Cytovision software

FISH analysis was performed on formalin-fixed, paraffin-embedded (FFPE) iCCA tissue samples pre-treated with the Paraffin Pretreatment Reagent Kit (Abbott Molecular Inc., Germany), following the manufacturer’s protocol. Two Empire Genomics probes (Empire Genomics, Buffalo, NY) were employed following the manufacturer’s protocol. Specifically, the FGFR2 gene probe (orange) maps on Chromosome 10q26.13 and the CEP10 probe maps to the centromeric region of Chromosome 10. Tumour cells were counterstained with 40,6-diamidino-2-phenylindole (DAPI) for nuclear detection. Analysis was performed using an Olympus BX53 microscopy equipped with the appropriate filter sets and CytoVision software (Leica Biosystems, Nussloch, Germany). Ratio of FGFR2 signals to CEP10 signals (FGFR2/CEP10 ratio) > 2 was interpreted as gene amplification, as previously reported^{26 (link)}.

Karyotyping was performed on cells at P5 by the Cytogenetics Department of the hospital. Briefly, hUCB-MSCs were cultured in medium supplemented with colcemid (10 μg/ml, PAA Laboratories). Then, the cells were re-suspended in a hypotonic solution of KCl and incubated at 37 °C for 10 min. After centrifugation, cells were resuspended in a fixative solution (3:1 methanol:acetic acid). Metaphase spreads were then prepared on glass microscope slides and analyzed using R- and Q-banding Giemsa staining. A total of 16 mitoses were analyzed per sample using CytoVision software (Leica Biosystems).

Fusions detected in our cohort were confirmed using fluorescence in situ hybridization (FISH) and reverse transcription–PCR (RT-PCR) followed by Sanger sequencing. FISH analyses were performed using dual-color probes EP300 and ZNF384 (Guangzhou LBP Medicine Science and Technology, China) according to the manufacturer’s instruction. The analyses were performed using a Zeiss Axioplan 2 microscope (Carl Zeiss AG, Oberkochen, Germany) and the CytoVision software (Leica Biosystems, Nussloch, Germany). For each patient, 200 interphase cells were analyzed.
For detection of the EP300-ZNF384 fusion transcript, RT-PCR was performed using PrimeScript^TM RT reagent Kit (Takara, Japan) and TransTaq® HiFi DNA Polymerase (Transgen, China). Two forms of EP300-ZNF384 fusion were amplified with the same forward primer (5′-AATCAGATGCCGACACAACC-3′) and reverse primer (5′-CAGCAAGGTGGGGTAGTGAG-3′). The resulting 321 bp and 261 bp amplicons were confirmed by Sanger sequencing (Sangon Biotech, China).

Chromosome analysis by G banding was performed on agnospheres. In order to increase the number of metaphases, cells were synchronized with Synchroset (Euroclone S.p.A.) according to manufacturer’s instructions and blocked with colcemid (10 μl/ml) for 1 h. Chromosome harvesting was carried out according to standard procedures. Briefly, cells were incubated in 0.075 M KCl hypotonic solution at 37 °C for 10 min, fixed in methanol–glacial acetic acid (3:1), dropped onto glass slides and dried using specific conditions for optimal chromosome spreading. G banding was performed by incubating slides in 2× SSC at 68 °C for 2 min and eventually stained with Wright’s solution for 2 min (ref. ^{57 (link)}). Metaphase images were captured using an Olympus BX61 microscope (Olympus Corporation) and analyzed by CytoVision software (Leica Biosystems). An average banding resolution of 300 bands was achieved. Aberrations were described according to the International System for Human Cytogenetic Nomenclature, 2016 (ref. ⁵⁸ ).

Dual-color FISH was performed on 4 µm paraffin-embedded tissue sections (Abbott Molecular: FOXO1 (Centromeric) SpectrumGreen Cat# 05J48-014; FOXO1 (Telomeric) SpectrumOrange Cat# 05J48-013). Probes were co-denatured with the target cells on a slide moat at 90 °C for 12 min. The slides were incubated overnight at 37 °C on a slide moat and then washed in 4 M Urea/2xSSC at 25 °C for 1 min. Nuclei were counterstained with DAPI (200 ng/ml) (Vector Labs) for viewing on an Olympus BX51 fluorescence microscope equipped with a 100-W mercury lamp; FITC, Rhodamine, and DAPI filters; 100X PlanApo (1.40) oil objective; and a Jai CV digital camera. Images were captured and processed using the Cytovision software from Leica Biosystems (Richmond, IL).

Dual-color FISH was performed on 4-µm-thick paraffin-embedded tissue sections. Probes were derived from BAC clones (BACPAC Resources, Oakland, CA) and labeled with either AlexaFluor-488 or AlexaFluor-555 fluorochromes. BAC clones were used to construct probes for the following genes: PDGFRA (laboratory-developed probe [RP11-231C18 & 601I15]; 4p control (CTD-2057N12 & CTD-2588A19), and CDK4 (Empire Genomics, Williamsville, New York, Cat# CDK4-CHR12-20-ORGR). Probes were co-denatured with target cells on a slide moat at 90 °C for 12 min. Slides were incubated overnight at 37 °C on a slide moat and washed in 4 M Urea/2× SSC at 25 °C for 1 min. Nuclei were counterstained with DAPI (200 ng/mL) (Vector Labs) for viewing on an Olympus BX51 fluorescence microscope equipped with a 100-watt mercury lamp; FITC, rhodamine, and DAPI filters; 100× PlanApo (1.40) oil objective; and a Jai CV digital camera. Images were captured and processed using the Cytovision software from Leica Biosystems (Richmond, IL)^{66 (link)}.