Date Published: November 4, 2009
Publisher: Public Library of Science
Author(s): Vassiliki Kotoula, Elpida Charalambous, Bart Biesmans, Andigoni Malousi, Eleni Vrettou, George Fountzilas, George Karkavelas, Joseph Alan Bauer. http://doi.org/10.1371/journal.pone.0007746
Abstract: Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.
Partial Text: Based on accumulated knowledge about tumor biology, newer drugs are meant to treat cancer in a more rational way than classic chemotherapy, i.e., by targeting specific molecules and pathways that are essential for promoting tumor growth, maintenance and metastasis. In this context, EGFR, a HER family receptor tyrosine kinase, has emerged as a major molecular target. Because EGFR was considered to be involved in the pathogenesis of most epithelial cancers , anti-EGFR drugs were anticipated to improve outcome for millions of patients worldwide. In fact, though, these drugs dramatically benefit only a small percentage of cancer patients, based on the alterations concerning EGFR itself (e.g., specific mutations targeted by small molecule tyrosine kinase inhibitors [TKIs]) or molecules in the EGFR effector pathways (for example, KRAS [official gene name: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; aliases: KRAS2, RASK2] mutations hampering therapeutic EGFR antibodies and possibly TKIs as well). Of note, success rates of as low as 5% correspond to hundreds thousands of patients worldwide for the major cancer types (breast, lung, colorectal). Hence, if aiming in a rational and beneficial use of molecule targeting drugs it is necessary to identify patients who will truly benefit from such treatments, thus limiting unnecessary toxicities, treatment delays ,  and health care costs .
This study shows that the assessment of DNA fragmentation provides important information on the amplification capacity of the extracted FFPE-DNA and on the reliability of the obtained results, in line with previous reports on methods involving relatively long PCR products , ,  but also short ones, as is the case with Q-PCR assays . Based on the degree of DNA fragmentation, FFPE samples can be distinguished into those with relatively well preserved DNA (favorable samples, roughly ¾ of our diagnostic cases) and those with very fragmented DNA (unfavorable samples, ¼ of our diagnostic cases). FFPE-DNA quality depends on numerous, oft imponderable parameters that can not be assessed in the diagnostic setting. For example, tissue block age, a multi-parameter involving at least storage conditions and continuous degradation of nucleic acids after embedding , , was vaguely related to the degree of DNA fragmentation in our series. If information on storage conditions can not be retrieved, the parameter can not be evaluated. In the same line, type of fixative, time-to-fixation, time-in-fixative, and tissue type are only few of the fixation-related parameters usually addressed for their influence on DNA integrity in FFPE tissues, whereby DNA is better preserved in buffered formalin as compared to simple formalin , , . Here we can only report on a limited number of biopsy samples (n = 7) that had been fixed overnight in buffered formalin and yielded favorable DNA, as expected. However, since the majority of the favorable DNA samples in this study derived from large colectomy specimens that had been fixed in simple formalin under unknown conditions, fixation in simple formalin can not be blamed as the major determinant of DNA fragmentation.