Molecular markers and new techniques in the evaluation of colorectal cancer

Colorectal cancer (CRC) is ranked in the Western world as the third leading cause of death from malignant tumors. Despite improvements in early detection of the disease, new surgical techniques and adjuvant and neoadjuvant chemoradiation, more than 50% of these patients die within 5 years. Patients suffering from colorectal polyps or ulcerative colitis run a considerable risk of developing CRC and may be used as models to study early cancer development. Furthermore, the origin of poorly differentiated pelvic adenocarcinomas often presents diagnostic difficulties and markers used for discrimination have to be interpreted with caution. Therefore, it is important to identify and evaluate new molecular markers and techniques used in diagnostics and for prognostic decision-making.

The overall aim of this study was to elucidate and investigate how specific factors in colorectal premalignant and malignant tissue act as early markers for risk assessment, diagnosis, and prognosis. Other goals included the characterization of the protein expression profile of colorectal tumors in order to improve diagnostic decisionmaking as well as the development of a new method for evaluating the three-dimensional structure of tumor vessels.

In ulcerative colitis, two surveillance groups with long-standing colitis and with similar risk factors were investigated. One group comprised patients with ulcerative colitis-associated colorectal carcinomas (UCC) who were compared to a control group. There was no difference in inflammatory activity and dysplasia between the two groups. One important finding was that aneuploid cell populations were scattered all over the colon and rectum early on during the observation period and almost exclusively in the group of patients finally developing UCC. Laminin-5 gamma2 chain (ln- 5)-positive cell populations were more frequently observed in the UCC group than in the controls. The ln-5 positive cells were distributed over the whole colon and rectum and did not correlate with dysplasia or inflammatory activity, but strongly with aneuploidy. According to the proliferation marker cyclin A, there was an increased expression in the UCC group during the surveillance period. Furthermore, the majority of the ln-5 and cyclin A-positive cells were aneuploid over a constructed cutoff point for high-risk level. Although this investigation is too small to yield reliable results, our data indicate that DNA assessment, In5 and cyclin A expression may help to identify patients at increased risk for cancer development in UC surveillance programs.

Ln-5 expression was evaluated as a marker for early invasion and was found to increase progressively from hyperplastic polyps (0%) to tubular adenomas (12.5%) and, finally, adenomas with a villous component (25%). Furthermore, ln-5 was upregulated during progression toward a more malignant stage of colon cancer. In total, 15% of the investigated polyps stained positive for ln-5 as compared to 96% of the colon adenocarcinomas. Ln-5 expression was also correlated with increasing size in both polyps and carcinomas. Univariate analysis identified ln-5, tumor differentiation, and Dukes grade as significant variables in predicting prognosis. Our interpretation is that ln-5 expression can be used as a sensitive marker to trace early malignant transition and may also serve as an indicator of aggressiveness in colon carcinomas.

We employed protein profiling to improve diagnostic decision-making in tumor samples where pathologic investigations did not give reliable results. A poorly differentiated adenocarcinoma of unclear origin in the pelvis served as a model for the evaluation of the method. Firstly, we characterized the protein expression profile in eight colon and seven ovarian carcinomas by using two-dimensional gel electrophoresis (2-DE). This was followed by a multivariate data analysis to match the protein profiles. Eighty-nine percent of the unclear tumor sample spots were matched with the colon standard gel, whereas only 63% of the spots could be matched with the ovarian standard. The degree of similarity was also assessed by correlation coefficient analysis (r-value calculation) in which the tested tumor sample showed an average r value of 0.70 (0.58-0.73) matched with the colon cancer, compared to 0.36 (0.3-0.41) matched with the ovarian cancer. Principal component analysis displayed the clustering of the unclear case within the colon cancer samples, whereas this case did not cluster at all within the group of ovarian adenocarcinomas. We conclude that these results show that 2-DE protein expression profiling combined with principal component analysis appears to be a sensitive method for diagnosing undifferentiated adenocarcinomas of unclear origin.

Current standard methods do not provide information about the three-dimensional structure of the vessel biopsy specimens and further, hampered by the experience of the investigator's ability to identify and quantify angiogenic "hot spots". We developed a new fast and easy method to visualize vessels in human biopsy specimens. By using a computerized deconvolution technique, it was possible to facilitate the identification of three-dimensional vascular structures in normal and tumor specimens. Furthermore, this technique does not destroy the tissue surrounding the vessels (i.e. the tumor) and therefore it could be used in experimental studies to analyze for instance, tumor vessel morphology in response to angiogenic therapy.

List of scientific papers

I. Habermann J, Lenander C, Roblick UJ, Kruger S, Ludwig D, Alaiya A, Freitag S, Dumbgen L, Bruch HP, Stange E, Salo S, Tryggvason K, Auer G, Schimmelpenning H (2001). Ulcerative colitis and colorectal carcinoma: DNA-profile, laminin-5 gamma2 chain and cyclin A expression as early markers for risk assessment. Scand J Gastroenterol. 36(7): 751-8.
https://pubmed.ncbi.nlm.nih.gov/11444475

II. Lenander C, Roblick U, Ost A, Tryggvason K, Auer G (2002). Laminin-5 gamma2 chain expression: a marker of early invasiveness in colorectal adenomas? [Manuscript]

III. Lenander C, Habermann JK, Ost A, Nilsson B, Schimmelpenning H, Tryggvason K, Auer G (2001). Laminin-5 gamma 2 chain expression correlates with unfavorable prognosis in colon carcinomas. Anal Cell Pathol. 22(4): 201-9.
https://pubmed.ncbi.nlm.nih.gov/11564896

IV. Roblick U, Lenander C, Becker S, Habermann J, Kronenwett U, Broll R, Bruch H-P, Franzen B, Alaiya A, Auer G (2002). Undifferentiated pelvic adenocarcinomas: Diagnostic potential of protein profiling and principal component analysis. [Manuscript]

V. Lenander C, Holmgren L (1999). A novel method of vizualizing vessels in human tumor biopsies. Angiogenesis. 3: 291-3.