Citation for published version (APA): Bleeker, W. A. (2001). Therapeutic considerations in Dukes C colon cancer s.n.

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1 University of Groningen Therapeutic considerations in Dukes C colon cancer Bleeker, Willem Aldert IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2001 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Bleeker, W. A. (2001). Therapeutic considerations in Dukes C colon cancer s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Chapter VI Impact of KRAS and TP53 mutations on survival in patients with left and right-sided Dukes C colon cancer Bleeker W.A. 1, Hayes V.M. 2, Karrenbeld A. 3, Hofstra R.M.W. 2, Hermans J. 4, Poppema S. 3, Buys CH.C.M. 2, Plukker J.Th.M. 1 Departments of Surgery 1, Medical Genetics 2, Pathology 3, University Hospital Groningen, and the department of Medical Statistics 4, Leiden University Medical Center, the Netherlands. American Journal of Gastroenterology: 2000;95(10):

3 Chapter VI Abstract Objectives: It has been suggested that KRAS and TP53 might influence the phenotypic behaviour of left and right-sided colon tumors. We investigated the incidence of these mutations in left and right-sided colon tumors and their possible influence on survival in a homogeneous group of patients with Dukes C colon cancers. Methods: The primary tumors of 55 patients with a sporadic Dukes C colon cancer, all treated with adjuvant chemotherapy were analysed for the presence of KRAS and TP53 mutations. Mutations detection of the KRAS and TP53 mutated tumors was performed on paraffin embedded tumor material, using denaturating gradient gel electrophoresis. Five-year survival rates of KRAS and TP53 mutated tumors were analysed regarding right sided tumors, defined as tumors up to the splenic flexure, and left sided tumors, defined as tumors from the splenic flexure to the rectosigmoid peritoneal reflection. Results: KRAS mutations occur more frequently in the right colon compared to the left colon (R=38%(10/26),L=10%(3/29): Chi-Square test:p=0,014). KRAS mutation did not influence survival in patients with riht-sided colon tumors. Patients with KRAS mutation negative tumors in the right colon, however, had a significantly worse survival than patients with leftsided KRAS mutation-negative tumors (5-year survival rate; R:34% vs L:65%, Log-rank test:p=0,007). TP53 mutations of a possible causative nature were found in 24 tumors (44%). Neither the incidence (R=42% (11/26); L=45% (13/29)) nor the survival of TP53 mutated tumors differed significantly between left and right-sided tumors. Furthermore, survival of patients with TP53mutation-negative tumors did not differ significantly between left and right-sided tumors. Introduction It has been suggested that left and right-sided colon tumors differ in their genetic susceptibilities to neoplastic transformation. 1-5 Genes which are thought to play an important role in the explanation of tumor genesis of left and right-sided colontumors are TP53 and KRAS. 3,6 K-RAS, a proto-oncogene, located on chromosome 12, is frequently mutated in colon cancers with an overall incidence that varies from 20% to 50%. Most studies that focused on tumor location report similar incidences of K-RAS mutations in left-sided tumors (22%-52%) 68

4 Impact of KRAS and TP53 mutations on survival in left and right sided Dukes C colon cancer and in right-sided colon tumors (18%-47%). 1,2,4,7,8 Whereas some authors found that patients with K-RAS mutated colon tumors have a worse survival compared to non-mutated tumors. 1,4,8-10, this is disputed by others. 7,11 K-RAS related patient survival data in combination with tumor location is scarce. Two studies reported on survival data of patients with KRAS mutations in combination with TP53 mutations. 1,12 They concluded that colon tumors with a combination of K-RAS and TP53 mutations lead to a worse survival, but differences in survival between left and right-sided colon tumors are not mentioned. TP53 is a tumor suppressor gene located on chromosome The presence of TP53 mutations carries an independent adverse prognostic value in colon cancer. 9,13 Recently Børresen-Dale et al. found a higher incidence of TP53 mutated tumors in the left colon and a shorter cancer-specific survival compared to right-sided colon tumors. 14 Unfortunately, the various stages of disease, the lack of specific survival data and the use of adjuvant chemotherapy make it difficult to compare these studies. 11 As we are involved in a randomised controlled trial of Dukes' C colon cancer patients treated with adjuvant chemotherapy, we were able to genotype KRAS and TP53 in a homogeneous group of patients and relate these data to tumor location and survival. Material and Methods Clinical Material Representative samples of the primary non-hereditary tumor of 55 consecutive patients were taken from original paraffin-embedded blocks and analysed for mutations in KRAS and TP53. All patients underwent a curative resection of a histological proven Dukes C adenocarcinoma of the colon and were treated with adjuvant chemotherapy between February 1991 and January Patient and tumor characteristics are summarized in table 1. Adequate followup data was available in all patients. The median follow-up of the patients still alive is 47 months. Twenty-six tumors were located at the right side i.e. caecum, ascending colon and transverse colon up to the splenic flexure and 29 tumors were situated at the left side of the colon i.e from the splenic flexure, descending colon and sigmoid. The left and right-sided group were comparable with respect to the important histological prognostic variables i.e. number of lymph nodes, degree of bowel wall invasion and tumor differentiation and there was no difference in survival regarding the given form of chemotherapy

5 Chapter VI Morphologic Analysis and Topographic selection Under microscopic control representative samples of 5 x 10m were taken from the deepest point of invasion of the paraffin-embedded blocks containing morphologically recognizable tumor cells. DNA was extracted using the chelex-boiling method. 16 Mutational Analysis Hotspot codons of the K-RAS oncogene (codons 12, 13 and 61), including adjacent codons, were amplified in two amplicons using a nested polymerase chain reaction (PCR). Amplicons were subjected to denaturing gradient gel electrophoresis (DGGE) containing a 20-60% UF denaturing gradient (100% UF = 7M Urea/ 40% deionised formamide). The evolutionarily conserved region of the TP53 gene (exon 5-8), including all splice site junctions, were amplified in four amplicons via a nested PCR and electrophoresed in a 35-75% UF denaturing gradient polyacrylamide gel. Electrophoresis was performed at 150 V for 7.5 hours at 59 º C. Primers and DGGE conditions used for both genes as previously described are available on request. Samples showing aberrant banding patterns were subjected to direct sequencing using single-stranded automated sequencing. All mutations were confirmed either by sequencing from the reverse primer or by restriction digestion. Definition of causative mutation In this study, mutations resulting directly in protein truncation (nonsense or frameshift mutations), or in a nonconservative amino acid substitution, or effecting the splicing mechanism are considered as presumably causative. Statistical Analysis Kaplan-Meier survival curves were generated for patients with left and right-sided colon tumors according to their genetic patterns of K-RAS and TP53 mutations. The Log-rank test was used to study the prognostic value of K-RAS and TP53 mutations alone or in combination with respect to cancer-specific survival. Cancer-specific survival was calculated as the time interval from randomisation to death due to cancer recurrence. Patients alive with no evidence of disease and patients who died from intercurrent disease are censored. The relationships between tumor characteristics and genetic alterations were determined by the chi-square test or Fisher s exact test. 70

6 Impact of KRAS and TP53 mutations on survival in left and right sided Dukes C colon cancer Results KRAS mutations were found in 13 of 55 tissue specimen (23%). Nine KRAS mutations were found in codon 12 and four in codon 13, no mutations were found in codon 61. Mutations are given in table 2. Causative KRAS mutations were more frequent in the right colon (R: 38%(10/26), L:10%(3/29), Fisher-exact test: p=0.003). These mutations were mainly aggressive mutations (alanine or asparatate substitutions in the second codon), as indicated by Pricolo et al (10/13; 77%: right colon and left colon 2/3). 12 Tumor specific survival, in relation to KRAS status and tumor location is shown in figure 1. KRAS mutations as a prognostic factor could be analysed in right-sided tumors only. In that group there was a trend towards a better survival in KRAS mutated tumors compared to KRAS non-mutated tumors (5- year survival; resp. 62% and 34%; log-rank test: p=0.07). The three patients with left-sided KRAS mutated tumors had a relatively poor-survival, but the numbers are to small to draw conclusions. In the group without a KRAS mutated tumors, patients with left-sided tumors had a better survival than those with right-sided tumors (5 year survival; R colon: 34%, L colon: 65%, log-rank test: p=0,007). Twenty-four tumors (24/55;44%) had a possible causative TP53 mutation. Mutations in the evolutionarily conserved region of the TP53 gene were evenly distributed between left and right-sided tumors (L; 45%(13/29), R; 42%(11/26); n.s.) table 3. Tumor-specific survival did not differ between TP53 mutated and TP53 non-mutated tumors in respectively the right and the left colon, figure 2 (Log rank test: p=0.21 and Log-rank test: p=0,66 resp.). Tumor location had also no influence on survival in these patients with respect to mutations in the TP53 gene. Table 1: Patient and tumor characteristics. Right colon Left colon N Age (range) 58 (31-76) 56 (39-74) Sex male/female 15/11 19/10 Histological details tumor depth muscularis 4 2 serosa differentiation good 2 5 moderate bad

7 Chapter VI unknown 4 5 number of lymph nodes < Table 2: Type of KRAS mutation and tumour location Right colon Left colon Total Total No KRAS mutation KRAS mutation Gly 13 -Asp;G-A transition 3 3 Gly12-Ala;G-C transversions 1 1 Gly12-Ala;G-T transversions 1 1 Gly12-Asp;G-A transition Gly12-Asp;G-A transition & pol:gly12-gly;t-c trans 1 1 Gly12-Cys;G-T transversions 2 2 Gly12-Val;G-T transversions Gly13-cys;G-T transversions 1 1 Table 3: Type of TP53 mutation and tumour location. Right colon Left colon Total Total No TP53 mutation TP53 mutation ACA-ATA 1 1 CCT-ACT 1 1 CGA-TGA 2 2 CGC-CAC CGG-CAG 2 2 CGG-TGG 5 5 CGT-TGT Deletion of C 1 1 Deletion of GA 1 1 GGG-GAG 1 1 TAC-TGC 1 1 TAT-TGT 2 2 deletion of CTC

8 Impact of KRAS and TP53 mutations on survival in left and right sided Dukes C colon cancer Right colon Left colon Total insertion of T 1 1 Figure 1: KRAS mutations and the relationship between left and right-sided Dukes C colon tumours. (n.e. = Not evaluable) 1,0,8 Left colon KRAS-,6 Right colon KRAS+,4 percentage of survival,2 0,0 Right colon: KRAS+:62% KRAS-:34% log rank test:p=0,07 Left colon: KRAS+:n.e. KRAS-:65% log rank test:p=n.e. Left colon KRAS+ Right colon KRAS follow-up in months Figure 2: TP53 mutations and the relationship between left and right sided Dukes C colon tumours. 100 Left TP Left TP53 - Right TP53-40 Right TP53 + percentage of survival 20 0 Right colon : TP53 +:38%, TP53 -:54%, Log rank test:p=0.21 Left colon : TP53 +:42%, TP53 -:56%, Log rank test:p= follow - up in months 73

9 Chapter VI Discussion In this group of 55 patients with Dukes C colon cancer, mutations in the KRAS gene we predominantly present in right-sided tumors. The overall percentage of KRAS mutated tumors and the higher incidence of KRAS mutations in right-sided tumors is comparable to that reported by others. 4,17 Despite the higher incidence and the occurrence of aggressive KRAS mutations in right-sided tumors, survival was equal compared to KRAS negative tumors. Elnatan found a significantly poorer prognosis in left-sided colon carcinomas with KRAS activation, data we could not confirm because of the low incidence of left-sided KRAS mutated colon tumors. 4 His data confirms that KRAS activation was not found to be associated with a poorer prognosis in tumors originating on the right of the colorectum. However Elnatan s study included several stages of colorectal tumors. 4 In this study of Dukes C colon tumors we endorse that KRAS mutations do not influence survival in rightsided tumors. On the other hand, we did find a clear difference in survival comparing patients with KRAS-negative tumors in the right colon to those with KRAS-negative tumors in the left colon. Pricolo et al were the first to demonstrate a strong correlation between TP53 mutations and survival in a homogeneous group of patients. They described the results of 70 Dukes C colon cancer patients, who did not receive adjuvant chemotherapy. 12,13 When we correlate TP53 mutated tumors with location and survival no significant difference was found. Neither the incidence of TP53 mutated tumors, nor the survival of patients with these tumors differed significantly with respect to the location of these tumors. These results are in contrast in contrast to Breivik et al who found more TP53 mutated tumors in the left colon. 2 Furthermore, survival of TP53 negative tumors in our study did not differ significantly between left and right-sided tumors. The absence of survival differences in TP53 mutated tumors might be the consequence of the given chemotherapy. Our data shows a difference between the presence of KRAS and TP53 mutated throughout the colon. TP53-mutated tumors are present through-out the entire colon, whereas KRAS mutated tumors are mainly present in the right colon. An explanation might be the presence of mutation factors present in the feaces which diminishes towards the end of the colon. 18 Because the incidence of KRAS mutations diminishes towards the left side of the colon, it is possible that the same mutating factors present in the right side of the colon also diminishes towards the left colon. It has been speculated that the frequency of TP53 mutations might influence the KRAS mutation 74

10 Impact of KRAS and TP53 mutations on survival in left and right sided Dukes C colon cancer frequency. 19 In this study, the frequency of TP53 mutations was, however, equal for both right and left-sided tumors. The activation of KRAS and TP53 mutations on the biological behaviour of colon tumors do not occur in isolation and are part of multiple genetic changes in the cell. Our results indicate that genes not subjected to analysis in this study, such as APC, DCC (deleted in colon cancer) and MCC (mutated in colorectal cancer), might explain the survival differences in patients with left and right sided sporadic Dukes C colon KRAS mutation negative tumors. It is, therefore, possible that these genes play a currently underestimated role in sporadic colorectal cancers. Although differences in survival in left and right-sided Dukes C colon cancer might be explained for the larger part by KRAS mutations, future research on genetic differences between right and left-sided sporadic colon cancer should also be focussed also on the mismatch repair genes. References 1. Bell SM, Scott N, Cross D, Sagar P, Lewis FA, Blair GE et al. Prognostic value of p53 overexpression and c-ki-ras gene mutations in colorectal cancer. Gastroenterology 1993;104: Breivik J, Meling GI, Spurkland A, Rognum TO, Gaudernack G. K-ras mutation in colorectal cancer: relations to patient age, sex and tumour location. Br.J.Cancer 1994;69: Bufill JA. Colorectal cancer: evidence for distinct genetic categories based on proximal or distal tumor location [see comments]. Ann.Intern.Med. 1990;113: Elnatan J, Goh HS, Smith DR. C-KI-RAS activation and the biological behaviour of proximal and distal colonic adenocarcinomas. Eur.J.Cancer 1996;32A: Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon [see comments]. Science 1993;260: Boland CR. The biology of colorectal cancer. Implications for pretreatment and followup management. Cancer 1993;71:

11 Chapter VI 7. Morrin M, Kelly M, Barrett N, Delaney P. Mutations of Ki-ras and P53 genes in colorectal cancer and their prognostic significance. Gut 1994;35: Bos JL. ras oncogenes in human cancer: a review. Cancer Res. 1989;49: Cerottini JP. The type of K-ras mutation determines prognosis in colorectal cancer. Am.J.Surg. 1998;175: Span M, Moerkerk PT, De Groeij AF, Arends JW. A detailed analysis of K-ras point mutations in relation to tumorprogression and survival in colorectal cancer. Int.J.Cancer 1996;69: Ahnen DJ, Feigl P, Quan G. Ki-ras mutations and p53 overexpression predict the clinical behavior of colorectal cancer: a Southwest Oncology Group Study. Cancer Res. 1998;58: Pricolo VE, Finkelstein SD, Wu TT, Keller G, Bakker A, Swalsky P et al. Prognostic value of TP53 and K-ras-2 mutational analysis in stage III carcinoma of the colon. Am.J.Surg. 1996;171: Pricolo VE, Finkelstein SD, Hansen K, Cole BF, Bland KI. Mutated p53 gene is an independent adverse predictor of survival in colon carcinoma. Arch.Surg. 1997;132: Borresen-Dale AL. TP53 and long-term prognosis in colorectal cancer: mutations in the L3 zinc-binding domain predict poor survival. Clin.Cancer Res. 1998;4: Bleeker WA, Mulder NH, Hermans J, Otter R, Plukker JT. The addition of lowdose leucovorin to the combination of 5-fluorouracil- levamisole does not improve survival in the adjuvant treatment of Dukes' C colon cancer. IKN Colon Trial Group. Ann.Oncol. 2000;11: Stein A, Raoult D. A Simple method for amplification of DNA from paraffinembedded tissue. Nulceic.Acids.Res. 1992;20: Delattre O, Olschwang S, Law DJ, Melot T, Remvikos Y, Salmon RJ et al. Multiple genetic alterations in distal and proximal colorectal cancer. Lancet 1989;2:

12 Impact of KRAS and TP53 mutations on survival in left and right sided Dukes C colon cancer 18. Morotomi M, Guillem JG, LoGerfo P, Weinstein IB. Production of diacylglycerol, an activator of protein kinase C, by human intestinal microflora. Cancer Res. 1990;50: Breivik J, Lothe RA, Meling GI, Rognum TO, Borresen-Dale AL, Gaudernack G. Different genetic pathways to proximal and distal colorectal cancer influenced by sexrelated factors. Int.J.Cancer 1997;74:

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