The sequential progression of colon tumorigenesis provides an excellent system to assess promising biomarkers for colon cancer screening and early detection. This adenoma-carcinoma sequence leading to CC is well described, and is characterized by multiple genetic and epigenetic events . However, to the best of our knowledge, there is not a single molecule uniformly up-regulated in all phases of CC development.
Colon Cancer Associated Transcript 1 (CCAT1) is a unique transcript up-regulated in CC compared to normal human tissues . Its role in tumorigenesis has yet to be defined. Previous observations demonstrated up-regulation of CCAT1 in over 90% of colonic adenocarinomas, with relatively little or no transcript expression in a panel of normal human tissues . We identified slightly higher expression of CCAT1 in colonic tissues obtained from patients with benign colonic disorders compared to normal, non-inflamed colonic tissue. This slight up-regulation was more prominent in the tissue samples of patients with inflammatory conditions and reached a 15-fold increase in patients with severe colonic inflammation. If this observation can be replicated in chronically inflamed colonic tissues such as those obtained from inflammatory bowel disease patients, this may suggest a role for CCAT1 in neoplastic transformation often observed in chronically inflamed tissues.
In this study we have shown again that CCAT1 is up-regulated in most (20/22) primary tumor tissues. Interestingly, CCAT1 was also up-regulated, albeit to a lesser degree, in many of the histologically normal appearing mucosa samples adjacent to the primary tumor site. This may be caused by a contamination of nearby cancer cells shed during surgery or during tissue handling. In to order address this potential issue of cellular contamination, we used formalin fixed paraffin-embedded tissue of one of the study subjects showing CCAT1 up-regulation in the primary tumor tissue and to a lesser degree in the adjacent normal tissue (T877) by qRT-PCR, and analyzed transcript expression by in-situ hybridization. Good correlation was observed between the qRT-PCR findings and the in-situ hybridization findings indicating that this is a real biological phenomenon rather than a false positive finding related to local tumor cell contamination. Currently we are in the process of performing in-situ hybridization on a large cohort of colon cancer patients to further study this phenomenon.
The up-regulation of a tumor marker in histologically normal-appearing tissue is a complex matter, a “double-edged sword.” On the one hand, this finding may provide a powerful tool to predict future risk of colon cancer by studying biomarker expression in random colonic mucosal biopsies in screening or high-risk populations. On the other hand, biomarker expression in clinically disease-free patients may be interpreted as a false positive finding, which amounts to reduced diagnostic accuracy of the test. If co-expression of CCAT1 and DNA-methylation abnormalities shown to appear in early phases of the adenoma-carcinoma sequence  can be found, this co-expression may be more predictive of adenoma-carcinoma sequence progression, and will serve as the basis for development of risk reduction or early curative intervention strategies.
A clinically important stage in the adenoma-carcinoma process is the formation of an adenomatous polyp. We analyzed tissue from adenomatous polyps for CCAT1 expression. We restricted our analysis to polyps larger than 10 mm in diameter, in order not to compromise histopathological diagnosis. The in-situ technique for CCAT1, described earlier , may provide a tool for future analysis of CCAT1 expression in smaller polyps, and for differential diagnosis of hyperplastic and adenomatous polyps, namely those with uniformly benign and those with possibly malignant natural history. We observed CCAT1 to be up-regulated in all but one of the 18 adenomatous polyps studied. In 61% of these adenomatous polyps the transcript expression exceeded 100-fold relative to that of normal colon tissue. This observation provides supportive evidence of a role of CCAT1 in the early neoplasia (adenoma formation) stage of colon carcinoma pathogenesis. The finding of mean CCAT1 expression in adenoma significantly exceeding that of carcinoma further supports this hypothesis, as it points to a down-regulating effect on CCAT1 expression once malignant transformation is attained.
CCAT1 up-regulation of 5-fold or higher compared to normal colon. Transcript up-regulation was seen in 90.1% of malignant primary tumor samples obtained from patients with Stage I-III colon adenocarcinoma. The fact that this non- coding RNA is located on chromosome 8q24.21, a “hot spot” for many cancer-related single nucleotide polymorphisms (SNPs), supports a role for CCAT1 in the tumorigenesis of colon carcinoma.
Current histopathological nodal staging techniques may overlook occult lymph node metastases amounting to pathological under-staging and under-treatment. Many investigators have tried to improve upon lymph node staging in patients with colon cancer [31–34]. We analyzed lymph nodes from patients with colon cancer having obvious macro-metastasis by standard histopathological staging for CCAT1 expression and compared this expression to that of negative lymph nodes by histopathology obtained from the same patients and to that of benign lymph nodes for patients without colon cancer. CCAT1 was highly up-regulated (over 100 fold) in all 10 metastatic lymph nodes studied. Such exceedingly high expression of CCAT1 may suggest an important role of this unique non-coding RNA in regional lymphatic and nodal dissemination of colon adenocarcinoma. Furthermore, this finding may be applied clinically for the detection of occult metastatic disease in seemingly disease-free regional lymph nodes of patients undergoing surgical resection of colon cancer with curative intent. This would improve staging accuracy and individualized treatment planning, specifically adjuvant systemic therapy in patients with nodal disease.
Two of the most common sites of metastatic spread of colon adenocarcinoma are the liver and peritoneum. Therefore, we included patients operated on for treatment of metastatic disease to these organ sites in our study. Unfortunately, all patients were previously treated by systemic therapy; therefore, treatment-related alterations in CCAT1 expression cannot be excluded in these pre-treated patients. However, the practical reality is that access to tissue of naïve (previously untreated) patients with colon cancer metastatic to the liver or peritoneum is limited, as it is a distinctly rare clinical scenario since most patients are treated, according to our evidence-based guidelines, with systemic therapy before surgery for metastatic disease.
Taking this potential bias into account, we showed that CCAT1 was up-regulated in liver as well as in peritoneal metastasis of colon cancer patients. The variability between the results may be due to the therapeutic efficacy of the previous chemotherapy, or may reflect true biological variability in CCAT1 expression. The only way to study this definitively is to obtain metastatic tissue before and after systemic therapy administration and demonstrate a decrease in CCAT1 expression in systemic treatment responders. Another non-coding RNA up-regulated in liver metastasis as well as in many cancer types is H19 . Interestingly, its expression was also shown to be higher in histologically normal-appearing liver surrounding metastasis . This correlates, in part, with our observation of CCAT1 up-regulation in normal colonic tissues adjacent to the primary tumor site.
Stein et al, recently discovered another transcript with potential clinical relevance, Metastasis-Associated in Colon Cancer-1 MACC . MACC1 has a regulatory role in the HGF/Met signaling pathway which has an important role in cell migration, invasion, and metastatic potential . MACC1 expression in the primary tumor and in plasma of CC patients was shown to be an independent risk factor for metastasis [38, 39]. The prognostic significance of CCAT1 is remains unclear. We are in the process of studying a large cohort of patients with early CC for level of CCAT1 expression, and will correlate expression of this transcript with overall survival.
Serum markers in clinical use for CC (CEA and CA 19-9) are neither sensitive nor specific . Therefore the most common application of CEA and CA-19-9 is to monitor patients for recurrent disease following treatment of CC or to monitor response to systemic therapy . If the measurement of CCAT1 levels in the plasma of CC patients should prove both feasible and reproductive, then it may be added to the current serum markers to monitor disease behavior and patient response to treatment.
Another interesting observation is that CCAT1 expression is higher in patients with peritoneal metastasis originating from colon cancer compared to peritoneal surface malignancy of appendiceal origin. The results did not reach statistical significance in this particular comparison, due to the large variability of transcript expression observed in the colon cancer patients. Nevertheless, we think that further investigation is warranted because appendiceal adenocarcinoma, as do some colon adenocarcinomas, demonstrates preferential spread to the peritoneal surface rather than to solid visceral organs.
The expression of CCAT1 in tissues of all stages of the adenoma-carcinoma sequence of colorectal cancer together with our previous preliminary observations  that CCAT1 can be amplified from the blood and stool samples of patients with CRC point to a promising, novel biomarker for CRC. CCAT1 can be used to enhance pathological staging in borderline cases by in-situ hybridization; it can be used in an RNA-based stool assay for the screening and early detection of CRC, and in blood tests for the diagnosis and follow-up of CRC patients.
In summary, we studied CCAT1 expression in human biospecimens spanning the biological spectrum of benign, pre-malignant and malignant colonic tissues and demonstrated CCAT1 up-regulation, which peaked in tissues from adenomas and colon adenocarcinoma lymph node metastases.