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© 2003 American Society for Clinical Oncology Genetic Tumor Markers With Prognostic Impact in Dukes’ Stages B and C Colorectal Cancer Patients
From the Department of Genetics, Institute for Cancer Research, and the Department of Surgery, The Norwegian Radium Hospital; Institute of Forensic Medicine, The National Hospital; and Section of Medical Statistics, University of Oslo, Oslo, Norway. Address reprint requests to Ragnhild A. Lothe, PhD, Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway; email: rlothe{at}radium.uio.no.
Purpose: To examine several genetic changes in primary colorectal carcinomas (CRCs) from patients with 10 years of follow-up and associate the findings with clinicopathologic variables. Material and Methods: DNA from 220 CRCs were analyzed for allelic imbalances at 12 loci on chromosome arms 1p, 14q, 17p, 18q, and 20q, and the microsatellite instability (MSI) status was determined. The clinical significance of the tumor protein 53 (TP53) mutations was re-evaluated. Results: Patients with tumors containing 17p or 18q deletions had shorter survival than those without these alterations (P = .021, P = .008, respectively). This was also significant for the Dukes’ B group (P = .025, P = .010, respectively). Furthermore, patients with tumors showing losses of both chromosome arms revealed an even poorer disease outcome than those with either 17p or 18q loss. Patients with low increase in 20q copy number in their tumors had longer survival compared with those without changes (P = .009) or those with a high increase of copy number (P = .037). This was also evident for the Dukes’ C group (P = .018, P = .030, respectively). MSI was seemingly a beneficial marker for survival (P = .071). A significant association between mutations affecting the L3 zinc-binding domain of TP53 and survival was confirmed in this cohort after 10 years of follow-up, and also was found to apply for patients in the Dukes’ B group. Several associations were found among genetic and pathologic data. Conclusion: The present study indicates that 17p, 18q, and 20q genotypes, and TP53 mutation status add information in the subclassification of Dukes’ B and C patients and may have impact on the choice of treatment.
COLORECTAL CANCER (CRC) is one of the most common causes of cancer-related death in Norway, as it is in most of the Western world, and despite major advances in the diagnosis and treatment of this disease, mortality has remained unchanged during the last 20 years.1,2 Currently, Dukes’ classification, or one of its many modifications, is the most commonly used predictor of prognosis for CRC patients. Thirty percent to 40% of Dukes’ stages B and C patients will experience relapse and die of the disease.1,2 Therefore, identification of additional prognostic markers to supplement standard clinical and pathologic staging could potentially subdivide patients in Dukes’ stages B and/or C into groups with high and low risk of relapse after surgery, and thus have impact on the choice of treatment. A minor subgroup of sporadic primary CRCs display microsatellite instability (MSI),3–6 a result of a defective DNA mismatch repair,7 but the majority of the carcinomas harbor numerous chromosome aberrations, and chromosomal instability seems to be quite pronounced in this tumor type.8 Although numerous studies have reported MSI in series of CRC, uncertainty remains whether positive MSI status is associated with prolonged survival for the patient.4,5,9–21 Deletions of the whole or part of chromosome arm 1p is found in early as well as in advanced stages of colorectal tumorigenesis.22–25 The smallest region of common deleted sequences of 1p is 1p32-pter,25–31 a region demonstrated to have functional importance in colorectal tumorigenesis.32 The target gene(s) for the reported deletions is unknown. Monosomy of chromosome 14 as well as allelic imbalance (AI) at 14q have been found both in primary and in advanced CRCs.23,24,33,34 Deletions at 17p and 18q are two of the most common changes in CRCs22,35–37 and often reflect inactivation of tumor protein 53 (TP53) at 17p13,38 and deleted in colorectal carcinoma (DCC)39 and mothers against decapentaplegic homolog 2, 4 (SMAD2, 4)40,41 at 18q21. Several studies have shown associations between deletion of 17p or 18q and reduced survival for patients with CRC,12,16,42–53 whereas other studies have not.9,45,52,54–56 The significance of TP53 for the clinical outcome of CRC is controversial, with reports of both favorable and unfavorable results in patients with overexpressed or mutated TP53.44,57,58 Gain of several chromosome regions that may pinpoint amplified oncogenes has been reported.26 In a large multicenter study of CRCs, mutations in the oncogene K-RAS were found associated with poor survival rate.59,60 Some studies have reported that gain of chromosome arm 20q is present more often in liver metastases from CRC patients than in primary carcinomas.61–66 We have analyzed several selected markers in a consecutive series of 220 CRCs in relation to clinical data after 10 years of patient follow-up. In addition, the new data have been compared with previously reported genetic data in the same series, and the impact of TP53 mutations status on survival after 10 years has been re-evaluated.
Patients and Samples Two-hundred twenty tumor and corresponding blood samples from a consecutive series of CRC patients prospectively collected from seven hospitals in the Oslo region between 1987 and 1989 were included in this study. Clinical data and pathologic characteristics of the tumors are given in Tables 1  and 2. The series consisted of 114 males and 106 females; the median age at diagnosis was 71 years (range, 26 to 94 years) for males and 69 years (range, 41 to 92 years) for females. The median survival time for these patients was 84.0 months (range, 0.5 to 147.2 months) and was established as time between surgery and death or the last update (July 1, 1999). Cause-specific deaths have been recorded from hospital and postmortem report. All of the patients underwent surgery alone as the curative treatment except for a few patients with rectal tumors who also received postoperative radiation therapy. At the time of relapse, approximately one third of the patients received palliative chemotherapy treatment. This study was approved by the Norwegian Data Inspectorate for use and storage of patient’s records.
The fresh frozen tumor tissues were mechanically minced in phosphate-buffered saline (pH 7.6), followed by nylon mesh filtration. The cells were fixed in 70% ethanol at 4°C. The degree of tumor cells versus normal cells in the tumor cell suspension was estimated as previously described,68 and a mean of 84% tumor cells was determined, ranging from 62% to 97%. Blood and tumor samples were extracted with chloroform/phenol followed by ethanol precipitation using a model 340 ABI nucleic acid extractor (PE Biosystems, Foster City, CA).69
Allelotype Analysis
The protocols for the radioactive and for the fluorescent labeling of microsatellites, followed by gel and capillary electrophoresis, respectively, were described in detail by Skotheim et al.73 Briefly, primers specific for each locus were used to amplify the repeat and flanking sequences in template DNA by polymerase chain reaction. In the radioactive protocol, the products were labeled with alpha-32-phoshporus deoxycytidine triphosphate (  -32P-dCTP, Amersham Pharmacia Biotech Inc, Piscataway, NJ), separated by electrophoresis in 6% denaturing polyacrylamide gels, and visualized through autoradiography. AI was scored by visually comparing the tumor DNA pattern with the constitutional (blood) DNA pattern of each patient. A skewed intensity ratio between the two alleles in tumor DNA was considered as AI.
In the fluorescent protocol, the products were amplified with a fluorescent (6-carboxyfluorescein [6'-FAM], hexachlorinated 6'-FAM, or tetrachlorinated 6'-FAM; PE Biosystems) labeled primer. The fluorescent products were separated by capillary electrophoresis and analyzed by ABI PRISM 310 Genetic Analyzer (PE Biosystems) and further analyzed with Genescan and Genotyper software (PE Biosystems). The two alleles of each locus were assigned according to the two highest peaks, and degree of AI, QLOH, was determined. QLOH is calculated from the measured peak heights by dividing the allele ratio in tumor DNA by the allele ratio in the corresponding constitutional (normal) DNA. To distinguish between deletion and degree of gain at chromosome arm 20q, tumors with known AI were evaluated against control loci. Gain was defined as an increase of up to three times in copy number, whereas an increase of three times or more in copy number was defined as amplification. Degree of alteration on each locus was determined by the alteration ratio (AR). AR was defined as the ratio between marker (M) allele and reference (R) allele in tumor (T) and the corresponding constitutional (B; blood) DNA; AR for allele x; x = 1 or 2: [MBx/(RB1+RB2)/2]/[MTx/(RT1+RT2)/2] (Fig 1
MSI Analysis In addition to the 12 microsatellites analyzed in this study, seven dinucleotide markers (D1S216, D5S404, D8S255, D10S197, D11S904, D13S175, and D17S787) and two mononucleotide markers (BAT25 and BAT26) have previously been analyzed for MSI in this series of CRCs.4,67 The following criteria were used to determine the MSI status: tumor samples showing MSI in  30% of the analyzed loci or having mutation in both BAT25 and BAT26 were classified as microsatellite high, whereas samples with MSI in at least one locus and in less than 30% of the loci (n = 21) analyzed were considered as microsatellite low. Samples with no MSI were classified as microsatellite stable (MSS).74 In total, the MSI status has been determined for 275 carcinomas from 275 patients (present study).4,67 Similarity between microsatellite low and MSS tumors have previously been shown, and therefore, these groups are dealt with as MSS throughout the text.4,74 Microsatellite high is referred to as MSI.
TP53 Mutations and 17p13 Losses By Southern blot analyses, AI has previously been found at pBHP53 and D17S30 in 231 CRCs, including 216 tumors from present series.36 Both loci are located at chromosome band 17p13. Deletions at one or both markers were found in 124 (68%) of 182 informative tumors included in the present study.
DNA Ploidy
Statistical Analysis
Analysis of AI and MSI Among the informative cases (defined as constitutional heterozygosity in the corresponding blood DNA) 84 (46%) of 184 carcinomas showed AI at one or more loci on chromosome arm 1p, 78 (39%) of 201 tumors showed AI on 14q, 129 (70%) of 184 tumors showed AI on 18q, and 138 (75%) of 185 tumors showed AI on 20q. The alterations on chromosome arm 20q included the following: 53 (39%) tumors with gain, 54 (39%) tumors with amplification, seven (5%) tumors with deletion (Fig 1  ), and 24 (17%) tumors with both gain and deletion. Tumors with deletion or both gain/amplification and deletion were excluded from the association analyses. The frequency and type of alteration at each locus are summarized in Table 3. All tumor DNAs were informative in at least five (median, 20 loci; range, five to 21 loci) of the microsatellite loci analyzed. Thirty-six (13%) of 275 informative tumors were classified as MSI. Exclusion of the few samples showing both MSI and AI did not have any statistical effects on the association or survival analyses; therefore, these tumors were included in the analyses.
Genetic and Clinicopathologic Associations
Survival Associations
Patients with tumors containing gain of 20q had a better clinical outcome than patients with retained heterozygosity and patients with amplification of this chromosome arm (P = .009 and P = .037, respectively; Fig 3A ). This relation held also for patients in the Dukes’ stage C group (P = .018 and P = .030, respectively; Fig 3B). Patients with tumors harboring MSI showed a trend toward a better survival compared with patients with MSS tumors, although it was not statistically significant (P = .071, Fig 3C).
A significant relationship between mutations affecting the L3 zinc-binding domain of TP53 and lower survival rate was found in the whole cohort as well as for patients in the Dukes’ stage B group (P = .016 and P = .032, respectively; Fig 4A and 4B). This trend was also seen for Dukes’ stage C patients but did not reach the chosen statistical significance level (P = .071). No associations were found between alterations on 1p, 14q, or TP53 mutation (independent of localization and type) and survival of the patients (P = .381, P = .207, and P = .184, respectively).
Previous studies26,27,34,63,71 have identified frequent deletions as well as the smallest regions of overlapping alterations within chromosome arms 1p, 14q, 17p, and 18q in CRC. Our series of CRCs was comparable with other series in that the frequencies of changes at 1p, 14q, 17p, and 18q (46%, 39%, 66%, and 70%, respectively) were in agreement with previous reports.22,24,31,36,75–77 To our knowledge, AI at 20q has not been reported in large CRC series, but the high frequency of 20q changes is consistent with results from comparative genomic hybridization (CGH) studies.62,66,71,78–83 If alterations were found, the different loci at the individual chromosome arms usually showed the same change, suggesting that the alterations most often reflected relatively large chromosomal changes in the tumor in question. It is well known that tumors located in the right versus the left part of the large bowel have different spectra of genetic alterations and have characteristic clinicopathologic profiles.3–6,53,84,85 The present report confirmed that MSI tumors were mainly right-sided, poorly differentiated, diploid, and rarely exhibited TP53 mutations. Furthermore, chromosome instable tumors with AI at 18q also contained TP53 mutations and 17p deletions. We found striking associations between aneuploidy and AI at 14q and amplification of 20q that were not previously reported. Both of these chromosomal alterations were frequently seen in tumors with TP53 mutation or 17p deletion, although statistical significance was only reached for tumors with 20q amplification. These findings suggest that, in addition to changes of 17p and 18q, deletion of 14q and amplification of 20q are part of the chromosomal instability phenotype that is characteristic for a major subgroup of CRCs.8 We have previously found an association between deletions of 1p sequences and adenomas located in the distal colon and rectum,25 and this has been reported by others in carcinomas.86 However, we could not confirm this in the present series of carcinomas. Various studies have aimed to find associations between genetic changes in tumor and the disease outcome for the patient. However, many of these studies were based on a small number of patients from nonconsecutive series.58,87 The present consecutive series included 220 white patients from the same geographic region who were treated with surgery and followed for 10 years. In 1989, Kern et al53 presented the first study that showed an association between allelic losses of 17p and 18q and poor survival. Several additional studies have confirmed this association12,16,42–52; however, others have not.9,45,52,54–56 These differences may be explained by variation in series size, selection of patients included, and variation in follow-up time. The present study demonstrated an impact of 17p and 18q deletions on survival for CRC patients who had undergone curative surgery. In addition, the Dukes’ stage B patients could be subdivided into two groups with significantly different prognosis dependent on the status of the 17p and 18q tumor markers. Finally, the combined status, in which both chromosome arms were affected, showed even a worse disease outcome. Several potential tumor suppressor genes have been mapped to chromosome band 18q21, including DCC,39 SMAD2,40 and SMAD4.41 DCC has rarely been found mutated in colorectal tumors, whereas SMAD2 and SMAD4 are found mutated in 11% and 30%, respectively.88,89 The tumor suppressor gene TP53 is the target gene at 17p13, and conflicting results exist with regard to whether TP53 mutations predict poor survival.57,58 Our initial report of the same series with 5 years of observation time showed that only mutations affecting the L3 zinc-binding domain were associated with poor survival.44 The present report confirmed this (P = .016) in the same cohort after 10 years of follow-up. Finally, subclassification of the patients into Dukes’ stages suggested that prognosis evaluation of both the B and C group could benefit from knowledge of the TP53 mutation status. For yet unknown reasons, gain (increase of up to three times in copy number) of 20q is associated with improved prognosis compared with amplification or no change at this chromosome arm. This was independent of Dukes’ classification and separated the Dukes’ stage C patients into two significantly different groups. The present study is the first that reports a clinical benefit for patients with tumors containing a low copy number increase of 20q. One previous report has studied 20q changes in association with clinical outcome in patients with CRC, and they reported that gain of 20q resulted in shortened patient survival.71 These different results may be explained by the different number of patients (220 in this study v 67 in the previous) included in the studies, sampling differences (consecutive v not), different methods used (microsatellite analysis v CGH), and different interpretation of the results (the former study did not distinguish between low and high copy number gain). In the present study, neither deletions of 1p nor of 14q sequences exhibit statistically significant associations to survival. To our knowledge, three reports have studied the relationship between alterations of 1p and survival.42,71,90 Two of these studies42,71 reported that loss of 1p was associated with shorter survival time. The third study90 did not find any relationship to survival. They found, however, prognostic impact of selected markers on 1p. All previous reports included a smaller number of patients (n = 47, n = 67, and n = 116) than the present study, and different methods have been used (restriction fragment length polymorphism, CGH, cytogenetic banding, and microsatellite analysis). De Angelis et al71 reported a relationship between deletion of 14q and survival, which cannot be confirmed in the present study. These different results may be explained by the same arguments as suggested for the 1p data above. Although we could not find that AI at 1p or 14q was associated with survival, the fact that these chromosome regions are frequently altered in the primary carcinomas as well as in their metastases23,26,63 suggests that they are important contributors to tumor progression but not likely contributors to aggressiveness. MSI in CRC was first described in 1993,3–6 and several studies have examined this phenomenon and clinical outcome. The conclusions of the different studies are conflicting; some find MSI to be associated with prolonged survival time for CRC patients,4,5,9–15,17,18 whereas others do not.16,19–21 In the present study, the patients with MSI seemed to have a better disease outcome than patients without MSI tumors (P = .071). This trend is in agreement with our initial report on the same cohort,4 in which we found significant prolonged survival for patients with microsatellite unstable tumors (P = .05). The present study indicated that changes at 17p, 18q, and 20q, and TP53 status add information in the subclassification of Dukes’ stages B and C patients and, thus, may have impact on the choice of treatment.
We gratefully acknowledge the technical assistance of Merete Hektoen.
Supported by the Norwegian Foundation for Health and Rehabilitation (C.B.D.) and the Norwegian Cancer Society (L.T., R.A.L.).
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ABSTRACT
INTRODUCTION
2 test or Fisher’s exact test. Cause-specific survival analysis (death by CRC) was performed by Cox regression and the Kaplan-Meier method, and the differences in survival were assessed using the log-rank test. The following variables were included in the Cox regression analysis: age, sex, Dukes’ stage, tumor site, histologic grade, DNA ploidy pattern, MSI status, and genotype changes at chromosome arms 1p, 14q, 17p, 18q, and 20q. Survival analysis of MSI status, 17p deletions, and TP53 mutations were performed for all patients included in the present and our previous studies.
