Limitations in molecular detection of lymph node micrometastasis from colorectal cancer

Colorectal cancer patients with lymph node metastasis have a shorter survival and may require adjuvant therapy after surgery of the primary tumor. It is supposed that a more reliable diagnosis can be achieved using tumor-specific DNA mutations for the detection of metastasizing cells. To design a practical approach for a molecular diagnosis of micrometastasis, we applied direct DNA sequencing to screen 48 early stage colorectal carcinomas for the most frequent mutations of the KRAS, P53, and APC tumor genes. KRAS mutations were detected as frequently as described earlier. In contrast, the frequency of P53 and APC hot spot mutations was unexpectedly low, compared with previous studies using other screening methods or including advanced tumor stages. Not more than 31% of early stage tumors showed a mutation in at least 1 of the selected hot spot codons. Applying mutant-enriched polymerase chain reaction (PCR), the mutation of the primary tumor was detected in lymph node DNA from 2 of the KRAS-positive patients. In 1 patient, the result was not verified by subtractive iterative PCR, a principally different molecular method with high sensitivity and specificity. Our data suggest that screening for suitable markers for a molecular detection of occult lymph node metastasis cannot be restricted to small-sized hot spot regions of a few tumor genes and possibly must include tumor-specific epigenetic changes. Furthermore, restriction enzyme-based methods such as mutant-enriched PCR are not suitable to detect any mutation with equal efficiency and they should be carefully controlled to avoid false-positive detection of marker mutations in lymph node DNA.