A high frequency of sequence alterations is due to formalin fixation of archival specimens

Abstract

Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.

Figure 1.

Direct sequencing data of exon 5 of the p53 gene from two independent PCR amplifications, each using 10 cells from, respectively, a frozen (top) and a formalin-fixed (bottom) part of the same tumor. The frozen sample displays the wild-type sequence, whereas the formalin-fixed sample exhibits two artificial mutations in codons 149 and 159.

Figure 2.

Schematic view of a plausible explanation for formalin-mediated artifacts. A: The results when intact target gene sequences (contiguous line), present in the original sample, quantitatively dominate the amplification process (bold line). In vitro repaired fragmented targets (broken line), including those with introduced errors (not represented), will be masked in the early cycles of the amplification process by the more efficiently copied intact target. The majority of the fragmented targets will remain unrepaired and will not be accessible for amplification (vertical bar). B: A case where there are no intact copies of target present in the original sample. The resulting amplicons will then originate from a repair event, leading to amplification of the wild-type sequence (sealed line) or a sequence with an artifact mutation (sealed and dotted line) or a combination thereof.