figure 3 Metaphase with one drain (arrow). morphic probes in 76% of common colonic adenocarcinomas 121]. Whereas, ill previous studies the short arm of chromosome #17 was selectively lost U21] we noted here that in five of the 13 cells one or both chromosome(s) #17 were totally missing. The X chromosome inactivation is the most common pattern of the clonal composition of h u m a n colonic adenomas and adenocarc:inomas [21]. Our cytogenetic study does not support this result because, in the present case, one chromosome X was missing in one and both were missing in two of 13 cells (Table 1). The role of rearrangements has not yet been established. Depression of a repressor effect has been considered [22[, however, in the case reported here, such a mechanism does not seem likely as the chromosome #1 marker was often associated with two normal chromosomes #1. Quantitative or qualitative modifications of oncogenic expression may also be involved: it is interesting to note that the cell oncogene ski is located in the q22---~q24 region and its role demonstrated in certain cancers [23]. Structural aberrations of chromosome #1 are usually considered to be late events in the course of cancer. Thus, although in endometrial cancer structural aberrations of chromosome #1 are almost always found, there is no such aberration in endometrial hyperplasia, although it is considered to be a precancerous lesion [24]. Double minute chromosomes sometimes responsible for gene amplification are
T u b u l o v i l l o u s A d e n o m a of the Colon
261
generally c o n s i d e r e d to be markers of t u m o r progression [25, 26l. T h e y are found in a p p r o x i m a t e l y 20% of c o l o n i c a d e n o c a r c i n o m a s and s e l e c t i v e l y o c c u r in i n v a s i v e forms (C and D in Duke's classification) [3]. The p r e s e n c e of s u c h markers in a benign t u m o r again raises the q u e s t i o n of the significance of these aberrations; a certain n u m b e r of d m i n c h r o m o s o m e s may consist of r e p e a t i n g but n o n f u n c t i o n a l DNA s e q u e n c e s that might not necessarily lead to genetic o v e r p r o d u c t i o n and w o u l d not affect tissue p h e n o t y p e [27]. Based on the m a c r o s c o p i c anti m i c r o s c o p i c data, the risk of n e o p l a s t i c transformation of the benign t u m o r we s t u d i e d was e s t i m a t e d at about 50%. C o n s i d e r e d alone, the c y t o g e n e t i c results suggest that it had already u n d e r g o n e transformation. C y t o g e n e t i c aberrations probably p r e c e d e changes in tissue p h e n o t y p e and c o u l d represent an i n v a l u a b l e m e t h o d for m o r e direct e v a l u a t i o n of the risk of n e o p l a s t i c transformation. C y t o g e n e t i c c h r o m o s o m a l studies or other m e t h o d s , flow c y t o m e t r y [28, 29], m i c r o n u c l e u s test [30, 31], c o u l d be w i d e l y used to assess the risk of transformation of p r e n e o p l a s t i c lesions in general.
REFERENCES 1. Mark J. Mitelman F, Dencker H, Norryd C, Tranberg KG (1973): Tbe specificity of the chromosomal abnormalities in human colonic polyps. Acta Pathol Microbiol Sl:and 81:85-90. 2. Mitelman (;. Mark J, Nilsson PG, 1)encker H, Norryd C. Tranberg KG (1974): Chromosomal banding pattern in human colonic polyps. Hereditas 78:63-68. 3. Couturier-Turpin M-H, ('outurier D. Nepveux P, I,ouvel A, Chapuis Y, Guerre J (1982): Human chromosome analysis in 24 cases of prilnary carcinoma of the large intestine: Contribution of the G-banding technique. Br i Cancer 46:856-869. 4. Morson BC (1974): Evolution of (:ancer of the colon and rectum. Cancer 34:845-849. 5. Couturie, r-Turpin M-H, Couturier D, Grandjouan S, Nepveux P, Forest M, Guerre J (1983): Cytog6,n6tique des polyad(~nomes coliques humains, relation avec la classification histologique (abstr). Gastroenterol Clin Biol 7:2A. 6. Couturier D. Couturier-Turpin M-H, Grandjouan S, Nepveux P, Guerre J (1983): Search for earlier chromosomal rearrangements in colonic polyadenomas and paradiploid adenocarcinomas [abstr). (;astroenterology 83:1130. 7. Reichmann A, Martin P, Levin B (1985): Chromosomal banding patterns in human large bowel adenomas, llum Genet 70:28-31. 8. Reicbmann A, Martin P, Levin B (1982]: Karyotypic findings in a colonic villous adenoma. Cancer Genet Cytogenet 7:51-57. 9. "l'eyssier J-R, Behar C. Pignon B, Caulet T, Patey M, Bajolle F, Adnet J-J (1986): Chromosoma[ changes in documented case of inalignant histiocytosis: Significance of polyploidy. Canc.er Genet Cytogenet 21:85 -91. 10. Rodenburg C], Cornelisse C], IIeintz PAM, Hermans J, Fleuren (;] (1987): Tumor ploidy as a major prognostic factor in advanced ovarian (:an(:er. Cancer 59:317-323. 11. Friedlander MI,, lledley DW, Taylor TW (1984): Clinical and biological signifi(:ance of aneuploidy in human tumors. J Clin Pathol 37:961. 12. l,evin B, Reichmann A (1986): Chromosome and large bowel tumors. Cancer Geuet Cytogenet 19:159-162. 13. Muleris M, Salmon RJ. Zafrani B, (;irndet J, Dutrillaux B [1985): Consistent deficiencies of chromosome 18 and of short arm of chromosome 17 in eleven cases of human large bowel cancer, a possible recessive determinisnl. Ann Genet 28:206-213. 14. Rowley JD (1978}: Abnormalities of chromosome 1 in hematalogical malignancies. I,ancet i:554 -555. 15. Kuvacs G (1978): Abnormalities of chramosome 1 in human solid malignant tumors, lnt J Cancer 21:688-694. 16. Brito-Babapulle V, Atkin NB (1981): Break points in chromosome I abnormalities of 218 human neoplasms. Cancer Genet Cytogenet 4:215-225.
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17. Reichmann A, Martin P, Levin B (1984): Chromosomes in human large bowel tumor, a study of chromosome 1. Cancer Genet Cytogenet 12:295-301. 18. Mitelman F {1984): Restricted number oi chromosomal regions implicated in aetiology of human cancer and leukaemia. Nature 310:325-327. 19. Solomon E, Voss R, llall V, Bodmer WF, Jass JR, leffreys AI. Lucibello FC, Patel I, Rider SM {1987): Chromosome 5 allele loss in human colorectal carcinomas. Natnre 328:616619. 20. Reichmann A, Martin P, Levin B (1985): Marker chromosomes in direct preparations of human large bowel tumors. Cancer Genet Cytogenet 16:229-233. 21. Fearon ER, Hamilton SR, Vogelstein B 11987/: Clonal analysis of human colorectal tumors. Science 238:193-197. 22. Stoler A, Bouck N (1985): Identification of a single chromosome in the normal human genome essential for suppression of hamster cell transformation. Proc Natl Acad Sci 82:570-574. 23. Chaganti RSK, Balazs I, ]hanwar SC. Murty VVVS, Koduru PRK, Greschik KH, Staunezer E (1986): The cellular homologue of the transforming gene of SKV avian retrovirus maps to human chromosome region lq22--~q24. Cytogenet Cell Genet 43:181-186. 24. Fujita 11, Wake N, Kutsuzawa T, lchinoe K, Hreschyshyn MM, Sandberg AA (1985): Marker chromosomes of the long arm of chromosome 1 in endometrial carcinoma. Cancer Genet Cytogenet 18:283-293. 25. Barker PE (1982): Double minutes in human tumor cells. Cancer Genet Cytogenet 5:8184. 26. Borst P (1984): DNA amplification and multidrug resistance. Nature 309:580. 27. Cnwell ]K 11982): Double minutes and homogenously staining regions: Gene amplification in mammalian cells. Ann Rev Genet 16:21-59. 28. Koss I,G, Greenebaum E 119861: Measnriug DNA in human cancer. I Am Med Assoc 255:3158-3159. 29. Van Den lngh tlF, Griffinen G, Cornelisse CJ 11985): Flow cytometric detection of aneuploidy in colo-rectal adenomas. Cancer Res 45:3392-3397. 30. Stich HF, Rosin MP (1984): Micronuclei in exfoliated human cells as a tool for studies in cancer risk and cancer intervention. Cancer I,ett 22:241-253. 31. Bird RP, Bruce WR (1986): Effect of dietary fat levels on the susceptibility of colonic cells to nuclear-damaging agents. Nutr Cancer 8:93-100. 32. ISCN [1978): An International System for Human Cytogenetic Nomenclature [1978). Birth Defects: Original Article Series, Vol. XIV, No. 8 (The National Foundation, New York, 1978): also in Cytogenet Cell (.;enet 21:304-404 (1978).
A s e s s i l e adenoma of the, left .f/exare of the colon wus studied after st~rgical colectamy. Specimens w e r e obtained ]or complete histalogic evaluation. The t u m o r consisted of glandular tubes with decreased m a t i n production and a papilla U, s t r u c t u r e on the, luminal aspect. The mllscularis F/It/CO,q(l l/cOs not involved: there was no (:ar(:inomat(~us fu(:us. Cytogenetic study was carried o u t on 56 (:el/s; n o n e w a s n o r m a l . 77% were byperdiploid (52-87 c h r o m o s a m o s ) , 16% w e r e hypodiploid 118-39 chrornosonles), a n d 7% w e r e paradiploid. The s u p e r n u m e r a r y chromosomes w e r e chromosomes #3. #6, #13, #19, a n d #20; chromosome # 1 8 w a s m i s s i n g in 80% of the ceils. A marker .for chromosome #1 restdting from a (t21.1-q21.2 brook with inversion of the centromere-bearing segment (pter-q21) was observed in 58'!4, of the cells. Twenty-five. p e r c e n t of the cells had double m i n u t e chromosomes. D e s p i t e the histologically benign nature of the t u m o r , all the cells showed significanl cytogenetic a b e r r o lions, some of which are considered to be m m ' k e r s of neoplastic transformation (polyploidy, double minutes, (:hromosome #1 markerl.
ABSTRA(~:
INTRODUCTION Cytogenetic results published to date on colonic adenomas concern a limited nulnber of cases because of the low mitotic index of these lesions I1. 21. Chromosomal number is normal or only slightly different from 46. Numerical anolnalies (trisomy} seem to preferentially involve chromosolnes -#7, #8. and -#13, whereas, structural aberrations most often involve groups A, C, and F chromosomes. Chromosomal rearrangements appear to be frequent in villous adenomas and adenomas with sev e r e dysplasia. Cytogenetic analysis of villous adenomas, which have a higher potential for neoplastic transforrnation than do other histologic forms, furnish an opportunity to test for cancer risk associated vvith these markers and to deterlnine the significance of chromosomal rearrangements in colonic cancer. From the Laboratoire d'Histoloy, ic-Biologie Ct~llulaire-Cytogl~netique. UEI?. X. Bichat (M-H (]-'1'.. Y. P.}, (;roupe fie Recherche en Patho/ogie Digestive, tl6pital Co(:hin {Pavilion G. Roussy) (D. C.. C. E., F. N.). and Service (l'Anatomie l~atholo~ique. H6pilal Co[:hin (A, L.). Paris, Frant:e.
Address r e q u e s t s for reprints to M-H. Couturier-Turpin. Luboratoire d'ttistologie-Biologie cellulaire-Cytog6.n(~.tique, UER X. Bichat, 16 r u e H e n r i tluchard, 75018 Paris. France. Iteceived July 15. 1987: accepted December 17. 1987.
253 ~(') 1988 Elsevier Scierlt:e Publishing Co., Inc. 52 Vanderhilt Ave., New York. NY 10017
C~mt:er (;enet Cytogenet 32:253-262 (1988) 9165-4608:'88;S03.50
254
M-H. Couturier-Turpin et al. In the case reported here, a complete pathologic study was available (because the tumor required surgical resection) as well as a detailed cytogenetic study because of the high quality of the cell cultures.
CASE REPORT The case concerned a 70-year-old female with a noninvasive sessile polypoid tumor, 3.5 cm in its largest diameter, on the descending colon (50-70 cm from the anal margin), discovered by colonoscopy. Endoscopic resection was attempted three times, several days apart (10/20/85, 10/30/85, 11/13/85), but total resection was not possible because the tumor had a broad base; the macroscopic appearance of the tumor suggested a villous tumor. Surgical resection was performed on 11/26/85 and consisted of left colectomy. The p o l y p o i d tumor, 2.5 cm in diameter, was located 15 cm from the distal site of the surgical resection. HISTOLOGIC STUDIES
Endoscopic Specimens On October 24, 1985, a forceps biopsy was performed. Six of the ten specimens consisted of a proliferation of glandular tubes b u d d i n g into a villous structure on the luminal aspect; mucin production was decreased. The nuclei, some of which were globular, were arranged in a pseudostratified manner. There were numerous mitoses. The other specimens consisted of edematous colonic mucosa. On October 30, 1985, three specimens were obtained by diathermal biopsy. The results were similar to the preceding and led to the conclusion that the tumor was a tubulovillous a d e n o m a with moderate dysplasia. The proliferation did not involve the axis of the polyp. The section passed through the tumor (incomplete resection). On November 15, 1985, one specimen was obtained by diathermal biopsy. The results were the same as in the two previous biopsies.
Colectomy Specimen A sessile polyp measuring 2.5 x I cm was obtained. The proliferation consisted of glandular tubules lined with cells exhibiting decreased mucin production. The cells were basophilic with oblong or round nuclei. The glandular tubes had a papillary structure oil the a d l u m i n a l aspect. There were a few erosions s u r r o u n d e d by inflammatory interstitial tissue. The tumor confined to the mucosa and submucosa was not extended beyond the muscularis mucosae. CYTOGENETIC STUDY
Materials and Methods Ten specimens were obtained a p p r o x i m a t e l y 15 minutes after surgical resection and opening of the surgical specimen, at the tip and periphery of the tumor. The specimens were treated according to a previously described technique 131. Briefly, the technique consists of washing the specimen, mincing it with scissors into small fragments for culture in McCoy's m e d i u m with gentamycin for 18 hours, incubation with colcemid (0.5 i~g/ml for 3 hours), filtration of the tissue through gauze, hypotonic shock with 75 mM KCI, fixation (methanol/glacial acetic acid), and preparation of slides. The Seabright G-banding technique was used.
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The results are expressed as r e c o m m e n d e d by the ISCN 1978 [32]. A cell clone was defined if at least two cells carried the same aberration. A single normal cell indicated the presence of a normal clone. The presence of at least two clones indicated a mosaic. Results Fifty-eight cells were examined. The modal number was established for 42 cells: complete results were obtained for 13 cells (Table 1). None of the cells studied were normal. Thirty-two cells (77%) had more than the normal number of chromosomes (52-81 chromosomes), three cells (7%) were pseud o d i p l o i d (Fig. 1), and seven (16%) were h y p o d i p l o i d (18-39 chromosomes). The supernumeraries observed in over 50% of the cells were chromosomes #3, #6, #13, #15, #19, and #20. Chromosome #18 was missing in 80% of the cells. A chromosome #1 marker resulting from a q21.1-q21.2 break with inversion of the centromere-bearing segment (pter-q21) was observed in 58% of the cells (Fig. 2). Twenty-five percent of the studied cells showed double minutes (dlnin) {Fig. 3). Despite the intercellular diversity of cytngenetic aberrations, a principal cell clone could be distinguished, wbich was characterized by a modal number of 67 (54-81 chromosomes): all the (:ells with the c h r o m o s o m e 1 marker and one or more supernumerary chromosomes #13: seven of eight cells showed a supernumerary chromosome #14, five of eight cells showed one or more DM, one or more supernulnerary chromosome #20, and c h r o m o s o m e #18 was missing in four of eight cells. DISCUSSION Until the present, evaluation of the neoplastic transformation potential of colonic adenomas rested oil indirect evidence: tumor size, histologic structure (tubular, tubulovillous, villous), and degree of dysplasia [4]. Ill the case reported here, the histologically benign character of the tumor demonstrated on three different histologic specimens and studies of the surgical resection specimen, contrast with tile demonstration of significant cytogenetic aberralions in the 56 metaphases studied. According to the theory of clonal evolution of cancer as a p p l i e d to the colonic a d e n o m a - a d e n o c a r c i n o m a sequence, adenomas should only show limited chromosomal aberrations, and in fact, most of the cytogenetic analyses of adenomas do show preservation of normal cell clones with only a few numerical (trisomy 7, 8, and 13) and structural aberrations (chromosomes #1, #7, #8} [5-71. In one case of colonic villous adenoma, aberrations included h y p e r d i p l o i d y {48-52 chromosomes), trisomy 8, loss of the Y chromosome, and presence of a chromosome #1 marker (8). in view of the histologically benign nature of the tumor that we studied, three types of aberrations draw particular attention: the p o l y p l o i d y of the majority of the cells, the presence of a chromosome #1 marker, and dmin. In certain neoplastic processes (histiocytosis), a n e u p l o i d y and more specifically p o l y p l o i d y are considered to be signs of malignant transformation 191. Generally speaking, a n e u p l o i d y is considered to be related to the severity of prognosis in puhnonary and ovarian tumors [101 and various solid tumors [111. The principal clone of colonic a d e n o c a r c i n o m a s is p o l y p l o i d in a p p r o x i m a t e l y 50% of cases: the noninvasive forms are more often p a r a d i p l o i d [3, 12]. Most colonic adenomas studled by cytogenetics show a near normal number of chromosomes. It has been observed previously that villous a d e n o m a s frequently have more than 48 chromosomes. The villous a d e n o m a studied in detail by Reichmann et al. [8] showed 4 8 -
256
257
15
14
13
dms
21
16
10
Karyotype of a pseudodiploid cell [cell 18) with particular numerical aberrations chromosome 1 marker): 48,XX,+X,+X,+2,+3,+7,-8,-12,+13,-14,-17,-17,-18,-18,+19,+19,+20,-21,-22,-22, + mar (X rearranged), + dmin.
Figure 1
8
7
6
and
17
11
two dmin
X
Xou (absence
18
12
M of
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r u b u l o v i l l o u s Adenoma of the Colon
q
Cell No.57
-/
Cell No. 25
Cell No.43 .o
Normal Chr I
Chr =1 Marker
Figure 2 Rearrangements of chromosome #1 in four different ceils. One or two normal chromosomes #1 are often found, 52 chromosomes; in the villous adenoma that we studied. 75% of the cells were polyploid, with some cells having mare than 69 chromosomes. The most common numerical aberrations were a supernumerary chromosome #13 and the loss of chromosome #18. The former was previously reported in a villous tumor [8J, the latter was reported to be one of the most frequent aberrations in colonic adenocarcinoma [13]. As concerns structural aberrations, a chromosome #1 marker was observed in ten of the 13 cells for which the karyotype was established. Rearrangements of chromosome #1 have been reported in a n u m b e r of different hematologic disorders and solid tumors [14, 15], the sites of the breaks and types of rearrangements vary [16]. Aberrations of chromosome #1 are observed in approximately 40% of colonic adenocarcinomas; the type varies from one case to another, but certain regions seem to be involved more often (i.e., q25--)q32; q24; q41--,q44) [17]. In the case reported here, the break was on q21, which is a site of chromosomal break often involved in neoplastic diseases [18]. The chromosome # 5 that bears the familial adenomatous polyposis gene near band 5q21-q22 is also implicated in 20% of the common colorectal cancers [19]. In the case reported here, one chromosome #5 was missing in only two of 13 cells. In some cytogenetic studies, a frequent deletion of the short arm of chromosome #17 has been reported, and a loss of one allele has been demonstrated using poly-
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M-H. Couturier-Turpin et al.
.r'., ",.t.
Figure 3 Metaphase with one drain (arrow). morphic probes in 76% of common colonic adenocarcinomas 121]. Whereas, ill previous studies the short arm of chromosome #17 was selectively lost U21] we noted here that in five of the 13 cells one or both chromosome(s) #17 were totally missing. The X chromosome inactivation is the most common pattern of the clonal composition of h u m a n colonic adenomas and adenocarc:inomas [21]. Our cytogenetic study does not support this result because, in the present case, one chromosome X was missing in one and both were missing in two of 13 cells (Table 1). The role of rearrangements has not yet been established. Depression of a repressor effect has been considered [22[, however, in the case reported here, such a mechanism does not seem likely as the chromosome #1 marker was often associated with two normal chromosomes #1. Quantitative or qualitative modifications of oncogenic expression may also be involved: it is interesting to note that the cell oncogene ski is located in the q22---~q24 region and its role demonstrated in certain cancers [23]. Structural aberrations of chromosome #1 are usually considered to be late events in the course of cancer. Thus, although in endometrial cancer structural aberrations of chromosome #1 are almost always found, there is no such aberration in endometrial hyperplasia, although it is considered to be a precancerous lesion [24]. Double minute chromosomes sometimes responsible for gene amplification are
T u b u l o v i l l o u s A d e n o m a of the Colon
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generally c o n s i d e r e d to be markers of t u m o r progression [25, 26l. T h e y are found in a p p r o x i m a t e l y 20% of c o l o n i c a d e n o c a r c i n o m a s and s e l e c t i v e l y o c c u r in i n v a s i v e forms (C and D in Duke's classification) [3]. The p r e s e n c e of s u c h markers in a benign t u m o r again raises the q u e s t i o n of the significance of these aberrations; a certain n u m b e r of d m i n c h r o m o s o m e s may consist of r e p e a t i n g but n o n f u n c t i o n a l DNA s e q u e n c e s that might not necessarily lead to genetic o v e r p r o d u c t i o n and w o u l d not affect tissue p h e n o t y p e [27]. Based on the m a c r o s c o p i c anti m i c r o s c o p i c data, the risk of n e o p l a s t i c transformation of the benign t u m o r we s t u d i e d was e s t i m a t e d at about 50%. C o n s i d e r e d alone, the c y t o g e n e t i c results suggest that it had already u n d e r g o n e transformation. C y t o g e n e t i c aberrations probably p r e c e d e changes in tissue p h e n o t y p e and c o u l d represent an i n v a l u a b l e m e t h o d for m o r e direct e v a l u a t i o n of the risk of n e o p l a s t i c transformation. C y t o g e n e t i c c h r o m o s o m a l studies or other m e t h o d s , flow c y t o m e t r y [28, 29], m i c r o n u c l e u s test [30, 31], c o u l d be w i d e l y used to assess the risk of transformation of p r e n e o p l a s t i c lesions in general.
REFERENCES 1. Mark J. Mitelman F, Dencker H, Norryd C, Tranberg KG (1973): Tbe specificity of the chromosomal abnormalities in human colonic polyps. Acta Pathol Microbiol Sl:and 81:85-90. 2. Mitelman (;. Mark J, Nilsson PG, 1)encker H, Norryd C. Tranberg KG (1974): Chromosomal banding pattern in human colonic polyps. Hereditas 78:63-68. 3. Couturier-Turpin M-H, ('outurier D. Nepveux P, I,ouvel A, Chapuis Y, Guerre J (1982): Human chromosome analysis in 24 cases of prilnary carcinoma of the large intestine: Contribution of the G-banding technique. Br i Cancer 46:856-869. 4. Morson BC (1974): Evolution of (:ancer of the colon and rectum. Cancer 34:845-849. 5. Couturie, r-Turpin M-H, Couturier D, Grandjouan S, Nepveux P, Forest M, Guerre J (1983): Cytog6,n6tique des polyad(~nomes coliques humains, relation avec la classification histologique (abstr). Gastroenterol Clin Biol 7:2A. 6. Couturier D. Couturier-Turpin M-H, Grandjouan S, Nepveux P, Guerre J (1983): Search for earlier chromosomal rearrangements in colonic polyadenomas and paradiploid adenocarcinomas [abstr). (;astroenterology 83:1130. 7. Reichmann A, Martin P, Levin B (1985): Chromosomal banding patterns in human large bowel adenomas, llum Genet 70:28-31. 8. Reicbmann A, Martin P, Levin B (1982]: Karyotypic findings in a colonic villous adenoma. Cancer Genet Cytogenet 7:51-57. 9. "l'eyssier J-R, Behar C. Pignon B, Caulet T, Patey M, Bajolle F, Adnet J-J (1986): Chromosoma[ changes in documented case of inalignant histiocytosis: Significance of polyploidy. Canc.er Genet Cytogenet 21:85 -91. 10. Rodenburg C], Cornelisse C], IIeintz PAM, Hermans J, Fleuren (;] (1987): Tumor ploidy as a major prognostic factor in advanced ovarian (:an(:er. Cancer 59:317-323. 11. Friedlander MI,, lledley DW, Taylor TW (1984): Clinical and biological signifi(:ance of aneuploidy in human tumors. J Clin Pathol 37:961. 12. l,evin B, Reichmann A (1986): Chromosome and large bowel tumors. Cancer Geuet Cytogenet 19:159-162. 13. Muleris M, Salmon RJ. Zafrani B, (;irndet J, Dutrillaux B [1985): Consistent deficiencies of chromosome 18 and of short arm of chromosome 17 in eleven cases of human large bowel cancer, a possible recessive determinisnl. Ann Genet 28:206-213. 14. Rowley JD (1978}: Abnormalities of chromosome 1 in hematalogical malignancies. I,ancet i:554 -555. 15. Kuvacs G (1978): Abnormalities of chramosome 1 in human solid malignant tumors, lnt J Cancer 21:688-694. 16. Brito-Babapulle V, Atkin NB (1981): Break points in chromosome I abnormalities of 218 human neoplasms. Cancer Genet Cytogenet 4:215-225.
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17. Reichmann A, Martin P, Levin B (1984): Chromosomes in human large bowel tumor, a study of chromosome 1. Cancer Genet Cytogenet 12:295-301. 18. Mitelman F {1984): Restricted number oi chromosomal regions implicated in aetiology of human cancer and leukaemia. Nature 310:325-327. 19. Solomon E, Voss R, llall V, Bodmer WF, Jass JR, leffreys AI. Lucibello FC, Patel I, Rider SM {1987): Chromosome 5 allele loss in human colorectal carcinomas. Natnre 328:616619. 20. Reichmann A, Martin P, Levin B (1985): Marker chromosomes in direct preparations of human large bowel tumors. Cancer Genet Cytogenet 16:229-233. 21. Fearon ER, Hamilton SR, Vogelstein B 11987/: Clonal analysis of human colorectal tumors. Science 238:193-197. 22. Stoler A, Bouck N (1985): Identification of a single chromosome in the normal human genome essential for suppression of hamster cell transformation. Proc Natl Acad Sci 82:570-574. 23. Chaganti RSK, Balazs I, ]hanwar SC. Murty VVVS, Koduru PRK, Greschik KH, Staunezer E (1986): The cellular homologue of the transforming gene of SKV avian retrovirus maps to human chromosome region lq22--~q24. Cytogenet Cell Genet 43:181-186. 24. Fujita 11, Wake N, Kutsuzawa T, lchinoe K, Hreschyshyn MM, Sandberg AA (1985): Marker chromosomes of the long arm of chromosome 1 in endometrial carcinoma. Cancer Genet Cytogenet 18:283-293. 25. Barker PE (1982): Double minutes in human tumor cells. Cancer Genet Cytogenet 5:8184. 26. Borst P (1984): DNA amplification and multidrug resistance. Nature 309:580. 27. Cnwell ]K 11982): Double minutes and homogenously staining regions: Gene amplification in mammalian cells. Ann Rev Genet 16:21-59. 28. Koss I,G, Greenebaum E 119861: Measnriug DNA in human cancer. I Am Med Assoc 255:3158-3159. 29. Van Den lngh tlF, Griffinen G, Cornelisse CJ 11985): Flow cytometric detection of aneuploidy in colo-rectal adenomas. Cancer Res 45:3392-3397. 30. Stich HF, Rosin MP (1984): Micronuclei in exfoliated human cells as a tool for studies in cancer risk and cancer intervention. Cancer I,ett 22:241-253. 31. Bird RP, Bruce WR (1986): Effect of dietary fat levels on the susceptibility of colonic cells to nuclear-damaging agents. Nutr Cancer 8:93-100. 32. ISCN [1978): An International System for Human Cytogenetic Nomenclature [1978). Birth Defects: Original Article Series, Vol. XIV, No. 8 (The National Foundation, New York, 1978): also in Cytogenet Cell (.;enet 21:304-404 (1978).