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Annals of Surgical Oncology 9:580-586 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Genetic Alterations in Normal Epithelium of Colorectal Cancer Patients May Be a Useful Indicator for Subsequent Metachronous Tumor Development

Minori Koshiji, MD, PhD, Yasuhiro Yonekura, MD, Takamichi Saito, MD, Noriko Sakaida, MD, Yoshiko Uemura, MD and Kazuhiko Yoshioka, MD

From the First (TS) and Second (MK, YY, KY) Departments of Surgery and the Department of Surgical Pathology (NS, YU), Kansai Medical University, Osaka, Japan.

Correspondence: Address correspondence and reprint requests to: Minori Koshiji, MD, PhD, Second Department of Surgery, Kansai Medical University, 10-15, Fumizono-cho, Moriguchi-City, 570-8507, Osaka, Japan; Fax: 81-6-6992-8475; E-mail: koshiji{at}takii.kmu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We attempted to identify areas of microsatellite alterations specific to histologically normal colorectal epithelium and to clarify the correlations among those molecular events and clinicopathologic features.

Methods: We conducted a prospective observation study on 51 colorectal cancer patients. Preoperative blood and microdissected histologically normal colorectal epithelium and neoplastic tissues were collected. Microsatellite analyses with seven microsatellite loci were performed to examine the genetic potential of individual tumors and histologically normal colorectal epithelium.

Results: In the sporadic colorectal cancer group, p53 LOH in the neoplastic epithelium had a significant correlation with the maximum tumor diameter and the preoperative serum cancer antigen 19-9 level, but not with the depth of invasion of the primary tumor. Among the patients who had p53 LOH in the histologically normal colorectal epithelium, four additional tumors were discovered within 30 months after curative surgery. For those patients, microsatellite alterations in normal colorectal epithelium were more sensitive than tumor markers.

Conclusions: For accurate LOH analysis, nonmalignant lymphocytes from blood should be used as the appropriate normal DNA sample. Focusing on the identification of high-risk patients for microsatellite alterations in histologically normal colorectal epithelium can be a useful indicator of subsequent metachronous tumor development after colorectal surgery.

For accurate LOH analysis, nonmalignant lymphocytes from blood should be used as the appropriate normal DNA sample. Focusing on the identification of high-risk patients for microsatellite alterations in histologically normal colorectal epithelium can be a useful indicator of subsequent metachronous tumor development after colorectal surgery.

Key Words: Loss of heterozygosity • Microsatellite analysis • Malignant potential • Prospective observation study


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer (CRC) is one of the most common human cancers and is a major cause of morbidity and mortality worldwide. Even if curative resections for primary tumors are performed, the metachronous regions of CRC are relatively frequent events compared with other tumor types,1,2 so additional factors that could reflect the potential of subsequent metachronous tumors would be advantageous.

The frequent occurrence of loss of heterozygosity (LOH) in CRC at APC (adenomatosis polyposis coli), p53, DCC (deleted in colorectal carcinoma), and hMLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2) and the chromosome 9p has been documented.3–19 However, most previous investigators have used histologically normal tissues as normal samples for LOH analyses.3–19 Because LOH is determined from the comparison of tumor samples with normal samples, previous reports may contain contradictory results when the normal tissues have genetic changes. Therefore, we believe that appropriate normal DNA samples for accurate LOH analysis are nonmalignant lymphocytes from blood.

In this study, we observed many microsatellite alterations in histologically normal epithelium, and we attempted to judge the potential genetic condition of normal colorectal epithelium of colon cancer patients. Besides classic markers for colorectal carcinogenesis (p53, APC, and hMLH1), we examined three microsatellite loci at chromosome 9p (D9S162, D9S171, and IFNA [interferon {alpha}]). The D9S171 locus is centromere to p15/INK4b and p16/INK4a, which may commit to downstream events of APC/ß-catenin pathways, and D9S162 and IFNA loci are telomere to those tumor suppressor genes. The previous analyses of markers at the chromosome 9p region and the DCC locus have demonstrated increased LOH in sporadic cancers of early onset in patients <50 years old.15,20

Our objectives were to identify areas of microsatellite alterations specific to histologically normal colorectal epithelium and to clarify the correlations among those molecular events and clinicopathologic features. This study recommends use of microsatellite alterations in histologically normal colorectal epithelium as a parameter for malignant potential to indicate subsequent metachronous tumor development.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Samples
Between January 1998 and December 2001, 383 primary CRC patients received operations in the second department of surgery of Kansai Medical University. Patients with inflammatory bowel disease, familial adenoma polyposis coli, and histories of other malignant diseases were excluded from this study. Under full institutional review board consent and with written, informed consent, 51 primary CRC patients who could clarify their family history of cancer at the time of surgery were prospectively enrolled. According to the Amsterdam criteria,21 51 patients were classified into the sporadic CRC group and the hereditary nonpolyposis colorectal cancer group (HNPCC). There were 18 men and 23 women with a median age of 63 years in the sporadic CRC group and 7 men and 3 women with a median age of 56 years in the HNPCC group. Before surgery, EDTA blood was collected; at the time of surgery, normal and neoplastic tissues were microdissected and stored as fresh-frozen tissues. All operations were performed by the same chief surgeon (K.Y.), and the neoplastic epithelium and normal colorectal epithelium were separately microdissected by Dr. Yoshioka in all cases. Normal colorectal epithelia were cut near the surgical margin, not adjacent to the colorectal tumor. The more distant margin from the tumor was taken for the normal colorectal epithelia. All patients received oral 5-fluorouracil therapy (800 mg/m2/day) for 2 years after surgical resection, were observed at our outpatient clinic every 2 weeks, and received monthly serum tumor marker examinations and occasional radiographical examinations (abdominal computed tomography and barium enema). No patient received radiotherapy.

DNA Preparation
Genomic DNA was extracted from surgical specimens by using the QIAamp tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s specifications. From blood, genomic DNA was extracted with a DNA extraction kit (Promega, Madison, WI). The concentrations of DNA were then measured at 260 nm with a spectrophotometer. When we observed high levels of protein contamination, we further purified the samples by using a GeneClean kit (Bio 101 Inc., Vista, CA).

Polymerase Chain Reaction
Fluorescent-labeled polymerase chain reaction (PCR) primers for p53, APC, DCC, and hMLH1 and three additional markers (D9S162, IFNA, and D9S171) on chromosome 9p were used. All primers were designed from published sequences (Table 1). PCR was performed in a 10-µl reaction volume in 1x PCR buffer, .25 mM of deoxynucleotide triphosphate solution, .45 U of Taq polymerase, 100 ng of genomic DNA, and .2 µM of primers. The optimal MgCl2 concentration and annealing temperature were determined for each set of primers (Table 1). Amplification was performed with a modified step-down protocol.22


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  		TABLE 1. TABLE 1. Microsatellite markers
 
Sample Preparation and Instrumentation
PCR amplicons were denatured with TAMRA standard (Bio Ventures, Inc., Murfreesboro, TN) as previously described.22 The denatured samples were then separated with an automated DNA sequencer (373A; Perkin-Elmer Cetus, Norwalk, CT) by following a previously described procedure.23 For each heterozygous locus, the ratio between the first and second allele peaks in tumor DNA was compared with the same ratio in normal DNA and was used to calculate a relative allele ratio, XLOH. LOH was defined for those samples that had XLOH values of <.80. Three different people repeated all reactions and analyses at least twice each (Fig. 1).



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  		FIG. 1. Representative results of microsatellite analysis. (a) Blood, (b) histologically normal colorectal epithelium, (c) neoplastic epithelium. (A) Normal, (B) loss of heterozygosity. The histologically normal colorectal epithelium had a microsatellite alteration. (C) The histologically normal colorectal epithelium showed the same pattern as nonmalignant lymphocytes.

 
Statistical Analysis
The Mann-Whitney U-test and the Kruskal-Wallis test were used to look for associations between LOH regions and clinicopathologic parameters.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Associations Between Microsatellite Alterations in Neoplastic Tissues and Clinicopathologic Parameters
Table 2 summarizes the frequency of LOH in p53, APC, DCC, hMLH1, D9S162, IFNA, and D9S171 in the sporadic CRC patients, and Table 3 summarizes those of the HNPCC patients. The appearance of novel alleles was regarded as microsatellite instability (MSI). Microsatellite alterations (LOH/MSI) were observed in histologically normal colorectal epithelium (highlighted samples; white characters). As shown in Table 4, in most of these loci, more frequent microsatellite alterations (LOH/MSI) in the neoplastic epithelium were observed in deeper-invasion groups (tumor-node-metastasis stages T3 and T4). As shown in Table 5, in most of those loci, more frequent microsatellite alterations (LOH/MSI) in the neoplastic epithelium were observed in larger-diameter groups (>50 mm). In the sporadic CRC patients, there were significant correlations between p53 LOH and the maximum diameter of tumors (P = .0005), between p53-LOH and preoperative serum cancer antigen 19-9 levels (P = .0479), and between APC LOH and age (P = .0062).


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  		TABLE 2.
 

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  		TABLE 3.
 

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  		TABLE 4. TABLE 4. The correlations between the depth of tumor invasion of primary tumors and microsatellite alterations
 

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  		TABLE 5. TABLE 5. The correlations between the maximum diameters of primary tumors and microsatellite alterations
 


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Associations Between Microsatellite Alterations in Histologically Normal Tissues and Clinicopathologic Parameters
Microsatellite alterations in the histologically normal colorectal epithelium were observed even in the early-stage patients, and more frequent microsatellite alterations in histologically normal colorectal epithelium were observed in the T3 group than the T4 group (Table 4). The correlations between the maximum diameters of primary tumors and microsatellite alterations are listed in Table 5. No significant correlation was observed in alterations in histologically normal colorectal epithelium and the maximum diameters or depth of invasions of primary tumors. No microsatellite alteration in histologically normal colorectal epithelium was observed in the DCC locus.

Clinical Outcomes
The median follow-up for the surviving patients was 27 months. Only one patient (patient H4) was dead from the disease after 12 months of surgical resection. Careful observation of those patients at high risk revealed that four patients who had p53 LOH in their normal epithelium developed another CRC within 30 months after curative surgery (patients H1, H5, S22, and S38). Their serum tumor marker levels (carcinoembryonic antigen and cancer antigen 19-9) remained within normal ranges in patients H1 and S22. Patients H1 and H5 had rectal cancer, and they were classified into the HNPCC group. The maximum diameters of their primary CRCs were 40 and 30 mm, respectively, and neither patient had lymph node metastases. The dissected tumors were diagnosed as well-differentiated adenocarcinoma, T3 (ss), L0, V3, stage 2; and T3 (ss), L1, V2, stage 2, respectively. The microsatellite analyses revealed that these patients (H1 and H5) had p53 LOH in histologically normal colorectal epithelium, so they were followed up carefully in our outpatient clinic. Within 2 years from the standard low anterior resection with D2 lymph node dissection, those patients developed another colon cancer in their ascending colon. At the same time, thyroid cancer was discovered in patient H5. Patients S22 and S38, who originally presented with sigmoid colon cancer, also developed another CRC in their remnant colon after sigmoidectomy with D2 lymph node dissection. Both cases had N1 lymph node metastases. The dissected tumors were diagnosed as well-differentiated adenocarcinoma, T4 (se), L1, V3, stage 3; and T3 (ss), L2, V2, stage 3, respectively.

The microsatellite analyses revealed that those patients (S22 and S38) had p53 LOH in histologically normal colorectal epithelium, and careful observation revealed secondary CRCs after 27 and 30 months of their operations, respectively. All secondary CRCs were discovered in a distant site from the anastomosis and had different histological grades from the primary tumors. Additional genetic alterations were observed in the secondary CRCs in patients H1 and S22 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since Cavenee et al.24 first described the use of LOH in cancer studies, the technique has been widely used in many tumor types.3–19 Even though several investigators have analyzed common markers such as p53, APC, DCC, and hMLH1 in CRC, the relationship between LOH in these markers and prognosis remains contradictory.3–19 The contradictory results of those previous LOH studies may result from the different methodologies. Many investigators used paraffin-embedded tissue,3,4,6,18 which leads to the admixture of tumor cells with normal cells. Some investigators neglected the step-down protocol and used higher PCR cycles,4,5,7 which leads to false-normal results. Also, various alleic ratios to determine LOH (XLOH) have been used,4,6 and several investigators have regarded only an entire alleic loss as LOH.5,17,18 Our cutoff value of .80 is in accordance with that suggested by Hahn et al.25 and has been further corroborated by quantitative mixing experiments as a value that corresponds to 30% of cells with LOH (data not shown).26 Furthermore, most previous investigators used histologically normal tissues as normal samples for LOH analyses.3–19

Because we focus on the practical prognostic factors for genetic staging, sensitive and specific markers are an advantage. Our results show more frequent LOH compared with previous studies; this is a product of the high cutoff value of XLOH (.8), few amplification cycles (fewer than 30 cycles), and blood-derived normal samples. It is interesting to note that p53 LOH in the neoplastic epithelium had a significant correlation with the maximum tumor diameter (Figs. 1–3), but not with the depth of invasion of the primary tumor. This suggests that tumor polymorphism of CRC may be associated with vertical expansion of tumor, and more clones may be involved in larger tumors.



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  		FIG. 2. The significant correlation between p53 loss of heterozygosity (LOH) and the preoperative serum cancer antigen (CA) 19-9 level.

 


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  		FIG. 3. The significant correlation between p53 loss of heterozygosity (LOH) and the maximum tumor diameter (d).

 
The significant correlation between patient age and APC LOH suggests that genetic alterations of APC may increase with aging (Fig. 4). No microsatellite alteration in histologically normal colorectal epithelium was observed in the DCC locus, suggesting that the genetic alteration of DCC may be a relatively late event in colorectal carcinogenesis.



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  		FIG. 4. The significant correlation between APC loss of heterozygosity (LOH) and the patient’s age.

 
Recently, Tabor et al.27 reported that microsatellite alterations were observed in the histologically normal epithelium of patients with head and neck squamous cell carcinoma, and they explained that the phenomenon reflects the process of field cancerization. However, all four patients with intramural recurrences developed the secondary CRCs in a distant site from the anastomosis, and the secondary tumors were different histological grades from the primary tumors, suggesting expansion of different subclones.

In this study, we demonstrated microsatellite alterations observed in histologically normal epithelium. Although microsatellite alterations in the neoplastic epithelium had correlations with the depth of invasion and the maximum tumor diameter, microsatellite alterations in histologically normal colorectal epithelium had no significant correlation with either, which suggests that histologically normal colorectal epithelium can be another pathologic indicator.

We expect for future investigators to conduct accurate LOH analysis with nonmalignant lymphocytes as normal DNA and to analyze histologically normal colorectal epithelium as a pathologic indicator of malignant potential. Further study may clarify the significance of each genetic alteration in normal colorectal epithelium.


   Acknowledgments
 
The authors thank Kahoru Yasaka for excellent laboratory assistance.

Received for publication December 10, 2001. Accepted for publication April 10, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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