Abstract
To explore intratumor heterogeneity in gene expression profiles from patients with cervical cancer. A total of 33 biopsies were obtained from 11 patients, sampling between two and five different areas for each tumor. The extracted RNA was hybridized onto the Affymetrix U133 Plus 2.0 oligonucleotide chip. The variance of expression within a patient (W), between patients (B) and the total variance (T = W + B) were calculated for each ProbeSet, and the ratio W/T was used as a measure of intratumor heterogeneity. Gene Ontology functional analysis was done to assess the function of genes that had high W/T (top 10%) and low W/T (bottom 10%) values. In total, 448 ProbeSets (2.2% of the total) had W/T < 0.10, indicating low intratumor heterogeneity, and 537 ProbeSets (2.7% of the total) had W/T > 0.90, indicating high intratumor heterogeneity. In total 14,473 ProbeSets (72.4%) had higher intertumor than intratumor heterogeneity (W/T < 0.5). Genes with low intratumor heterogeneity were characterized by a statistically significant enrichment of immune-related functions (P < 0.0001). Genes with high intratumor heterogeneity were characterized by a significant tendency towards nuclear localization and nucleic acid binding (both P < 0.0001). For genes with W/T > 0.5, more than six biopsies would be required to minimize the intratumoral heterogeneity to <0.15; if W/T is 0.3 to 0.4, four biopsies are required; and for low W/T of 0.16 to 0.3, only two to three biopsies would be needed. Although the intratumor heterogeneity was low for the majority of the tested ProbeSets, for many genes, multiple biopsies are required to obtain a reliable estimate of gene expression.
Highlights
Genes with high intratumor heterogeneity were characterized by a significant tendency towards nuclear localization and nucleic acid binding
Conclusion: the intratumor heterogeneity was low for the majority of the tested
Sixty-six flash-frozen punch biopsies were obtained from 16 patients with cervical cancer who were undergoing examination under general anesthesia as part of their pretreatment evaluation for cervical cancer
Summary
When these samples were re-examined in greater detail, we went back to the original QRT-PCR data, samples labeled ‘‘12.5’’ and ‘‘4.1’’ gave unreliable results, indicating that RNA may have been damaged after repeated thawing and freezing After excluding those two samples, the correlation coefficients were 0.828 for DAF, 0.834 for IRAK1, 0.770 for VEGF, 0.717 for DUSP1, 0.822 for IL8, and 0.669 for. Except one, clustered perfectly with the corresponding patient, indicating greater similarity in gene expression pattern within patients, than between different patients (A).This is even more clearly shown for ProbeSets whose expression heterogeneity is limited to W/T values of. The model predicts that as the W/T value for a gene increases (greater intratumoral heterogeneity), a larger number of biopsies would be necessary to obtain a representative ‘‘whole tumor’’ expression for that specific gene. For highly heterogeneously expressed genes with W/T values >0.5, six or more biopsies would be required
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