Genomic instability plays an essential role in the development and progression of human colorectal cancer (CRC)[ ¹]. Two major types of genetic instability have been described in CRC: chromosomal instability and microsatellite instability[ ¹]. About 60% of CRCs develop through the chromosomal instability pathway, which is characterized by losses and gains of chromosomes (aneuploidy), as well as losses of heterozygosity[ ¹].

CRC progresses through four distinct clinical stages that are described as dysplastic crypts, small benign tumors, malignant tumors invading surrounding tissues, and finally metastatic cancer. This progression involves several genetic changes such as inactivation of tumor suppressor genes and activation of oncogenes[ ²]. Mutations of the adenomatous polyposis coli ( APC) gene are considered the earliest[ ³] and most prevalent genetic changes in colorectal tumorigenesis. More than 85% of colon cancers are estimated to have a somatic mutation of APC[ ⁴]. Furthermore, a large number of genes that trigger chromosomal instability have been identified in yeast in the past[ ⁵]. The underlying mechanisms leading to chromosomal instability in colorectal cancer remain to be characterized. The DNA double-strand break (DSB) is regarded as the most critical of all DNA lesions[ ^{6 , 7}], and it has been shown that defects in the cellular response to DSBs can lead to genetic alteration, chromosomal instability, and ultimately malignant transformation[ ⁸].

The genome damage to the lymphocytes of peripheral blood has been widely used as a biomarker of genotoxic environmental factors, and long-term studies have demonstrated its validity and high clinical productivity[ ⁹]. Micronucleus (MN) is an acentric chromosome fragment or whole chromosome that is left behind during mitotic cellular division and appears in the cytoplasm of interphasic cells as a small additional nucleus[ ¹⁰]. The formation of MN in dividing cells is the result of chromosome breakage due to unrepaired or mis-repaired DNA lesions, or chromosome malsegregation due to mitotic malfunction. These events may be induced by oxidative stress, exposure to clastogens or aneugens, genetic defects in cell cycle checkpoint and/or DNA repair genes, as well as deficiencies in nutrients required as co-factors in DNA metabolism and chromosome segregation machinery[ ^{11 - 14}]. All these events can cause the formation of MN through chromosomal rearrangements, altered gene expression or aneuploidy, effects associated with the chromosome instability phenotype often seen in cancer[ ^{15 , 16}].

The MN frequency test, widely accepted for in vitro and in vivo genotoxicity investigations, is a sensitive marker of genomic damage[ ^{17 , 18}]. The presence of an association between MN induction and cancer development is supported by a number of observations. The most substantiated include: the high frequency of MN in untreated cancer patients and in subjects affected by cancer-prone congenital diseases, e.g. Bloom syndrome or ataxia telangiectasia[ ^{15 , 19}], the presence of elevated MN frequencies in oral mucosa, used as a surrogate biomarker of cancer in clinical chemo-prevention trials[ ²⁰], the correlation existing between genotoxic MN-inducing agents and carcinogenicity, e.g. ionizing and ultraviolet radiation[ ^{21 , 22}].

CRCs progress through a series of clinical and histopathological stages ranging from dysplastic crypts through small benign tumors to malignant cancers. This progression is the result of a series of genetic changes that involve activation of oncogenes and inactivations of tumor suppressor genes[ ²⁶]. In colorectal cancer, chromosomal instability is the major form of genetic instability[ ²⁷]. It is generally agreed that colorectal cancers develop as a consequence of accumulation of mutations in key genes such as K-Ras, Apc, and p53 that are critical for regulating cell proliferation or cell cycle checkpoint control. In humans, the development from early adenomas to metastatic carcinomas takes somewhere from 20 to 40 years; it is believed that genetic instability plays a key role in accelerating the rate of mutation in cancerous cells[ ²⁸].

CRCs exhibit a defect in chromosome segregation, leading to frequent gains or losses of chromosomes (> 10 ² per chromosome per generation)[ ²⁸]. Chromosome instability has been detected in the smallest adenoma, suggesting that chromosome instability may occur at very early stages of colorectal carcinogenesis[ ²⁹]. Extensive research during the past has led to the identification of genes that play a major role in the development of colorectal cancer. For example, mutations or deletions of the adenomatous polyposis coli ( APC) gene, encoding a 310-kDa cytoplasmic protein[ ^{30 , 31}], are commonly found in inherited familial adenomatous polyposis patients and in sporadic colorectal cancers[ ^{32 , 33}]. Such mutations appear to be an early event during colorectal tumorigenesis[ ⁴].

The most commonly affected gene in sporadic colon cancer with defective DNA mismatch repair (MMR) is hMLH1, with the primary mechanism of gene inactivation being hypermethylation of the promoter[ ³⁴]. These tumors account for approximately 15% of sporadic colon cancers. The majority of sporadic colon cancers (85%), however, are proficient in DNA MMR but show another form of genomic instability at the gross chromosomal level, which has been called chromosomal instability. Such chromosomal instability represents the end result of a number of processes, including mutations in mitotic checkpoint genes, microtubule spindle defects, and telomere dysfunction[ ³⁵].

Two types of genetic instability have been identified, with chromosomal instability predominating[ ^{1 , 36}]. The molecular basis for chromosomal instability is just beginning to be explored[ ³⁷]. A large number of gene alterations can give rise to chromosomal instability in Saccharomyces cerevisiae[ ^{5 , 38}]. These genes include those involved in chromosome condensation, sister-chromatid cohesion, kinetochore structure and function, and microtubule formation and dynamics as well as checkpoints that monitor the progress of the cell cycle. To date, the only genes implicated in aneuploidy in human tumor cells are those of the latter class. Heterozygous mutations in the mitotic spindle checkpoint gene hBUB1 were detected in a small portion of colorectal tumors with the chromosomal instability[ ³⁹].

The identification of aneuploidy at early stages of tumor formation in MYH- and APC-mutant polyps is interesting also in view of previous reports showing that loss of APC function in primary mouse cell lines results in chromosomal instability due to a kinetochore attachment defect at mitosis[ ³⁶]. It is generally accepted that APC’s main tumor suppressing activity resides in its capacity to bind and regulate Wnt/β-catenin signal transduction[ ⁴⁰]. However, additional APC functions in cytoskeletal organization, mitotic spindle assembly, cell migration, and apoptosis may play important roles in tumor progression and malignant transformation[ ^{40 , 41}].

It has been demonstrated that chromosomes display nonrandom changes in cancer cells. These include structural rearrangements, e.g. deletions, amplifications or translocations that arise from breaks in DNA, as well as alterations in the number of intact chromosomes, known as whole-chromosome missegregations, originating from errors in cell division (mitosis). As a result of the accumulation of such processes, chromosomal instability is thought to play a key role in tumor development[ ⁴⁰].

In the present study, we investigated whether cytogenetic abnormalities participate in the pathogenesis of early CRC. Cytogenetic endpoints are sensitive biomarkers that are widely accepted to evaluate chromosome damage[ ^{42 , 43}]. MN assay provides a measure of both chromosome breakage and chromosome loss or nondisjunction in clastogenic and aneugenic events, respectively[ ^{11 , 13}].

MN assay is a sensitive indicator of exogenously or endogenously caused genetic damage and MN frequency has become an important end point in genotoxicity testing both in vivo and in vitro[ ^{17 , 18}]. Elevated levels of MN are indicative of defects in DNA repair and chromosome segregation which could result in generation of daughter cells with altered gene dosage, or deregulation of gene expression that could lead to the evolution of the chromosome instability phenotype often seen in cancer[ ^{10 , 11 , 15 , 21}]. These considerations give mechanistic support to a possible causal association between MN frequency and the risk of cancer. A recently published cohort study linking the frequency of micronuclei in lymphocytes of healthy subjects to the risk of cancer reported stomach cancer among the sites most specifically associated with micronuclei frequency[ ⁴⁴]. Similar findings have also been reported for preneoplastic lesions of colon[ ⁴⁵], esophagus[ ⁴⁶] and cervix[ ⁴⁷]. In particular, the higher risks noted for stomach and intestinal cancers, are in agreement with the literature, which emphasizes the role of chromosome rearrangements in the early stages of these tumours[ ^{47 , 48}].

Our study, which showed increased MN frequencies in the lymphocytes of CRC and colon polyp patients, could support these observations, as the induction of changes in DNA that lead to mutations plays a role in carcinogenicity. Establishment of inherited susceptibility factors is important in recognizing individuals at a higher risk of developing CRC, so that they may benefit from early detection and prevention programs. Many investigators have demonstrated genomic instability and abnormalities in patients with CRC[ ^{49 - 51}]. Further, experimental evidence shows that early colorectal adenomas have allelic imbalance[ ⁵²]. hCDC4 mutations have been shown to occur early in colorectal tumorigenesis[ ⁵³].

An association between MN and cancer has been reported[ ¹⁹]. The causes of this association may be structural chromosomal aberrations and aneuploidy[ ¹⁹]. The presence of an association between the frequency of micronuclei in lymphocytes and cancer risk has been suggested[ ^{13 , 44}]. Our findings of a high level of MN frequency in patients with CRC or NP seem to support this association. Thus, MN assay may be performed in lymphocytes as an indicator of genomic instability relevant to colorectal tumorigenesis.