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. 2004 Nov-Dec;6(6):761-7.
doi: 10.1593/neo.04301.

Stability and heterogeneity of expression profiles in lung cancer specimens harvested following surgical resection

Fiona H Blackhall  1 , Melania Pintilie, Dennis A Wigle, Igor Jurisica, Ni Liu, Nikolina Radulovich, Michael R Johnston, Shaf Keshavjee, Ming-Sound Tsao
Affiliations

Stability and heterogeneity of expression profiles in lung cancer specimens harvested following surgical resection

Fiona H Blackhall et al. Neoplasia. 2004 Nov-Dec.

Abstract

One of the major concerns in microarray profiling studies of clinical samples is the effect of tissue sampling and RNA extraction on data. We analyzed gene expression in lung cancer specimens that were serially harvested from tumor mass and snap-frozen at several intervals up to 120 minutes after surgical resection. Global gene expression was profiled on cDNA microarrays, and selected stress and hypoxia-activated genes were evaluated using real-time reverse transcription polymerase chain reaction (RT-PCR). Remarkably, similar gene expression profiles were obtained for the majority of samples regardless of the time that had elapsed between resection and freezing. Real-time RT-PCR studies showed significant heterogeneity in the expression levels of stress and hypoxia-activated genes in samples obtained from different areas of a tumor specimen at one time point after resection. The variations between multiple samplings were significantly greater than those of elapsed time between sampling/freezing. Overall samples snap-frozen within 30 to 60 minutes of surgical resection are acceptable for gene expression studies, thus making sampling and snap-freezing of tumor samples in a routine surgical pathology laboratory setting feasible. However, sampling and pooling from multiple sites of each tumor may be necessary for expression profiling studies to overcome the molecular heterogeneity present in tumor specimens.

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Figures

Figure 1
Figure 1
Unsupervised clustering analysis by the BTSVQ algorithm. Component planes of SOMs illustrate the gene expression, based on data from two arrays, at each time point. Finding a visually similar pattern across multiple samples indicates that the same set of genes is similarly expressed.
Figure 2
Figure 2
The effect of time from surgical resection on the expression of selected stress/hypoxia genes measured by real-time RT-PCR.
Figure 3
Figure 3
Expression of stress/hypoxia genes by real-time RT-PCR. For each gene, the measurements for two time course studies at each time point (left) are compared to the gene measurements for four tumors sampled from six different sites at the same time point from resection (right). The Y-axis values represent the ΔCT (CT[gene]-CT[18S]). The numbers on the left side are for the two longitudinal time course specimens, whereas the numbers on the right are for the four multiple sampling specimens. The differences in the ΔCT values of the two groups of specimens were due to different dilutions of cDNA used to perform 18S realtime RT-PCR measurements. X, central samples; O, peripheral samples.
Figure 4
Figure 4
The effect of increasing the number of samples per tumor on the width of the 95% confidence interval for expression of selected stress/hypoxia genes.
See this image and copyright information in PMC

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