COMPARISON OF GENETIC CHANGES IN FROZEN BIOPSIES AND MICRODISSECTED ARCHIVAL MATERIAL FROM THE SAME COLORECTAL LIVER METASTASES

Microdissection of tissue sections from formalin-fixed, paraffin-embed ded tumor material allows separation of microscopic sites within a sam ple. DNA can easily be extracted, and polymerase chain reaction (PCR) technology makes it possible to perform different molecular biologic a nalyses on small cell populations. The presence of normal cells or tum or heterogeneity may cause false negatives in allelic imbalance (AI) s tudies. Microdissected well-defined cell populations from a tumor sect ion are assumed to increase the sensitivity of AI analyses. The presen t study has evaluated this in colorectal liver metastases by comparing genotypes in frozen biopsies with genotypes in microdissected archiva l samples from the same patients. Constituional genotypes were obtaine d from corresponding peripheral blood leukocytes as well as normal liv er tissue. Archival samples (n = 43) from 16 patients were analyzed af ter microdissection with 2-5 of 10 selected microsatellite markers. Fr ozen biopsies from one metastasis of each patient had previously been investigated at numerous microsatellite loci. From those results we se lected, for the comparable analysis of archival samples, 41 tumors gen otypes at 10 loci representing 11 heterozygotes, 13 AI, 7 losses of he terozygosity (LOHs), 8 homozygotes, and 2 microsatellite unstable case s. The microdissected samples revealed AI or a complete loss of one al lele (LOH) in 5 of 11 (45%) genotypes that were previously evaluated a s unchanged (retained heterozygosity) in the frozen biopsies, and LOH in 8 of 13 (62%) genotypes at loci known to exhibit AI in the frozen b iopsies. Microsatellite instability, LOH, and homozygosity found in th e frozen samples were all confirmed by analyses of the archival materi al. Intertumoral genetic heterogeneity was found in samples from two p atients. The same allelic intensities were seen in DNA from tumor-clos e liver tissue as in blood DNA from the same patient except in one sam ple. The present study shows a 54% increase in sensitivity of genetic alterations if pure tumor cell components are used (five ''new'' AIs a nd LOHs and eight ''new'' LOHs among previously scored heterozygotes [ n = 11] and AI [n = 13], respectively). In total, a 93% success rate ( 108/115 analyses) was obtained using standard PCR conditions for the 1 0 selected markers. The fact that standard PCR conditions and 5-mu m t umor sections are used shows how easy these analyses are to perform, a nd that only minor amounts of Valuable archival material is used.