Frozen/FFPE correlations.

<p><b>a</b>. Correlation in all the patients and for all the genes between the two tissue preparation methods. The adjusted Pearson correlation between FFPE and frozen tissue for all tested genes was greater (Pearson coefficient = 0.88 p<0,0001). <b>b</b>. Two examples of correlations determined individually for each patient measured in all genes.</p

RNA analyses.

<p><b>a</b>. Representative total RNA integrity analysis paired frozen and FFPE tissue specimens using Capillary electrophoresis Agilent 2100 bioanalyzer, shows that in the FFPE samples RNA exists primarily as fragments between 200 and 100 bases in length. Left panel: fresh frozen human melanoma tissue. Right panel: matched FFPE tissue. <b>b</b>. Boxplot represents the mean mRNA levels and gene expression between frozen and FFPE samples. In red, reference genes.</p

Corrected FFPE/Frozen correlations for each individual.

<p>The correlation test tries the hypothesis «the correlation is useless”. The threshold represents the threshold reject alpha = 5% (correct for the multiple test by Bonferroni). The individuals to spread are the ones who are below the threshold.</p

Effect of PCs inhibition on gelatinase activity and MMP-2/TIMP-1 and MMP-2/TIMP-2 ratios.

<p>(<b>A</b>) Serum-free media derived from M10 and M10/PDX cells were collected and analyzed for gelatinase enzymatic activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. Following total RNA extraction, real-time PCR analysis was performed using specific primers for MMP-2 (<b>B</b>), TIMP-1 (<b>C</b>), TIMP-2 (<b>D</b>) or β2-microglobulin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. During PCR, the transcription of β2-microglobulin that was evaluated in each sample was used as endogenous control. Results are expressed as mRNA transcript ratios. Data are shown as means ± S.E of three experiments performed in duplicate. (<b>E, F</b>) The ratios of MMP-2/TIMP-1 and MMP-2/TIMP-2 are deduced from data obtained in (B), (C) and (D). (<b>G, H</b>), Tumor cells were incubated at 37°C in serum-free media. After 24 hours, media were collected and analyzed for the presence of TIMP-1 and TIMP-2 using ELISA kit. Results shown are representative of 3 experiments. Data are mean ± SEM (<i>n = </i>6 per group). Student's <i>t</i> test was used for statistical analysis ***, <i>P</i><0.001.</p

Furin, PACE4, PC5 and PC7 expression and activity in human primary melanoma cells.

<p>(<b>A</b>) Expression of the indicated PCs was analyzed in M10 cells using specific primers for the PCs found in the secretory pathway (Furin, PACE4, PC5, PC7) and reverse transcription-PCR analysis assay. Note that all these PCs are expressed in the M10 cells. (<b>B</b>) Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for Furin, PACE4, PC5, PC7 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. During PCR, the transcription of β2-microglobulin that was evaluated in each sample was used as endogenous control. Results shown in the bar graph are expressed as ratio of PCs mRNA transcripts (M10)/(M10/PDX) deduced from values derived from M10 and M10/PDX cells mRNA analysis. Data are shown as means ± S.E of three experiments performed in duplicate. Real-time PCR analysis revealed that expression of α1-PDX in M10 cells did not affect significantly the expression levels of these PCs in M10 cells. Processing of proPDGF-A (<b>C</b>) and proIGF-IR (<b>D</b>) analyzed by Western blotting revealed that expression of α1-PDX in M10 cells (M10/PDX) completely inhibited the processing of pro-IGF-1R and proPDGF-A. (<b>E</b>) PCs activity in M10 cells and M10/PDX cells was assessed by evaluating the cell extracts for their ability to digest the universal PCs substrate, the fluorogenic peptide pERTKR-MCA at the indicated time points. Expression of α1-PDX in M10 cells reduced their PCs activity. (<b>F</b>) Results shown in the bar graph represent the PCs activity at 2 hours of incubation of the indicated tumor cells. Results are representative of three experiments and data are mean ± S.E performed in triplicate. ***p<i><</i>0.0001.</p

Schematic representation of the effects of PCs inhibition on migration and invasion of human melanoma cells with altered <i>CDKN2A</i>, <i>p53</i> and <i>Ras</i> genes.

<p>The <i>CDKN2A</i> locus codes for the tumor suppressors p16 and ARF that regulate cell cycle progression, DNA repair and cell invasion via activating pRb, and the p53 pathway via inhibiting the activity of MDM2, respectively. Mutations or deletions of the <i>CDKN2A</i> gene result in altered MMPs/TIMPs and/or uPA/uPAR/PAI-1 expression and activity, which in turn lead to tumor cell invasion. The <i>Ras</i> gene is important for regulating the ERK activity, and hence expression of MMPs and uPA/uPAR. An activating mutation in the Ras gene can result in uncontrolled activity of MMPs and the urokinase system, which can lead to increased melanoma invasion. Blockade of PCs activity can reduce the expression and/or generation of active MMPs, uPA and uPAR leading to reduced tumor cells invasiveness. The asterisks indicate altered genes.</p

Effect of PCs inhibition on uPA, uPAR and PAI-1 expression.

<p>(<b>A–C</b>), Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for indicated genes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. Results are shown in the bar graph and are expressed as the mRNA transcript ratios. Data are shown as means ± S.E of three experiments performed in duplicate. Student's <i>t</i> test was used for statistical analysis. *, <i>P</i><0.05 and ***, <i>P</i><0.001. (<b>D</b>), Tumor cells were incubated at 37°C in serum-free media. After 24 hours, media were collected and analyzed for the presence of uPAR using ELISA kit. Results shown are representative of 3 experiments. Data are mean ± SEM (<i>n = </i>6 per group). Student's <i>t</i> test was used for statistical analysis ***, <i>P</i><0.001</p

Mechanisms Underpinning Increased Plasma Creatinine Levels in Patients Receiving Vemurafenib for Advanced Melanoma

<div><p>Context</p><p>Serum creatinine has been reported to increase in patients receiving Vemurafenib, yet neither the prevalence nor the mechanism of this adverse event are known.</p><p>Objective</p><p>We aimed to evaluate the frequency and the mechanisms of increases in plasma creatinine level in patients receiving Vemurafenib for advanced melanoma.</p><p>Methods</p><p>We performed a retrospective monocentric study including consecutive patients treated with Vemurafenib for an advanced melanoma. We collected clinical and biological data concerning renal function before introduction of Vemurafenib and in the course of monthly follow-up visits from March 2013 to December 2014. Cystatin C-derived glomerular filtration rate was evaluated before and after Vemurafenib initiation, as increase in serum cystatin C is specific to a decrease in the glomerular filtration rate. We also performed thorough renal explorations in 3 patients, with measurement of tubular secretion of creatinine before and after Vemurafenib initiation and a renal biopsy in 2 patients.</p><p>Results</p><p>70 patients were included: 97% of them displayed an immediate, and thereafter stable, increase in creatinine (+22.8%) after Vemurafenib initiation. In 44/52 patients in whom Vemurafenib was discontinued, creatinine levels returned to baseline. Serum cystatin C increased, although proportionally less than serum creatinine, showing that creatinine increase under vemurafenib was indeed partly due to a renal function impairment. In addition, renal explorations demonstrated that Vemurafenib induced an inhibition of creatinine tubular secretion.</p><p>Conclusion</p><p>Thus, Vemurafenib induces a dual mechanism of increase in plasma creatinine with both an inhibition of creatinine tubular secretion and slight renal function impairment. However, this side effect is mostly reversible when Vemurafenib is discontinued, and should not lead physicians to discontinue the treatment if it is effective.</p></div

Main clinical and biological data of the 70 patients at baseline and during the treatment.

<p>Main clinical and biological data of the 70 patients at baseline and during the treatment.</p

Flow-chart of patients included.

<p>Flow-chart of patients included.</p