MicroRNA Expression and Outcome in Resected NSCLC—Response
Authors: Johannes Voortman, Aaron J. Schetter, Curtis C. Harris, Giuseppe Giaccone
Journal: Cancer Research (2011)

We are grateful to Kageyama and colleagues for their interest in our article and for their comments concerning several methodologic aspects of our study.It is argued that accuracy of obtained results could be increased by the use of multiple tissue sections per patient as well as microdissection of tumor tissue for RNA isolation.Unfortunately, for the purpose of this study, only 1 tissue section per patient was allocated by the International Adjuvant Lung Cancer Trial (IALT) Bio committee. Tumor tissue was macrodissected after pathologic review, ensuring high tumor content. We think that further microdissection would not greatly improve results. In case the authors refer to laser capture microdissection, this would be an impractical biomarker to implement in a clinical setting on hundreds of samples.The second concern regards the use of U66 as normalization control for microRNA expression. The authors acknowledge that no standard method for normalization has been established but state accurate normalization is essential for data interpretation.In our laboratory, U66 has been shown to be a reasonable control for these types of experiments (1). In the absence of a universal, standardized method for normalization, we selected U66 prior to the start of experimental procedures. We think that altering the normalization method on the basis of the fact that we did not see strong associations with the clinical outcomes we were expecting would likely introduce artifacts.Finally, it is indicated that the study was challenging because the samples were collected from 28 centers in 14 countries, as there may be differences in tissue quality and sample preparation that are critically important. The authors have indicated that microdissection (removal of “normal” tissue) and “more accurate” normalization would be better but fail to indicate any details concerning alternative methods.We acknowledge that conducting a large-scale study is a challenge and it is almost inevitable to obtain specimens derived from multiple centers. In the case of the IALT-Bio initiative, numerous studies have been previously published on the same unique patient cohort (2–4). The potential heterogeneity introduced by the presence of different centers and conditions are part of the experimental design and represent the reality. In theory, the data could be reanalyzed adjusting for each center to determine any predictive or prognostic association with the data, however this was not done. As for heterogeneity caused by tumor content of the slide, this issue would be sufficiently eliminated by the fact that we only used the tumor portion of the formalin-fixed, paraffin-embedded (FFPE) slide for the main analysis.In regard to the “clear standard” that should be established for normalizing, we tend to agree with the ideal of this control, but it may be impractical as well. One control might work well in one tissue type but not in other tissue types. Furthermore, one control may be superior when using high-quality RNA from frozen tissue but not reliable when using FFPE tissue in which RNA is considerably more degraded. We would like to urge the community to come to some conclusion concerning optimal controls for all occasions, driven by research rather than opinion or how it fits with a certain data set. Unfortunately, we are far from that consensus and must continue to carry out research using the “best” methods we can.No potential conflicts of interest were disclosed.See the original Letter to the Editor, p. 5357