Associated with Longstanding Ulcerative Colitis - NCBI

Americani Journal of Patholqgy, Vol. 144, No. 4, April 1994 Copyright © Anmeicani Society for Investigative Pathology,

K-ras Mutations and p53 Alterations in Neoplastic and Nonneoplastic Lesions Associated with Longstanding Ulcerative Colitis

Pascal Chaubert,* Jean Benhattar,* Emilia Saraga,* and Jose Costa* From the Institut Universitaire de Pathologie,*

Lausanne, Switzerland

We have analyzed K-ras mutations and p53 alterations in 39 tumor and nontumor samples taken from nine patients with longstanding ulcerative colitis and colorectal carcinoma. Two of nine invasive carcinomas contained a K-ras mutation. By a combination of immunohistochemistry and single-strand conformation polymorphism analysis, p53 alterations were found in three of nine carcinomas. Five of 13 dysplastic lesions harbored a mutated K-ras gene, even in the absence of detectable changes in associated invasive tumors. One single focus of dysplastic mucosa harbored concomitant K-ras and p53 gene alterations. In two patients, a K-ras mutation was detected in epithelial lesions considered to be devoid of malignant potential (vilous regeneration, active colitis). Our results indicate that: 1) the prevalence of K-ras and p53 genetic alterations found in ulcerative colitis-associated colonic carcinomas appears to be lower than in sporadic carcinomas; 2) K-ras mutations can be detected in dysplasia, vilous regeneration, and active colitis and affect a subpopulation of the ceUs composing the lesions; 3) diverse genetic alterations can be detected in the same patient and the dysplastic lesions can exhibit a different genotype than the carcinomas; and 4) at least part of active colitis and villous regeneration lesions should be considered as preneoplastic in ulcerative colitis. (Am J Pathol 1994,

fined cytological and architectural alteration of the colon mucosa, is considered to be a neoplastic lesion and widely accepted as a marker of increased cancer risk in UC.2 Villous regeneration occurs after repeated mucosal damage and results in structural changes in the colon mucosa without the cytological features of dysplasia. This regeneration is generally considered to be nonpreneoplastic.3 The biological significance of regenerative and other morphological nonneoplastic lesions in UC remains to be clarified. Mutational activation of the K-ras oncogene and inactivation of the p53 tumor suppressor gene play a role in the genesis of sporadic colorectal carcinomas.4 K-ras mutations are present in 40 to 50% of these cancers5,6 but only in 8 to 25% of the carcinomas arising in UC patients.7-9 In previous studies from other laboratories, only 6 of 46 dysplastic lesions from UC patients have contained K-ras mutations.7 10 Mutations in the p53 gene, found in more than 70% of the sporadic cancers,11'12 are present in a large proportion of UC-associated neoplasms13 and seem to be associated in most cases with p53 allele loss.13-16 To determine the frequency of K-ras mutations and p53 alterations in UC-associated cancers and precancerous and nonneoplastic lesions, we examined archival colon specimens from nine patients with longstanding UC who underwent colectomy for colorectal carcinoma. We applied the allele specific polymerase chain reaction (ASPCR) to detect K-ras mutations and we developed a procedure sequentially using PCR cloning, ASPCR screening, and DNA sequencing to confirm the presence of mutations and quantify the proportion of mutated alleles present in the population of cells analyzed. To detect p53 alterations, we used both immunohistochemistry and non-

144:767-775)

Supported by grants from La Ligue Suisse contre le Cancer and

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology. Patients with longstanding UC have a 20- to 40-fold increased risk of developing colorectal carcinoma.1 Dysplasia, a well de-

La Fondation Veillon. Accepted for publication December 17, 1993. Dr. Costa's current address is Department of Pathology, Yale University School of Medicine, New Haven, CT. Address reprint requests to Dr. Pascal Chaubert, Institut Universitaire de Pathologie, Bugnon 25, CH-101 1 Lausanne, Switzerland.

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radioactive single-strand conformation polymorphism (SSCP) analysis. In each of the above specimens, we investigated the carcinomas and, when available, dysplastic, hyperplastic, inflammatory, and normal mucosae.

Materials and Methods Patients and Specimens Archival formalin-fixed and paraffin-embedded colorectal tissues from nine patients with longstanding UC who underwent colectomy for carcinoma were studied. The carcinomas were staged in accordance with Dukes' classification.17 Age and sex of the patients, duration of the UC at the time of colectomy, and localizations and stages of the carcinomas are summarized in Table 1. We reviewed all the hematoxylin and eosin (H & E)-stained sections and classified the histological lesions according to Riddell et al.2 The term villous regeneration was used in accordance with the definition given by Lee.3 In each specimen, we selected one sample corresponding to the invasive carcinoma and one or several samples corresponding to different noncancerous lesions, all geographically distinct from each other. The 39 samples selected for study corresponded to the following lesions: 9 (from the 9 patients) corresponded to invasive carcinomas, 13 (from 5 of the 9 patients) to low-grade or high-grade dysplasia, 6 (from 3 of the 9 patients) to villous regeneration, and 7 (from 5 of the 9 patients) to active colitis. Four samples of histologically normal mucosa were also studied.

et ae18 with slight modifications as described previously.19 We used three different anti-p53 antisera with a relatively good efficiency on fixed tissues: the mouse monoclonal antibody NCL-p53-DO-7 (diluted 1/200) and the rabbit polyclonal antiserum NCC-CMl (diluted 1/500) were obtained from Novocastra Laboratories (Newcastle, UK), whereas the mouse monoclonal antibody OM-1 1-918 (diluted 1/500) was from Cambridge Research Biochemicals (Cambridge, UK). These three antibodies detect both wild-type and mutated forms of the p53 protein.

Microdissection and DNA Extraction For each sample, the histological lesion of interest was first identified on H & E-stained slides. The most representative area was delineated and microdissected by scraping the tissue directly from the paraffin blocks to maximize the percentage of epithelial (neoplastic or nonneoplastic) cells analyzed. In the noncancerous mucosae, we estimated that the proportion of epithelial cells contained in the microdissected sample ranged from 30 to 40%. In the carcinomas, the tumoral/stromal cells ratio ranged from 30 to 80% (Table 2). The presence of the lesion of interest was also verified after microdissection on another histological section. DNA was extracted by a modification of the Coates et al procedure.20 Briefly, the lesion-enriched tissue chips were boiled 10 minutes at 100 C in 40 to 200 p1 of TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0). Three microliters of this solution were immediately subjected to ASPCR or PCR-SSCP. Each microdissection was performed at least twice to exclude con-

tamination problems.

p53 Immunohistochemical Staining All the selected tissues were examined by immunohistochemistry using the PAP method of Sternberger Table 1. Clinical and Pathological Data Duration Patient of Disease Localization Dukes' No. Age Sex (years)* of Carcinoma Stage 1 2 3 4 5 6 7 8 9

67 26 39 46 51 71 37 40 50

F M M M M M M F F

30 12 14 20 19 >29 15 22 21

Ascending Descending

Sigmoid Rectum Ascending Ascending Descending Transverse Rectum

B C B C B C A A A

* Time in years between the onset of the UC and discovery of a colorectal carcinoma.

Detection of K-ras Mutations by ASPCR The procedure for amplifying DNA by ASPCR has been described previously.21 For each DNA, nine separate reactions were performed using oligonucleotides specific for nine different mutations in the codons 12 and 13 of the K-ras gene.21 Three microliters of DNA template were amplified in a reaction volume of 30 pl. Five cycles with incubations of 1.5 minutes at 94 C, 1.75 minutes at 59 C, and 2 minutes at 73 C were followed by 45 cycles with lower annealing temperature (55 C). Each reaction was performed at least twice to avoid false positives due to the fact that primers with mismatches at the 3' end are still able to extend to some degree. Negative control DNAs from nonneoplastic or normal colonic tissues

K-ras and p53 Alterations in Ulcerative Colitis 769 AJP April 1994, Vol. 144, No. 4

Table 2. K-ras Mutations and p53 Alterations in UC-Associated Cancers and Related Nonneoplastic Tissues

Patient No. 1 2

3

4 5

6 7

8

9

Histological Lesions*

% ECt

Carcinoma Active colitis (D) Carcinoma LG dysplasia, villous architecture (D) LG dysplasia, villous architecture (D) LG dysplasia, villous architecture (D) Villous regeneration (P) Villous regeneration (D) Active colitis (D) Carcinoma Active colitis (D) Active colitis (D) Active colitis (D) Normal mucosa (D) Normal mucosa (D) Carcinoma LG dysplasia, villous architecture (D) Carcinoma Active colitis (D) LG dysplasia (D) Carcinoma Normal mucosa (D) Minute carcinoma** HG dysplasia, villous architecture (P) HG dysplasia, villous architecture (P) HG dysplasia, villous architecture (P) HG dysplasia, villous architecture (D) Villous regeneration (D) Villous regeneration (D) Minute carcinoma** HG dysplasia, villous architecture (P) HG dysplasia, villous architecture (P) HG dysplasia, villous architecture (D) HG dysplasia, villous architecture (D) Villous regeneration (D) Villous regeneration (D)

50 40 60 30 30 30 30 40 30 60 30 30 30 30 30 70 40 50 30 30 80 30 30 30 30 30 40 30 30 40 30 30 40 30 30 30 60 30 30

Carcinoma Active colitis (D) Normal mucosa (P)

K-ras mutations* Ratio§ Type 0 0 0 0

p53 Analysis SSCP Shift Expressioni None detected -

None detected

12GAT

12GATm 0 12GAT1 0 0 12GTT-

12GATn1 0 0 0 0 0 0 0 0 0 0

12TGT 12TGT 12TGT 0 0 0 0 0

12TGT 0 0 0 0 0 13GAC 0 0

6%

+++

Exon 8

14% -

None detected None detected

-

None detected

-

Exon 6

-

+

None detected None detected

-

None detected

-

None detected

6% 4%

++

-

Exon 6 None detected

D or P after the lesion indicate its geographical position with respect to the carcinoma (P, peritumoral; D, distant from carcinoma). Each lesion examined was geographically distinct from each other and was sampled from a different histological paraffin block. LG, low grade; HG, high grade. t Percentage of epithelial cells (EC) obtained by microdissection, estimated on H&E-stained slides. * K-ras mutations are detected by ASPCR (0 = no K-ras mutation detected). § Ratio was derived from the number of mutated clones counted among 50 clones of K-ras exon 1 PCR product as detailed in Materials and Methods. It corresponds to the percentage of mutated alleles contained in the microdissected tissue sample. I p53 expression by immunohistochemistry (-, negative; +, 50% of positive cells). Mutations confirmed by DNA sequencing (See Materials and Methods). Minute carcinoma designates an invasive carcinoma measuring less than 5 mm.

obtained from the same and from other patients were analyzed at the same time.

Quantification of K-ras Mutations by PCR

Cloning and ASPCR To evaluate the frequency of the K-ras mutations detected in some dysplastic and nonneoplastic lesions, we amplified exon 1 by PCR using primers external to the codons 12 and 13. The PCR products were purified on a 3% NuSieve (FMC Bioproducts, Rockland, ME) agarose gel and inserted into a modified Blue-

script SK phagemid vector22 using the T4-Readyto-Go ligation kit (Pharmacia, Milwaukee, WI). Escherichia coli competent bacteria transformed with recombinant vectors were plated on LB-ampicillinagar containing X-gal and IPTG. After an overnight growth, 50 clones containing inserts were randomly selected and each clone inoculated into separate microfuge tubes containing 300 pl LB-ampicillin and grown overnight. The 250 pl of each culture were reserved and kept in 20% glycerol at -70 C (for eventual further growing, plasmid extraction and sequencing), whereas the remaining 50 pi was centrifuged quickly.

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The cellular pellet was resuspended in 2 ml of TE, pH 8, buffer and heated at 100 C for 5 minutes. The DNA from each expanded clone was tested by ASPCR with the corresponding specific primers to evaluate the proportion of mutated clones. For each sample containing a number of clones showing a K-ras mutation by ASPCR, the presence of the mutation was confirmed by sequencing two independent clones by the dideoxy termination method using the Taq polymerase and the T7 primer (5'-AATACGACTCACTATAG3') on a Applied Biosystems model 373A automatic sequencer (Applied Biosystems, Foster City, CA).

p53 PCR Amplification Exons 5, 6, 7, and 8 of the p53 gene were amplified by PCR using the following primers: 5A: TTCCTCTTCCTGCAGTAC and 5B: GCCCCAGCTGCTCACCA for exon 5; 6A: GGGCTGGTTGCCCAGGGT and 6B: TAGTTGCAAACCAGACCTC for exon 6; 7A: GTGTTG(A)TCTCCTAGGTTG and 7B: TGGCAAGTGGCTCCTGAC for exon 7; 8A: CCTATCCTGAGTAGTGGT and 8B: GTCCTGCTTGCTTACC for exon 8. The reactions were performed in a volume of 30 pl. The thermal cycle profile was 1.5 minutes at 94 C, 1.75 minutes at 55 C (for exons 5, 7, and 8) or 60 C (for exon 6), and 2 minutes at 73 C. This cycle was repeated 45 times.

Detection of p53 Gene Alterations by Nonradioactive SSCP We modified the method described by Orita et al23 and Spinardi et a124 as previously reported.25 Briefly, 2 to 10 ng of each PCR product were denatured in 50 mM NaOH and 1 mM EDTA at 50 C for 10 minutes (volume 10 p1l). These conditions allow an almost complete denaturation of the DNA. After addition of 1.5 pi of formamide dye the samples were analyzed immediately in an 8 to 10% polyacrylamide gel (49:1 acrylamide to bisacrylamide) with or without 5% (volume/volume) glycerol. Electrophoresis was performed at 20 V/cm in 0.5x TBE for 4 hours and the temperature was maintained between 20 and 23 C. The gels were silver stained (Silver Stain Plus, Bio-Rad, Richmond, CA) according to the manufacturer's specifications.

Results The 39 samples of neoplastic and nonneoplastic tissues taken from the nine patients were examined for K-ras mutations at codons 12 and 13 by ASPCR.

In some noncancerous lesions, exon 1 of the K-ras gene was amplified by conventional PCR, cloned in a plasmid, and the frequency of mutated alleles evaluated by examining 50 separate clones by ASPCR and confirming the mutation by sequencing two of the identified mutated clones. For p53 analysis, the nine carcinomas were systematically examined by both PCR-SSCP and immunohistochemistry. In cases positive for p53 alterations, one sample of nonneoplastic tissue from the same patient was also analyzed by SSCP to exclude a constitutive DNA polymorphism. The 30 samples of precancerous and nonneoplastic mucosa were first examined by immunohistochemistry to localize precisely the p53 overexpressing cells. Then, the immunohistochemically positive regions were microdissected, exons 5 to 8 of the p53 gene were amplified separately by PCR, and the products analyzed by

SSCP.

Carcinomas Two of the 9 (22%) invasive carcinomas contained a K-ras mutation. The mutation was located at codon 12 in one case (patient 7: mutation GGT-*TGT) and at codon 13 in the other (patient 9: mutation GGC-*GAC) (Figure 1B). The histological appearance of the carcinoma of patient 9 is shown in Figure 1A. The p53 gene alterations and/or protein overexpression were observed in three of nine (33%) invasive carcinomas. Two tumors (cases 5 and 9) contained an alteration within exon 6, which was detected by PCR-SSCP (Figure 1 D, patient 9). In addition, the tumor of case 9 showed a strong nuclear immunoreactivity with the NCC-CM 1 anti-p53 antiserum (Figure 1 C). In the third carcinoma (no. 6), the nuclei of a small number of tumor cells were reactive for all three antip53 antisera by immunohistochemistry, suggesting the presence of a mutation that was not detected by SSCP (Table 2).

Dysplasia Among the 13 samples (from 5 patients) showing lowgrade or high-grade dysplasia, 5 (38%) belonging to three patients contained a K-ras mutation and 1 (7%) contained an altered p53 gene (Table 2). In one patient (no. 2) two separate foci of dysplasia contained the same K-ras mutation (12 GGT-*GAT) (Figure 2B). One of them, measuring approximately 5 mm, corresponded histologically to low-grade dysplasia (Figure 2A). To confirm the presence of mutated alleles in this focus and at the same time gain


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1. Carcinoma positive for both K-ras and p53 alterations (patient 9). A: HLstology by 1&E staining (x240). B: ASPCR analysis. Amplificalion is obtained nitb primem specific for the nild-tve K-ras gene (lane 3) and for the 13 GAC mutation (lane 2). There is no amplification using primers specific for a 12 GTT mutation (lane 1). M, marker (pUC 19/HaeIII). C: Immu.nohistochemistry. Strong nuclear positivity of all carcinomatouis cells for the NCC-CM1 anti-p53 antiserum (PAP, X240). D: Fxon 6-SSCP analysis. Presence of an additional banid indicated bV the arrouw (lante 1). Sequenced DNA from a p53 unild-type carcinoma is a negative control (lane 2).

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quantitative estimate of the number of mutated alleles in the same tissue sample, we proceeded to amplify the first exon of K-ras with amplimers outside of the codons 12 and 13. Fifty independent clones of the product were retested by allele-specific amplification to determine the proportion of mutated and wild-type alleles present in the product as described in detail in Materials and Methods. To confirm the authenticity of the sequences detected by the GAT primers, two of the positive clones by ASPCR were sequenced and confirmed to be GAT. In this way, the tissue sample corresponding to this focus of dysplasia has been estimated to contain approximately 6% of mutated K-ras alleles. In the same area, more than 70% of the epithelial cells were strongly immunoreactive for p53 (Figure 2C). By PCR-SSCP analysis, the DNA extracted from this minute zone showed a shift confirming the presence of an altered allele in exon 8 (Figure 2D). Thus, one focus of low-grade dysplastic mucosa accumulated alterations of both K-ras and p53 genes. Interestingly, the carcinoma was devoid of K-ras and p53 mutation in this case. a

In another patient (no. 7) the K-ras mutation present in two foci of high-grade dysplasia was identical to the one present in the adjacent invasive carcinoma (12 GGT-*TGT), whereas in the same individual a third sample with peritumoral dysplasia and a sample from a distant dysplasia were wild type. In patient no. 8, a K-ras mutation was found in one of two samples with peritumoral high-grade dysplasia (mutation GGT--TGT in codon 12), but in the same patient a minute focus of invasive carcinoma and a distant focus of dysplastic mucosa both showed a wild-type K-ras gene.

Villous Regeneration One of six (16%) samples showing villous regeneration without dysplasia contained a 12 GGT--GAT mutation of the K-ras gene detected by ASPCR (patient 2). To confirm this mutation and estimate the proportion of mutated alleles in this tissue sample, we amplified the first exon of the gene using primers outside

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Chaubert et al


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Figure 2. Area of low-grade dysplasia positive for both K-ras anid p53 alterations (patient 2). A: H&E staining. Arrows inidicate the limits of the posititve area (x 13). B: ASPCR analysis. Amplificationi is obtained uith pimers specificfor the uild-type K-ras gene (lane 3) andfor the 12 GAT mutation (lane 1). Negative conitrol uising a primer spec ic for a 12 TGT mnttationi (lane 2). M, mtarker (pUIC191HaeIII). C: Immunohistochemistry. Strong nuclear positiviiy of most oJ the dysplastic epithelial cells for the NCC-CM1 anti-p53 antisenrm within the same area (PAP, X 190). D: Fvon 8-SSCI' analysis. DNA from the microdissected area immunoreactiwe.forp53 (lane 1) shows an additional (b), a decreasing (c), and an increasinig band (a). Sequenced DNA fromn a p53 wild-ye carcinoma is a negative conitrol (lane 2).

of the codons 12 and 13 and cloned the product into a plasmid. Seven of 50 independent clones screened by ASPCR (see Materials and Methods) contained the GAT mutation indicating that the proportion of mutated K-ras alleles was approximately 14% in this tissue sample. Two of the positive clones were sequenced and confirmed to be GAT. From the same patient, two samples with dysplasia contained an identical K-ras mutation (12 GGT-*GAT), whereas the corresponding invasive carcinoma did not have a K-ras mutation (Table 2). No p53 overexpression was observed in villous regeneration.

Active Colitis Seven samples with active inflammation were examined for K-ras and p53 mutations, all lacking any cy-

tological features of dysplasia. Two of them, obtained from the same patient (case 3), contained a K-ras mutation at the second base of codon 12 detected by ASPCR (Table 2). It was a GGT--GTT mutation in one lesion (Figure 3B) and GGT-*GAT in the other lesion. In both samples, we amplified the first exon of the gene using primers outside of the codons 12 and 13 and cloned the product into a plasmid. For each lesion, we retested 50 independent clones by ASPCR (see Materials and Methods) using the specific primers. In the first lesion, 3 of 50 clones contained the GTT mutation (Figure 3C) indicating that the proportion of mutated K-ras alleles was approximately 6% in the tissue sample. In the second lesion, 2 of 50 clones were found to be GAT (4% of mutated alleles). Both mutations were verified by sequencing two clones positive by ASPCR from each lesion. The histological

K-ras and p53 Alterations in Ulcerative Colitis

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AJP April 1994, Vol. 144, NVo. 4

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Figure 3. Foci(s qf active colitis conitctiiliiig a K-r.ts tnlttcationi ( patient 3) A: Histologyb!h H51 stcaining. Presence oJan imipor-tanit active

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appearance of the second lesion is illustrated in Figure 3A. We observed no p53 overexpression in active colitis.

Histologically Normal Mucosa The samples obtained from histologically normal mucosa showed no evidence of K-ras or p53 mutations (Table 2).

Discussion In this study we assessed the frequency of p53 alterations in colorectal carcinomas occurring after longstanding UC. Using a combination of immunohistochemical phenotypic and PCR-SSCP genotypic analysis to minimize the risk of missing a mutation, we found only 3 of 9 (33%) UC-related carcinomas showing any evidence of p53 alteration. Interestingly, two

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1io0 is obtainied itith primers spec f/ic Jor the itild-tipc Ka-ras genie (la)te 4u) andl fr the 12 G7T muttattttiotn ( httte 3) Th)ere is 00o amtiJlicatio) 1isi03g primnrs speci/ic J/br a 12 AGT niutationi (loate 2) Lsing sequenced DANA frot ca K-ras dild-t)pe ccarcinomtat Cas C teinplate, there is tio ctetpl/ificcttiott with pvimen2sspecific /br the /2 (GTT itlttattio)t (lanie 1 ) C: Qliatitijicatioti of thce 12 G7TE unittationi itt the sattie /bc its K- i.s exoti 1 was amplified by PCI iusitgl )rittets oittside of the cocloIns 12 aticl 13 cand thbe p)toClttLt twas cloned ititoCa plasotid. Efi y indepeticlcent clottes itere retested by, ASPICR utsitg

the GTT primtens. The 50 clotnes wtere fist screetned bv grottps of five clonues (top). 7Ibree grottps had at least otte clotIe conitaitnitng the GTEtInItcItiott (lattes 1, 4. aticl 10 top) Bli testit) sepcarately thefive clotiesftot ecich oJ the three positive gr-otlps ( bottotti). tht-ee clottes colItitisiig the GTE ,ittttatioti were identtf/ied (latits 5, 7, citict iS, bottottt) The G Y iibtct -

tioni wacts cot)tfirred by, DNA sequtetncitngi ott twt o

of th'e three positive clottes.

carcinomas that appeared to contain a mutated p53 gene gave a conformation polymorphism located in exon 6 that is not frequently mutated in sporadic cancers.1 1 In both cases, a constitutive dimorphism was excluded because the SSCP shifts were not found in the nonneoplastic tissues from the same patients. The positive p53 staining in the carcinoma that showed no alteration in SSCP pattern can be explained by stabilization of the p53 protein by a mechanism other than mutation.26-28 K-ras mutations were found in 2 of 9 (22%) UC-associated carcinomas, a percentage that is consistent with previous reports in the literature.7-9 Thus, using a more sensitive technique than previous reports (ASPCR), we confirm that K-ras mutations are less frequent in UC-related carcinomas in comparison with sporadic ones. Detection of genetic alterations in histologically precancerous, ie, dysplastic mucosae, is useful to assess their role during carcinogenesis. In such tissues,

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small amounts of point mutations cannot be detected by direct sequencing because this technique is not sensitive enough. Systematically cloning and sequencing DNA from such lesions even after microdissection is not routinely feasable because it represents an enormous amount of work. It would be necessary to sequence hundreds of clones for each sample to detect a mutation. Therefore, we have applied the ASPCR, which is, with some precautions (see Materials and Methods), a reliable and sensitive technique to detect mutations in cases where DNA sequencing is not easily possible. Using this technique, we found K-ras mutations in 5 of 13 (38%) dysplastic lesions. In other words, in 3 of the 5 (60%) patients who had available dysplasia, we found at least one focus of dysplasia containing a K-ras mutation. This frequency was even higher than in invasive carcinomas (22%), suggesting that the cells bearing K-ras mutations found in dysplasia are not the most likely to progress toward cancer in UC. Indeed, in one patient the invasive carcinoma contained only wild-type K-ras and p53 genes, whereas a dysplastic lesion in the same specimen had accumulated a K-ras mutation and a p53 gene alteration. Thus, it is likely that the carcinoma arose from cells containing other genetic alterations. In another case, the cancer showed no evidence of mutation, despite the presence of a mutated K-ras gene in the adjacent dysplasia. A similar case was recently reported by Chen et al.10 The possibility that K-ras mutations may be lost during the dysplasia-carcinoma sequence is unlikely in view of data suggesting that acquired K-ras mutations are fairly stable throughout the natural history of conventional sporadic colorectal carcinomas.21 There are probably various pathways leading from inflammatory mucosa to neoplasm in UC. Although the classical dysplasia-*carcinoma sequence resulting from cumulative K-ras and p53 mutations occurs in some UC-associated cancers, most cases seem to be the consequence of genetic events other than the ones we have studied. We found that in carcinomas alterations of the p53 gene had roughly the same prevalence as K-ras mutations, whereas they were noticeably less frequent in noncancerous mucosae. This difference could be due to the different sensitivities of the methodologies used. By ASPCR we can detect about one cell harboring a K-ras mutation among 100 normal cells in fixed tissues, as determined by a dilution assay (P. Chaubert, unpublished data). In contrast, SSCP is in our conditions at least 1 0-fold less sensitive to detect the presence of mutated alleles than ASPCR.25 To detect minute clones, it is essential to select p53 im-

munoreactive cells making the PCR substrate rich in overexpressing cells. However, p53 immunohistochemistry on fixed tissues fails to detect some mutated cases, even when three different antibodies are used. Thus, we cannot exclude that some p53 mutations escape detection in some nontumoral tissues. On the other hand, in cancers that represent large clonal expansions a negative p53 result, using both immunohistochemistry and PCR-SSCP, is more significant. There are only limited data supporting the existence in UC of an affiliation between histologically nonneoplastic lesions and carcinoma.10'15 We found unambiguously that K-ras mutations can be present in histological lesions not considered as preneoplastic, ie, regenerative (villous regeneration) and even inflammatory (active colitis) mucosae. In these foci, the proportion of mutated K-ras alleles, as evaluated by a PCR cloning procedure, was even far higher than expected. Considering the estimated percentage of epithelial cells contained in the analyzed tissue samples (Table 2), it is clear that the K-ras-mutated cells present in active colitis and villous regeneration constitute authentic clones with a yet unknown biological significance. From a genetic point of view, these clones should be considered as preneoplastic. They may represent the precursors of dysplasia and carcinoma in UC. In one patient we observed that the K-ras mutation present in one carcinoma was also found in the surrounding dysplastic mucosa, confirming that clones in the histologically defined dysplasia represent a precancerous lesion in UC. Although these observations were made in specimens from different patients, they suggest, as previously proposed,29 that a sequence active colitis-*villous regeneration--dysplasia---carcinoma is possible in UC. Moreover, these findings bring up the question of the biological significance of histopathologically defined inflammation and regeneration.

Acknowledgments We thank S. Burki and J. Maillardet for photography, J. Bertoncini and P. Martin for technical assistance, and Drs. E. Offord, P. Shaw, and E. Fearon for critical reading of the manuscript.

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