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

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 (HE 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