Replication Study: Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma

Abstract

As part of the <ext-link ext-link-type="uri" xlink:href="https://osf.io/e81xl/wiki/home/">Reproducibility Project: Cancer Biology</ext-link>, we published a Registered Report (Repass et al., 2016), that described how we intended to replicate an experiment from the paper 'Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma' (Castellarin et al., 2012). Here we report the results. When measuring Fusobacterium nucleatum DNA by qPCR in colorectal carcinoma (CRC), adjacent normal tissue, and separate matched control tissue, we did not detect a signal for F. nucleatum in most samples: 25% of CRCs, 15% of adjacent normal, and 0% of matched control tissue were positive based on quantitative PCR (qPCR) and confirmed by sequencing of the qPCR products. When only samples with detectable F. nucleatum in CRC and adjacent normal tissue were compared, the difference was not statistically significant, while the original study reported a statistically significant increase in F. nucleatum expression in CRC compared to adjacent normal tissue (Figure 2; Castellarin et al., 2012). Finally, we report a meta-analysis of the result, which suggests F. nucleatum expression is increased in CRC, but is confounded by the inability to detect F. nucleatum in most samples. The difference in F. nucleatum expression between CRC and adjacent normal tissues was thus smaller than the original study, and not detected in most samples.

Keywords: Fusobacterium nucleatum; Reproducibility Project: Cancer Biology; cancer biology; colorectal carcinoma; human; metascience; replication; reproducibility.

Figure 1.. Relative abundance of F. nucleatum in colorectal carcinoma versus adjacent normal biopsies in samples with detectable F. nucleatum.

Only samples in which F. nucleatum was detected in both colorectal carcinoma (CRC) and adjacent normal tissue by quantitative PCR (qPCR) and confirmed by sequencing of the qPCR product are shown (n = 6). The mean relative abundance of F. nucleatum (normalized to PGT expression) in CRC tissue versus adjacent normal tissue of both independent runs is reported for each patient sample and error bars represent SEM. The y-axis represents mean fold gene expression change (2^-ΔΔCt) while the x-axis represents patient samples. Exploratory analysis: two-tailed paired t test: t(5) = 1.067, p = 0.335, r = 0.43, 95% CI [−0.59, 0.92]. Additional details for this experiment can be found at https://osf.io/rb4yq/.

Figure 1—figure supplement 1.. First independent qPCR run.

First of two independent qPCR runs performed in technical duplicate. (A) Distribution of raw Ct values for PGT in colorectal carcinoma (CRC) tumor, adjacent normal tissue, or matched normal samples. (B) Distribution of raw Ct values for F. nucleatum in each sample type (CRC tumor, adjacent normal tissue, or matched normal). The y-axis represents a binned count of each Ct score and the x-axis represents the value of Ct scores. Bin size = 0.1. Additional details can be found at https://osf.io/rb4yq/.

Figure 1—figure supplement 2.. Second independent qPCR run.

Second of two independent qPCR runs performed in technical duplicate. (A) Distribution of raw Ct values for PGT in colorectal carcinoma (CRC) tumor, adjacent normal tissue, or matched normal samples. (B) Distribution of raw Ct values for F. nucleatum in each sample type (CRC tumor, adjacent normal tissue, or matched normal). The y-axis represents a binned count of each Ct score and the x-axis represents the value of Ct scores. Bin size = 0.1. Additional details can be found at https://osf.io/rb4yq/.

Figure 2.. Relative abundance of F. nucleatum by qPCR in colorectal carcinoma versus adjacent normal biopsies.

Fold change values are shown for all paired specimens based on differences in Ct values, irrespective of whether the qPCR products were confirmed to be specific or non-specific upon sequencing. Tissue was collected from colorectal carcinoma (CRC) and adjacent normal tissue (n = 40). qPCR was performed independently two times and averaged. The mean relative abundance of F. nucleatum (normalized to PGT expression) in CRC tissue versus adjacent normal tissue of both independent runs is reported for each patient sample and error bars represent SEM. The y-axis represents mean fold gene expression change (2^-ΔΔCt) while the x-axis represents patient samples. Two-tailed Wilcoxon signed-rank test: Z = 2.14, p = 0.032. Additional details for this experiment can be found at https://osf.io/rb4yq/.

Figure 2—figure supplement 1.. Independent qPCR runs.

Fold change values are shown for all paired specimens based on differences in Ct values, irrespective of whether the qPCR products were confirmed to be specific or non-specific upon sequencing. (A) First of two independent qPCR runs performed in technical duplicate. Mean relative abundance of F. nucleatum (normalized to PGT expression) in colorectal carcinoma (CRC) tissue versus adjacent normal tissue is reported for each patient sample. The y-axis represents mean fold gene expression change (2^-ΔΔCt) and the x-axis represents patient samples. The median fold change across all samples was 1.11 (IQR = 0.89–2.18) and the mean fold change was 3.62 [SD = 7.21]. (B) Second of two independent qPCR runs performed in technical duplicate. Mean relative abundance of F. nucleatum (normalized to PGT expression) in CRC tissue versus adjacent normal tissue is reported for each patient sample. The y-axis represents mean fold gene expression change (2^-ΔΔCt) and the x-axis represents patient samples. The median fold change across all samples was 1.13 (IQR = 0.90–2.54) and the mean fold change was 8.27 [SD = 32.2]. Additional details can be found at https://osf.io/rb4yq/.

Figure 3.. Meta-analyses of each effect.

Effect size (r) and 95% confidence interval are presented for Castellarin et al. (2012), this replication attempt (RP:CB), and a meta-analysis to combine the two effects. To directly compare and combine the results of both studies, the qPCR results for RP:CB were used irrespective of whether the amplicon was confirmed to be F. nucleatum by sequencing. The effect size r is a standardized measure of the correlation (strength and direction) of the association between tissue type and normalized F. nucleatum DNA levels, with a larger positive value indicating CRC tissue is correlated with a higher F. nucleatum expression level. Sample sizes used in Castellarin et al. (2012) and this replication attempt are reported under the study name. Random effects meta-analysis of the abundance of F. nucleatum in CRC tissue compared to adjacent normal tissue (meta-analysis p = 5.86 × 10⁻⁴). Additional details for this meta-analysis can be found at https://osf.io/kup8d/.