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10.1245/ASO.2003.03.004
Annals of Surgical Oncology 10:1095-1099 (2003)
© 2003 Society of Surgical Oncology
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ORIGINAL ARTICLES

Identifying a Region of Interest in Site- and Stage-Specific Colon Cancer on Chromosome 13

Nala S. Sivarajasingham, FRCS, Richard Baker, MRCS, Jonathan V. Tilsed, FRCS, John Greenman, PhD, John R. T. Monson, MD and Lynn Cawkwell, PhD

From the Academic Surgical Unit (NSS, RB, JVT, JRTM) and Cell and Molecular Medicine (JG, LC), Division of Cancer, Postgraduate Medical Institute of the University of Hull, in association with the Hull and York Medical School, Castle Hill Hospital, Castle Road, Cottingham, United Kingdom.

Correspondence: Address correspondence and reprint requests to: L. Cawkwell, PhD, R&D Building, Castle Hill Hospital, Castle Road, Cottingham HU16 5JQ, UK; Fax: 01-48-262-2398; E-mail: l.cawkwell{at}hull.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The role of genes on chromosome 13q has not been confirmed in colorectal tumors, in part because most series that have been studied are heterogeneous in terms of tumor site, stage, and replication error (RER) status. Using a highly homogenous series of colon tumors, our aim was to identify areas of interest on 13q that are important in carcinogenesis.

Methods: Twenty-three RER-negative tumor specimens from patients with right-sided Dukes’ stage C colon tumors were selected for analysis with 10 microsatellite markers spanning 13q. The polymerase chain reaction–amplified products were analyzed by using a standard fluorescent loss of heterozygosity/allele imbalance assay.

Results: Markers showing the highest frequency of allelic imbalance were as follows: D13S175 (31%), D13S289 (27%), D13S263 (25%), and D13S265 (27%). The overall resolution of the map was approximately 11.4 to 11.7 cM.

Conclusions: This study of right-sided, RER-negative, Dukes’ stage C colon tumors showed the highest area of allelic imbalance corresponding to 13q11.2–11. This region includes LATS2 (large tumor suppressor 2 gene) and FGF9 (fibroblast growth factor 9), which may be involved in carcinogenesis.

Key Words: Colorectal cancer • Chromosome 13 • Loss of heterozygosity • Bowel tumorigenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancer is the second most common cause of cancer death in the United Kingdom, with approximately 35,000 new cases of cancer diagnosed each year. It is thought to arise through the accumulation of genetic events1,2 and can be either hereditary or sporadic.

Two pathways have been defined for colorectal tumorigenesis: the classic and the alternative pathways.3 The classic pathway is characterized by chromosomal abnormalities, involving both oncogenes and tumor-suppressor genes, that usually result in aneuploid tumors. Several common oncogenes and tumor-suppressor genes have been identified, e.g., APC on 5q, p53 on 17p, and the DCC region (SMAD2 and SMAD4) on 18q. The alternative pathway is characterized by defective mismatch repair genes (hMLH1 and hMSH2), microsatellite instability, and uncorrected nucleotide sequences.

Comparative genomic hybridization studies on a cohort of 50 mixed-stage colorectal tumors from our unit identified a gain of genetic material on chromosome 13 in 58% of tumors. These findings are in agreement with previous studies.4–8 Chromosome 13 has also been found to be involved in progression from adenoma to carcinoma5 and in metastatic disease.6 Various genes, including BRCA2, BRCA3, Rb1, CDX2, CDX3, and LATS2 (large tumor suppressor 2), have been found to reside on chromosome 13, but none has been implicated in colon cancer tumorigenesis.

Right-sided colon cancer is believed to behave differently from that arising in the left side in terms of presentation, prevalence, and prognosis.9–11 This can be partly explained by the distinct embryological origin and blood supply; however, distinct genetic mechanisms have also been hypothesized.9–11 Archival specimens of Dukes’ C, right-sided colon cancer specimens were selected for microsatellite polymerase chain reaction (PCR) studies to generate a medium-resolution map of chromosome 13 from which areas of interest that demonstrate allelic imbalance in colon cancer could be identified.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A cohort of 23 replication error (RER)-negative archival specimens was studied. The series was homogeneous because all samples originated from patients with right-sided, Dukes’ stage C tumors, as confirmed by an experienced histopathologist. All 23 tumors were confirmed as being RER-negative by using both a PCR-based microsatellite instability assay and immunohistochemistry with antibodies against hMLH1 and hMSH2.12 Each set included a normal colon specimen taken from the resection margin, a primary tumor, and involved lymph nodes. All the selected tumor specimens were microdissected and contained at least 50% malignant cells.

DNA was extracted by using a standard proteinase K kit (Nucleon; Tepnel, Manchester, UK) according to the manufacturers’ protocol. Polymorphic microsatellite markers were chosen from human genome databases; selection depended on documented heterozygosity, base-pair sizes, and map position (Table 1 and Fig. 1). Ten markers were obtained from Applied Biosystems (Warrington, UK).


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  		TABLE 1. Markers used and their position on chromosome 13
 


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  		FIG. 1. Markers with their corresponding positions on chromosome 13 (mapped from the Human Genome Project).

 
Polymerase Chain Reaction
Microsatellite PCR analysis was performed per Cawkwell et al.12 The target DNA sequence from each specimen was amplified with the premade True allele mix (Applied Biosystems) according to the manufacturer’s instructions. PCR studies were performed on the normal and tumor specimens from each of the 23 sets by using all 10 markers. The amplified fragments were analyzed with an ABI Prism 310 genetic analyzer (Applied Biosystems).

Analysis
Allelic imbalance between primary tumor and normal specimens was performed as described previously.12 The ratio of the heights of the two normal alleles was compared with the ratio of the heights of the two tumor alleles. If the resulting number (normal:tumor) was <=.5, allelic imbalance or loss of heterozygosity (LOH) was said to have occurred.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Figure 2 shows a pair of heterozygous peaks representing the genetic locus of a normal specimen (Fig. 2A) and corresponding tumor specimen (Fig. 2B). The height of each peak represents an arbitrary unit of fluorescence, which reflects the amount of DNA per allele. Using the 10 markers to study chromosome 13 (114–117 cM in length) gave a map resolution of 11.4 to 11.7 cM.



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  		FIG. 2. (A) Allelogram of normal DNA locus for marker D13S175 showing heterozygosity, i.e., a pair of alleles of 107 and 114 base pairs (bp). The height of each allele represents the amount of DNA. x-axis, base-pair size; y-axis, arbitrary unit of fluorescent intensity. (B) Allelogram of tumor DNA showing loss of DNA in the shorter allele.

 
The number of tumors showing allelic imbalance per marker against the total number of interpretable/informative results for that marker gave the frequency of allelic imbalance. The results for all tumors are listed in Table 2. Tumor samples 3 and 10 display an amplification or loss of the whole of chromosome 13. In other tumors, however, there are distinct areas that show allelic imbalance or LOH, e.g., the area mapped by D13S175 and D13S289 in tumor 12.


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  		TABLE 2. Result table showing results obtained for primary tumors against the marker used
 
Markers showing the highest allelic imbalance or LOH were D13S175 (31%), D13S289 (27%), D13S265 (27%), and D13S263 (25%). The other markers used were D13S218 (13q12.2–13), D13S219 (13q12.3–13), D13S153 (13q14.1–14.3), D13S170 (13q31–31), D13S159 (13q32–32), and D13S285 (13q34–34).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The lowest frequency of allelic imbalance seen in our cohort of tumors was 13%. This gives an estimate of the background LOH in a series of sporadic, Dukes’ stage C colon tumors. The lowest frequency of allelic imbalance/background LOH in other colorectal cancer series has been similar, at approximately 11% to 14%.13–15

The highest frequency of allelic imbalance in this study was found at marker D13S175. This marker maps to the 13q11–12 region, which includes the LATS2 gene, a human homolog of the drosophila tumor-suppressor gene. LATS2 inactivation in esophageal cancer has been investigated, although the results were equivocal.16 No involvement in colon cancer has been previously reported; however, the relatively high allelic imbalance raises the possibility that this gene may be a possible target for LOH in colon cancer. Fibroblast growth factor 9 (FGF9) is also found in region 13q11–12. It is a glia activating factor that acts through tyrosinase kinase receptors and is involved in various biological processes. FGF9’s involvement in tumorigenesis is not known.

Markers D13S289 (13q12.1) and D13S265 (13q31–32) both demonstrated an allelic imbalance frequency of 27%. No putative tumor genes have been identified in these locations.

Rb1, the retinoblastoma gene found at 13q14.1–14.2, is homogeneously lost in retinoblastoma and is the classic example used to explain Knudson’s two-hit hypothesis. In our study, there was a 25% allelic imbalance in this chromosomal region, demonstrated by marker D13S263. Such a result is supported by previous studies, because gains of 25% to 28% have been identified in colorectal tumors.17,18 However, both of these earlier studies analyzed a mixed group of tumors, i.e., tumors of different stage, site, and/or mismatch repair status rather than a defined cohort.

BRCA2 is located at 13q12.3. An allelic imbalance of only 13% was found at this region (D13S218) in our cohort of tumors. This confirms previous studies on BRCA2, in which no correlation was described between colorectal cancer and the gene.19 At least two other putative cancer genes are located at 13q12.3: CDX2 and CDX3. Drummond et al.20 and Ee et al.21 propose that a CDX2 mutation is an essential event in the origin of colorectal cancers. The caudal-type homeobox gene encodes a transcription factor, which is expressed in intestinal epithelial cells and plays a role in proliferation and differentiation. The CDX2 protein also positively regulates carbonic anhydrase 1, which plays a major role in sodium chloride absorption. Expression of both carbonic anhydrase 1 and CDX2 is markedly reduced in colorectal dysplasia and is absent in many carcinomas.20,22 Mice with a heterozygous null mutation of this gene developed multiple intestinal polyps.20 Drummond et al.20 cloned human CDX2 complementary DNA and showed it to be on 13q12–13; more recently, the Human Genome Project has mapped the gene to 13q12.3. According to our study, D13S218 (13q12.2–13) showed an allelic imbalance of only 13%; therefore, it is likely that the loss of CDX2 expression is not due to LOH. Other mechanisms, which were not investigated, such as homozygous deletion23 of the CDX2 gene, a null mutation,22 or promoter hypermethylation, may be responsible for decreased CDX2 expression. Alternatively, the position of CDX2 might be more telomeric than the D13S218 marker region.

ING1, inhibitor of growth 1, which is believed to play a role in regulating cell progression and susceptibility to apoptosis, is retained in colorectal cancer.24 ING1 is located on 13q34, and our study has confirmed minimal allelic imbalance for the marker at that position (D13S285).

Because a number of potential areas involved in tumorigenesis have been identified, several options exist. First, these areas should be further analyzed by using more specific markers, e.g., by using an intragenic LATS2 marker to study D13S175. In addition, the resolution of the 13q map could be increased by studying the same tumor specimens with more markers, e.g., region 14.3–31. The BRCA3 gene, which may be involved in non-BRCA1/2 breast cancer, is found in this region, and a role in colon cancer has not been investigated. Finally, the next logical step is to detect any regions of interest on chromosome 13 in terms of disease spread using the involved nodes; this is currently under way.

In summary, a medium-resolution (11.4- to 11.7-cM) map of chromosome 13 from a cohort of right-sided, Dukes’ stage C, RER-negative tumors showed the highest frequency of allelic imbalance in the regions marked by D13S175, D13S265, D13S289, and D13S263. To elucidate the subtle genetic differences between tumors of different sites, larger studies with similarly homogeneous cohorts of tumors to those used here are now required. Such studies will help to unravel the vast amounts of data and, it is hoped, lead to better-tailored therapies.


   ACKNOWLEDGMENTS
 
ACKNOWLEDGMENTS

The authors thank the Henry Chatterton Cancer Research Fellowship for supporting the cost of the consumable reagents, and they also thank the technicians in the department for their invaluable help in processing the data.

The acknowledgments are available online at www.annalssurgicaloncology.org.


   FOOTNOTES
 
A medium-resolution deletion map of chromosome 13q in Dukes’ stage C, replication error–negative, right-sided colon cancer was produced. A number of areas showing allelic imbalance were detected.

Received for publication March 6, 2003. Accepted for publication July 6, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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N. S. Sivarajasingham, L. Cawkwell, R. P. Baker, S. L. O'Kane, E. F. Smyth, J. V. T. Tilsed, M. B. Watson, J. Greenman, and J. R. T. Monson
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