Formalin-fixed paraffin-embedded tissue (FFPE) sections of the tumours, and spheroids were cut at 2.5-μm thickness, unmasked, and deparaffinised in citrate buffer (10 mM, pH 6.0) at 98 °C for 15 min. After blocking, the tumour sections were incubated with anti-Ki67 (Dako), anti-VEGF (Santa Cruz Biotechnology, USA), and anti-CD31 (Dako) at 4 °C overnight and then, automated IHC was performed on Omnis platform (Agilent, USA). The FFPE sections of the liver, lung and kidney were deparaffinised and stained for haematoxylin and eosin for histological evaluation. The sections were inspected in an optic microscope (Axiocam MRc5, Zeiss).
Omnis platform
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The Omnis platform is a flexible and configurable laboratory automation solution designed to streamline and optimize various analytical workflows. It provides a modular and customizable architecture to support a range of applications and instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
22c3 pharmdx antibody, by Agilent Technologies (2 mentions)
Ras extension pyro kits, by Qiagen (2 mentions)
Cobas 4800 braf v600 mutation test, by Roche (2 mentions)
Axiocam mrc5, by Zeiss (1 mentions)
Anti cd31, by Agilent Technologies (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Anti vegf, by Santa Cruz Biotechnology (1 mentions)
Envision flex detection kit, by Agilent Technologies (1 mentions)
Cd31 antibody, by Agilent Technologies (1 mentions)
Ndp view, by Hamamatsu Photonics (1 mentions)
Nanozoomer xr, by Hamamatsu Photonics (1 mentions)
Cd8 c8 144b, by Agilent Technologies (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Envision flex dab kit, by Agilent Technologies (1 mentions)
Envision flex, by Agilent Technologies (1 mentions)
Imagescope software, by Leica (1 mentions)
Aperio cs2 slide scanner, by Leica (1 mentions)
Halo image analysis software, by Indica Labs (1 mentions)
15 protocols using omnis platform
1
Immunohistochemical Analysis of Tumor Samples
2
Tumor Microvessel Density Quantification
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Tumor MVD was assessed by IHC using standard methods. Tissue sections (3 μm) were stained using the CD31 antibody (Agilent Technologies) following the manufacturer's instructions. The staining was performed on the Omnis platform (Agilent Technologies) with the EnVision Flex+ detection kit (GV800) using the RTU format. Incubation time was 20 min, and sections were counterstained with hematoxylin.
Stained slides were scanned using the Hamamatsu Nano Zoomer‐XR at a magnification equivalent to ×20 magnification. Tumor MVD was assessed using the method introduced by Weidner (Kraby et al., 2019 (link); Rogatsch et al., 1997 (link); Weidner et al., 1993 (link)) and manually quantified using the Hamamatsu viewing software NDP.View (version 2.6.13, Hamamatsu Photonics). First, tumor sections were scanned at low magnification (×4 and ×10) to identify areas of high neovascularization (“hot spots”). Second, the quantification was performed in one visual field (0.17 mm2) from three randomly chosen neovascular hot spots at high (×40) magnification. The MVD was expressed as both the mean and the highest MVD from the three fields.
Stained slides were scanned using the Hamamatsu Nano Zoomer‐XR at a magnification equivalent to ×20 magnification. Tumor MVD was assessed using the method introduced by Weidner (Kraby et al., 2019 (link); Rogatsch et al., 1997 (link); Weidner et al., 1993 (link)) and manually quantified using the Hamamatsu viewing software NDP.View (version 2.6.13, Hamamatsu Photonics). First, tumor sections were scanned at low magnification (×4 and ×10) to identify areas of high neovascularization (“hot spots”). Second, the quantification was performed in one visual field (0.17 mm2) from three randomly chosen neovascular hot spots at high (×40) magnification. The MVD was expressed as both the mean and the highest MVD from the three fields.
3
PD-L1 and Ki-67 Expression in Cancer
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The expression of PD-L1 on the surface of cancer cells was assessed in all cases by means of the Dako Omnis Platform and the 22C3 pharmDx antibody (Agilent, Santa Clara, CA) using FDA-scoring guidelines [14 ]. Briefly, a minimum of 100 viable tumor cells were assessed for membranous staining of any intensity for the 22C3 antibody. The percentage of viable tumor cells showing partial or complete membrane staining relative to all viable tumor cells present in the sample (positive and negative) was then used to derive a tumor proportion score (TPS). PD-L1 levels were scored by a board-certified anatomic pathologist as per published guidelines [15 (link)], with a TPS ≥ 50% considered as a strongly positive result for different comparisons, while a result of ≥1% considered as positive result for different comparisons. PD-L1 TPS ≥ 1% to < 50% were considered weakly positive for additional comparative purposes. PD-L1 TPS < 1% was considered as negative. Ki-67 positivity amongst neoplastic and immune cells was scored upon nuclear staining, regardless of intensity, with the M7240 (clone MIB1) antibody from Dako (Carpentaria, CA) with the percentage of each cell type recorded.
4
Immunohistochemical Evaluation of BAP1 in MPM
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All selected MPM histological samples were reviewed by an experienced mesothelioma pathologist (F.B.) and divided into epithelioid, sarcomatoid (including desmoplastic) and biphasic MPM according to the 2015 World Health Organization (WHO) classification (21 (link)). To include the MPM sample in the b-MPM subgroup, the presence of both sarcomatoid and epithelioid components was required at least in the ten percent of the tumor. All samples were FFPE and a single 4-mm-thick paraffin section was cut from the sample with the greatest amount of tumor tissue for each patient. All sections were deparaffinized and rehydrated in graded concentrations of xylene and ethanol. Sections were coated with 1:50 mouse monoclonal BAP1 antibody (clone C4:sc-28383; Santa Cruz Biotechnology, USA) and incubated at 4 °C overnight. Then, automated IHC was performed on Omnis platform (Agilent, USA). BAP1 IHC status was considered as “positive/retained” if there was an unambiguous positive nuclear staining in any number of tumor cells, and “negative/loss” if the nuclear staining was absent in neoplastic cells. Tumor cells with cytoplasmic reactivity without a clear nuclear staining were considered negative. Non-neoplastic cells, such as vascular endothelium, fibroblasts or inflammatory cells, were considered as internal positive control.
5
PD-L1 Expression Assessment in Cancer
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The expression of PD-L1 on the surface of cancer cells was assessed in all cases using the Dako Omnis Platform and the FDA-approved 22C3 pharmDx antibody (Agilent, Santa Clara, CA). PD-L1 levels were scored by a board-certified anatomic pathologist as per published FDA-approved guidelines,17 (link) with a tumor proportion score (TPS) in neoplastic cells of ≥1% considered positive. PD-L1 was also scored as a combined positive score (CPS)18 (link),43 (link),44 evaluating both neoplastic and immune cells with a value of ≥1 considered positive, whereby the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total viable tumor cells is multiplied by 100.
6
Automated Immunohistochemistry for PD-L1 and CD8
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Automated immunohistochemistry was performed on Omnis platform (Agilent, USA). The following antibodies were used: PD-L1 (22C3, dilution 1/40, Dako-Agilent) and CD8 (C8/144B, dilution 1/100, Dako-Agilent). Appropriate external (tonsil) and internal controls (mononuclear cells) were used.
7
Immunohistochemical Profiling of Lung Cancer
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All diagnoses were reviewed in light of the WHO 2021 classification [12 ]. Anti-CK7 and CK20 immunohistochemistry never exhausted the sample for further techniques such as immunohistochemistry or molecular biology. Automated immunohistochemistry was performed on 4 μm sections using the Omnis platform (Dako-Agilent, Courtaboeuf, France) according to the manufacturer’s instructions. Immunohistochemistry was performed on OMNIS (Dako-Agilent) using anti-TTF-1 antibodies (8G7G3/1, 1/50, Dako-Agilent). TTF-1 expression was considered positive if more than 5% of the tumor cells were nuclear stained. In the case of negativity, anti-CK7 (OV-TL 2/30, 1/600, Dako-Agilent) and CK20 (Ks20.8, 1/100, Dako-Agilent) were used. Mucin stain was used to confirm the adenocarcinoma in the case of negative TTF-1 and Napsin A (IP64, Leica) immunohistochemistry was perfomed in negative TTF-1.
ALK (D5F3, 1/100, Cell Signaling Technology, Danvers, MA, USA) and ROS1 (D4D6, 1/40 Cell Signaling Technology) antibodies were used for the screening of ALK and ROS1 rearrangements. When positive or doubtful, another technique was used to confirm ALK or ROS1 rearrangement.
ALK (D5F3, 1/100, Cell Signaling Technology, Danvers, MA, USA) and ROS1 (D4D6, 1/40 Cell Signaling Technology) antibodies were used for the screening of ALK and ROS1 rearrangements. When positive or doubtful, another technique was used to confirm ALK or ROS1 rearrangement.
8
Comprehensive Genomic and Phenotypic Analysis of CCR
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Mutational analysis for BRAF, KRAS, and NRAS genes was performed on FFPE CCR samples by pyrosequencing and PCR‐based assay. Briefly, DNA was isolated from 20 μm of representative tumor tissue. KRAS and NRAS were studied by pyrosequencing using the therascreen KRAS and RAS Extension Pyro Kits (Qiagen, Venlo, The Netherlands), following the manufacturer's recommendations. BRAF was assayed by the PCR‐based Cobas 4800 BRAF V600 Mutation Test (Roche, Basel, Switzerland).
The MSI phenotype was studied by testing the expression of the four MMR proteins (MLH1, MSH2, MSH6, and PMS2) by immunohistochemistry on Omnis platform (Dako, Glostrup, Denmark), using conventional 3‐μm tissue sections from the same specimens. Interpretation of staining was performed by qualified pathologists. Finally, proliferation was estimated as percentage by labeling Ki67 expression in tumor cells by immunohistochemistry on Omnis platform.
The MSI phenotype was studied by testing the expression of the four MMR proteins (MLH1, MSH2, MSH6, and PMS2) by immunohistochemistry on Omnis platform (Dako, Glostrup, Denmark), using conventional 3‐μm tissue sections from the same specimens. Interpretation of staining was performed by qualified pathologists. Finally, proliferation was estimated as percentage by labeling Ki67 expression in tumor cells by immunohistochemistry on Omnis platform.
9
Somatic Mutation Profiling in Colorectal Cancer
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Mutational analysis for BRAF, KRAS, and NRAS genes was performed on FFPE CC samples by pyrosequencing and PCR‐based assay. DNA was isolated from 20‐µm FFPE sections of representative tumor tissue. KRAS and NRAS were studied by pyrosequencing using the therascreen KRAS and RAS Extension Pyro Kits (Qiagen, Venlo, the Netherlands), following the manufacturer’s recommendations. BRAF was assayed by the PCR‐based Cobas 4800 BRAF V600 Mutation Test (Roche, Basel, Switzerland). The microsatellite instability (MSI) phenotype was studied by testing the expression of the four mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, PMS2) by immunohistochemistry on an Omnis platform (Dako, Glostrup, Denmark), using conventional 3‐µm tissue sections from the same specimens. Interpretation of staining was performed by qualified pathologists. Finally, proliferation was estimated as percentage Ki67‐labeled tumor cells by immunohistochemistry on an Omnis platform.
10
Quantifying TTF1 Expression in Lung Cancers
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Immunostaining for TTF1 was obtained in LM-uADC and LM-uLMS using a Dako Omnis platform. Antigen retrieval was performed using Target Retrieval Solution High pH (Dako-Agilent) at 97° for 30 min prior to incubation with a mouse monoclonal anti-TTF1 antibody (clone: SPT24, Gennova) for 30 min. EnVisionTM Flex Mouse Linker (Dako) was added for 10 min followed by EnVision FLEX/HRP (Dako) for 20 min and incubation with diaminobenzidine (DAB) chromogen for 5 min. QuPath v0.2.3 open-source digital pathology software was used for TTF1 quantification. A positive cell detection algorithm was employed to detect every nucleus that expressed TTF1. The number of positive cell nuclei per mm2 (density) at the ITF regions (1 × 1 mm) was calculated for each ROI in LM-uADC and LM-uLMS (Supplementary Table S1 ) and statistical differences were obtained by Student’s t-Test.
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