Douglass, Eugene

Abstract Triple-Negative Breast Cancer (TNBC) presents a significant clinical challenge due to its heterogeneity and lack of targeted treatment options, with chemotherapy and immunotherapy combinations currently serving as the main therapeutic strategy. Efforts to address TNBC heterogeneity have largely focused on classifying intrinsic cancer subtypes based on differential tumor mRNA expression, a strategy that has proven effective in hormone receptor-positive breast cancers but has yet to yield a clinically useful predictor of survival or treatment response in TNBC. We hypothesize that both the intrinsic characteristics of TNBC and the surrounding immune microenvironment influence treatment outcomes and that immune cell infiltration affects TNBC subtype classification and response variability. To explore this hypothesis, we compared the predictive and prognostic capabilities of cancer subtype-based (TNBC-type) gene signatures and immune cell deconvolution methods (CIBERSORT) within the same TNBC datasets. We found that immune cell abundance outperformed TNBC subtype-signatures and multicellular immune cell aggregates showed the highest performance of all. More specifically, aggregate immune cells associated with tertiary lymphoid structures and tumor associated macrophages/monocytes demonstrated statistically significant predictive value. These findings were confirmed in an independent cohort of 67 TNBC patients treated with neoadjuvant chemotherapy. Further, single-cell RNA sequencing analysis revealed that the predictive power of cancer-subtype could be partially explained by immune- and stromal features. Examination of single-cell resolution spatial transcriptomic data confirmed presence of TLS-like, TAM- and cancer-stromal niches within TNBC biopsy samples that were associated with treatment response. Overall, our results highlight that immune cell aggregates, which capture the spatial organization of the TME, outperform cell-type specific gene signatures in predicting TNBC outcomes. Our novel approach provides a robust framework for interpreting spatial relationships in bulk RNA-seq data, offering a pathway for reconciling past data with current advancements in spatial profiling technologies. This work paves the way for future studies to leverage the multi-cellular complexity of TNBC, enhancing diagnostic precision and facilitating the development of therapies that strategically modulate the tumor microenvironment for improved anti-cancer responses.

Abstract The histologic heterogeneity of human Muscle Invasive Bladder Cancer (MIBC) is one of the main contributing factors to poor treatment outcomes. Recent reports on MIBC histological variants have highlighted increased levels of transcription factors (TF) associated with epithelial-mesenchymal transformation (EMT) and epigenetic regulation in the sarcomatoid histology variant of MIBC. Dogs with spontaneously developing MIBC have been shown to display similar histological and molecular features, in addition to closely mimicking the clinical behavior of human MIBC. Here, we add to previously reported data on histological variants in canine MIBC by evaluating 21 tumor biopsies for variant histology and performing bulk RNAseq analyses on 3 organoid cell lines derived from one sarcomatoid subvariant and two conventional urothelial carcinoma (UC). MIBC biopsies from 21 dogs were reviewed for variant histology by a human urologic pathologist. Organoids derived from the urine of canine MIBC patients were cultured, preserved in RNA later, and sequenced for mRNA expression to identify TF associated with sarcomatoid variants versus conventional UC. All canine MIBC biopsies were classified as invasive, high-grade UC. Histologic variants were identified as glandular differentiation in 7/21, sarcomatoid differentiation in 3/21, squamous differentiation in 3/21, micropapillary carcinoma in 1/21 MIBC samples and small nested variants in 1/21 samples; with the rest of the tumors classified as conventional UC. Raw RNAseq analyses revealed no clear clustering of expression profiles between the three organoid cell lines, indicating distinct RNA expression profiles between the sarcomatoid variants and conventional UC of canine MIBC. To evaluate differences in the expression of TF targets between the organoid cell lines, pathway analyses and VIPER algorithms were used. Organoids derived from sarcomatoid variant UC showed increased expression of Snail familiy transcription repressor 1 (SNAI1) along with decreased expression of cadherin 1 (CDH1), indicating a signature of EMT, similar to that previously identified in human MIBC organoids with sarcomatoid variant histology. Canine MIBC organoids derived from conventional UC histology samples showed a TF profile associated with proliferation, metastasis, and progression, such as increased Zinc finger E-box binding homeobox 1 (ZEB1), E2F transcription factor 3 (E2F3), and centromere protein F (CENPF), as previously described in humans with high grade, invasive UC. In conclusion, the frequency of some histologic variants in canine MIBC, such as sarcomatoid UC, appear to be higher than reported in human patients. TF analyses of canine MIBC-derived organoid cell lines showed evidence of EMT, similar to the sarcomatoid variants of MIBC in humans. Organoids derived from canine MIBC could potentially serve as a valuable model for evaluating the efficacy of novel treatment modalities for the rare histologic variants of UC. Citation Format: Karin Allenspach, Mohamed Elbadawy, Hannah Nocholson, Christopher Zdyrski, John Cheville, Eugene Douglass, Elizabeth W. Howerth, Lilian Oliveira, Jonathan P. Mochel. Sarcomatoid Histological Variants of Canine Muscle-Invasive Bladder Cancer: Transcription Factor Activation Highlights Pathways of Epithelial-Mesenchymal Transformation Similar to Humans [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR002.