Abstract A050: Validation of β-catenin as a tumor segmentation marker for delineating tumor from stromal tissues in quantitative multiplex immunofluorescence analysis of formalin-fixed, paraffin-embedded biopsy specimens
Authors: Navas Tony , Kinders Robert J. , Makhlouf Hala , Lawrence Scott M. , Chuaqui Rodrigo , Fino Krisitin , Dull Angie , Ferry-Galow Katherine , Wilsker Deborah , Chen Alice P. , Kummar Shivaani , Parchment Ralph E. , Doroshow James H. 
Published: March 2020

Abstract
Background: Immunofluorescence assay (IFA) analysis of pharmacodynamic biomarkers in tumor biopsy tissue is limited by lack of a tumor histology–agnostic methodology for segmenting tumor tissue from the surrounding stroma. We have developed a tumor segmentation methodology utilizing β-catenin staining in combination with cell morphology to manually segment tumor regions from surrounding mesenchyme. Here, we show extensive validation of the use of β-catenin as a general tumor segmentation marker through the examination of various tumor tissue microarrays (TMAs), including both carcinoma and sarcoma specimens. Methods: We examined TMAs comprised of tumors from 4 different histologies: colorectal carcinoma (CRC), non-small cell lung cancer (NSCLC), breast cancer, and sarcoma. For each TMA specimen, we quantified tumor content via hematoxylin and eosin (H&E) staining and then assessed tumor expression of β-catenin and other established phenotypic markers of tumor differentiation by IFA and immunohistochemical (IHC) analysis, respectively. To assess b-catenin expression in tumor vs. stroma, we used a pathologist-guided annotation of specific tumor marker staining to segment tumor from stroma on sections adjacent to those stained with an immunofluorescence-conjugated β-catenin antibody; tissue overlay and area reconciliation enabled determination of the ratio of β-catenin expression in tumor vs. stroma in regions of interest (ROIs) within each tumor core. The tumor markers utilized for each histology were: carcinoembryonic antigen (CEA) and pan-cytokeratin (pan-CK) for CRC and NSCLC; MUC1, CEA, and pan-CK for breast; and desmin and pan-CK for sarcoma. Results: We detected β-catenin immunofluorescence in 169 of the 185 (91%) carcinoma TMA tumors (58/61 CRC, 57/62 NSCLC, 54/62 breast). The mean β-catenin fluorescence intensity threshold ratios in tumor vs. stroma were: CRC (3.6), NSCLC (1.5) and breast (1.1). Although IHC staining of pan-CK was detected in 183/185 (99%) carcinoma tumors (59/61 CRC, 61/62 NSCLC, 61/62 breast), pan-CK immunofluorescence is more diffuse and cytoplasmic in nature compared to β-catenin and therefore less robust as a marker for IFA-based automated tumor tissue segmentation. In addition, we examined β-catenin expression in sarcoma TMAs, detecting β-catenin in 45 of 98 sarcoma TMAs examined (46%), which was superior to both pan-CK (9 of 98; 9%) and the known sarcoma marker desmin (37 of 98 (38%). Conclusions: Together, these results demonstrate that our β-catenin–based tumor segmentation method is useful for the majority of carcinoma specimens and allows automated co-registration of tumor and pharmacodynamic biomarkers defined by tumor versus stromal areas. The same approach worked for certain sarcoma specimens. We have since developed a Definiens-based algorithm that allows automated β-catenin–based tumor segmentation for specimens from entire biopsy cohorts of NCI-sponsored clinical trials, including those examining DNA damage response and immuno-oncology biomarkers. Funded by NCI Contract No HHSN261200800001E.
Citation Format: Tony Navas, Robert J. Kinders, Hala Makhlouf, Scott M. Lawrence, Rodrigo Chuaqui, Krisitin Fino, Angie Dull, Katherine Ferry-Galow, Deborah Wilsker, Alice P. Chen, Shivaani Kummar, Ralph E. Parchment, James H. Doroshow. Validation of β-catenin as a tumor segmentation marker for delineating tumor from stromal tissues in quantitative multiplex immunofluorescence analysis of formalin-fixed, paraffin-embedded biopsy specimens [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A050. doi:10.1158/1535-7163.TARG-19-A050