EpCAM Research Articles

Comparative Immunohistochemical Analysis of Clinicopathological Subgroups in Hepatocellular Carcinomas from Japan and Indonesia

BackgroundStandardized pathological evaluation based on immunohistochemical (IHC) analysis could improve HCC diagnoses worldwide. We evaluated differences in clinicopathological subgroups in HCCs from two academic institutions in Tokyo-Japan, and Jakarta-Indonesia. MethodsClinicopathological parameters and molecular expression patterns were evaluated in 35 HCCs from Indonesia and 41 HCCs from Japan. IHC analysis of biliary/stem-cell (B/S) markers (cytokeratin 19, sal-like protein 4, epithelial cell adhesion molecule) and Wnt/β-catenin (W/B) signaling-related molecules (β-catenin, glutamine synthetase) could determine the IHC-based subgroups. For immunosubtypes categorization, CD3/CD79α double immunohistochemistry was done to evaluate the infiltration of T and B cells. CD34 staining allowed identification of vessels that encapsulated tumor clusters (VETC). ResultsIndonesian HCC patients were mostly <60 years old (66%) with a hepatitis B virus (HBV) background (82%), in contrast to Japanese HCC patients (8% and 19%, respectively, both P<0.001). In comparison with Japanese, Indonesian cases more frequently had >5 cm tumor size (74% vs 23%, P=0.001), poor differentiation (40% vs 24%), portal vein invasion (80% vs 61%), and AFP levels >500 ng/ml (45% vs 13%, P=0.005). No significant differences were found in the proportions of B/S, W/B, and -/- subgroups from both countries. No Immune-high tumors were observed among Indonesian cases, and Immune-low tumors (66%) were more common than in Japanese cases (54%). VETC-positive tumors in Indonesia were significantly more common (29%), and most were in the HBV (90%) and -/- subgroups (90%), whereas Japanese VETC cases (10%, P=0.030) were nonviral (100%) and W/B subgroups (75%). ConclusionIHC-based analysis more precisely reflected the clinicopathological differences of HCCs in Japan and Indonesia. These findings provide new insights into standardization attempts and HCC heterogeneity among countries.

Regulation of the Function and Expression of EpCAM.

The epithelial cell adhesion molecule (EpCAM) is a single transmembrane protein on the cell surface. Given its strong expression on epithelial cells and epithelial cell-derived tumors, EpCAM has been identified as a biomarker for circulating tumor cells (CTCs) and exosomes and a target for cancer therapy. As a cell adhesion molecule, EpCAM has a crystal structure that indicates that it forms a cis-dimer first and then probably a trans-tetramer to mediate intercellular adhesion. Through regulated intramembrane proteolysis (RIP), EpCAM and its proteolytic fragments are also able to regulate multiple signaling pathways, Wnt signaling in particular. Although great progress has been made, increasingly more findings have revealed the context-specific expression and function patterns of EpCAM and their regulation processes, which necessitates further studies to determine the structure, function, and expression of EpCAM under both physiological and pathological conditions, broadening its application in basic and translational cancer research.

Epithelial Cell Adhesion Molecule (EpCAM) Expression in Human Tumors: A Comparison with Pan-Cytokeratin and TROP2 in 14,832 Tumors.

EpCAM is expressed in many epithelial tumors and is used for the distinction of malignant mesotheliomas from adenocarcinomas and as a surrogate pan-epithelial marker. A tissue microarray containing 14,832 samples from 120 different tumor categories was analyzed by immunohistochemistry. EpCAM staining was compared with TROP2 and CKpan. EpCAM staining was detectable in 99 tumor categories. Among 78 epithelial tumor types, the EpCAM positivity rate was ≥90% in 60 categories-including adenocarcinomas, neuroendocrine neoplasms, and germ cell tumors. EpCAM staining was the lowest in hepatocellular carcinomas, adrenocortical tumors, renal cell neoplasms, and in poorly differentiated carcinomas. A comparison of EpCAM and CKpan staining identified a high concordance but EpCAM was higher in testicular seminomas and neuroendocrine neoplasms and CKpan in hepatocellular carcinomas, mesotheliomas, and poorly differentiated non-neuroendocrine tumors. A comparison of EpCAM and TROP2 revealed a higher rate of TROP2 positivity in squamous cell carcinomas and lower rates in many gastrointestinal adenocarcinomas, testicular germ cell tumors, neuroendocrine neoplasms, and renal cell tumors. These data confirm EpCAM as a surrogate epithelial marker for adenocarcinomas and its diagnostic utility for the distinction of malignant mesotheliomas. In comparison to CKpan and TROP2 antibodies, EpCAM staining is particularly common in seminomas and in neuroendocrine neoplasms.

Single-cell SERS imaging of dual cell membrane receptors expression influenced by extracellular matrix stiffness

Membrane receptors perform a diverse range of cellular functions, accounting for more than half of all drug targets. The mechanical microenvironment regulates cell behaviors and phenotype. However, conventional analysis methods of membrane receptors often ignore the effects of the extracellular matrix stiffness, failing to reveal the heterogeneity of cell membrane receptors expression. Herein, we developed an in-situ surface-enhanced Raman scattering (SERS) imaging method to visualize single-cell membrane receptors on substrates with different stiffness. Two SERS substrates, Au@4-mercaptobenzonitrile@Ag@Sgc8c and Au@4-pethynylaniline@Ag@SYL3c, were employed to specifically target protein tyrosine kinase-7 (PTK7) and epithelial cell adhesion molecule (EpCAM), respectively. The polyacrylamide (PA) gels with tunable stiffness (2.5–25 kPa) were constructed to mimic extracellular matrix. The simultaneous SERS imaging of dual membrane receptors on single cancer cells on substrates with different stiffness was achieved. Our findings reveal decreased expression of PTK7 and EpCAM on cells cultured on stiffer substrates and higher migration ability of the cells. The results elucidate the heterogeneity of membrane receptors expression of cells cultured on the substrates with different stiffness. This single-cell analysis method offers an in-situ platform for investigating the impacts of extracellular matrix stiffness on the expression of membrane receptors, providing insights into the role of cell membrane receptors in cancer metastasis.

Identification of functional and diverse circulating cancer-associated fibroblasts in metastatic castration-naïve prostate cancer patients.

In prostate cancer (PCa), cancer-associated fibroblasts (CAFs) promote tumor progression, drug resistance, and metastasis. Although circulating tumor cells are studied as prognostic and diagnostic markers, little is known about other circulating cells and their association with PCa metastasis. Here, we explored the presence of circulating CAFs (cCAFs) in metastatic castration-naïve prostate cancer (mCNPC) patients. cCAFs were stained with fibroblast activation protein (FAP), epithelial cell adhesion molecule (EpCAM), and receptor-type tyrosine-protein phosphatase C (CD45), then FAP+EpCAM- cCAFs were enumerated and sorted using fluorescence-activated cell sorting. FAP+EpCAM- cCAFs ranged from 60 to 776 (389 mean ± 229 SD) per 2 × 108 mononuclear cells, whereas, in healthy donors, FAP+ EpCAM- cCAFs ranged from 0 to 71 (28 mean ± 22 SD). The mCNPC-derived cCAFs showed positivity for vimentin and intracellular collagen-I. They were viable and functional after sorting, as confirmed by single-cell collagen-I secretion after 48 h of culturing. Two cCAF subpopulations, FAP+CD45- and FAP+CD45+, were identified, both expressing collagen-I and vimentin, but with distinctly different morphologies. Collectively, this study demonstrates the presence of functional and viable circulating CAFs in mCNPC patients, suggesting the role of these cells in prostate cancer.

Single-cell transcriptomics reveal metastatic CLDN4+ cancer cells underlying the recurrence of malignant pleural effusion in patients with advanced non-small-cell lung cancer.

Recurrent malignant pleural effusion (MPE) resulting from non-small-cell lung cancer (NSCLC) is easily refractory to conventional therapeutics and lacks predictive markers. The cellular or genetic signatures of recurrent MPE still remain largely uncertain. 16 NSCLC patients with pleural effusions were recruited, followed by corresponding treatments based on primary tumours. Non-recurrent or recurrent MPE was determined after 3-6 weeks of treatments. The status of MPE was verified by computer tomography (CT) and cytopathology, and the baseline pleural fluids were collected for single-cell RNA sequencing (scRNA-seq). Samples were then integrated and profiled. Cellular communications and trajectories were inferred by bioinformatic algorithms. Comparative analysis was conducted and the results were further validated by quantitative polymerase chain reaction (qPCR) in a larger MPE cohort from the authors' centre (n=64). The scRNA-seq revealed that 33590 cells were annotated as 7 major cell types and further characterized into 14 cell clusters precisely. The cell cluster C1, classified as Epithelial Cell Adhesion Molecule (EpCAM)+ metastatic cancer cell and correlated with activation of tight junction and adherence junction, was significantly enriched in the recurrent MPE group, in which Claudin-4 (CLDN4) was identified. The subset cell cluster C3 of C1, which was enriched in recurrent MPE and demonstrated a phenotype of ameboidal-type cell migration, also showed a markedly higher expression of CLDN4. Meanwhile, the expression of CLDN4 was positively correlated with E74 Like ETS Transcription Factor 3 (ELF3), EpCAM and Tumour Associated Calcium Signal Transducer 2 (TACSTD2), independent of driver-gene status. CLDN4 was also found to be associated with the expression of Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A) and Vascular Endothelial Growth Factor A (VEGFA), and the cell cluster C1 was the major mediator in cellular communication of VEGFA signalling. In the extensive MPE cohort, a notably increased expression of CLDN4 in cells from pleural effusion among patients diagnosed with recurrent MPE was observed, compared with the non-recurrent group, which was also associated with a trend towards worse overall survival (OS). CLDN4 could be considered as a predictive marker of recurrent MPE among patients with advanced NSCLC. Further validation for its clinical value in cohorts with larger sample size and in-depth mechanism studies on its biological function are warranted. Not applicable.

Hybridized quantum dot, silica, and gold nanoparticles for targeted chemo-radiotherapy in colorectal cancer theranostics

Multimodal nanoparticles, utilizing quantum dots (QDs), mesoporous silica nanoparticles (MSNs), and gold nanoparticles (Au NPs), offer substantial potential as a smart and targeted drug delivery system for simultaneous cancer therapy and imaging. This method entails coating magnetic GZCIS/ZnS QDs with mesoporous silica, loading epirubicin into the pores, capping with Au NPs, PEGylation, and conjugating with epithelial cell adhesion molecule (EpCAM) aptamers to actively target colorectal cancer (CRC) cells. This study showcases the hybrid QD@MSN-EPI-Au-PEG-Apt nanocarriers (size ~65 nm) with comprehensive characterizations post-synthesis. In vitro studies demonstrate the selective cytotoxicity of these targeted nanocarriers towards HT-29 cells compared to CHO cells, leading to a significant reduction in HT-29 cell survival when combined with irradiation. Targeted delivery of nanocarriers in vivo is validated by enhanced anti-tumor effects with reduced side effects following chemo-radiotherapy, along with imaging in a CRC mouse model. This approach holds promise for improved CRC theranostics.

Abstract 5310: KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, demonstrates antitumor effects in combination with standard therapies for NSCLC

Abstract CD40 is a key molecule expressed on antigen presenting cells (APCs), whose signaling activates APCs and generates antitumor immune responses. Although CD40 agonists have been investigated in clinical trials, systemic toxicities have limited their full potential in providing adequate antitumor effects. To overcome this limitation, we developed KK2269, a bispecific antibody, using REGULGENT™ technology. KK2269 binds to CD40 and tumor-expressed epithelial cell adhesion molecule (EpCAM) to allow activation of CD40+ APCs only at EpCAM+ tumor sites, thus avoiding systemic toxicity. EpCAM is overexpressed in many types of epithelial cancers, including non-small cell lung cancer (NSCLC). KK2269 exerts synergistic antitumor activity and tumor-specific immune responses in combination with docetaxel, a common chemotherapy for NSCLC, in an EpCAM-expressing tumor mouse model. Hence, we aimed to evaluate KK2269 for the treatment of NSCLC. Docetaxel is used as a combination therapy with ramucirumab, an anti-VEGF receptor 2 (VEGFR2) antibody, for previously treated advanced NSCLC. In addition, many clinical studies are evaluating the add-on effects of PD-1/PD-L1 blockade combined with standard chemotherapies (including docetaxel) in NSCLC, which may become the standard of care for advanced NSCLC in the future. Therefore, we investigated the antitumor effect of KK2269 in combination with docetaxel + anti-mouse VEGFR2 (study 1) and with docetaxel + anti-mouse PD-1 (study 2) using a mouse EpCAM-expressing B16F10 tumor-bearing human CD40 transgenic mouse model. The prolongation of TTP5X (the day on which the tumor volume is observed to be ≥5-fold of that observed on day 0) was evaluated. In study 1, the median TTP5X (95% confidence interval [CI]) with the control, docetaxel + anti-VEGFR2, and triple combination (KK2269, docetaxel, anti-VEGFR2) was 10.0 days (7.0, 10.0), 26.0 days (17.0, 28.0), and 52.0 days (42.0, not calculable), respectively. The TTP5X was significantly prolonged with the triple combination compared with the control (p=0.000007, log-rank test) and docetaxel + anti-VEGFR2 (p=0.000004, log-rank test). In study 2, the median TTP5X (95% CI) was 7.0 days (not calculable), 9.0 days (6.0, 11.0), 16.0 days (7.0, 18.0), and 26.5 days (7.0, 32.0) with the control, KK2269, docetaxel + anti-PD-1, and triple combination (KK2269, docetaxel, anti-PD-1), respectively. TTP5X was significantly prolonged with the triple combination compared with the control, KK2269, and docetaxel + anti-PD-1 (p&amp;lt;0.0001, p=0.0003, and p=0.0023, respectively, log-rank test). Thus, a significant antitumor effect was demonstrated when KK2269 was added to the standard treatment of NSCLC (docetaxel + anti-VEGFR2 antibody) and to docetaxel + anti-PD-1 antibody. Our findings strongly suggest that KK2269 provides improved treatment options for patients with NSCLC. Citation Format: Yuta Tezuka, Kyoko Kawasaki, Yoshiki Sumitomo, Masato Saito, Akari Yao, Munetoshi Ando. KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, demonstrates antitumor effects in combination with standard therapies for NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5310.

Abstract 1882: DXC024, a novel anti-TROP2/EGFR bispecific antibody and tubulysin conjugate, for targeted treatment of highly TROP2- or EGFR-expressing tumors

Abstract Antibody drug conjugates (ADCs) which combine the precision of targeted therapy with the cytotoxic effects of chemotherapy have become a promising drug class in cancer therapy. Epidermal growth factor receptor (EGFR) is overexpressed in a wide diversity of epithelial tumors, promoting cell proliferation and survival pathways. Tumor-associated calcium signal transducer 2 (Trop2) is also expressed in a wide range of solid tumors and has been used as a transport gate for cytotoxic agents into cells in antibody-drug conjugate constructions for clinic applications. However, the approved Trop2 ADC, sacituzumab govitecan, only showed an objective response rate (ORR) of 17% and median OS of 9.5 months for lung cancers. In addition, both EGFR-ADC and Trop2-ADC had exhibited strong side effects in clinical trials due to their expression on some normal tissues, such as skin, mouth, eyes, etc. We generated bispecific Fab/scFv antibodies targeting both TROP2 and EGFR using the knob-into-hole technology, which presumably have wider efficacy, less on-target toxicity in comparison to the ADCs constructed based on the fully individual EGFR and Trop2 IgG antibodies having strong affinity to either EGFR or Trop2 antigens. Indeed, many of generated anti-TROP2/EGFR BsAbs showed strong binding activity in TROP2highEGFRhigh cells and demonstrated over 60% internalization rate within 60 min in vitro. The selected BsAbs were subsequently conjugated with tubulysin B analogs with varieties of peptidyl linkers to generate bispecific ADC (DXC024) candidates, two of which exhibited strong killing activity in A431, MDA-MB-468, Calu3, MCF-7 cells with IC50 of single to ten digital pM. In vivo, they showed very good durable antitumor response at dose as low as 4.6 mg/Kg for one injection in Calu3 (TROP2highEGFRmoderate) xenograft model, and demonstrated enhanced anti-tumor efficacy in comparison to those of the same payload conjugated to the monoclonal TROP2 or EGFR ADCs. These results indicated that DXC024 would be a promising ADC candidate for targeted treatment of highly either TROP2- or EGFR-expressing tumors. Citation Format: Jingjing Zhu, Junxiang Jia, Huihui Guo, Xiangfei Kong, Yong Du, Zhicang Ye, Lingli Zhang, Yongxiang Chen, Lu Bai, Yunxia Zheng, Wei Zheng, Jun Zheng, Juan Wang, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Mengmeng Liu, Binbin Chen, Meng Dai, Fang Du, Miaomiao Chen, Zhixiang Guo, Qingliang Yang, Robert Y. Zhao. DXC024, a novel anti-TROP2/EGFR bispecific antibody and tubulysin conjugate, for targeted treatment of highly TROP2- or EGFR-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1882.

Abstract 7504: Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients

Abstract Introduction: Anaplastic lymphoma kinase positive non-small cell lung cancer (ALK+NSCLC) represents a distinct subtype of NSCLC characterized by oncogenic ALK gene mutations leading to ALK overexpression and activation in tumor cells. ALK+ NSCLC will frequently develop mutations during treatment, leading to nonresponse and eventually cancer relapse. Therefore, there is a critical need for sensitive technologies to monitor ALK+ NSCLC disease status in patients over time. To facilitate longitudinal monitoring, there has been a rapid evolution in diagnostic technologies leveraging circulating tumor cells (CTCs) as a liquid biopsy tool. CTCs can be collected non-invasively at various intervals throughout patient treatment. Moreover, they shed from solid tumors and disseminate into the bloodstream, which makes isolating CTCs a valuable source of genomic, transcriptomic, and proteomic insights pertaining to the primary tumor. In this study, we present a comprehensive workflow for conducting multi-omic analysis on CTCs derived from ALK+ NSCLC patients. Methods: Blood samples were collected from a cohort of ALK+ NSCLC patients, and CTCs were isolated using a high throughput, label free microfluidic device, Labyrinth. Enriched CTCs were immunofluorescently stained for cytokeratin, CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers. Heterogeneous CTC populations with hybrid epithelial/mesenchymal (EM) phenotypes are reported. Fluorescence in situ hybridization (FISH) was performed on enriched CTC samples. DNA was extracted and sequenced using a 523 gene targeted sequencing panel. Single cell RNA-seq technology was performed in parallel on CTC samples to investigate the gene expression throughout treatment. Results: The median concentration of CTCs in the ALK+ NSCLC cohort (n=33) is 137 CTCs per ml of blood, and 64% of CTCs are in an EM hybrid state. In ALK+ NSCLC patients (n=3) with an average of 4.6 visits per sample, we observed a trend of decreasing CTC concentration for patients with partial response to the treatment. The presence of ALK fusion in ALK+ NSCLC patient’s CTC is confirmed by FISH. Oncogenic mutations (JAK1, SPEN, ERBB4, NTRK3) are observed in bulk CTC samples from two NSCLC patients. Single cell RNA sequencing was performed on three ALK+ NSCLC patients; 58, 199, and 700 potential CTCs were detected with positive cytokeratin expression and no white blood cell marker expression. Conclusions: This study examined the feasibility of using CTCs for studying and monitoring the clinical status of the ALK+ NSCLC patients as well as the genomic and transcriptomic signatures within their tumors. More patients will need to be processed in order to validate our workflow and reinforce our conclusions. Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Zhaoping Qin, Albert Liu, Xu Cheng, Liwei Bao, Habib Serhan, Bryce Vandenburg, Varun Kathawate, Larua Goo, Peter Ulintz, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7504.

Abstract 5311: KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity

Abstract Several anti-CD40 agonist antibodies have been evaluated in clinical trials based on their preclinical antitumor activity via generation of antitumor innate and adaptive immunity through activation of antigen-presenting cells. However, conventional monospecific anti-CD40 agonist antibodies have faced limitations in terms of safety margins and clinical efficacy. To address this problem, we developed KK2269, a novel bispecific antibody (REGULGENT™ format) that binds to both CD40 and epithelial cell adhesion molecule (EpCAM), which is highly expressed in several types of tumors. KK2269 demonstrated EpCAM-dependent activation of CD40 signals both in vitro and in vivo, confirming that it is a tumor-targeting CD40 agonist. Liver metastasis is common in EpCAM-positive cancers, including colon, stomach, and lung cancers. Intrahepatic tumors can attenuate the efficacy of immune checkpoint inhibitors due to the liver tumor-specific suppressive immune milieu. Although CD40 agonists are expected to overcome the immunosuppressive environment, non-tumor-targeting CD40 agonists cause hepatotoxicity. In the present study, we used a syngeneic intrahepatic tumor model to examine whether KK2269 exhibits antitumor efficacy without causing hepatotoxicity. Firstly, we confirmed that KK2269 administration did not increase aspartate aminotransferase (AST) or cause pathological abnormalities in the liver despite Il12b induction in the EpCAM-positive tumor. Additionally, gene expression analysis of peritumoral normal liver revealed that KK2269 did not induce inflammatory cytokines. In contrast, a conventional anti-CD40 agonist antibody increased AST, caused pathological abnormalities, and upregulated inflammatory cytokines in peritumoral normal liver, as reported in the clinical setting. Secondly, the antitumor effect of KK2269 in combination with docetaxel was evaluated. The combination therapy synergistically reduced intrahepatic tumor volume, and the effect persisted even 21 days after the administration. Gene expression analysis revealed a lower immune infiltration in the intrahepatic tumor versus subcutaneous tumor and that KK2269 in combination with docetaxel increased T-cell infiltration and activation even in the immunosuppressive intrahepatic tumor. In summary, our study demonstrated that KK2269 activated dendritic cells in an EpCAM-positive tumor in the liver but did not affect peritumoral normal liver tissues. In addition, KK2269 in combination with docetaxel showed potent antitumor effect accompanied by T-cell infiltration and activation against the intrahepatic tumor, which has a lower immune infiltration. These results suggest that the combination therapy of KK2269 and docetaxel is promising for metastatic EpCAM-positive tumors. Citation Format: Yoshiki Sumitomo, Kyoko Kawasaki, Yuta Tezuka, Masato Saito, Akari Yao, Munetoshi Ando. KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5311.

Abstract 6939: Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program

Abstract Background: In HER2+ breast cancer (BC), residual disease (RD) after neoadjuvant HER2 inhibition is associated with increased risk of recurrence. However, the genetic and non-genetic resistance mechanisms underlying RD remain incompletely characterized. Here we used single nuclei (sn) RNA and DNA sequencing to profile genetic and transcriptional changes in growing, residual, and recurrent tumors from an inducible mouse tumor model of HER2+ breast cancer. Methods: We used mammary tumors/tumor bed (n=2-3/time point) from female MMTV-rtTA; TetO-ERBB2 mice at 9 weeks DoxOn (HER2on), 7 days DoxOff (HER2off) and 12-21 weeks Doxoff (recurrent). Samples were snap frozen and single nuclei isolation was performed. snRNAseq was performed using 10 × 5’ v2 kits for library preparation and sequencing. snDNAseq was performed using a custom amplicon panel targeting the mouse genome and human ERBB2 locus via the MissionBio Tapestri platform. Results: For snRNAseq analysis, tumor cell clusters were identified by Epcam and ERBB2 gene expression. Tumor cells were further subclustered into 18 unique clusters that separated by ERBB2 expression. Seven subclusters of HER2on tumor cells were characterized by low ERRB2 expression: 1.38 vs 2.94 (median normalized log counts) in HER2on-ERBB2high cells. Top 5 differentially expressed genes (DEG) per condition (log2 fold change &amp;gt;1.5, adjusted p value &amp;lt; 0.05) were: HER2on: Erbb2, Csn3, Lcn2, Csn2, Wfdc18; HER2on-ERBB2high: Csn3, Igfbp5, Csn2, Lcn2, Trf; HER2on-ERBB2low: Lars2, Gm47283, Cmss1, Gm42418, Neat1; HER2off: Lars2, Hexb, Cmss1, Gphn, Gm42418; Recurrent: Capza2, Vim, Lgals1, Crabp1, S100a6. HER2on-ERBB2low cells were more closely related in transcriptional space to HER2off residual tumor cells than HER2on-ERBB2high tumor cells. Trajectory analysis suggested that HER2off RD evolved from HER2on-ERRB2low cells and formed the basis of recurrent disease. To determine clonal evolution over time, we used common top 10 DEGs between HER2on vs HER2off vs HER2low snRNAseq data and intersected that set with significantly enriched single nucleotide variants (SNVs) from our snDNAseq data. We identified 16 clones containing at least 1 SNV at &amp;gt;1% frequency across all samples. We observed clonal persistence, relative enrichment and extinguishment between HER2on, HER2off and recurrent samples but without clonal dominance. Conclusion: Time-series analysis of genetic and transcriptional evolution in a mouse model of HER2+ BC RD suggests that a tumor cell subpopulation characterized by an ERBB2low-associated transcriptional program drives RD after HER2 inhibition. Citation Format: Sheheryar K. Kabraji, Weihua Wang, Yali Zhang, Johann Bergholz, Tao Jiang, Vittal Kurisetty, Maia Homsi, Jianmin Wang, Jean Zhao. Residual disease after HER2 inhibition is driven by a primary tumor subpopulation expressing an ERBB2-low associated transcriptional program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6939.

Abstract 3136: Towards development of a novel biparatopic trop2-ADC

Abstract Background: Trophoblast cell surface antigen 2 (TROP-2) is a member of Tumor-associated Calcium Signal Transducer family (TACSTD2), which is a 35kDa transmembrane protein with 4 N-linked glycosylation sites. The overexpression of TROP2 in various cancers is associated with poor prognosis and increased risk of metastasis. Anti-TROP2 antibody drug conjugates (ADCs) are emerging as an innovative therapeutic intervention for cancer treatment. While these efforts were rewarded by the FDA approval of Trodelvy (Sacituzumab govitecan), a Trop2 targeted ADC, in 2020 for the TNBC patients, tumor heterogeneity could present one of the limitations for the Trop2-targeting therapy, leading to drug resistance. Here, we report our effort in developing a novel biparatopic TROP2 ADC which demonstrate superior cell killing activity compared with the monospecific TROP2 ADC, including certain resistant cell lines for the approved therapy. Methods: Various biparatopic Abs were engineered using two different mAbs targeting the two non-overlapping epitopes of Trop2. The physical stability and biological activities were characterized. Binding competition assay was utilized for epitope binning using SPR. Cell binding affinity was determined using flow cytometry. Cellular internalization activity was measured using Incucyte system, and tumor cell killing efficacy was evaluated in multiple cancer cell lines. CDX mouse models were utilized to investigate the TROP2-ADC in vivo efficacy. Results: We identified a biparatopic TROP2 mAb using two monospecific TROP2 mAbs with A recognizing the same epitope as Trodelvy, and B binding to a non-overlapping epitope vs Sacituzumab’s. mAb-B demonstrates superior ability to recognize a few resistance cell lines to mAb-A. The biparatopic TROP2 mAb-A/B displays a KD of 0.81 nM protein binding compared with 6.42 nM and 0.26 nM, respectively for mAb-A and mAb-B. Importantly, the biparatopic mAb-A/B demonstrates significantly faster cellular internalization activity compared to those of the individual mAbs, signifying the biparatopic scaffold could drive receptor clustering and subsequently enhance the internalization, trafficking to the lysosome and degradation of the target. As expected, the ADC of the biparatopic mAb-A/B conjugated with a TOPi payload shows cancer cell killing efficacy in both in vitro and in vivo experiments. Furthermore, we tested mAb-A, -B and biparatopic mAb-A/B for their binding affinity to 14 missense mutations in TROP2 ECD expressed in HEK293 cells. Interestingly, mAb-A showed decreased binding in 5 mutant cell lines, while mAb-B and biparatopic mAb-A/B maintains activity. Conclusion: We have engineered a novel TROP2 biparatopic antibody with unique biological features as well as superior in vitro and in vivo activities. Such a biparatopic ADC presents its potential to be a best-in-class ADC therapy by overcoming the limitation of the monospecific ADC therapy such as drug resistance. Citation Format: Yinan Wu, Brittany Jiang, Julliet Rivera, Fang Wang, Jing Zhou, Lin Liu, Silvia Hudson, Will Draper, Jennifer Li, Xiaoshan Min, Hang Chen, Zhulun Wang. Towards development of a novel biparatopic trop2-ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3136.

Abstract 1542: Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal cancer

Abstract Epithelial cell adhesion molecule (EpCAM) is a pleiotropic type-1 transmembrane glycoprotein and known cancer stem cell marker, yet its tumorigenic mechanisms remain elusive. Cancer stem cells (CSC) have been implicated in cancer progression and recurrence, but targeting them with therapeutics is a major challenge. It is known that EpCAM is highly expressed in CSCs of different cancer types. Here, we studied the role of EpCAM in colorectal cancer (CRC) stemness. First, we show that the extracellular domain of EpCAM (EpEX) functions as a ligand for Wnt receptors, frizzled6/7 and LRP5/6, while the intracellular domain (EpICD) upregulates transcription of these receptors. Further, EpEX-induced Wnt signaling activates TACE and γ-secretase enzymes to augment shedding of EpEX and EpICD, establishing a positive feedback loop. Importantly, we show that the EpCAM-neutralizing antibody, EpAb2-6, targets the Wnt pathway to attenuate stemness characteristics in colorectal cancer (CRC). Our EpCAM-neutralizing antibody (EpAb2-6) and a porcupine inhibitor (LGK974) each partially attenuated cancer stemness, while their combination abolished stemness-related endpoints, induced apoptosis in vitro and markedly diminished tumor progression in animal models of human CRC. From these findings, we conclude that EpCAM stimulates Wnt signaling to promote cancer stemness, and the combination of EpAb2-6 and porcupine inhibitors may represent an effective CRC treatment, including for KRAS-mutant cancers. Our findings reveal an opportunity to combat drug resistance in tumor stem cells. Thus, the mechanistic insights gained from our study may be useful to improve existing treatments or to develop novel anticancer therapeutics. Citation Format: Han-Chung Wu, Sushree Shankar Panda, Kai-Chi Chen, Chi-Chiu Lee, Hsing-Pang Hsieh, Yi-Jen Su. Epithelial cell adhesion molecule induces canonical Wnt pathway to promote stemness in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1542.

Diagnostic Performance of Epithelial Cell Adhesion Molecule for Early Detection of Hepatocellular Carcinoma Among HCV High-Risk Patients.

Hepatocellular carcinoma (HCC) is often diagnosed at advanced stage where hopeless for effective therapies. Identification of more reliable biomarkers for early detection of HCC is urgently needed. Circulating tumor cells (CTCs) represent a unique liquid biopsy carrying comprehensive biological information of the primary tumor. Herein, we sought to develop a novel score based on the combination of the most significant CTCs biomarkers with routine laboratory tests for accurate detection of HCC. Epithelial cell adhesion molecule (EpCAM), α-fetoprotein, albumin, and platelets count were assayed in HCC patients (98), liver cirrhosis patients (77). Areas under receiving operating curve (AUCs) were calculated and used for construction on novel score. A novel score named EpCAM-HCC = AFP (U/L) × 0.11 - Albumin (g/dl) × 1.5 + EpCAM % × 2.9 - Platelets count (×109)/L× 0.75 - 93. EpCAM-HCC score produce AUC of 1 for differentiate patients with HCC from those with liver cirrhosis with sensitivity and specificity of a cut-off 1.7 (i.e., less than 1.7 the case is considered cirrhotic, whereas above 1.7 it is considered HCC. EpCAM-HCC score could replace AFP during screening of HCV patients and early detection of HCC.

Abstract IA016: Molecular diversity of uterine serous carcinoma

Abstract Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer with a high recurrence rate and poor prognosis disproportionately affecting black women. Whole-exome-sequencing analysis revealed that up to 10% of USC harbor features of ultra/hypermutation due to a deficiency of polymerase e (POLE) and/or mismatched-repair genes (MMRd). These tumors are notable for having a high number of single nucleotide variants (SNV), no LOH segments or copy-number variants (CNV) and a peculiar genetic signature characterized by an increased rate of C &amp;gt; A transversions. In contrast, the overwhelming majority of USC (~ 90%) are POLE/MMR proficient, have a low number of SNV and demonstrate alteration in genes including TP53, PIK3CA mutation/amplification, ERBB2 amplification, CCNE1 amplification, FBXW7 mutation/deletion, PPP2R1A mutation, and somatic mutations involving homologous recombination genes. Frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling-complex, and TAF1, an element of the core TFIID transcriptional machinery are also detected in a significant number of USC. Somatic CNVs, a sign of genomic instability, are identified in the majority of USC with multiple copy number gains and copy number losses, with the most frequent events taking place in pathways similar to those found from analysis of SNVs. In addition to loss of TP53, frequent deletion of a small segment of chromosome 19 containing MBD3, a member of the NuRD-chromatin-modification-complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (encoding for the HER2/neu receptor) and CCNE1 (a target of FBXW7-mediated ubiquitination) are commonly detected. Overall, these findings suggest that both somatic point mutations (SNV) and somatic CNV play a major role in the pathogenesis of USC. Importantly, the frequent mutation of DNA damage, chromatin remodeling, cell cycle and cell proliferation pathways in USC including the overexpression/amplification of HER2/neu, CCNE1 and the tumor-associated calcium signal transducer 2 (TACSTD2/TROP2) genes, suggest novel targets for therapy against this lethal variant of endometrial cancer. Citation Format: Alessandro D. Santin. Molecular diversity of uterine serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA016.

Síndrome de Lynch. Cáncer de colon familiar no sindrómico

Lynch syndrome is the most common hereditary colorectal cancer (CRC) syndrome. It accounts for 2% to 4% of all CRC cases globally. It follows an autosomal dominant inheritance pattern and is defined by the presence of a pathogenic germline variant of one of the genes that encodes for DNA repair proteins (MMR), MLH1, MSH2, MSH6, PMS2, or at the 3’ end of the EPCAM gene. This syndrome entails a greater risk of developing CRC and endometrial cancer, in addition to other tumors. Its diagnosis is based on clinical criteria that take into account the family and personal history of cancer, the detection of a deficient MMR phenotype in the tumor tissue, and a genetic study of the germline of individuals at risk of having it. Its early diagnosis allows for establishing screening strategies to try to prevent or detect the presence of tumors in early stages which, in the case of CRC, has been shown to increase survival. Nonsyndromic familial CRC includes cases that have a familial component without a well-established molecular mechanism. They may benefit from more intensive follow-up than what is established for the general population.

Quantitative Evaluation by Digital Pathology of Immunohistochemical Expression of CK7, CK19, and EpCAM in Advanced Stages of NASH.

(1) Background: Nonalcoholic Steatohepatitis/Nonalcoholic Fatty Liver Disease (NASH/NAFLD) is the most recurrent chronic liver disease. NASH could present with a cholestatic (C) or hepatic (H) pattern of damage. Recently, we observed that increased Epithelial Cell Adhesion Molecule (EpCAM) expression was the main immunohistochemical feature to distinguish C from H pattern in NASH. (2) Methods: In the present study, we used digital pathology to compare the quantitative results of digital image analysis by QuPath software (Q-results), with the semi-quantitative results of observer assessment (S-results) for cytokeratin 7 and 19, (CK7, CK19) as well as EpCAM expression. Patients were classified into H or C group on the basis of the ratio between alanine transaminase (ALT) and alkaline phosphatase (ALP) values, using the "R-ratio formula". (3) Results: Q- and S-results showed a significant correlation for all markers (p < 0.05). Q-EpCAM expression was significantly higher in the C group than in the H group (p < 0.05). Importantly ALP, an indicator of hepatobiliary disorder, was the only biochemical parameter significantly correlated with Q-EpCAM. Instead, Q-CK7, but not Q-CK19, correlated only with γGlutamyl-Transferase (γGT). Of note, Stage 4 fibrosis correlated with Q-EpCAM, Q-CK19, and ALP but not with γGT or ALT. Conclusions: Image analysis confirms the relation between cholestatic-like pattern, associated with a worse prognosis, with increased ALP values, EpCAM positive biliary metaplasia, and advanced fibrosis. These preliminary data could be useful for the implementation of AI algorithms for the assessment of cholestatic NASH.

Functionalized tetrahedral DNA frameworks for the capture of circulating tumor cells.

Identification and characterization of circulating tumor cells (CTCs) from blood samples of patients with cancer can help monitor parameters such as disease stage, disease progression and therapeutic efficiency. However, the sensitivity and specificity of current multivalent approaches used for CTC capture is limited by the lack of control over the ligands' position. In this Protocol Update, we describe DNA-tetrahedral frameworks anchored with aptamers that can be configured with user-defined spatial arrangements and stoichiometries. The modified tetrahedral DNA frameworks, termed 'n-simplexes', can be used as probes to specifically target receptor-ligand interactions on the cell membrane. Here, we describe the synthesis and use of n-simplexes that target the epithelial cell adhesion molecule expressed on the surface of CTCs. The characterization of the n-simplexes includes measuring the binding affinity to the membrane receptors as a result of the spatial arrangement and stoichiometry of the aptamers. We further detail the capture of CTCs from patient blood samples. The procedure for the preparation and characterization of n-simplexes requires 11.5 h, CTC capture from clinical samples and data processing requires ~5 h per six samples and the downstream analysis of captured cells typically requires 5.5 h. The protocol is suitable for users with basic expertise in molecular biology and handling of clinical samples.

Prognostic evaluation of a multi-target magnetic bead-enriched circulating tumor cell-enriched identification system for colorectal cancer.

Colorectal cancer (CRC) is the third most prevalent cancer in the world. Traditional tissue biopsy cannot provide dynamic monitoring of patients' tumors or reflect the characteristics of tumors in real time because the sampling process is invasive and accompanied by risks. Circulating tumor cells (CTCs) are considered a major cause of tumor metastasis, and investigating CTCs helps to understand the biology and vulnerability of malignant tumors during hematogenous metastasis. We sequentially used epithelial cell adhesion molecule (EpCAM)-coated immunoliposomal magnetic beads (Ep-IMBs) and vimentin-coated immunoliposomal magnetic beads (Vi-IMBs) to capture and characterize CTCs from 110 CRC patients. We further constructed a Cox risk regression model, optimized the model composition using backward stepwise regression, and finally applied nomograms to show the effect of each variable on survival risk. The specificity of the CTCs enrichment and identification system was 100% and the sensitivity was 79.0%. Multivariate analysis indicated total CTC number was an important predictor for bad survival, whereas American Joint Committee on Cancer (AJCC) stage, lymph node metastasis, and carcinoembryonic antigen (CEA) level were associated with prognosis, and the risk of mortality was associated with the AJCC stage of the CRC. The CTC enrichment and identification system constructed in this research demonstrated superior accuracy. In addition, CTCs can be used as an important predictor for prognosis of patients with CRC, and the combination of other clinical predictive factors can help clinicians to better design individualized treatment regimens, which is of great clinical application value.