Dexamethasone effects on primary tumor cells.

<p>(A) Whisker-box plot of PNMT mRNA relative ratio from PGL cells (tumors P16, P30, P35 and P42) incubated with <i>vs</i>. without dexamethasone 1μM for 24 hrs (reference). Reporter genes included the following: EEFIA1, GAPDH and TBP, ns; not statistically different. (B) Whisker-box plot of TH, DBH and PNMT mRNA relative ratio from PHEO cells (tumors P23, P36, P44, P45, P52, P53, P56, P61 and P62) incubated with <i>vs</i>. without dexamethasone 1μM for 24 hrs (reference). TH: FC 2.8, SE 1.28–7.55, CI 0.68–17.38, reporter genes included the following: EEFIA1, GAPDH and TBP. ** <i>P</i><0.01. (C) Western blot of PHEO cell incubated with (+) or without (-) dexamethasone (Dexa.) 1μM for 24 hrs.</p

mRNA and protein quantification of TH, DBH and PNMT in PGL <i>vs</i>. mixed and NorAD PHEO.

<p>(A) Whisker-box plot of mRNA relative ratio from PGL (n = 11) <i>vs</i>. mixed PHEO (n = 28) (reference). TH: reaction efficiency (RE) 0.99, fold change to reference (FC) 0.29, Standard error (SE) 0.04–1.65, 95% confidence Interval (CI) 0.003–48.29; DBH: not statistically significant (ns); PNMT: RE 0.98, FC 0.024, SE 0.001–0.41, CI 0–4.82. Reporter genes included the following: EEFIA1, RE 0.99; GAPDH, RE 1.04, TBP, RE 0.97. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, (B) Ratio from PGL (n = 11) <i>vs</i>. NorAd PHEO (n = 22) (reference). (C) Ratio from NorAd PHEO (n = 22) <i>vs</i>. mixed PHEO (n = 28) (reference) PNMT: FC 0.07, SE 0.005–0.93, CI 0.001–10.99.</p

Patients and tumors characteristics.

<p>Localisation: PHEO were found either in the left (L) or right (R) adrenal gland. PGL were excised from the following location: abd: abdominal; 1: paraortal right; 2: paraortal left; 3: Glomus vagal right; 4: retroperitonal left, paraortal; 5: abdominal, paraortal. NT: non-tested, Sp*: sporadic, RET, SDHB, -D and VHL negative for mutation</p><p>Sp**: RET and SDHB negative. SP¹: SDHA, -B, -C, -D, MAX, VHL negative. SP²: SDHB, -D, RET, VHL, Men1 negative, SP³: SDHB, -C, -D, RET, negative, SP⁴: VHL negative, SP⁵: SDHB, -C, -D, Men1, RET negative. For CAT and metabolites in plasma, upper reference limits are (in nmol/l); NE (norepinephrine): 6.55, E (epinephrine): 1.23, free NMN (normetanephrine): 1.39, free MN (metanephrine): 0.85, P65 was later shown not to be a PHEO and do not appear in this table</p><p>Patients and tumors characteristics.</p

Flowchart of the patients and tumor samples included in the study.

<p>For CAT and MNs quantification in tissue 63 patients were enrolled and 66 tumors were collected (two patients had bilateral tumors (P21 and P43) and one had both a PHEO and a PGL (P25)*, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.t001" target="_blank">Table 1</a>. Tumors consisted of 53 PHEO and 13 PGL. Quantification of CAT in plasma was performed on 72 patients from 50 PHEO and 23 PGL patients, (including P25 with both a PHEO and a PGL). The plasma CAT analysis was performed on the same patient recruited for tissue CAT and MNs study with the following exceptions: two plasma were not available (P14 and P27) and two tumors were bilaterally excised at the same time (P43 and P44), finally, 10 additional plasma from PGL patients were added in the study without tissue sample available. For MNs quantification in plasma, sample P62 was not available (instead of P27 for CAT). Three tissue samples for PHEO and two for PGL were stored in perchloric acid, not compatible for mRNA extraction for qPCR assay**. 15 PHEO tissue sample were no longer available for WB assays***.</p

Quantification of E, NE, MN and NMN in plasma and tumor tissue.

<p>(A) Quantification of epinephrine (E), norepinephrine (NE), total CATs (E+NE) and NE/E ratio from 53 PHEO (filled circles) and 13 PGL (open circles) samples. Results are given in nanomoles per gram of tissue and represented on a logarithmic scale. Geographic mean and standard deviation for each group are reported in supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.s002" target="_blank">S2 Table</a>). (B) Same as in A, quantification of CAT in plasma for 50 PHEO and 23 PGL. (C) Quantification of metanephrine (MN), normetanephrine (NMN), sum of metanephrines (MN+NMN) and NMN/MN ratio from 53 PHEO (filled circles) and 13 PGL (open circles).(D) Same as in C, quantification of metanephrines in plasma for 51 PHEO and 23 PGL. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns: not statistically significant. For CAT and metanephrines in tumor tissue, minimal values were set at 0.1 nmol/g representing limit of quantification.</p

PNMT expression in NorAd PHEO <i>vs</i>. mixed tumors.

<p>(A) Whisker-box plot of PNMT mRNA relative ratio from NorAd tumors (including PHEO and PGL, n = 29) <i>vs</i>. mixed tumor (PHEO and PGL, n = 32, reference). Fold change to reference (FC) 0.06, Standard error (SE) 0.003–0.29, 95% confidence Interval (CI) 0–22.36; Reporter genes included the following: EEFIA1, GAPDH and TBP. (B) Quantification of the signal obtained by western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.g006" target="_blank">Fig 6</a> for PNMT signal in mixed and NorAd tumor (independently of PHEO or PGL). Results are given as QL (corresponding band pixel values)-BG (background pixel value) and calculated value is divided by the β-actin signal value and reported on the y-axis. Geo. mean 0.64 <i>vs</i>. 0.18, standard deviation: 0.7 and 0.14 for respectively mixed and NorAd tumor.</p

Quantification of E, NE, MN and NMN in plasma and tumor tissue of PGL, mixed and NorAd tumors.

<p>(A) Quantification of epinephrine (E), norepinephrine (NE), total CAT (E+NE) and NE/E ratio from 30 mixed PHEO (Mix, open diamonds); 23 noradrenergic PHEO (NorAd, filled diamonds) and 13 PGL (open circles) samples. Results are given in nanomoles per gram of tissue and represented on a logarithmic scale. Geographic mean and standard deviation for each group are reported in supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125426#pone.0125426.s004" target="_blank">S4 Table</a>). (B) Same as in A, quantification of CAT in plasma for 28 mixed PHEO, 22 NorAd PHEO and 23 PGL. (C) Quantification of MN and NMN sum of MNs (MN+NMN) and NMN/MN ratio from 30 mixed PHEO (Mix, open diamonds); 23 NorAd PHEO (filled diamonds) and 13 PGL (open circles) samples. (D) Same as in C, quantification of MNs in plasma for 28 mixed PHEO, 22 NorAd PHEO and 23 PGL. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns: not statistically significant. For CAT and MNs in tumor tissue, minimal values were set at 0.1 nmol/g representing limit of quantification.</p