Sporadic colorectal carcinoma microdissection: a novel laboratory technique to identify tumor suppressor genes

Abstract

Purpose: Multiple tumor suppressor genes have been found by loss of heterozygozity (LOH) analysis. Surgical samples are frequently contaminated with normal tissue elements, making molecular analysis with polymerase chain reaction–based techniques problematic to carry out and analyze accurately. Therefore, the aim of this study was to use a laser dissecting microscope to remove contaminating tissues, re-evaluate pathologic staging, and identify tumor suppressor genes after microdissection. This technique is sensitive but expensive, costing $50,000 to $75,000 for the microscope alone.Methods: At Roswell Park Cancer Institute, all colorectal tumors (CRC) are stored in a tumor data bank. The pathologist identified tumor tissue that was frozen in liquid nitrogen and archived it for research analyses. The remainder of the tissue was used for pathologic staging. Sixty-seven CRC were microdissected with a 15-blade scalpel, a 22-gauge needle, and an inverted dissecting microscope. Each tumor was weighed, placed in a cryogel, and cut into 30-μm shavings using a Cryostat machine. The tumors were kept at −24 C. Hematoxylin and eosin (H&E)-stained sections were taken at the first, center, and last shavings and labeled A, B, and C, respectively. The shavings between A and B were collected into a Petri dish and those between B and C into a second dish. A pathologist analyzed the H&E slides. Invasive carcinoma was classified as H&E with greater than 95% invasive carcinoma.Results: Invasive carcinoma was found in 34 samples (51%) and villous adenoma in 17 (25%); 8 samples had less than 50% carcinoma and 8 contained no carcinoma.Conclusions: These results indicate that tumors should be microdissected and serially stained with H&E to give a representation of the carcinoma present through the tumor prior to any biologic analysis. This technique is inexpensive, sensitive, and specific. Furthermore, it allows picking the best shavings from AB or BC prior to any LOH investigation, which is crucial when looking for tumor suppressor genes. (Funded by National Institutes of Health Grant T32 CA09581.)