Impact of chromosomal instability on colorectal cancer progression and outcome

Genetic instability is a hallmark of cancer cells and has been proposed to act as a catalyst of cancer development from early stages on [1, 2]. It is generally agreed that tumor progression occurs according to a scheme of gradual accumulation of genetic anomalies and that genetic instability is highest in most aggressive and metastatic forms of the disease. In colorectal cancer (CRC), genetic instability is subdivided into three classes; (i) mismatch repair deficiency (MIN), often of hereditary origin but also sporadically acquired, associated with base slippage mostly at poly(A) or poly(C) tracks and near diploid genomes, 15% of CRC (ii) chromosomal instability (CIN) resulting in severely rearranged karyotypes and aneuploidy, 65% of CRC (iii) non-MIN/non-CIN showing a methylator phenotype, 20% of CRC [3]. Major genetic mutations found and acting as key events in CRC, affect the WNT/APC/CTNNB1, KRAS/BRAF, FBXW7, PTEN, SMAD4, TGFBRII, and TP53 genes [4, 5]. Interestingly, patterns of mutated genes vary according to the class of CRC. BRAF mutations seem prevalent in MIN, whereas TP53 mutations are essentially found in CIN. Interestingly, genes promoting DNA repair, DNA damage checkpoint as well as translesional DNA replication are mostly down-regulated in CRC tumors compared to proliferating normal adjacent tissues, probably favoring the overall genetic instability at the nucleotide level [6]. In addition to these functionally validated aberrations, CGH based studies have identified widespread copy number alterations (CNA), some of which highly recurrent. Typical CNA patterns in CRC involve gains at 8q, 13q and 20q as well as losses at 5q, 8p, 17p and 18q [7]. These observations were confirmed in higher resolution array-CGH analyses and the boundaries of these regions of CNA defined with greater precision. Moreover, a number of focal events were pointed out [8]. The number of genetic anomalies linked to CRC pathogenesis is elevated and has risen with recent large scale sequencing efforts [9]. However, questions remain as to the role of widespread chromosomal instability in the course of the disease, in particular how these relate to progression of CRCs and patient relapse.

Although the sequential order originally proposed by Fearon and Vogelstein for CRC progression has been disputed the overall model is still regarded as valid [10]. Stepwise progression from normal epithelium, through dysplasia to carcinoma builds on a gradual accumulation of genetic anomalies. Recent work showed that copy number alterations (CNA) set in early in adenomas and reached in progressed adenomas a level similar to that found in carcinomas [6, 11]. It has also been suggested on the basis of a meta-analysis of chromosome CGH [7] and array-CGH [8] that progression from invasive cancer to metastasis was accompanied by an increase in the number of CNAs.

However, no clear cut results were proposed ascertaining the existence of a molecular progression scheme between early carcinoma (stage 1), invasive (stage 2 and 3) and metastatic (stage 4) CRC. In this work we wanted to verify whether we could relate CRC progression (from stage 1 to stage 4 and, eventually, to distal metastasis) to a gradual increase of genetic instability and sketch out a sequence of CNA increment. Moreover, we wanted to determine whether genetic instability correlated with patient outcome. To this aim we analyzed a set of 162 CIN CRCs comprising 131 primary carcinoma evenly distributed through stage 1 to 4 and 31 metastases (28/31 formed a primary-tumor/matched-metastasis pair) and 14 adenomas by array-CGH. Our data showed that stage 1, 2, 3 and 4 tumors did not differ significantly at the level of their CNA profiles. This led us to ask whether the level of genomic instability, as illustrated by array-CGH, was linked to disease outcome. Based on the Global Genomic Index (GGI), which corresponds to the fraction of the genome involved in CNA and the number of breakpoints (nbBP), which were determined as chromosomal sites where copy number shifts occurred, we defined two classes of tumors showing strong differences in outcome and hazard risk. CNAs correlating with early relapse or death in stage 2 or 3 patient were searched and two regions of copy number loss could be selected due to their strong association to negative outcome.

In this work, using array-CGH as an analytical approach, we aimed at determining whether genetic instability was related to progression of colorectal cancer and verify whether it could be used as a prognostic indicator. Colorectal cancer has served as a model of stepwise progression from normal epithelium, through benign growth, into malignant cells that eventually become invasive and acquire metastatic properties [5]. We were interested in determining the existence of a progression scheme between superficial carcinomas (stage 1), invasive carcinomas (stage 2 and 3) and metastatic carcinomas (stage 4) based on a gradual accumulation of genomic alterations, with alterations occurring specifically at each step of progression.

Main regions of gain and loss observed in this work were concordant with previously reported observations on CRC [8, 13]. Our data showing a marked increase in the number of CNAs in the transition from adenomas to carcinomas are in concordance with previously reported array-CGH work on early and advanced colorectal adenomas [11]. Most significant changes between adenomas and stage 1 CRCs were the occurrence of gains at 8q, 13q, 20 and losses at 8p, 15p, 17p and 18q in keeping with published works [7, 11, 19]. However, in contrast to Diep and coworkers [7] who proposed that transition from Duke’s B to C stage was associated to increased occurrence of gain at 1q and that Duke’s C to D to that of gains at 20q and Xq and loss at 21q, we could not identify CNAs whose occurrence was assigned to the transition from one stage to the next. This was exemplified by loss at chromosomes 14 and 15 which were present in stage 2, absent in stage 3 and present again in stage 4. We found that the number of CNAs in metastases was higher than in primary tumors. Analyzing concomitantly 28 pairs of primary tumors and corresponding metastases, we detected small regions of gains on chromosome 1q, 6p21, 10p and 17q21 and loss at chromosome 8p12 that occurred more frequently in metastases than in primary tumors (see Additional file 5). However, we could not infer the existence of anomalies specifying metastatic invasion as previously proposed [20]. Along similar lines we could not determine differences in CNA profiles between TP53 wild type and mutated tumors [8]. The absence of specific changes associated to the TP53 status in our dataset may be related to sampling differences. As a matter of fact, our series was restricted to CIN CRCs, while that in the work by Sheffer and colleagues (2009) comprised MSI cases that show TP53 mutations at a lower frequency and present fewer CNAs and at different locations than in CIN tumors [21].

We could not associate any qualitative change of CNA to disease progression, but the increase of genetic instability between stage 1 and stage 4 tumors and between primary CRCs and metastases suggested that the global level of genetic instability could be of clinical or prognostic significance in CIN colorectal cancer. We were interested to note that whereas genetic instability was lowest in stage 1 and highest in stage 4 CRC, its level in stage 2, genetic instability was in keeping with that in stage 4. Taken together, our data were not consistent with a model where CRCs progress gradually from stage 1 to stage 4, because of the resemblance of CNA profiles and genetic instability levels in stage 2 and stage 4 tumors. This similarity suggested that a large part of stage 4 may arise directly from most unstable stage 2 tumors. The difficulty to define a progression scheme with increasing numbers of CNAs according to disease stages may be related to the fact that CRC staging is a clinical progression scale which takes local and distal invasion into account and no tumor intrinsic characteristics.

We, hence, asked the question of whether the level of genetic instability could be a prognostic indicator in stage 2 and 3 CRCs. Using the fraction of the genome involved in CNA (GGI) and the number of breakpoints (nbBP) detected in the array-CGH profiles, we defined 5 groups of CRC. These groups differed in their level of genetic instability, but also their profile of anomalies. Indeed, tumors with low nbBP presented CNA involving large chromosomal regions (whole chromosomes or chromosomal arms), whereas those with elevated nbBP showed fractional gains or losses. The group with the lowest instability (low-GGI/low-nbBP) was expectedly associated to good prognosis, whereas those with high-nbBP correlated with increased risk of relapse or shortened overall survival in stage 2 and 3 CRCs. Our results suggest that genetic instability could be an interesting tumor specific prognostic variable in CIN colorectal cancer. Along similar lines, Poulogiannis and coauthors [17] defined 4 groups of instability in CRC, with low levels of instability associated with good outcome and high levels with bad prognosis. It is of note that this study was performed on a series comprising both MSI and MSS CRC and it is likely that their group of low instability was largely composed of MSI cases, which are of better prognosis than MSS. Other studies have proposed to relate genetic CNA patterns to outcome [22] or response to chemotherapy [13]. Remarkably, in our study the group presenting an elevated fraction of the genome involved in CNA, but low breakpoint numbers (high-GGI/low-nbBP), was the other group associated with low risk. This group was representative of tumors with large regions of CNA or whole chromosomal arm copy variations and contrasted in terms of prognosis with tumors with high-nbBP which were of bad prognosis. These results were reminiscent of observations by Janoueix-Lerosey et al., [23], who showed that in neuroblastoma tumors with whole chromosome CNA displayed good survival, while those with fractionated CNA presented a high risk of relapse.

These data led us to search for specific copy number changes correlated to the bad outcome groups. To this aim we used two convergent strategies to identify regions of CNA correlated to adverse outcome in our stage 2 and 3 series. Copy number loss at 19q13.3 and/or 16p13.3 was clearly associated to worsened disease course, as shown by strong correlation with either shortened RFS or OS in stage 2 and 3 tumors. The 16p13.3 region has already been described as a prognostic region in CRC [15]. This region bears a total of 167 known genes among which AXIN1 appears a serious candidate as it had been shown to be mutated in colorectal cancers and wild-type axin 1 can induce apoptosis in colorectal cancer cells [24, 25]. The 19q13.3 region comprises 285 genes and among them the BAX pro-apoptotic gene and the polymerase delta gene POLD1. Low expression of BAX protein in stage 3 colorectal cancers has been linked to shorter RFS and 5-FU-based treatment resistance [26]. POLD1 deletion could be involved in impaired DNA replication generating breaks and high rate of mutations [27]. As 16p13.3 (AXIN1) and 19q13.3 (BAX) are frequently co-deleted in our high risk group, apoptotic pathway in these tumors could be severely impacted giving significant resistance to apoptosis and growth advantage in malignant cells of these tumors. It was of note that we could confirm the association with adverse outcome for only 5/87 genes whose gain or loss had been previously shown to be of prognostic significance. Similarly, a large number of prognostic gains or losses could not be confirmed in our dataset [8, 15–18].

CNA profiles in CIN CRC are not consistent with the conventional scheme stating a stepwise progression from stage 1 to stage 4.

The level and pattern of genetic instability has been found to correlate with disease outcome, as tumors with fractionated gains and losses were of worse prognosis than tumors showing low breakpoint levels.

We identified that recurrent loss at 16p13 and 19q13 were significantly associated to bad outcome in stage 2 and 3 CRCs. Both regions were co-occurring in the high risk genetic instability groups.