OBJECTIVES:

Colorectal cancer (CRC) occurs rarely in young individuals (<45 yr) and represents one of the criteria for suspecting hereditary cancer families. In this study we evaluated clinical features and molecular pathways (chromosomal instability [CIN] and microsatellite instability [MSI]) in early-onset CRC of 71 patients.

METHODS:

Detailed family and personal history were obtained for each patient. Expression of APC, -catenin, p53, MLH1, MSH2, and MSH6 genes was evaluated by immunohistochemistry. MSI analysis was performed and constitutional main mutations of the mismatch repair (MMR) genes were searched by gene sequencing.

RESULTS:

Fourteen (19.7%) out of the 71 cases showed both MSI and altered expression of MMR proteins. In the 57 MSI-negative (MSI-) lesions altered expression of APC, -catenin, and p53 genes were found more frequently than in MSI-positive(MSI+) tumors. Seven (50%) out of the 14 patients with MSI+ tumors presented clinical features of Lynch syndrome (hereditary non-polyposis colorectal cancer [HNPCC]) and in all but one, constitutional mutations in MLH1 or MSH2 genes could be detected. The same mutations were also found in other family members.

CONCLUSIONS:

Our study demostrates the involvement of CIN in a majority of early-onset colorectal tumors. Furthermore, we identified Lynch syndromes in seven cases (50%) of early-onset colorectal carcinomas with impairment of the MMR system. These results suggest that patients with early-onset CRC should be screened for hereditary cancer syndrome through clinical and molecular characterizations.

INTRODUCTION

Early-onset colorectal cancers (i.e., those diagnosed in patients younger than 45 yr) are relatively rare, accounting for 2–8% of the whole burden of large bowel neoplasms (^1,2). This cohort represents an heterogeneous group which includes patients with strong familial clustering of colorectal cancer as well as evidence for other non-hereditary or sporadic cases (^3,4).

Recent lines of evidence have shown that colorectal cancer can arise at least through two distinct pathways: chromosomal instability (CIN) and microsatellite instability (MSI). The first, and more common, is characterized by loss of heterozigosity and by chromosomal changes leading to alterations of tumor-suppressor genes, such as APC (chromosome 5q), p53 (chromosome 17p), DCC, and SMAD 2 and 4 (chromosome 18q) (^5,6). APC gene product is a large protein with multiple functions, such as the binding with -catenin (⁷). Mutations of the APC gene result in increased cytoplasmic levels of -catenin, which ultimately interacts with transcriptional factors, with a consequent upregulation of several cancer-related genes (⁸). APC alterations have also been studied by immunohistochemistry, using antibodies directed to the site involved in the binding with -catenin (C-terminus region) (^9,10,11). -catenin expression can be evaluated by using monoclonal antibodies that identify either the normal membranous or the altered cytoplasmic and nuclear localization of the protein (^10,11,12).

A second pathway is typical of hereditary non-polyposis colorectal cancer (HNPCC), i.e., Lynch syndrome, an autosomal dominant condition that accounts for 2–3% of all colorectal carcinomas (¹³). This "mutator" pathway is characterized by repeated nucleotide sequences called "microsatellites." MSI may result from defects in the DNA mismatch repair (MMR) machinery (^14,15) consequent to constitutional mutations of the MMR genes (^13,16). MSI has also been identified in 10–15% of sporadic colorectal carcinomas in which constitutional mutations of hMLH1 and hMSH2 are rarely found (^17,18), and the "mutator phenotype" is frequently due to MLH1 promoter hypermethylation (^19,20). It has been widely shown that loss of MMR protein expression in tumor tissue, detected by immunohistochemistry, may correlate with MSI (^21,22,23). CIN and MSI pathways can be distinguished by their molecular characteristics, although there is evidence of some degree of overlapping; moreover, there are tumors that do not show evidence of implications in either of the mutational pathways (²⁴).

We evaluated the expression of APC, -catenin, p53 proteins, and MMR proteins (MLH1, MSH2, and MSH6) by immunohistochemical studies and microsatellite instability in colorectal malignancies in individuals younger than 45 yr. More specifically, our purpose was to detect which of the best-known molecular pathways (CIN and MSI) are more frequently involved in early-onset colorectal cancer, and the possibility that these features might be useful markers for screening HNPCC.

MATERIALS AND METHODS

Patients and Family History of Malignancy

A series of 71 early-onset colorectal cancers was identified from 1992 to 2001 through the files of the Department of Pathology (University of Modena) and the Service of Pathology (Hospital of Carpi). Tumor specimens were taken from patients who had undergone surgical resection of the large bowel. All the slides were reviewed by two pathologists (LL and CDG). Pathological features of colorectal carcinomas and clinical data of patients are summarized in Table 1.

Table 1 - Clinicopathological Features of Patients with Early-Onset Colorectal Carcinomas.

Full table

Detailed family history was obtained for each case by interviewing the patients and/or their relatives. Verification of cancer occurrence among family members could be obtained in most cases through clinical charts, pathological records, or death certificates. Families were classified as HNPCC when they met the Amsterdam criteria II, as defined by Vasen et al. (²⁵). More specifically: (i) a family should include at least three patients with histologically verified colorectal cancer, or HNPCC-related malignancies; (ii) one of the affected patients should be a first-degree relative of the other two; (iii) at least two different generations should be affected; and (iv) in at least one of the affected individuals, colorectal cancer should be diagnosed before the age of 50 yr; (v) FAP should be excluded.

Microsatellite Analysis

Paraffin-embedded tumor tissue and adjacent normal mucosa were microdissected with sterile scalpels into polypropylene tubes. Samples were incubated for 2 h in xylene (room temperature) and pelletted for 5 min. After a wash in ethanol, samples were dried and DNA was extracted as previously described (²⁶). MSI was evaluated with five microsatellite markers (BAT25, BAT26, BAT40, D2S123, D5S346), using a fluorescence-based polymerase chain reaction (PCR) method. DNA from normal and tumor tissues was amplified in a 10-L volume containing 30–50 ng of DNA; 5 ng of dye-labeled forward and unlabeled reverse primers; 200 m dGTP, dTTP, dATP, and dCTP; 1.5 mM MgCl₂; 50 mM KCl; 10 mM Tris (pH 8.3), and 0.3 units of Taq polymerase. All samples were run on a CEQ 8000 sequencer, and analyzed using the Fragment Analysis System by Beckman Coulter. MSI+ tumors were defined as those in which the instability was detected with at least two microsatellite markers.

Immunohistochemistry

For each case, a representative paraffin-embedded block containing tumor tissue and normal mucosa was sectioned at 4 m. Slides were submitted to microwave antigen retrieval in 10 mM citrate buffer (pH 6) at 350 W for 30 min.

Mouse monoclonal antibodies used were: APC (Ab120, Abcam, Cambridge, UK) at 1:500 dilution for 2 h, -catenin (Transduction Labs, BD Biosciences, Milano) at 1:100 dilution for 30 min, p53 (Cellmarque, Austin, TX) at 1:60 dilution for 30 min; mouse monoclonal antibodies against MLH1 protein (clone 168–15) and MSH2 protein (clone G129–1129, Pharmingen, San Diego, CA) at 1:100 dilution, and MSH6 protein (Transduction Labs, BD Biosciences, Milano) at 1:2,000 dilution, overnight. Immunoperoxidase staining using diaminobenzidine as chromogen was carried out with the NEXES Automatic Staining System (Ventana, Strasbourg, France). The slides were counterstained with Hematoxylin.

APC immunoreactivity was present in the cytoplasm of goblet cells of normal colonic mucosa. Altered expression of APC was considered when absence of cytoplasmic staining was seen in all neoplastic cells. -catenin expression was predominantly localized in the cell membrane of normal colonocytes. Altered expression of -catenin was considered when greater than 10% of the tumor cells showed nuclear and/or cytoplasmic immunoreactivity (²⁷). Staining for p53 protein was evaluated by counting at high-power magnification (400) the number of positive nuclei for 1,000 tumor cells, with a cut-off of 10% for positivity. Lack of expression of MLH1, MSH2, and MSH6 proteins was defined as complete absence of detectable nuclear staining in tumor cells. Intact nuclear staining of the colonic crypts of the peritumoral normal mucosa, stromal cells, and lymphocytes served as internal positive control, and was required for adequate evaluation. All immunohistochemical slides were evaluated separately by two pathologists (LL and CDG).

Analysis of Germline Mutations in hMLH1 and hMSH2

In patients whose tumors had MSI and no MMR protein expression, a search for germline mutation in the corresponding genes was executed by direct genomic sequencing of DNA derived from blood leukocytes. This analysis could not be carried out in one patient without family history who died suddenly, and in four patients who refused genetic testing. Amplification products were generated with primers located in the flanking introns approximately 50 base pairs from the respective intron/exon borders in order to detect all possible splice junction mutations, as previously described (²⁸). Direct sequencing of the PCR products was performed using cycle sequencing with Big Dye Terminator kit (Perkin-Elmer) and reactions were run on an ABI 310 capillary sequencer (Perkin-Elmer) according to the manufacture's instructions.

MLH1 Promoter Methylation Analysis

Sodium bisulphite conversion of genomic DNA was obtained as previously described (²⁹). The modified DNA was subjected to methylation-specific PCR (MSP) with primers encompassing a small region proximal to the transcription start of the MLH1 promoter, critical for gene silencing (³⁰).

Statistical Analysis

Pearson's ² and Fisher's exact tests were used to assess the statistical significance of differences in immunohistochemical staining and molecular data. The analysis was carried out with the Statistical Package for the Social Sciences (SPSS) software.

RESULTS

Clinical Data

In the present study, a total of 71 unrelated patients who were diagnosed with colorectal cancer at less than 45 yr of age were included. The median age at diagnosis was 40.6 yr (range: 19–45 yr) and there were 43 males and 28 females. Primary tumors were localized in the colon in 58 cases (23 in the proximal and 35 in the distal) and in the rectum in 13 cases. Well and moderate differentiation were detected in 12 and 51 tumors, respectively, whereas poor differentiation was present in eight cases. Tumor-infiltrating lymphocytes (TIL), medullary pattern, and Crohn's-like reaction were reported in 16, 1, and 9 cases, respectively. The clinical profiles and pathological features are summarized in Table 1.

A careful evaluation of family history allowed the diagnosis of HNPCC in 7 out of 71 (10%) patients. In this "hereditary" group, colorectal neoplasms were more frequently located in the right colon (from cecum to the splenic flexure) and the median age at diagnosis was 39.2 (range: 29–43 yr). Besides colon cancers, an increased incidence of multiple cancers of the endometrium and ovary was clearly evident in the HNPCC families (Table 2).

Table 2 - Germline Mutations and Clinical Features in HNPCC Patients.

Full table

MSI Analysis

Fourteen (19.7%) out of the 71 cases, were classified as MSI positive (MSI+) (Fig. 1). These tumors showed absence of MMR protein expression with a sensitivity of 100%. All 57 cases found to be MSI negative (MSI-) showed normal expression of the MMR proteins (specificity 100%).

Figure 1.

Electropherogram of BAT25 marker in normal mucosa and in cancer tissue of the same patient.

Full figure and legend (47K)

Immunohistochemistry Analysis

The results of immunohistochemical analysis in our series of tumors are shown in Table 3. Altered expression of APC, -catenin, and p53 proteins was more frequent in MSI- than in MSI+ tumors, though the differences did not reach the statistical significance. Altered protein expression—or microsatellite instability—was not directly related to clinical or morphological parameters (Table 1). However, proximal location of lesions was found to be more frequently associated with microsatellite instability and altered expression of MMR proteins (p = 0.002). Examples of immunohistochemistry are shown in Figure 2 (A–D).

Figure 2.

(A) Loss of MLH1 protein expression in neoplastic cells of a signet ring cell colorectal carcinoma. The nuclei of normal colonic epithelial cells served as positive internal controls (20). (B) Lack of expression of APC protein in neoplastic cells of a signet ring cell colorectal carcinoma. The internal positive control is represented by muscularis layer of the microvessels (20). (C) Nuclear expression of -catenin in colorectal adenocarcinoma (20). (D). Overexpression of p53 protein in the majority of the neoplastic nuclei of a colorectal adenocarcinoma (10).

Full figure and legend (354K)

Table 3 - Results of Immunohistochemical Analysis in Early-Onset Colorectal Carcinomas.

Full table

In all MSI+ tumors, lack of expression of the main MMR proteins could be observed: in five of these cases there was a parallel altered expression of both MSH2 and MSH6 proteins whereas in one case MLH1 and MSH6 proteins were both altered. Clinical features of Lynch syndrome were found in seven MSI+ cases, in whom APC, -catenin, and p53 tended to be unaltered (Table 4).

Table 4 - Clinical and Genetic Features of the 14 Cases Whose Tumors Showed Microsatellite Instability.

Full table

Mutational Analysis

Mutational analysis could be executed in nine probands out of 14 (64%) MSI+ cases and in five relatives (Table 4). Six HNPCC cases showed germline mutations of the MMR gene corresponding to the altered protein expression; in particular, a deletion of TT at nucleotide 880 in exon 5 of MSH2 (case 1), a transition of C to T at nucleotide 1459 in exon 13 of MLH1 (case 2), a deletion of AATG at nucleotide 727 in exon 9 in MLH1 (case 5, Fig. 3). All these alterations resulted in truncated proteins. Missense mutations were found in other two cases: a transition of G to A at nucleotide 2005 in exon 13 in MSH2 (Gly to Asp, case 3) and a transition of T to A at nucleotide 2246 in exon 19 in MLH1 (Leu to Pro, case 6); a "frame" mutation in which deletion of AAT at nucleotide 1786 in exon 12 of MSH2 was detected in case 7. In the group without clinical HNPCC diagnosis, only one case (case 9) showed a deletion of GGGA at nucleotide 1952 in exon 17 of MLH1, again resulting in a truncated protein. In the remaining two patients who could be assessed (n. 4 and 10 of Table 4), no constitutional mutation could be detected. However, hypermethylation of the MLH1 promoter region was observed.

Figure 3.

Example of germline mutation represented by deletion of AATG at nucleotide 727 in exon 9 in MLH1 gene (case 5).

Full figure and legend (30K)

In four HNPCC cases, we studied some proband's relatives and we found the same MMR genes mutation of the affected case (Table 2). In case 1, an MSH2 mutation was found also in the mother of the proband affected by synchronous endometrium and ovary cancers at age 40 and in the cousin who developed a carcinoma of the ovary at age 34. In case 3, the same MLH1 missense mutation was detected in the proband's mother affected by colorectal adenocarcinoma. In case 6, the same MLH1 missense mutation was detected also in the proband's mother. The same "frame" MSH2 mutation detected in the proband of family seven was reported in the cousin affected by metachronous adenocarcinoma (sigma and cecum).

DISCUSSION

The results of the present study can be summarized as follows: (i) early-onset colorectal carcinomas, as far as their molecular pathogenesis is concerned, can broadly be subdivided into two groups: those showing MSI and lack of expression of the MMR proteins and those with stable lesions not associated with altered protein expression; (ii) in the former group, HNPCC can be diagnosed in approximately 50% of the cases; and (iii) in the latter group, the frequent lack of expression of APC, -catenin, and p53 suggests the involvement of the "CIN" pathway (characterized by loss of heterozygosity) in cancer development.

The "loss of heterozygosity" pathway seems to be the most common pathway in the multistep process of colorectal carcinogenesis in individuals younger than 45 yr as well as in older patients (^31,32,33,34). In our series, 57 out of 71 tumors (80%) appeared MSI stable, and the large majority (66–79%) showed loss or altered expression of APC, -catenin, or p53. These data are similar to those previously reported by other authors (^{35,36,37,38,39}). Along this sequence, cancer develops in a stepwise fashion, from small adenomas to large adenomas, to infiltrating carcinoma. The Basil Morson's genial intuition (⁴⁰) was subsequently confirmed by various observations of Vogelstein et al. (⁴¹) and Boland et al. (⁴²) who demonstrated that APC mutations were the rate-limiting step in the passage from normal mucosa to small adenomas, whereas the inactivation of p53 was involved in the late phases of tumorigenesis. However, the fact that these alterations are not found in all MSI- tumors indicates that the matter is probably more complex, and that other genetic changes are presumably implicated (²⁴). It cannot be excluded that what we at present know as "loss of heterozygosity" sequence is indeed a complex of genetic pathways having in common the fact of developing in MSI- tumors. Clearly, more studies are needed to better define this condition.

We found that 14 (20%) of early-onset colorectal carcinomas are MSI+, and that all these lesions lacked the expression of one or two MMR proteins. APC, -catenin, and p53 expression could be altered, but not as frequently as in MSI- tumors. On a clinical ground, male sex and location in the right colon were more frequent; more important, in seven of these patients the pedigree was consistent with HNPCC, according to the Amsterdam criteria. In the remaining individuals (50%), however, as in the HNPCC cases, there was a similar preponderance of male sex, and predilection for the proximal colon. We found germline mutations in one of the MMR genes in six cases with family history compatible with HNPCC and in one case with a strong suspicion of HNPCC (Amsterdam criteria not fulfilled). Affected relatives of patients with MLH1 mutations showed a significantly higher frequency of colorectal cancer but a lower frequency of endometrium and ovarian cancers than individuals with MSH2 mutations. In tumors of patients with MSH2 mutation, altered expression of both MSH2 and MSH6 proteins was seen. MSH2 and MSH6 form a heterodimeric protein complex and have sequence-specific recognition of mismatches. These genes cooperate in some biomolecular pathways and often the mutation of MSH2 gene could be responsible for functional alteration of MSH6 in immunohistochemical analysis. The mutation in the MLH1 gene of patient with suspected HNPCC could be a "de novo" mutation (first mutation in a family, which will show the full-blown clinical features of HNPCC in subsequent generations). Therefore, early-onset colorectal cancer could be a useful marker for the identifications of HNPCC. In all MSI+ cases without family history there is, presumably, the involvement of both molecular pathways (CIN and MSI). The coexistence of different genetic alterations has already been reported (²⁴), thus suggesting that the two mechanisms are not mutually exclusive. It is possible that the loss of MMR function in this subset of patients was caused by allelic losses of one of the MMR genes (²⁴) and not by mutational events.

Other studies reported several differences between MSI-positive sporadic tumors (without family history) and HNPCC. In particular, a greater preponderance among females, predilection for the proximal colon, the near exclusive involvement of the MLH1 gene, a lower frequency of APC and -catenin mutations, and a higher frequency of DNA methylation were reported in MSI positive sporadic tumors (^{38,39,43,44,45}). Therefore, early-onset MSI+ tumors represent an heterogeneous group of neoplasms, with truly hereditary factors implicated in part of these lesions and presumably epigenetic factors (DNA methylation?) responsible for others. Although "de novo" mutations cannot be excluded, this event seems unlikely; indeed, other studies (^46,47,48,49) in patients with early-onset colorectal neoplasms detected mutations in only a fraction of the investigated cases.

Various studies suggested that the systematic search for MSI and/or lack of expression of MMR proteins can be adopted as a screening procedure for HNPCC among patients with colorectal neoplasms (^22,23,32,49). Our observations confirm the validity of this approach in patients with early-onset carcinomas. Indeed, half of the patients showing MSI+ tumors had features of HNPCC, and in all but one constitutional mutations were detected. Therefore, we would recommend to assess MSI and/or expression of MMR proteins in all patients developing colorectal cancer before the age of 45 yr.

In conclusion, the occurrence of early-onset colorectal cancers is an intriguing biological event that must be investigated in detail. The combined use of clinical features and MSI and IHC analysis can be useful for the characterization of these peculiar early cancers and recognition of Lynch syndrome. This approach allows adeguate and tempestive family surveillance programs in these heritable disorders through early-age colon cancer screening and in some cases surveillance for extracolonic tumors of the Lynch spectrum (uterus, ovary, and others neoplasms).

References

1. Parramore, JB, Wei, JP, Yeh, KA. Colorectal cancer in patients under forty: Presentation and outcome. Am Surg 1998;64: 563–568.
2. Turkiewicz, D, Miller, B, Schache, D, et al. Young patients with colorectal cancer: How do they fare? ANZ J Surg 2001;71: 707–710.
3. Guillem, JG, Baster, AL, Ng, J, et al. Clustering of colorectal cancer in families of probands under 40 years of age. Dis Colon Rectum 1996;39: 1004–1007.
4. Hall, NR, Bishop, DT, Stephenson, BM, et al. Hereditary susceptibility to colorectal cancer. Relatives of early onset cases are particularly at risk. Dis Colon Rectum 1996;39: 739–743. | PubMed | ISI | ChemPort |
5. Lengauer, C, Kinzler, KW, Vogelstein, B. Genetic instability in colorectal cancers. Nature 1997;386: 623–627. | Article | PubMed | ISI | ChemPort |
6. Rajagopalan, H, Nowak, MA, Vogelstein, B, et al. The significance of unstable chromosomes in colorectal cancer. Nat Rev Cancer 2003;3: 695–701. | Article | PubMed | ISI | ChemPort |
7. Rubinfeld, B, Albert, I, Porfiri, E, et al. Binding of GSK3 to the APC--catenin complex and regulation of complex assembly. Science 1996;272: 1023–1025. | Article | PubMed | ISI | ChemPort |
8. Behrens, J, Leung, TC, Ernst, H, et al. Regulated binding of a PTP1B-like phosphatase to N-cadherin: Control of cadherin-mediated adhesion by dephosphorylation of -catenin. J Cell Biol 1996;134: 801–803. | Article | PubMed | ISI | ChemPort |
9. Midgley, CA, White, S, Howitt, R. APC expression in normal human tissues. J Pathol 1997;181: 426–433. | Article | PubMed | ISI | ChemPort |
10. Iwamoto, M, Ahnen, DJ, Franklin, WA, et al. Expression of -catenin and full-length APC protein in normal and neoplastic colonic tissues. Carcinogenesis 2000;11: 1935–1940.
11. Takayama, T, Ohi, M, Hayashi, T, et al. Analysis of K-ras, APC and -catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis. Gastroenterology 2001;121: 599–611. | Article | PubMed | ChemPort |
12. Hao, XP, Tomlinson, IP, Ilyas, M, et al. Reciprocity between membranous and nuclear expression of -catenin in colorectal tumours. Virchows Arch 1997;431: 167–172. | Article | PubMed | ISI | ChemPort |
13. Lynch, HT, Smyrk, T, Lynch, JF. Overview of natural history, pathology, molecular genetics and management of HNPCC (Lynch syndrome). Int J Cancer 1996;69: 38–43. | Article | PubMed | ISI | ChemPort |
14. Chung, DC, Rustgi, AK. DNA mismatch repair and cancer. Gastroenterology 1995;109: 1685–1699. | Article | PubMed | ISI | ChemPort |
15. Rhyu, MS. Molecular mechanisms underlying hereditary nonpolyposis colorectal carcinoma. J Natl Cancer Inst 1996;88: 240–251. | PubMed | ChemPort |
16. Liu, B, Parsons, R, Papadopoulos, N, et al. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat Med 1996;2: 169–174. | Article | PubMed | ISI | ChemPort |
17. Liu, B, Nicolaides, NC, Markowitz, S, et al. Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nat Genet 1995;9: 48–55. | Article | PubMed | ISI | ChemPort |
18. Wu, Y, Nystrom-Lahti, M, Osinga, J, et al. MSH2 and MLH1 mutations in sporadic replication error-positive colorectal carcinoma as assessed by two-dimensional DNA electrophoresis. Genes Chromosomes Cancer 1997;18: 269–278. | Article | PubMed | ChemPort |
19. Cunningham, JM, Christensen, ER, Tester, DJ et al. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 1998;58: 3455–3460. | PubMed | ISI | ChemPort |
20. Kuismanen, SA, Holmberg, MT, Salovaara, R, et al. Genetic and epigenetic modification of MLH1 accounts for a major share of microsatellite-unstable colorectal cancer. Am J Pathol 2000;156: 1773–1779. | PubMed | ISI | ChemPort |
21. Thibodeau, SN, French, AJ, Roche, PC, et al. Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res 1996;56: 4836–4840. | PubMed | ISI | ChemPort |
22. Cawkwell, L, Gray, S, Murgatroyd, H, et al. Choice of management strategy for colorectal cancer based on a diagnostic immunohistochemical test for defective mismatch repair. Gut 1999;45: 409–415. | PubMed | ISI | ChemPort |
23. Lindor, NM, Burgart, LJ, Leontovich, O, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol. 2002;20: 1043–1048. | Article | PubMed | ISI | ChemPort |
24. Goel, A, Arnold, CN, Niedzwiecki, D, et al. Characterization of sporadic colon cancer by patterns of genomic instability. Cancer Res 2003;63: 1608–1614. | PubMed | ISI | ChemPort |
25. Vasen, HFA, Watson, P, Mecklin, JP, et al. The International Collaborative Group on hereditary non-polyposis colorectal cancer (ICG-HNPCC). Gastroenterology 1999;116: 453–456.
26. Pedroni, M, Tamassia, MG, Percesepe, A, et al. Microsatellite instability in multiple colorectal tumors. Int J Cancer 1999;81: 1–5. | Article | PubMed | ISI | ChemPort |
27. Lim, S, Lee, HS, Kim, HS, et al. Alteration of E-cadherin-mediated adhesion protein is common, but microsatellite instability is uncommon in young gastric cancer. Histopathology 2003;42: 128–136. | Article | PubMed | ISI | ChemPort |
28. Viel, A, Genuardi, M, Capozzi, E, et al. Characterization of MSH2 and MLH1 mutations in Italian families with Ereditary Non Polyposis Colorectal cancer. Genes Chromosomes Cancer 1997;18: 8–18. | Article | PubMed | ISI | ChemPort |
29. Menigatti, M, Di Gregorio, C, Borghi, F, et al. Methylation pattern of different regions of the MLh1 promoter and silencing of gene expression in hereditary and sporadic colorectal cancer. Genes Chromosomes Cancer 2001;31: 357–361.
30. Deng, G, Chen, A, Hong, J, et al. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res 1999;59: 2029–2033. | PubMed | ISI | ChemPort |
31. Suh, JH, Lim, SD, Kim, JC, et al. Comparison of clinicopathologic characteristics and genetic alterations between microsatellite instability-positive and microsatellite instability-negative sporadic colorectal carcinomas in patients younger than 40 years old. Dis Colon Rectum 2001;44: 219–228.
32. Percesepe, A, Borghi, F, Menigatti, M, et al. Molecular screening for hereditary nonpolyposis colorectal cancer: A prospective, population-based study. J Clin Oncol 2001;19: 3944–3950. | PubMed | ISI | ChemPort |
33. Kakar, S, Burgart, LJ, Thibodeau, SN, et al. Frequency of loss of hMLH1 expression in Colorectal Carcinoma increases with advancing age. Cancer 2003;97: 1421–1427. | Article | PubMed | ISI | ChemPort |
34. Liang, JT, Huang, KC, Cheng, AL, et al. Clinicopathological and molecular biological features of colorectal cancer in patients less that 40 years of age. Br J Surg 2003;90: 205–214.
35. Konishi, M, Kikuchi-Yanoshita, R, Tanaka, K, et al. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111: 307–317. | Article | PubMed | ISI | ChemPort |
36. Olschwang, S, Hamelin, R, Laurent-Puig, P, et al. Alternative genetic pathways in colorectal carcinogenesis. Proc Natl Acad Sci U S A. 1997;94: 12122–12127. | Article | PubMed | ChemPort |
37. Losi, L, Ponz de Leon, M, Jiricny, J, et al. K-ras and p53 mutations in hereditary non-polyposis colorectal cancers. Int J Cancer 1997;74: 94–96.
38. Salashor, S, Kressner, U, Pahlman, L, et al. Colorectal cancer with and without microsatellite instability involves different genes. Genes Chromosomes Cancer 1999;247–252. | Article | PubMed | ISI | ChemPort |
39. Miyaki, M, Iijima, T, Kimura, J, et al. Frequent mutation of -catenin and APC genes in primary colorectal tumors from patients with Hereditary Nonpolyposis Colorectal Cancer. Cancer Res 1999;59: 4506–4509. | PubMed | ISI | ChemPort |
40. Morson, BC. The pathogenesis of colorectal cancer. In Bennington JL, consulting ed: Major problems in pathology, Vol. 10. WB Saunders Co: Philadelphia, (1978).
41. Vogelstein, B, Fearon, ER, Hamilton, SR, et al. Genetic alterations during colorectal-tumor development. N Engl Med 1988;319: 525–532.
42. Boland, CR, Santo, J, Appelman, HD, et al. Microallelotyping defines the sequence and tempo of allelic losses at tumour suppressor gene loci during colorectal cancer progression. Nat Med 1995;1: 902–909. | Article | PubMed | ISI | ChemPort |
43. Young, J, Simms, LA, Biden, KG, et al. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings. Parallel pathways of tumorigenesis. Am J Pathol 2001;159: 2107–2116. | PubMed | ISI | ChemPort |
44. Jass, Jr, Walsh, MD, Barker, M, et al. Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability. Eur J Cancer 2002;38: 858–866. | Article | PubMed | ISI | ChemPort |
45. Yamamoto, H, Min, Y, Itoh, F, et al. Differential involvement of the hypermethylator phenotype in Hereditary and Sporadic Colorectal Cancers with high-frequency microsatellite instability. Genes Chromosomes Cancer 2002;33: 322–325. | Article | PubMed | ISI | ChemPort |
46. Liu, B, Farrington, SM, Petersen, GM, et al. Genetic instability occurs in the majority of young patients with colorectal cancer. Nat Med 1995;1: 348–352. | Article | PubMed | ISI | ChemPort |
47. Chan, TL, Yuen, ST, Chung, LP, et al. Frequent microsatellite instability and mismatch repair gene mutations in young Chinese patients with colorectal cancer. J Natl Cancer Inst 1999;91: 1221–1226. | Article | PubMed | ChemPort |
48. Farrington, SM, Lin-Goerke, J, Ling, J, et al. Systematic analysis of hMSH2 and hMLH1 in young colon cancer patients and controls. Am J Hum Genet 1998;63: 749–759. | Article | PubMed | ISI | ChemPort |
49. Lamberti, C, Kruse, R, Ruelfs, C, et al. Microsatellite instability- a useful diagnostic tool to select patients at high risk for Hereditary non-polyposis colorectal cancer: A study in different groups of patients with colorectal cancer. Gut 1999;44: 839–843. | PubMed | ISI | ChemPort |

Acknowledgements

The authors are grateful to Professor Gian Paolo Trentini for providing helpful suggestions, Dr. Filippo Schepis for skilful discussion, Mrs Paola Manni and Elisa Toni, and Mr Luca Fabbiani for their excellent technical assistance. This work has been supported by Consiglio Nazionale delle Ricerche (PSO, CNR-MIUR), Associazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of University (COFIN 2001), Fondi per gli Investimenti della Ricerca di Base (FIRB), Legna Tumori della Regione Emilia Romagna, Progetti di Ricerca Avanzata.