Oncogene mutations in cancer and dysplasia associated with inflammatory bowel disease

Chronic inflammatory bowel disease (IBO) predisposes affected individuals to the development of invasive colon cancer. Increased cancer risk has been seen in ulcerative colitis and Crohn's disease in Western societies as well as in schistosomal colitis in China. In this study it was found that the frequency of the 12th codon of c-Ki-ras, as well as the specific amino acid substitutions, were similar in sporadic colon cancers, cancers associated with ulcerative colitis in the United States, and colon cancers found in patients with schistosomal colitis in Jiangsu province in China. Further, activating mutations of codon 12 of e-Ki-ras were found in high grade dysplastic lesions in chronic ulcerative colitis. The latter finding indicates that some dysplasias are clonal proliferative lesions with genetic characteristics associated with invasive cancer. Can J Gastroenterol 1990;4(7):384,389

I T HAS BEEN RECOGNIZED FOR MANY years that patients with longstanding ulcerative colitis are at increased risk for develo ping invasive colon carcinoma.This association is most evide nt in patien ts with extensive disease of more than 10 yea rs' Jurat1on ( l-4).Population -based studies indicate that cancer risk is approx imate ly 0.5 ro I % per year beginning 10 years after diagnosis.Epithelial dysplas1a is considered a precancerous lesion m ulcerative colit is and is use<l as a h1stologic predictor of cancer development ( 5).The rela t1onsh1p between dysplasia and cancer in ulcera tive colins has been inferred from the observation that colons resecte<l for cancer m ulcerative colit is almost always have regions of associated dysplasia (6).Also, specimen s resecre<l because of <lysplasia freque ntly ha rbour occult carcino mas (7).Because dysplasia may be difficult to distinguish fro m reparative ch anges in ulcerative colitis, a stan<la r<lized classification h as been developed and used co grade the severity of the dysplasttc cha nges (8).Ir is presumed , but not unequivocally proven , tha t neoplastic transfonnation invo lves the progression of a colonic epithe lial cell from norma l morpho logy through increasing grades of dysplasia to invasive carcinoma, and that this process is associated with the accumula tion of specific genetic lesions.Colectomy specimens from patients with chronic ulcerative colitis are an excellent resource to study the biology and genetics of the precancerous phase of colon cancer, since specimens often have carcinoma, varying grades of dysplasia and relatively normal or regenerative epithelium.
Cancer associated with inflammatory bowel disease (IBD) is of con-s1Jerable importance in other regions of the worlJ as well, particularly in southeastern China.Schistosoma japonicum infection 1:; endemic m Jiangsu province and leads to a chronic sch1stosomal colitis.The colon cancer mcidence rates in J iangsu province, 44 cases per 100,000, are the highest in Asia.Dr Chen Ming-Chai an<l his colleagues (9-12) have dc::.cribed the epidemiology anJ clinical aspects of schistosomal colitis in this region and presented the histologic evolution of inflammatory lesions progressing to invasive cancer.The histopathology of chronic schistosomal colitis is remarkably similar to that found m chronic ulcerative colitis and, in both disorders, flat dysplastic changes appear to be the precancerous lesion.
Invasive colon cancer is a genetically complex disease, evidenced by the finding of multiple karyotypic abnormalities and a high frequency of allelic loss at multiple chromosomal sites in colon cancer cells (l 3-16).Nevertheless, a scnes of nonrandom generic changes have been identified which occur frequently and appear to p lay a significant role in the molecular pathogenesis of this disease.These include alterations of dominantly acting oncogenes (ras, c-myc and c-src) and of presumed 'suppressor' genes located on chromosomes Sq, l 7p and 18q.
Of the dominantly acting oncogenes, the ras genes have been studied in greatest detail, and approximately 50% of colon cancers have been found to have an oncogenic ras mutation (17,18).The majority of mutations of ras genes in colon cancer occur at coJon 12 or 13 of c-Ki-ras (approximately 90%), the remainder occurring at codon 61 of c-Ki-ras and at similar positions in N-ras.These mutations are associated with JimmisheJ GTPase ac-t1v1ty and enhanced transforming properties of the p21 molecule in vitro.pp60c-src tyrosyl kinase activity has been found to be increased, compared to normal colonic mucosa, in the majority of colon cancers, with some tumours demonstrating disproportionately high kinase activity compared to pp60c-src protein levels (19)(20)(21)(22).This has given rise to the notion that pp6if•src IS 'activated' in colon cancer, as can be seen with the neural form of pp60vsr', oncogemc mutations of c-src (eg, at position::.338, 378, 441, 527 of chicken c-src, and 3) when a high proportion of pp60'-•src molecules are phosphorylated at Tyr 416 relative to Tyr 527.In previous studies the author was unable to detect activating mutations of c-src in colon tumours expressing high levels of pp6oc•$TC kinase activity (23).The author's interpretation of these data 1s that colon cancer may represent the clonal expansion of a colonic epithelial cell at a specific stage in differentiation at which the cell normally expresses high levels of pp60<.:-mryrosyl kmase, and this activity is not necessarily linked to cell transformation.
Both cytogenenc and molecular studies have shown that a portion or all of chromosomes 5, I 7 and 18 are frequently lost in advanced colon carcinoma.This suggests that recessive genes on these chromosomes may be ac, tivated by loss of a normal balancing allele.A locus on Sq (APC) has been linked to familial adenomatous polyposis of the colon and has been mapped to Sq21-22 (24,25).Nineteen to 36% of sporadic colon carcinomas have been reported to have hemizygous loss of Sq (14, [26][27][28][29].lf genetic markers closely flanking the APC locus are used, approximately 50% of sporadic colon cancers will demonstrate loss of a portion of Sq, indicating the importance of this locus in the molecular pathogenesis of sporadic colon cancer (30).A high incidence of allelic loss at chromosome l 7p has a lso been found in a number of studies of colon cancers (13-15).In a number of instances this has been associated with mutations of the p53 gene in the remaining allele (30-32).The described mutations occur in two evolutionary conserved regions of p53 and suggest that these mutations alter or inactivate the normal function of the gene product.Allelic loss at chromosome 18q occurs in approximately 75% of colon cancers, determined by karyotypic analysis and molecular studies (11,27).A candidate 'suppressor' gene involved in the allelic deletion on 18q has recently been described (33).The gene on 18q shows sequence similarity to neural cell adhesion molecules and has been shown co have diminished expression as well as being mutated in a number of colon carcmomas.
The high frequency of specific genetic alterations m colon cancer allowed Vogelstein and his colleagues (27) to define the sequence of these genetic changes in the polyp to cancer sequence.They found that ras mutations and Sq deletions occurred with equal frequency in polyps and cancers but that l 7p and 18q deletions occurred more frequently in invasive carcinomas.Their data suggest that the ras mutation and Sq deletion occur early in this sequence (but after the polyp has developed) and that the l 7p and 18q deletions occur at the transition to, or after carcinoma has developed.
In this work, the author addresses a number of issues related to the role of oncogene mutations in cancers and dysplasias associated with lBD.First, the frequency and type ofras mutations in sporadic colon cancers (evolved from polyps) are determined and contrasted with colon cancers associated with IBD both ulcerative colitis in the United States and schistosomal colitis in Chma.Second, evidence is presented that the ras mutation may occur in the dysplastic phase of lBD.The 3' end primer contains an internal BstNl site (CCTGG).5' end primer: DNA from eight colon cancers were assayed and adjacent lanes represent polymerase chain reaction-amplified DNA which is uncuc (U) or cut (C) with Bsc NI.Four w.moun (I, 2, 6 and 8) have a mutant band indicating a codon 12 mutation mutations at codon L2.The aspartic acid mutation of codon 13 (G to A at position 2) creates an Hph I recognition site.Therefore, this mutation which 1s the most common mutation of codon 13, was scored by using primers as shown below for codon 12 followed by digestion with Hph I.

5'GGGCCTCACCTCTATGGTGG3'
DNA ( 1 mg) was added to 50 mL of PCR reaction mix containing 1 mM dNTPs, 5 mM Tris hydrochloride, pH 7.5, 75 mM sodium chloride, 5 mM magnesium chloride and 1 µg o ligomer primer.Two and one-half units of Taq I polymerase was added and the mixture overlaid with mineral oil.The DNA was amplified in a thermal cycler using the following conditions: denaturation 1 min at 95°C; annealing 1 min at 55°C; and elongation 1 min at 72°C.The sample was subjected co 30 cycles of amplification, the oil removed and the reaction mixture extracted with chloroform.One-tenth of the reaction mix was added directly to the appropriate restriction enzyme buffer.Restriction enzyme was used at a ratio of five units enzyme per microgram of DNA, and digested for 2 h.The digested products were subjected to electrophoresis in nondenaturing 8% polyacrylamide gels and analyzed after staining with ethidium bromide.In a number of instances the oligonucleotide primers were end labelled with [ 32 P]-A TP and polynucleoc1Je kinase.Following amplification, enzyme Jigestion and electrophoresis, digestion products were analyzeJ by autoradiography.
DNA sequencing: Amplified DNA products were extracted with chloroform, diluted with 2.5 ml water and subjected to centrifuge-driven d1alys1s using a Centricon C-30 filter (Amicon, Massaschusetts), to remove excess primers.The double-stranded DNA was sequenced by the dideoxy-terminator method (36) using the 3' end amplification primer and the modified T7 DNA polymerase, Sequenase (US Biochemicals, Ohio) as previously described (37).

RESULTS
Restnction fragment polymorphism assays were performeJ to detect mutations of codon 12 and 13 of c-Ki-ras and N-ras in 42 sporadic colon cancers, 15 colon cancers associated with schistosomal colitis and three tumours associated with ulcerative colitis.The majority of mutations detected in the cancers in this study was at codon 12 of c-Ki-ras (Figure 1).These were scored using an artificially generated Bst N 1 site at codon 12 using the PCR as described in the 'Patients and Methods' section.Twenty-three of 47 sporadic cancers, 12 of 15 colon cancers associated with schistosomiasis, and two of three cancers associated with ulcerative colitis were positive for ras mutations (Table l).Eight of the mutations in cancers associated with schisto-

TABLE 1 c-Kl-ras mutations In sporadic inflammatory bowel disease-associated colon cancers
assay was used on five contiguous samples of high grade dysplasia; those results were previously reported.Subsequent to that manuscnpt a carcinoma was found on the same specimen.RFLP analysis and DNA sequencing were performed on both cancer and dysplasia.Multiple RFLP assays were performed on the second specimen.In case 1 the invasive cancer and two contiguous regions of h igh grade dysplasia had a glycine to aspartate mutation of the c-Ki-ras codon 12 (Figure 2).In case 2, RFLP analysis demonstrated c-Ki-ras codon 12 mutations of the cancer and two regions of high grade dysplasia, but a nonnal c-Ki--ras gene was found in DNA from normal appearing mucosa distant from the tumour (Figure 2).somiasis and one associated with ulcerative colitis were subjected to DNA sequencing of the PCR-generated ras amplification product.Of the sch1stosomiasis-associated cancers, six were found to have glycine to aspartare substitutions and two had glycine to valine sub,titutions.The one cancer found in an ulcerative colitis patient that was sequenced had a glycine to aspartate substitution.
The PCR-based assay was used for c-Ki-and N-ras mutations to sample small biopsies of mucosa from patients with chronic ulcerative colitis (Table 2).No mutations were detected in multiple biopsies from ulcerative colitis that were negative for dysplas1a.Two patients in this group were at increased risk for colorectal cancer as they had active ulcerative coliti& for 10 and 20 years, respectively.However, samples from three of five patients with high grade dysplasia contained c-Ki-ras codon 12 mutations.Samples from one patient were sequenced and found to contain a glycine to aspartate mutation.
Multiple areas from two colectomy specimens were assayed which contained invasive cancer, areas of

D I SCUSSION
In this study the frequency of ras mutations in sporadic cancers (approximately 50%) was similar to that found in a number of previous studies (17,18).The authors were unable tO  ras mutations may represent a true higher incidence of mutation in this disease or more likely a selection bias in a small series.The assay of additional cancers associated with IBO will clarify this issue.The c-Ki-ras mutations were primarily of the 12th codon and were either aspartate or valine substitutions for glycine.These are two frequent substitutions reported in sporadic cancers in the United States although serine and cystine mutations, which are also found in sporadic cancers, were not detected in these tumours.
Of interest was that four carcinomas had faint mutation bands in the RFLP assay on repeated analysis using ethidium staining or radioisotopically labelled oligonucleotide primers.The authors have examined the histologic sections immediately adjacent to the tissue assayed for mutations, and more than 80% of the cells were identified as tumour cells in all four tumours (data not shown).The lower limit of sensitivity of the RFLP assay is the detection of a mutation in approximately 5% of cells.le appears that the ras mutation was present in only a subpopulation of tumour cells, probably 5 to 10% of the cells.Therefore, it seems likely that the ras mutation occurred late in the evolu-tion of these cancers or, less likely, that the mutated allele was lost from a major population of tumour cells in the progression of the tumor.Other work has suggested that the ras mutation occurs early in most sporadic cancers, ie, in the adenoma stage (27).
The finding of activating c-Ki-ras mutations in high grade dysplasia is consistent with the finding of ras mutations in some polyps (27) and supports che notion that this mutation can frequently occur prior to the invasive stage of colon cancer development.The assay of additional specimens with low and intermediate grades of dysplasia will elucidate what the earliest stage in the transformation process is m which the ras mutation can be detected.Further, these studies will indicate whether activating mutations of ras will be a useful adjunct to histopathology in the assessment of cancer risk in chronic IBO.
In two instances the authors were able to score ras mutations in multiple samples throughout colectomy specimens from patients with ulcerative colitis with both Lancer and dysplasia.It was found chat dysplastic cells containing the identical mutation covered a wide area of mucosa, suggesting chat dysplasia is not a focal lesion but rather it is a clonal.proliferative abnormality n.ConcerteJ nonsynrcmc ,1llel1c loss in which spreads horizontally but is not vertically invasive.These data are supported by recent studies of cellular DNA content using flow cytometry and cytophotometric techniques demonstrating aneuploid clones of dysplastic epithelial cells in ulcerative colitis (34,35).Further, it was found chat there were regions of high grade dysplasia which lacked ras mutations even though they were contiguous to morphologically identical epithelium which scored posinve in ras mutation assays.These data suggest chat either the mutation occurs late in the evolution of the dysplastic clone or that there are multiple dysplastic clones in longstanding IBO.In two instances carcinoma contiguous to a region of dysplasia has been detected in which both cancer and dysplasia had the identical activating c-Ki-ras mutation.
In these instances it is likely that the cancer evolved from the area of dysplasia.ln summary, these studies have shown that activating mutations of c-Ki-ras represent a common genetic pathway for transformation of colonic epithelial cells irrespective of etiology or ethnic background.Further, this genetic lesion can be found in the noninvasive stage in the evolution of colon carcinoma associated with IBO.
of inflammatory mucosa devoid of dysplasia.Multiple histologic sections were obtained to ensure chat dysplastic areas were devoid of invasive carcinoma.In the study of the first specimen.an RNase protection 5c m obtain a sufficient number of samples of sporadic colon cancer from China to compare the mutation frequencies in sporadic cancers from China and the United States.The finding of 12 of 15 ( 80%) schistosomias1s-associated colon r---, : :-( -) L ---J 11111 Cancer EZ] Dysplosio c::::J Normal