Flow cytometry data were acquired in a FACSCanto cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc.). The following primary BPTU antibodies were used for immunoblots: phospho-Rb (Ser780), Rb (clone D20), FGFR-1 (clone D8E4), pFRS2 (Tyr196), pFRS2 (Tyr436) (all from Cell Signaling Technology), CCND1 (clone A12, Santa Cruz), FRS2 (Proteintech), and pFGFR1 (Tyr653/654) (Invitrogen). (Rb) phosphorylation and active cell cycle despite the double blockade. FGFR1-amplified or -overexpressing models treated with hormones plus palbociclib were fully eradicated only when rogaratinib (a pan-FGFR1-4 inhibitor that displays activity in different tumors with diverse molecular alterations in FGFR1-4) [46, 47] was added. Patients and methods Patients Female patients with a diagnosis of primary, non-metastatic breast cancer with expression of estrogen and/or progesterone receptor ?1% and lack of HER2 amplification diagnosed between January 2001 and December 2002 at Hospital 12 de Octubre were eligible for this study (H12O cohort). The data cutoff for the follow-up of patients was 10?years later (2012), MYH9 although some patients discontinued clinical follow-up earlier and thus were lost-to-follow-up. The study protocol was approved by the Institutional Review Board of Hospital 12 de Octubre (Ref: 11/137). Access to the METABRIC dataset was granted by Drs. Rueda and Caldas. Fluorescence in situ hybridization (FISH) determination and RNAscope FISH chromosome enumeration probes specifically recognizing FGFR1 were purchased from ZytoVision (ZytoLight SPEC FGFR1/CEN8 Dual Color Probe). FISH analyses were performed according to the manufacturers instructions. FISH images were captured using a CCD camera (Photometrics SenSys camera) connected to a PC running the Zytovision image analysis system (Applied Imaging Ltd., UK). Signals were counted in at least 200 cells using the appropriate filters. Results were expressed as the ratio of gene signal to centromere (control) using the following ratios: FISH ratio lower than 1.8 indicates no gene amplification (negative), a ratio higher than 2.2 as gene amplification (positive), and a ratio between 1.8 and 2.2 as equivocal cases. The gene/chromosome copy number alterations were also recorded in the cells as four gene and control signals as moderate polysomy and more than four gene and control signals as high polysomy. Regarding RNAscope, tissue samples were fixed in 10% neutral buffered formalin (4% formaldehyde in solution), paraffin-embedded and cut at 4?m, mounted in superfrost?plus slides, and dried overnight. RNAscope staining method was performed in an automated immunostaining platform (Ventana Discovery ULTRA, Roche). Antigen retrieval was first performed with the appropriate buffer and protease (RNAscope VS Universal Sample Prep ReagentV2, 323740, ACD), and endogenous peroxidase was blocked (peroxide hydrogen at 3%). Then, slides were incubated with the human FGFR1 probe, transcript variant 1, mRNA (ACD, 310079). Slides were then incubated with the corresponding Probe Amplification kit (RNAscope VS Universal HRP Detection Reagent, 323210, ACD), conjugated with horseradish peroxidase and reaction was developed using 3, -diaminobenzidinetetrahydrochloride (DAB Detection Kit, 760-224, Ventana, Roche); nuclei were counterstained with Hematoxylin II and slides were mounted. Positive control sections were included for each staining run using Positive Control Probe_Hs-PPIB (313,909, ACD). Samples were acquired and digitalized using the AxioScan.Z1 system (Zeiss). Digitalized images were analyzed with the ZEN 2.3 lite software (Zeiss), and tumoral areas were categorized in the different scores: score 0 (no staining or ?1 dot/10 cells), score 1 (1C3 dots/cell), score 2 (4C9 dots/cell and none or very few are in clusters), score 3 (10C15 dots/cell and ?10% dots are in clusters), and score 4 ( ?15 dots/cell and ?10% dots are in clusters). Scores of 3 and 4 were considered RNAscope-positive. In vitro experiments MCF7, T47-D, and HCC1428 cells were acquired from the American Type Culture Collection (ATCC). Cells were maintained following the ATCC recommendations and routinely tested for mycoplasma using the MycoalertTM Mycoplasma Detection Kit (Lonza). Cell lines were authenticated every 6?months using short-tandem repeat profiling. Cell collection clones resistant to estrogen deprivation were generated following a method explained by Martin et al [48, 49]. Briefly, the method consisted BPTU of weekly passage and tradition of cells in medium comprising 10% dextran charcoal-stripped (DCC) fetal bovine serum (FBS) (Sigma) instead of full FBS, which removes steroids. The medium was changed.?Fig.1f).1f). keeping a resilient retinoblastoma protein (Rb) phosphorylation and active cell cycle despite the double blockade. FGFR1-amplified or -overexpressing models treated with hormones plus palbociclib were fully eradicated only when rogaratinib (a pan-FGFR1-4 inhibitor that displays activity in different tumors with varied molecular alterations in FGFR1-4) [46, 47] was added. Individuals and methods Individuals Female individuals with a analysis of main, non-metastatic breast malignancy with manifestation of estrogen and/or progesterone receptor ?1% and lack of HER2 amplification diagnosed between January 2001 and December 2002 at Hospital 12 de Octubre were eligible for this study (H12O cohort). The data cutoff for the follow-up of individuals was 10?years later (2012), although some individuals discontinued clinical follow-up earlier and thus were lost-to-follow-up. The study protocol was authorized by the Institutional Review Table of Hospital 12 de Octubre (Ref: 11/137). Access to the METABRIC dataset was BPTU granted by Drs. Rueda and Caldas. Fluorescence in situ hybridization (FISH) dedication and RNAscope FISH chromosome enumeration probes specifically recognizing FGFR1 were purchased from ZytoVision (ZytoLight SPEC FGFR1/CEN8 Dual Color Probe). FISH analyses were performed according to the manufacturers instructions. FISH images were captured using a CCD video camera (Photometrics SenSys video camera) connected to a Personal computer operating the Zytovision image analysis system (Applied Imaging Ltd., UK). Signals were counted in at least 200 cells using the appropriate filters. Results were indicated as BPTU the percentage of gene transmission to centromere (control) using the following ratios: FISH percentage lower than 1.8 indicates no gene amplification (negative), a percentage higher than 2.2 as gene amplification (positive), and a percentage between 1.8 and 2.2 while equivocal instances. The gene/chromosome copy number alterations were also recorded in the cells as four gene and control signals as moderate polysomy and more than four gene and control signals as high polysomy. Concerning RNAscope, tissue samples were fixed in 10% neutral buffered formalin (4% formaldehyde in answer), paraffin-embedded and slice at 4?m, mounted in superfrost?plus slides, and dried over night. RNAscope staining method was performed in an automated immunostaining platform (Ventana Finding ULTRA, Roche). Antigen retrieval was first performed with the appropriate buffer and protease (RNAscope VS Common Sample Prep ReagentV2, 323740, ACD), and endogenous peroxidase was clogged (peroxide hydrogen at 3%). Then, slides were incubated with the human being FGFR1 probe, transcript variant 1, mRNA (ACD, 310079). Slides were then incubated with the related Probe Amplification kit (RNAscope VS Common HRP Detection Reagent, 323210, ACD), conjugated with horseradish peroxidase and reaction was developed using 3, -diaminobenzidinetetrahydrochloride (DAB Detection Kit, 760-224, Ventana, Roche); nuclei were counterstained with Hematoxylin II and slides were mounted. Positive control sections were included for each staining run using Positive Control Probe_Hs-PPIB (313,909, ACD). Samples were acquired and digitalized using the AxioScan.Z1 system (Zeiss). Digitalized images were analyzed with the ZEN 2.3 lite software (Zeiss), and tumoral areas were categorized in the different scores: score 0 (no staining or ?1 dot/10 cells), score 1 (1C3 dots/cell), score 2 (4C9 dots/cell and none or very few are in clusters), score 3 (10C15 dots/cell and ?10% dots are in clusters), and score 4 ( ?15 dots/cell and ?10% dots are in clusters). Scores of 3 and 4 were regarded as RNAscope-positive. In vitro experiments MCF7, T47-D, and HCC1428 cells were acquired from your American Type Tradition Collection (ATCC). Cells were maintained following a ATCC recommendations and routinely tested for mycoplasma using the MycoalertTM Mycoplasma Detection Kit (Lonza). Cell lines were authenticated every 6?weeks using short-tandem repeat profiling. Cell collection clones resistant to estrogen deprivation were generated following a method explained by Martin et al [48, 49]. Briefly, the method consisted of weekly passage and tradition of cells in medium containing.
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