Journal: Molecular Oncology

Article Title: In situ expression of ERG protein in the context of tumor heterogeneity identifies prostate cancer patients with inferior prognosis

doi: 10.1002/1878-0261.13225

Figure Lengend Snippet: Heterogeneity in expression of ERG protein and ETS RNA in prostate cancer. (A) Representative images of positive (top) and negative (bottom) staining for ERG protein on tissue microarrays (10x magnification; scale bar = 100 μm). Endothelial cells were used as an internal positive control. A patient was classified as ERG‐positive if at least one tissue core had any percentage of malignant cells with positive nuclear staining. In total, 51% (244/480) of patients, and 43% (625/1447) of malignant samples, were ERG‐positive. (B) Whole‐mount prostatectomy specimen illustrates two malignant tumor foci (dashed lines). R and L indicate right and left sides, respectively. Green circles illustrate areas sampled from formalin‐fixed paraffin‐embedded tissue for protein analyses, whereas areas marked in yellow represent where fresh‐frozen tissue samples were collected. From the fresh‐frozen tissue, RNA was isolated and analyzed. Intrafocal heterogeneity was defined as the same focus having at least one positive and one negative sample, either for ERG protein or ETS RNA overexpression. Interfocal heterogeneity was defined as the patient having at least one positive and one negative malignant focus and was assessed in patients with samples from at least two malignant foci. Scale bar = 1 cm. (C) Intrafocal heterogeneity of ERG protein expression within a single tissue core (10x magnification; scale bar = 100 μm). (D) Intrafocal heterogeneity of ERG protein expression and RNA overexpression of ERG and ETS genes combined. Intrafocal heterogeneity was identified in 29% (50/170) of patients for ERG protein, and in 39% of patients for both ERG RNA (19/49) and ETS overexpression (24/61). (E) Interfocal heterogeneity of ERG protein expression in two different malignant foci from the same patient. The top picture shows a negative malignant focus, and the bottom a positive malignant focus (10x magnification; scale bar = 100 μm). (F) Interfocal heterogeneity of ERG protein expression and RNA overexpression of ERG and ETS genes combined. Interfocal heterogeneity was identified in 33% (52/156) of patients for ERG protein, 27% (15/56) for ERG RNA, and 34% (19/56) for ETS RNA overexpression. Scale bars were generated using imagej (version 1.53 K; National Institutes of Health, Bethesda, MD, USA).

Article Snippet: Semi‐quantitative RNA expression levels of four ETS genes ( ERG , ETV1 , ETV4 , and FLI1 ) and one reference gene ( ABL1 ) were determined with real‐time RT polymerase chain reaction (PCR), in a reaction volume of 10 μL, using TaqMan Universal Master Mix II, with UNG (Thermo Fisher Scientific) and TaqMan Gene Expression assays (Thermo Fisher Scientific): Hs01554630_m1 ( ERG ), Hs00231877_m1 ( ETV1 ), Hs00944562_m1 ( ETV4 ), Hs00956709_m1 ( FLI1 ), and Hs01104728_m1 ( ABL1 ).

Techniques: Expressing, Staining, Positive Control, Formalin-fixed Paraffin-Embedded, Isolation, Over Expression, Generated

Journal: Molecular Oncology

Article Title: In situ expression of ERG protein in the context of tumor heterogeneity identifies prostate cancer patients with inferior prognosis

doi: 10.1002/1878-0261.13225

Figure Lengend Snippet: Clinicopathological characteristics of the cohort, stratified by the patient's ERG protein status. The χ 2 test of independence was used for categorical variables and the Wilcoxon rank‐sum test for continuous variables. Asterisks (*) indicate statistical significance, P < 0.05. IQR, interquartile range; N , number of patients; pN‐stage, pathological lymph node stage; PSA, prostate‐specific antigen; pT‐stage, pathological tumor stage; RP, radical prostatectomy.

Article Snippet: Semi‐quantitative RNA expression levels of four ETS genes ( ERG , ETV1 , ETV4 , and FLI1 ) and one reference gene ( ABL1 ) were determined with real‐time RT polymerase chain reaction (PCR), in a reaction volume of 10 μL, using TaqMan Universal Master Mix II, with UNG (Thermo Fisher Scientific) and TaqMan Gene Expression assays (Thermo Fisher Scientific): Hs01554630_m1 ( ERG ), Hs00231877_m1 ( ETV1 ), Hs00944562_m1 ( ETV4 ), Hs00956709_m1 ( FLI1 ), and Hs01104728_m1 ( ABL1 ).

Techniques:

Journal: Molecular Oncology

Article Title: In situ expression of ERG protein in the context of tumor heterogeneity identifies prostate cancer patients with inferior prognosis

doi: 10.1002/1878-0261.13225

Figure Lengend Snippet: Prognostic value of ERG protein expression. Patients with persistently elevated PSA levels and/or who received adjuvant treatment postradical prostatectomy were excluded from time‐to‐event analyses ( N = 63). Kaplan–Meier plots were truncated when the number at risk in a specific group was less than five. (A) Fraction of patients without biochemical recurrence, stratified by ERG protein status. There was no statistically significant association between ERG status and biochemical recurrence (log‐rank test, P = 0.1). (B) Fraction of patients without clinical recurrence, stratified by ERG protein status. A statistically significant association between ERG status and clinical recurrence was identified (log‐rank test, P = 0.008). (C–D) Fraction of patients without (C) biochemical recurrence or (D) clinical recurrence, stratified by ERG status and Grade Group. BCR, biochemical recurrence; CR, clinical recurrence; ERG‐, ERG‐negative; ERG+, ERG‐positive; GG, Grade Group; PSA, prostate‐specific antigen.

Article Snippet: Semi‐quantitative RNA expression levels of four ETS genes ( ERG , ETV1 , ETV4 , and FLI1 ) and one reference gene ( ABL1 ) were determined with real‐time RT polymerase chain reaction (PCR), in a reaction volume of 10 μL, using TaqMan Universal Master Mix II, with UNG (Thermo Fisher Scientific) and TaqMan Gene Expression assays (Thermo Fisher Scientific): Hs01554630_m1 ( ERG ), Hs00231877_m1 ( ETV1 ), Hs00944562_m1 ( ETV4 ), Hs00956709_m1 ( FLI1 ), and Hs01104728_m1 ( ABL1 ).

Techniques: Expressing

Journal: Molecular Oncology

Article Title: In situ expression of ERG protein in the context of tumor heterogeneity identifies prostate cancer patients with inferior prognosis

doi: 10.1002/1878-0261.13225

Figure Lengend Snippet: RNA expression of ETS genes and concordance with ERG protein. (A) Relative RNA expression levels of four ETS genes ( ERG , ETV1 , ETV4 , and FLI1 ) in 419 benign and 359 malignant samples from 165 prostate cancer patients, as determined by real‐time RT‐PCR. Individual thresholds for overexpression were calculated for each gene. Twenty patients were excluded from further analysis due to only having benign samples. In total, 48% (70/145), 8% (12/145), and 3% (5/145) of patients had overexpression of ERG , ETV1 , and ETV4 in at least one malignant sample, respectively. No patients overexpressed FLI1 . Samples with no expression are shown as data points at the bottom of the plot. (B) An example gel image demonstrating the result of RT‐PCR followed by gel electrophoresis. Samples with visible bands on the gel were scored as positive for TMPRSS2 ‐ ERG expression. In total, 56% (81/145) of patients had expression of this fusion gene in malignant samples. TMPRSS2 ‐ ERG was also detected in benign tissue from 31% of patients (50/161) with available benign samples. A 100 bp size marker was included on both ends on all well rows, with the shortest fragment being 100 bp and the longest 1000 bp. (C) Three TMPRSS2 ‐ ERG fusion breakpoint sequences identified with Sanger sequencing. Sanger sequencing was performed for 15 samples from 10 patients. The sequences correspond to TMPRSS2 exon 1 (ENST00000332149.10) and ERG exon 4, 3, and 2 (ENST00000442448.5). The fusion breakpoint between TMPRSS2 exon 1 and ERG exon 4 was found to be the most common (13 samples). The exons were annotated with Ensembl release 97. (D) Concordance between ERG protein, ETS RNA overexpression, and TMPRSS2 ‐ ERG RNA expression could be determined for 137 patients where both protein and RNA data were available. Each column represents one patient. Gray indicates that the patient is negative for ERG protein, ETS RNA overexpression, or TMPRSS2 ‐ ERG RNA. RT‐PCR, reverse transcription polymerase chain reaction.

Article Snippet: Semi‐quantitative RNA expression levels of four ETS genes ( ERG , ETV1 , ETV4 , and FLI1 ) and one reference gene ( ABL1 ) were determined with real‐time RT polymerase chain reaction (PCR), in a reaction volume of 10 μL, using TaqMan Universal Master Mix II, with UNG (Thermo Fisher Scientific) and TaqMan Gene Expression assays (Thermo Fisher Scientific): Hs01554630_m1 ( ERG ), Hs00231877_m1 ( ETV1 ), Hs00944562_m1 ( ETV4 ), Hs00956709_m1 ( FLI1 ), and Hs01104728_m1 ( ABL1 ).

Techniques: RNA Expression, Quantitative RT-PCR, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Marker, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Charge Conversion Polymer–Liposome Complexes to Overcome the Limitations of Cationic Liposomes in Mitochondrial-Targeting Drug Delivery

doi: 10.3390/ijms23063080

Figure Lengend Snippet: Characterization of synthesized copolymers. 1 H–NMR spectrum of ( a ) mPEG–b–P(MAAc) –NH 2 copolymers and ( b ) mPEG–b–P(MAAc–g–His) –NH 2 copolymers. ( c ) FT–IR spectra of copolymers ( n = 3, showing one measurement in the data).

Article Snippet: Methacrylic acid (MAAc) (No. AC168310250) was purchased from ACROS.

Techniques: Synthesized

Journal: International Journal of Molecular Sciences

Article Title: Charge Conversion Polymer–Liposome Complexes to Overcome the Limitations of Cationic Liposomes in Mitochondrial-Targeting Drug Delivery

doi: 10.3390/ijms23063080

Figure Lengend Snippet: Copolymer desorption and morphology changes of polymer–liposome complexes at low pH. GPC analysis of polymer–liposome complexes coated by ( a ) mPEG-b-P(MAAc-g-His)-NH 2 and ( b ) mPEG-b-P(MAAc)-NH 2 copolymers. TEM images of polymer–liposome complexes coated by ( c ) mPEG-b-P(MAAc-g-His)-NH 2 and ( d ) mPEG-b-P(MAAc)-NH 2 copolymers at pH 5.0 for 6 h ( n = 3, displaying one of the measurements from the data).

Article Snippet: Methacrylic acid (MAAc) (No. AC168310250) was purchased from ACROS.

Techniques: Polymer