IHC Analysis of HNF1alpha, SMAD4, and p53 in Genetic Variants

Immunohistochemistry (IHC) analysis was performed automatically with a Benchmark XT^® immunostainer (Ventana Medical Systems) HNF1alpha antigen. The immunohistochemical analysis was validated through positive controls (as an external positive control put on the slide according to Bragoni et al. [13 (link)]) and negative controls (by omitting the primary antibody). Cases carrying predicted damaging variants in HNF1alpha and cases with no variants in the gene were evaluated by IHC from FFPE surgical specimens. IHC was performed for HNF1alpha and scoring was carried out by two independent expert pathologists, blindly with respect to the mutation status.
IHC analysis for SMAD4 and p53 was carried out as described previously [12 (link)]. Evaluation of SMAD4 immunostains was performed by two expert pathologists. For each case, the percentage of neoplastic cells with SMAD4 preserved or lost immunosignal was collected.

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Orsini A., Mastracci L., Bozzarelli I., Ferrari A., Isidori F., Fiocca R., Lugaresi M., D’Errico A., Malvi D., Cataldi-Stagetti E., Spaggiari P., Tomezzoli A., Albarello L., Ristimäki A., Bottiglieri L., Krishnadath K.K., Rosati R., Fumagalli Romario U., De Manzoni G., Räsänen J., Martinelli G., Mattioli S, & Bonora E. (2023). Correlations between Molecular Alterations, Histopathological Characteristics, and Poor Prognosis in Esophageal Adenocarcinoma. Cancers, 15(5), 1408.

Publication 2023

Benchmark XT® immunostainer

Antibody Antigen Gene Hnf1alpha Immunohistochemistry Mutation Neoplastic Pathologists Smad4 Surgical

Corresponding Organization : University of Bologna

Other organizations : Ospedale Policlinico San Martino, University of Genoa, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Istituti di Ricovero e Cura a Carattere Scientifico, Maria Cecilia Hospital, Azienda USL di Bologna, Humanitas University, Azienda Ospedaliera Universitaria Integrata Verona, San Raffaele University of Rome, University of Helsinki, Helsinki University Hospital, European Institute of Oncology, Antwerp University Hospital, Vita-Salute San Raffaele University, University of Verona

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Variable analysis

independent variables

Presence or absence of predicted damaging variants in HNF1alpha gene

dependent variables

HNF1alpha protein expression level (measured by immunohistochemistry (IHC))
SMAD4 protein expression level (measured by IHC)
P53 protein expression level (measured by IHC)

control variables

Use of positive controls (external positive control on the slide) for HNF1alpha IHC
Use of negative controls (omitting primary antibody) for HNF1alpha IHC
Evaluation of IHC by two independent expert pathologists, blinded to mutation status

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