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Replication Study: Melanoma genome sequencing reveals frequent PREX2 mutations

  1. Stephen K Horrigan
  2. Pascal Courville
  3. Darryl Sampey
  4. Faren Zhou
  5. Steve Cai
  6. Reproducibility Project: Cancer Biology  Is a corresponding author
  1. Noble Life Sciences, United States
  2. BioFactura, United States
  3. TACGen, United States
Replication Study
Cite this article as: eLife 2017;6:e21634 doi: 10.7554/eLife.21634
5 figures

Figures

Figure 1 with 1 supplement
Sequencing of endogenous PREX2 gene in NRASG12D melanocytes.

Representation of sequencing coverage of PREX2 by Sanger sequencing. Reference gene isoforms shown in blue, sequence coverage seen in black, and sequencing run name shown on the y-axis. Vertical red lines represent exon/intron breaks. White arrows indicate strand of the sequence. Image created using UCSC Genome Browser’s custom track feature in multi-region, exon only view, GRch38/hg38 (http://goo.gl/JLezy5). Additional information can be found at https://osf.io/r53z8/.

https://doi.org/10.7554/eLife.21634.002
Figure 1—figure supplement 1
Endogenous PREX2 sequence evaluation.

Summary of sequencing results of PREX2 in NRASG12D melanocytes. Additional information can be found at https://osf.io/r53z8/.

https://doi.org/10.7554/eLife.21634.003
Expression of PREX2 isoforms.

NRASG12D melanocytes were transduced to express GFP or PREX2 isoforms (PREX2WT, PREX2G844D, PREX2Q1430*). (A) Representative Western blot using an anti-V5 antibody (top panel) or an anti-α-Tubulin antibody (bottom panel). Lanes 1–4 are from cells transduced with GFP, PREX2WT, PREX2G844D, or PREX2Q1430*, respectively, generated during the original study (Berger et al., 2012). Lanes 5–8 are from cells transduced with GFP, PREX2WT, PREX2G844D, or PREX2Q1430*, respectively, made during this replication attempt (RP:CB). Membranes were cut at ~70 kDa so that V5 and α-Tubulin could be probed in parallel. (B) Relative protein expression (V5/Tubulin) of ectopically expressed PREX2 isoforms are presented for each stable cell line and normalized to PREX2WT cells made during the original study. Means represented by red bars, circles indicate individual biological replicates [n = 3], and error bars indicate 95% CI. Green circles represent protein expression reported in Figure S6A of the original study. Two-way ANOVA main effect of PREX2 isoform(F(2,12) = 4.608, p=0.033), the main effect of study (F(1,12) = 8.701, p=0.012), and isoform:study interaction (F(2,12) = 7.52, p=0.008). (C) Representative Western blot using an anti-PREX2 antibody (top panel) or an anti-α-Tubulin antibody (bottom panel). Lanes 1–4 are from cells generated during the original study transduced with GFP, PREX2WT, PREX2G844D, or PREX2Q1430*, respectively. Lanes 5–8 are from cells made during this replication attempt transduced with GFP, PREX2WT, PREX2G844D, or PREX2Q1430*, respectively. Membranes were cut at ~70 kDa so that PREX2 and α-Tubulin could be probed in parallel. (D) Relative protein expression (PREX2/Tubulin) of ectopically expressed PREX2 isoforms are presented for each stable cell line and normalized to PREX2WT cells generated during the original study. Means represented by red bars, circles indicate individual biological replicates [n = 3], and error bars indicate 95% CI. Two-way ANOVA main effect of PREX2 expression (F(3,16) = 15.033, p=6.47×10−5), the main effect of study (F(1,16) = 13.08, p=0.002), expression:study interaction (F(3,16) = 4.50, p=0.018). Additional details for this experiment can be found at https://osf.io/4c8tu/.

https://doi.org/10.7554/eLife.21634.004
Figure 3 with 1 supplement
Tumor-free survival and tumor histopathology.

(A) Kaplan-Meier plot of tumor-free survival (TFS). Female athymic nude mice subcutaneously injected in the right flank with NRASG12D melanocytes harboring GFP [n = 14] or PREX2 variants (PREX2WT[n = 7], PREX2G844D[n = 14], or PREX2Q1430*[n = 8]) were monitored every other day for tumor growth. Cox proportional hazards regression model (CPH) with a priori Bonferroni adjusted significance threshold = 0.0125. GFP vs. PREX2G844D, uncorrected p=0.847 (Bonferroni corrected p>0.99); GFP vs. PREX2Q1430*, uncorrected p=0.944 (Bonferroni corrected p>0.99); PREX2WT vs. PREX2G844D, uncorrected p=0.652 (Bonferroni corrected p>0.99); PREX2WT vs. PREX2Q1430*, uncorrected p=0.529 (Bonferroni corrected p>0.99). Median TFS for all groups = 1 week. (B–E) Representative histological section of (B) GFP xenograft, (C) PREX2WT xenograft, (D) PREX2G844D xenograft, and (E) PREX2Q1430* xenograft stained with hematoxylin and eosin. Additional details for this experiment can be found at https://osf.io/anf2s/.

https://doi.org/10.7554/eLife.21634.005
Figure 3—figure supplement 1
Table summarizing incidence rates for both Berger et al. and the current study by week after injection with NRASG12D melanocytes harboring PREX2 isoforms.

Additional details for this experiment can be found at https://osf.io/anf2s/.

https://doi.org/10.7554/eLife.21634.006
Figure 4 with 1 supplement
Tumor growth.

Female athymic nude mice were subcutaneously injected in the right flank with NRASG12D melanocytes expressing GFP or PREX2 isoforms (PREX2WT, PREX2G844D, or PREX2Q1430*). Following tumor detection, caliper measurements were taken every seven days and used to calculate tumor volume. (A) Line graph of first 6 tumor volume measurements of mice before tumors began to reach 1.5 cm3 and mice were euthanized (y-axis is natural log scale). Two mice (one GFP and one PREX2G844D) are excluded because there were euthanized during the reported timeframe. Means reported and error bars represent SD. Number of mice: GFP [n = 13], PREX2WT [n = 7], PREX2G844D [n = 13], and PREX2Q1430* [n = 8]. (B) The first six tumor volume measurements were used to calculate area under the curve (AUC) for each mouse. Box and whisker plot with median represented as the line through the box, individual animal AUC values represented as dots, and whiskers representing values within 1.5 IQR of the first and third quartile (y-axis is natural log scale). One-way ANOVA on AUC (natural log-transformed); F(3,37) = 2.411, p=0.0824, ηP2 = 0.164, 90% CI [0,0.290]. Four contrasts were performed: GFP vs. PREX2G844D: t(37) = 1.217, uncorrected p=0.231, Bonferroni corrected p=0.925, Cohen’s d = 0.477, 95% CI [−0.308, 1.253]; GFP vs. PREX2Q1430*: t(37) = 2.568, uncorrected p=0.014, Bonferroni corrected p=0.058, Cohen’s d = 1.154, 95% CI [0.188, 2.094]; PREX2WT vs. PREX2G844D: t(37) = 0.752, uncorrected p=0.457, Bonferroni corrected p>0.99, Cohen’s d = 0.352, 95% CI [−0.578, 1.274]; PREX2WT vs. PREX2Q1430*: t(37) = 1.988, uncorrected p=0.054, Bonferroni corrected p=0.217, Cohen’s d = 1.029, 95% CI [−0.076, 2.099]. Additional details for this experiment can be found at https://osf.io/anf2s/.

https://doi.org/10.7554/eLife.21634.007
Figure 4—figure supplement 1
Individual tumor xenografts.

This is the same experiment as in Figure 4, but with the tumor volume plotted for each animal rather than averages. Female athymic nude mice subcutaneously injected in the right flank with NRASG12D melanocytes expressing GFP [n = 14] or PREX2 isoforms (PREX2WT [n = 7], PREX2G844D [n = 14], or PREX2Q1430* [n = 8]). Following tumor detection, tumor measurements were taken every seven days and used to calculate tumor volume. (A–D) Growth curves (natural log scale) for each mouse in each condition is shown. Mice were euthanized when tumor volume reached 1.5 cm3. Additional details for this experiment can be found at https://osf.io/anf2s/.

https://doi.org/10.7554/eLife.21634.008
Meta-analyses of each effect.

Effect size and 95% confidence interval are presented for Berger et al. (2012), this replication attempt (RP:CB), and a random effects meta-analysis of the two effects. Sample sizes used in Berger et al. (2012) and this replication attempt are reported under the study name. Hazard Ratio (HR) of tumor-free survival in mice bearing tumors from NRASG12D melanocytes expressing PREX2 isoforms or GFP are shown. GFP vs. PREX2G844D (meta-analysis p=0.344), GFP vs. PREX2Q1430* (meta-analysis p=0.376), PREX2WT vs. PREX2G844D (meta-analysis p=0.477), PREX2WT vs. PREX2Q1430* (meta-analysis p=0.493). Additional details for this experiment can be found at https://osf.io/ys8hm/.

https://doi.org/10.7554/eLife.21634.009

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