It is hypothesized that genomic alterations leading to ulcerative colitis neoplasia result from long-standing chronic inflammatory mucosal injury. However, only an estimated 10% minority of the ulcerative colitis population ever develops neoplasia. As such, improved markers to identify this at risk group are greatly needed. This study identifies promising molecular markers using array-based comparative genomic hybridization.
Discoveries in the last several years have advanced our understanding of the molecular biology of ulcerative colitis. Widespread genomic alterations have been demonstrated in ulcerative colitis progressors with neoplasia, but not nonprogressors, using assays of telomere shortening and anaphase bridge development (2
), DNA-fingerprinting by arbitrarily primed PCR (13
), preclonal chromosomal gains and losses by fluorescence in situ hybridization (FISH) (1
), metaphase-comparative genomic hybridization (26
), and estrogen receptor gene promoter hypermethylation (29
To our knowledge, this is the first report of array comparative genomic hybridization in ulcerative colitis. Our goal was to interrogate the genome broadly to: 1) identify specific alterations or patterns of chromosomal alterations that distinguish ulcerative colitis progressors from nonprogressors, and 2) to better understand the early events that lead to neoplastic progression in ulcerative colitis by analyzing mucosa without dysplasia. Array technology permits high resolution genomic scanning for chromosomal losses and gains over many thousands of genomic targets. The much higher resolution afforded by array technology is a major advance over earlier comparative genomic hybridization technology using metaphase chromosomes (26
). The higher density 4,298 feature BAC array used in this study has a mean intermarker spacing of 675 kilobase in comparison to the approximate five megabase resolution of metaphase chromosomes. Metaphase-comparative genomic hybridization also necessitates considerable cytogenetic expertise for the morphologic evaluation of hybridization signals along physical chromosomes rather than the automated signal detection of known chromosomal sites spotted onto systematic arrays. Regardless of the differences in these technologies, it is encouraging that a few differential chromosomal sites identified by earlier metaphase-comparative genomic hybridization were also observed in our array results, specifically chromosomal losses on 15, 5q, and 18q (26
An initial low-resolution array (287 clones) provided proof of principle that a greater degree of clonal genomic alteration was in fact present in non-dysplastic mucosa from 33% the progressors (three of nine) in contrast to none of the eight nonprogressors or two normal controls. The low-density array data additionally provided preliminary data regarding locations of highest frequency genomic alterations. As anticipated, subsequent analysis of the identical DNA samples, using markedly higher-density arrays (4,153 clones), demonstrated this phenomenon on a much larger scale, distinguishing all nine progressors from all of the eight nonprogressors and two normal controls, when analyzed by a combination of statistical measures.
In addition, analysis of the higher-density arrays identified specific and unique genomic losses associated with neoplastic progression in dysplasia-free colonic samples from ulcerative colitis progressors. Of importance, the biopsy samples were obtained far distant from their respective tumors, highlighting the widespread nature of the genomic alterations within the colon. However, given the 4,153 clones analyzed, it is not unlikely that due to chance alone a few targets in the progressors will all have gains or losses below some threshold and nonprogressors above, or vice-versa, which could thus falsely discriminate perfectly among the groups. If the responses are not large in magnitude, then it is likely that the group differences are a result of experimental noise and not due to consistent chromosomal gains or losses. To eliminate these from consideration, we restricted our search for potential discriminating targets to the subset of targets having a significantly higher variance in response than the overall sample variance in response. This selection was independent of the progressor/nonprogressor status of the cases. The reasoning behind this selection criterion is as follows: for most genomic locations, most of the 19 cases do not have genomic abnormalities, and have array response intensity deviations that are relatively small in magnitude and centered around zero. In contrast, if many cases have abnormalities at a location corresponding to a given clone, then many of the 19 responses will be large in magnitude and the sample variance for that target will be large. By selecting for consideration only those bacterial artificial chromosomes with high variance, we are selecting targets for which there is evidence of genomic aberration across some of the 19 cases, although no consideration of group membership is taken into account, eliminating bias.
While a portion of the chromosomal gains and losses were seen in both progressors and nonprogressors, most of them were random and not consistent between biopsies. However, a smaller subset of changes with great variance was non-random, as shown by t-tests comparing their distribution among progressors and nonprogressors ( lists the eleven most significant). Such non-random losses of several genomic regions were observed in progressors, with the most powerful discriminator being a chromosomal loss on RP11-196B3, which maps to the distal tip of the 18q arm (18q23). Two adjacent and overlapping targets at 18q were also highly discriminant. The 18q23 loss was detected in eight of nine (89%) progressors and was not seen in any of the eight nonprogressors or two normal controls. Other than several sequence tagged sites, there is only one known gene in this area, named “nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1” (NFATC1), according to the NCBI clone registry. Of interest, this transcription factor initially identified as an important modulator of T-cell cytokine production (30
) has been found to have increasing roles outside the immune system. A recent study by Duque and colleagues documents its importance in COX-2 regulation in colorectal carcinoma cell lines (31
). COX-2 upregulation is well-described in colorectal tumorigenesis, both sporadic and ulcerative colitis forms (12
It is interesting that this 18q as well as other highly discriminant loci (5p15.33 and 10q26.3) by array comparative genomic hybridization are located in subtelomeric chromosomal regions. This finding further implicates a chromosomal bridge-fusion-breakage mechanism in relation to telomere shortening in ulcerative colitis neoplastic progression (1
) and in other cancers (33
). Subtelomeric regions may be inherently more prone to breakage after telomere attrition-mediated end-to-end fusions with breakage at anaphase.
It is also noteworthy that the highest variance non-random chromosomal alterations were all genomic losses (). This implicates loss of tumor suppressor genes as an important mechanism of the molecular pathogenesis of ulcerative colitis neoplasia.
These array comparative genomic hybridization data provide evidence that genomic changes associated with neoplastic progression in ulcerative colitis are sufficiently advanced to be clonal, which is intriguing since they are present in non-dysplastic single random biopsies from ulcerative colitis progressors. That they are clonal derives from the knowledge that the detection threshold for comparative genomic hybridization gains or losses requires the majority of cells to demonstrate a given alteration (34
Three progressors failed to simultaneously demonstrate all three statistical measures of array comparative genomic hybridization alteration. Specifically, one of the tested ulcerative colitis progressors (P3) in our small series of nine patients did not exhibit the 18q alterations (), and an additional two ulcerative colitis progressors (P1 and P2) failed to demonstrate the random global alterations assessed by extreme 5th percentile deviation and standard deviation analyses (). This could be related to the still limited resolution of ~ 1 MB afforded by even the higher density 4,298 bacterial artificial chromosome arrays utilized in this study. The improved sensitivity gained by the higher density array supports this hypothesis. Improved resolution afforded by SNP, oligo and tiling or submegabase-resolution tiling (SMRT) arrays may improve sensitivity even further.
These three patients’ results may also relate to sampling error within the colon, namely that more than one biopsy may be necessary to identify progressors and nonprogressors. Future mapping studies of the distribution of CGH alterations throughout the colon of both progressors and nonprogressors will be important.
A final potential explanation for why three ulcerative colitis progressors were not detected by all three statistical measures in this study could be that these patients differed clinically. This was true for the two that lacked global genomic instability, namely P1 and P2 (). Progressor P2 had incident high-grade dysplasia that was detected during a prolonged 20 years of surveillance endoscopy, in comparison to the much more advanced prevalent adenocarcinomas in the majority of the remaining progressors. Further, progressor P1 was the only ulcerative colitis progressor without ulcerative pancolitis. Instead, progressor P1 had disease limited to the rectum rather than the pancolonic ulcerative colitis in the remaining progressors. Numerous studies document that ulcerative proctitis appears to be a different disease with limited to no increased cancer risk. Surveillance is, in fact, not recommended for the ulcerative proctitis variant of ulcerative colitis (35
). The biopsy analyzed by comparative genomic hybridization for patient P1 derived from the unaffected sigmoid colon without changes of ulcerative colitis. It is possible that patient P1, who was also 74 years of age, actually developed a sporadic-type rectal cancer rather than an ulcerative colitis cancer. This remains speculation but this patient was certainly clinically distinct from the remaining ulcerative colitis progressors. Thus, it is possible that these clinical differences may explain at least part of the variant array comparative genomic hybridization results for these three ulcerative colitis progressors that were not detected by all three statistical measures.
The fact that far removed single biopsies lacking dysplasia in our small series of 17 ulcerative colitis cases were able to distinguish progressors and nonprogressors is remarkable. From a clinical perspective, this adds further credence to the concept that full colonoscopy with extensive biopsy sampling, currently the standard of care, could possibly be eliminated in the future. This follows from the finding that even single biopsies may harbor the genomic signature identifying ulcerative colitis progressors that could focus surveillance efforts onto the subset most likely to benefit. Natural history data on the development of genomic alterations during ulcerative colitis progression remain to be determined, as the current study only evaluated biopsies only at the time of synchronous high-grade dysplasia or carcinoma. Longitudinal and prospective validation studies are underway.
In summary, array comparative genomic hybridization is a powerful discovery technique for genomic markers of ulcerative colitis cancer risk to better identify the minority subset of patients in need of intensive colonoscopic surveillance. By sequentially utilizing low- and high-resolution arrays, we demonstrate that the power of this approach increases with the increasing array target density. Comparing the patterns of genomic alteration in ulcerative colitis progressors with nonprogressors, random and specific alterations identify progressors with 100% sensitivity and specificity in our small series of 17 ulcerative colitis patients. These results were achieved using only single non-dysplastic biopsies far distant from the patients’ neoplasms. Of the changes identified, loss at the subtelomeric region of chromosome 18q appears to be the most powerful marker, along with several other subtelomeric losses, further implicating the telomere shortening mechanism with bridge-breakage-fusion cycles previously identified in ulcerative colitis neoplastic progression. These array data represent an important additional step toward understanding of molecular events in neoplastic development in ulcerative colitis, and further demonstrate the great promise of molecular markers on even single biopsy samples to advance ulcerative colitis cancer surveillance.