Trophoblast Cell Surface Research Articles

An assembly of TROP2-mediated signaling events.

Trophoblast cell surface antigen 2 (TROP2) is a calcium-transducing transmembrane protein mainly involved in embryo development. The aberrant expression of TROP2 is observed in numerous cancers, including triple-negative breast cancer, gastric, colorectal, pancreatic, squamous cell carcinoma of the oral cavity, and prostate cancers. The main signaling pathways mediated by TROP2 are calcium signaling, PI3K/AKT, JAK/STAT, MAPKs, and β-catenin signaling. However, collective information about the TROP2-mediated signaling pathway is not availablefor visualization or analysis. In this study, we constructed a TROP2 signaling map with respect to its role in different cancers. The data curation was done manually by following the NetPath annotation criteria. The described map consists of different molecular events, including 8 activation/inhibition, 16 enzyme catalysis, 19 gene regulations, 12 molecular associations, 39 induced-protein expressions, and 2 protein translocation. The data of the TROP2 pathway map is made freely accessible through the WikiPathways Database ( https://www.wikipathways.org/index.php/Pathway:WP5300 ). Development of TROP2 signaling pathway map.

Abstract 4396: TROP2 expression in non-small cell lung cancer

Abstract Introduction: Non-small cell lung cancer (NSCLC) is a major cause of cancer-related deaths globally with high unmet need. A variety of novel therapeutic strategies are being explored to improve patient outcomes including the use of antibody drug conjugates (ADCs) to deliver highly potent, cytotoxic payloads to tumors. Trophoblast cell-surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), has emerged as an attractive target for ADCs. Sacituzumab govitecan-hziy is a novel ADC composed of a TROP2 antibody coupled to SN-38 and is being evaluated in NSCLC. High TROP2 mRNA expression is observed in many tumor types, however, TROP2 protein expression in NSCLC is not well-established. Here we characterized TROP2 expression across three independent datasets to explore the TROP2 relationship to baseline characteristics, molecular features of interest, and its prognostic value in NSCLC. Methods: We analyzed three independent datasets: (1) The Cancer Genome Atlas (TCGA) NSCLC adenocarcinoma (LUAD, N=660) and squamous cell carcinoma (LUSC, N=484) mRNA expression data, (2) a NSCLC adenocarcinoma FFPE tumor sample set (“Translational data set”; N=107), (3) an independent clinical NSCLC adenocarcinoma (N=103) and squamous cell carcinoma (N=37) data set. TROP2 IHC was performed on tumor sample sets (2) and (3) using the SP295 antibody (Robust Prototype Assay, Ventana) with H-Score and assessment of the percentage of membrane-positive tumor cells as readouts. Wilcoxon rank-sum and Kruskal-Wallis tests (continuous variables), Kaplan-Meier method (survival probability curves) and log-rank test (time to event outcome) were used (GraphPad Prism 8.1.2 and R version 4.0.5). Results: TROP2 mRNA was highly expressed in NSCLC and expression was similar in adenocarcinoma and squamous cell carcinoma in the TCGA data set. TROP2 expression did not vary with patient’s age, gender, race or tumor stage. Furthermore, TROP2 expression was not associated with TP53, KRAS or driver mutation status (including EGFR, ALK, ROS, RET, MET). TROP2 expression in the Translational sample set was consistent with the findings from the TCGA data analysis: TROP2 mRNA and protein expression were high and independent of patient baseline demographics, tumor stage and TP53 and KRAS mutation status. Finally, in an independent clinical NSCLC dataset, TROP2 protein was highly expressed and there was no relationship between TROP2 and baseline demographics including gender and age. TROP2 membrane H-scores and percentage of membrane-positive tumor cells also highly correlated in the two independent datasets. Conclusions: Three large independent datasets confirmed high expression of TROP2 in NSCLC both, as mRNA and protein. TROP2 expression in tumors did not differ between histological subtypes, baseline characteristics or clinically relevant driver alterations. Based on the TCGA data set, TROP2 expression was not prognostic for survival. Citation Format: Peiwen Kuo, Emon Elboudwarej, Lauri Diehl, Jilpa Patel, Sabeen Mekan, Juliane M. Jürgensmeier. TROP2 expression in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4396.

Overview of Trop-2 in Cancer: From Pre-Clinical Studies to Future Directions in Clinical Settings.

Trophoblast cell surface antigen-2 (Trop-2) is a glycoprotein that was first described as a membrane marker of trophoblast cells and was associated with regenerative abilities. Trop-2 overexpression was also described in several tumour types. Nevertheless, the therapeutic potential of Trop-2 was widely recognized and clinical studies with drug-antibody conjugates have been initiated in various cancer types. Recently, these efforts have been rewarded with the approval of sacituzumab govitecan from both the Food and Drug Administration (FDA) and European Medicines Agency (EMA), for metastatic triple-negative breast cancer patients. In our work, we briefly summarize the various characteristics of cancer cells overexpressing Trop-2, the pre-clinical activities of specific inhibitors, and the role of anti-Trop-2 therapy in current clinical practice. We also review the ongoing clinical trials to provide a snapshot of the future developments of these therapies.

TROP2 expression and SN38 antitumor activity in malignant pleural mesothelioma cells provide a rationale for antibody-drug conjugate therapy

ObjectivesMalignant pleural mesothelioma (MPM) is an aggressive cancer which at large is not amenable to curative surgery. Despite the recent approval of immune checkpoint inhibitor therapy, the response rates and survival following systemic therapy is still limited. Sacituzumab govitecan is an antibody-drug conjugate targeting the topoisomerase I inhibitor SN38 to trophoblast cell-surface antigen 2 (TROP-2)-positive cells. Here we have explored the therapeutic potential of sacituzumab govitecan in MPM models. Materials and methodsTROP2 expression was analyzed in a panel of two well established and 15 pleural effusion derived novel lines by RT-QPCR and immunoblotting, TROP2 membrane-localization was studied by flow cytometry and immunohistochemistry. Cultured mesothelial cells and pneumothorax pleura served as controls. The sensitivity of MPM cell lines to irinotecan and SN38 was studied using cell viability, cell cycle, apoptosis and DNA damage assays. Drug sensitivity of cell lines was correlated with RNA expression of DNA repair genes. Drug sensitivity was defined as an IC50 below 5 nM in the cell viability assay. ResultsTROP2 expression was detected at RNA and protein level in 6 of the 17 MPM cell lines, but not in in cultured mesothelial control cells or in the mesothelial layer of the pleura. TROP2 was detectable on the cell membrane in 5 MPM lines and was present in the nucleus in 6 cell models. Ten of 17 MPM cell lines showed sensitivity to SN38 treatment, among those 4 expressed TROP2. High AURKA RNA expression and high proliferation rate correlated with sensitivity to SN38-induced cell death, DNA damage response, cell cycle arrest and cell death. Sacituzumab govitecan treatment effectively induced cell cycle arrest and cell death in TROP2-positive MPM cells. ConclusionTROP2 expression and sensitivity to SN38 in MPM cell lines support biomarker-selected clinical exploration of sacituzumab govitecan in patients with MPM.

Abstract P4-07-12: Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations

Abstract Background: Sacituzumab govitecan (SG, Trodelvy®) is a human trophoblast cell surface antigen 2 (TROP-2) directed antibody drug conjugate (ADC) coupled to an active form of irinotecan (SN-38) via our novel hydrolyzable linker (CL2A). SG is the only FDA-approved ADC treatment for TNBC patients in the second-line setting. TROP-2 is a transmembrane protein encoded by the tumor-associated calcium signal transducer 2 (TACSTD2) gene and highly expressed in TNBC, an aggressive type of cancer accounting for approximately 15% of all breast cancers. TROP-2 overexpression is associated with poor survival and relapse, but its biological function in TNBC remains poorly understood. Hypothesis/rationale: To better understand TROP-2 and TROP-2-directed ADC biology, we developed and characterized TROP2high vs TROP2low TNBC syngeneic tumors and an SG surrogate directed to murine TROP-2. Experimental design: We established 2 syngeneic TNBC models with differential TROP-2 expression: 4T1 cells were flow sorted into high (>95%) vs low (< 7%) TROP-2 expressors and EMT6 cells were transduced with a murine TACSTD2-encoding lentivirus. Balb/c mice were subcutaneously implanted with 0.5 × 10 E6 TROP-2high, TROP-2low, or parental tumor cells (4T1 or EMT6). Tumor immunophenotyping and transcriptomic analyses were performed 15 and 24 days after implantation. An SG mouse surrogate was engineered to mimic SG, using an anti-TROP-2 antibody (Rab64) that cross-reacts with human and murine TROP-2 covalently attached to SN-38 by the CL2A linker. SG surrogate activity was characterized in vitro and in 4T1 syngeneic models. Results: SG surrogate demonstrated high affinity for human and mouse TROP-2 (KD=1.1 and 1.4 nM, respectively) with SN-38 release rates and PK similar to that of SG. Flow cytometry analysis after bulk cell sorting of 4T1 or lentivirus transduction of EMT6 confirmed high TROP2 expression after at least 3 in vitro passages. Fifteen days after subcutaneous implantation, flow cytometry analysis of tumor single-cell suspensions revealed significant differences in immune infiltrates between 4T1-derived tumor groups (n=5/group; mean percentages in TROP2high vs TROP2low 4T1-derived tumors of cells expressing CD45: 65% vs 10%, P < 0.0001; CD8: 5.5% vs 1%, P = 0.0033; CD4: 22% vs 4%, P = 0.0055; macrophages: 12.5% vs 2.5%, P = 0.0002; myeloid cells: 52% vs 75%, P = 0.0066). In addition, TROP2high 4T1-derived tumors were smaller and had significantly less necrosis than TROP2low and unsorted 4T1-derived tumors 25 days after implantation. Finally, transcriptomics analyses of TROP2high vs TROP2low 4T1-derived tumors demonstrated the association of TACSTD2 expression levels with regulation of distinct molecular pathways. Conclusion: Syngeneic tumors derived from 4T1 cells with differential TROP2 expression levels are associated with differential cellular states and tumor microenvironment composition. In contrast, no significant phenotypic changes were observed in tumors derived from TACSTD2-transduced compared with mock-transduced EMT6 cells. Taken together, these results suggest that expression of the TACSTD2 gene is associated with, but not causative of, different tumor phenotypic states. Additional studies to investigate TROP-2 expression as a correlative marker of patient prognosis and the antitumor immune response are warranted. The effects of in vivo treatment with an SG surrogate on 4T1 tumor growth and immune phenotype will be discussed at the time of the presentation. Citation Format: Chih-Chien Chou, Jordan Kardos, Becky Yang, Jessica Orf, Rutwij Dave, Yurong Lai, Chingwei V. Lee, Giuseppe A. Papalia, Kelli Boyd, Lauri Diehl, Nathalie Scholler. Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-12.

Robust protein-based engineering of hepatocyte-like cells from human mesenchymal stem cells.

Cells of interest can be prepared from somatic cells by forced expression of lineage-specific transcription factors, but it is required to establish a vector-free system for their clinical use. Here, we report a protein-based artificial transcription system for engineering hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (MSCs). MSCs were treated for 5 days with 4 artificial transcription factors (4F), which targeted hepatocyte nuclear factor (HNF)1α, HNF3γ, HNF4α, and GATA-binding protein 4 (GATA4). Then, engineered MSCs (4F-Heps) were subjected to epigenetic analysis, biochemical analysis and flow cytometry analysis with antibodies to marker proteins of mature hepatocytes and hepatic progenitors such as delta-like homolog 1 (DLK1) and trophoblast cell surface antigen 2 (TROP2). Functional properties of the cells were also examined by injecting them to mice with lethal hepatic failure. Epigenetic analysis revealed that a 5-day treatment of 4F upregulated the expression of genes involved in hepatic differentiation, and repressed genes related to pluripotency of MSCs. Flow cytometry analysis detected that 4F-Heps were composed of small numbers of mature hepatocytes (at most 1%), bile duct cells (~19%) and hepatic progenitors (~50%). Interestingly, ~20% of 4F-Heps were positive for cytochrome P450 3A4, 80% of which were DLK1-positive. Injection of 4F-Heps significantly increased survival of mice with lethal hepatic failure, and transplanted 4F-Heps expanded to more than 50-fold of human albumin-positive cells in the mouse livers, well consistent with the observation that 4F-Heps contained DLK1-positive and/or TROP2-positive cells. Taken together with observations that 4F-Heps were not tumorigenic in immunocompromised mice for at least 2 years, we propose that this artificial transcription system is a versatile tool for cell therapy for hepatic failures.

Abstract OT1-03-05: TROPION-Breast02: Phase 3, open-label, randomized study of first-line datopotamab deruxtecan versus chemotherapy in patients with locally recurrent inoperable or metastatic TNBC who are not candidates for anti-PD-(L)1 therapy

Abstract Introduction: Despite recent treatment advances, the prognosis for patients diagnosed with locally recurrent inoperable or metastatic triple-negative breast cancer (TNBC) remains poor. Single-agent chemotherapy has been the mainstay of treatment for metastatic TNBC for many years, with very limited treatment options for patients who are not candidates for anti-PD-1/PD-L1 therapy; consequently, there remains an urgent unmet need. Trophoblast cell surface protein 2 (TROP2) is a type I transmembrane glycoprotein highly expressed on various solid tumors, including breast. Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) composed of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to an exatecan derivative (DXd) – a highly potent topoisomerase I inhibitor payload – via a stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd has previously been evaluated in TROPION-PanTumor01 (NCT03401385) – an ongoing Phase I clinical trial across multiple advanced solid tumors, including heavily-pretreated, metastatic TNBC. As of the July 30, 2021 cut-off date, the objective response rate (ORR) by blinded independent central review (BICR) was 34% (15/44) in all patients with TNBC and 52% (14/27) in those patients treatment-naïve to topoisomerase I inhibitor-based ADC therapies, with measurable disease (per RECIST 1.1) at baseline. Furthermore, a manageable safety profile was reported, with no new safety signals identified; low grade nausea and stomatitis were most frequent, with neutropenia and diarrhea being uncommon. The aim of the phase 3 TROPION-Breast02 trial is to evaluate the efficacy and safety of Dato-DXd versus investigator’s choice of chemotherapy (ICC) in patients with TNBC who are not candidates for PD-1/PD-L1 inhibitor therapy. Methods: TROPION-Breast02 (NCT05374512) is an ongoing Phase 3, open-label, randomized study of Dato-DXd versus ICC in first-line treatment of patients with locally recurrent inoperable or metastatic TNBC. Approximately 600 patients will be randomized 1:1 to receive either Dato-DXd 6 mg/kg IV every three weeks or ICC (paclitaxel, nab-paclitaxel, carboplatin, capecitabine, or eribulin mesylate). Patients ≥18 years with histologically/cytologically confirmed TNBC who have not received prior chemotherapy or targeted systemic therapy for metastatic or locally recurrent inoperable disease are eligible for inclusion. Patients are also required to have an ECOG PS of 0 or 1 and be eligible for treatment with one of the ICC options per investigator assessment. Patients must have ≥1 measurable lesion per RECIST v1.1 not previously irradiated, along with a formalin-fixed and paraffin-embedded tumor sample available. Those with a previously treated neoplastic spinal cord compression or clinically inactive brain metastases can be included. Key exclusion criteria are history of another primary malignancy; persistent toxicity from previous anti-cancer treatments; uncontrolled infections; current/prior interstitial lung disease/pneumonitis or clinically severe pulmonary function compromise; clinically significant corneal disease; and prior treatment with topoisomerase I inhibitors, TROP2-targeted therapy, or the same chemotherapy agent chosen for on-study ICC. The dual primary endpoints are progression free survival (PFS) per RECIST 1.1 by BICR, and OS. Secondary endpoints include ORR, duration of response, PFS by investigator assessment, time to deterioration for patient-reported outcomes, time to first subsequent therapy, time to second subsequent therapy, time to second progression or death, pharmacokinetics and immunogenicity of Dato-DXd, and safety. Recruitment for this study is ongoing as of June 2022. Citation Format: Rebecca Dent, David W. Cescon, Thomas Bachelot, Kyung Hae Jung, Zhi-Ming Shao, Shigehira Saji, Tiffany A. Traina, Petra Vuković, Darlington Mapiye, Micah Maxwell, Peter Schmid, Javier Cortés. TROPION-Breast02: Phase 3, open-label, randomized study of first-line datopotamab deruxtecan versus chemotherapy in patients with locally recurrent inoperable or metastatic TNBC who are not candidates for anti-PD-(L)1 therapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-03-05.

Abstract P4-08-15: Identification of target kinases whose inhibition enhances antitumor efficacy of sacituzumab-govitecan in metastatic HER2-negative breast cancer

Abstract BACKGROUND HER2-negative (HER2−) breast cancer (BC) subtypes, such as luminal A and triple-negative breast cancer (TNBC), express human trophoblast cell-surface antigen 2 (Trop-2). A high level of Trop-2 expression is associated with a poor prognosis in solid tumors. Sacituzumab-govitecan (SG), a Trop-2–directed antibody conjugated with SN-38 (topoisomerase Ⅰ inhibitor) significantly improves survival in patients with metastatic TNBC. Also, the IMMU-132-01 phase Ⅰ/Ⅱ study revealed that SG has antitumor activity in previously treated hormone receptor (HR)+/HER2− metastatic BC. In a phase Ⅲ trial (TROPiCS-02), SG demonstrated statistically significant progression-free survival in patients with heavily pre-treated HR+/HER2− endocrine-resistant metastatic BC. To augment the efficacy of SG, we sought to identify potential kinase targets whose inhibition enhances the antitumor efficacy of SG to formulate an SG-based combination therapy. MATERIALS AND METHODS Fluorescence-activated cell sorting analysis and Western blotting were used to evaluate the expression of Trop-2 in 24 TNBC and 8 HR+ BC cell lines. Non-biased high-throughput kinome-wide RNAi screening was performed with BT-20 (TNBC) and T47D tamoxifen/abemaciclib double-resistant (HR+) cell lines to identify target kinases whose inhibition produces synergistic antitumor efficacy of SG. To validate the combination antitumor effect of SG and selected kinase target inhibitors, sulforhodamine B staining proliferation assays, cell cycle analysis, and caspase 3/7 activity assays were performed in Trop-2+ TNBC and HR+ BC cell lines, including cell lines resistant to tamoxifen and double-resistant to tamoxifen and CDK4/6 inhibitor (palbociclib or abemaciclib). RESULTS In vitro proliferation assays revealed that the half-maximal inhibitory concentration (IC50) of SG in tested TNBC cell lines ranged from 10 nM to 84 nM, and that of HR+ cell lines ranged from 12.5 nM to >250 nM. We identified 69 kinase targets in TNBC cell line BT-20 and 44 kinase targets in HR+ BC cell line T47D (tamoxifen/abemaciclib double-resistant) by the sensitivity index analysis (>0.15) of RNAi screening results, and further pathway analysis revealed DNA damage response (DDR), (drug: BAY1895344), PI3k/Akt/mTOR (drug: copanlisib), and MAPK (drug: trametinib) as potential target canonical pathways whose targeting enhanced the cytotoxic effect of SG in both TNBC and HR+ BC. Among these strategies, inhibition of the DDR pathway with ATR inhibitor (BAY1895344) yielded a synergistic antiproliferative effect in combination with SG compared to SG or BAY1895344 alone in all tested TNBC and HR+ BC cell lines. Growth inhibition by SG combined with BAY1895344 averaged 97.1% in TNBC cell lines and ranged from 30.7% to 81.3% in HR+ BC cell lines, with P< 0.001. Other combinations with SG showed partially synergistic antiproliferative effects (copanlisib: effective in 3 of 5 cell lines, trametinib: effective in 2 of 5 cell lines). CONCLUSIONS RNAi screening uncovered DDR, PI3k/Akt/mTOR, and MAPK pathways as potential targets for combination with SG. Among inhibitors of these targets, DDR pathway inhibitor BAY1895344 had the most robust synergy with SG, compared to two other target inhibitors, against Trop-2+ TNBC and HR+ BC cell lines. These in vitro data warrant future in vivo validation studies of the synergistic antitumor effect of SG and BAY1895344 in metastatic HER2– breast cancer. Citation Format: Nakyung Oh, Jon A. Fuson, Huey Liu, Debu Tripathy, Naoto T. Ueno, Jangsoon Lee. Identification of target kinases whose inhibition enhances antitumor efficacy of sacituzumab-govitecan in metastatic HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-15.

Antibody-Drug Conjugates in Prostate Cancer: A Systematic Review.

The prognosis in the setting of metastatic castration-resistant prostate cancer patients (mCRPC) remains limited. Therefore, novel treatment strategies remain an unmet need. Antibody-drug conjugates (ADC) emerged as a new drug concept with the potential to deliver a cytotoxic payload with limited off-target toxicity and potentially bystander effect. Following the success of ADCs in breast cancer and urothelial tumours, their activity in prostate cancer is now under investigation. Thus, the aim of this systematic review was to identify published and ongoing prospective clinical trials regarding ADC treatment in prostate cancer. A systematic search of PubMed, MEDLINE, and Web of Science was conducted as per PRISMA guidelines to identify prospective clinical trials of ADCin prostate cancer. Trials are currently ongoing on ClinicalTrials.gov and in the EU. The Clinical Trials Register was also identified. Abstracts, publications in languages other than English, review articles, retrospective analyses, and phase I trials were excluded. A total of six phase I/II prospective clinical trials already published were included. Seven ongoing trials were also identified. All studies were in the refractory/advanced tumour setting, and two included only mCRPC patients. The ADC targets were prostate-specific membrane antigen (PSMA), trophoblast cell surface antigen-2 (TROP-2), six-transmembrane epithelial antigen of prostate-1 (STEAP-1), tissue factor (TF), delta-like protein 3 (DLL-3), B7-H3 family of proteins (B7-H3), and human epidermal growth factor receptor 2 (HER2). Regarding the efficacy of PSMA ADC treatment in the second-line or beyond mCRPC setting, a PSA ≥ 50% decline rate in 14% of all treated patients was reported. One patient achieved a complete response with TROP-2 ADC. Overall, a wide range of safety issues were raised, particularly in connection with neuropathy and hematologic toxicity. Novel therapies have been changing the scope of treatment in mCRPC. ADCs seem to provide efficacy benefits, even with potential toxicity. The results of most prospective ongoing studies are still awaited, and a longer follow-up time is warranted to evaluate the real impact of ADCs in PCa.

The association between trophoblast cell surface antigen 2 expression and nivolumab monotherapy in patients with advanced gastric cancer.

438 Background: Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein which is associated with cancer growth. Overexpression of TROP2 in advanced gastric cancer (AGC) is known as a poor prognostic factor. However, the association between TROP2 expression and prognosis in patient with AGC treated with nivolumab (NIVO) is not known. Methods: Patients who received NIVO monotherapy as third- or later-line treatment (NIVO group) and those who received only fluoropyrimidine, platinum and taxanes (non-NIVO group) at our hospital between 2015 and 2019 were enrolled. High TROP2 expression was defined as ≥50% positive tumor cells with weak to moderate membranous staining or ≥10% tumor cells with strong membranous staining using immunohistochemistry. Overall survival from first-line chemotherapy (OS) and progression-free survival (PFS) from starting of NIVO were compared according to TROP2 expression and combined positivity score (CPS). Results: Of 111 patients, 61 were in the NIVO group and 50 were in the non-NIVO group. Patient characteristics in the NIVO/non-NIVO groups were median age 67 (range, 41-83)/64.5 (29-80) years; performance status 0, 20 (33%)/23 (46%); lymph node metastasis 41 (67%)/23 (46%); peritoneal metastasis 32 (53%)/26 (52%); liver metastasis 11 (18%)/6 (12%); lung metastasis 4 (7%)/4 (8%); TROP2 high 55 (90%)/43 (86%). Patient characteristics at starting of NIVO did not differ between TROP2 high and low in the NIVO group. Of 55 patients with TROP2 high, 22 patients (40%) were CPS≥1. In the non-NIVO group, OS from first-line chemotherapy did not differ between TROP2 high and low (median, 16.2 months [95%CI, 13.6-19.2] vs. 26.2 months [95%CI, 7.7-NA], HR 0.97, p=0.946). Meanwhile, in the NIVO group, patients with TROP2 high had significantly longer OS from first line chemotherapy than those with TROP2 low (median survival, 23.5 months [95%CI, 17.2-31.2] vs. 19.2 months [95%CI, 10.7-NA], HR 0.40, p=0.04). Progression-free survival (PFS) from starting of NIVO tended to be longer in TROP2 high group than those in TROP2 low group (median, 1.9 months [95%CI, 1.6-2.3] vs. 0.6 months [95%CI, 0.2-NA], HR 0.62, p=0.26). OS from first-line chemotherapy and PFS from starting of NIVO in patients with TROP2 high did not differ between CPS≥1 and CPS<1 (OS median, 24.0 months [95%CI, 13.8-39.7] vs. 23.2 months [95%CI, 17.0-32.4], HR 0.87, p=0.69; PFS median, 1.9 months [95%CI, 1.6-3.5] vs. 2.0 months [95%CI, 1.4-2.8], HR 0.90, p=0.72). Conclusions: Higher TROP2 expression related to longer survival in the patients with AGC treated with NIVO monotherapy.

A Phase IIb, single arm, multicenter trial of sacituzumab govitecan in Chinese patients with metastatic triple-negative breast cancer who received at least two prior treatments.

Refractory or relapsing metastatic triple-negative breast cancer (mTNBC) has a poor prognosis. Sacituzumab govitecan (SG) is a novel antibody-drug conjugate, targeting human trophoblast cell-surface antigen 2 (Trop-2). This is the first report of SG's efficacy and safety in Chinese patients with mTNBC. EVER-132-001 (NCT04454437) was a multicenter, single-arm, Phase IIb study in Chinese patients with mTNBC who failed ≥2 prior chemotherapy regimens. Eligible patients received 10mg/kg SG on Days 1 and 8 of each 21-day treatment cycle, until disease progression/unacceptable toxicity. The primary endpoint was objective response rate (ORR) assessed by the Independent Review Committee. Secondary endpoints included: duration of response (DOR), clinical benefit rate (CBR), progression-free survival (PFS), overall survival (OS) and safety. Eighty female Chinese patients (median age 47.6 years; range 24-69.9 years) received ≥1 SG dose with a median of 8 treatment cycles by the cutoff date (August 6, 2021). Median number of prior systemic cancer treatments was 4.0 (range 2.0-8.0). ORR and CBR were reported 38.8% (95% confidence interval [CI]: 28.06-50.30) and 43.8% (95% CI, 32.68-55.30) of patients, respectively. The median PFS was 5.55 months (95% CI, 4.14-N/A). SG-related Grade ≥3 treatment-emergent adverse events (TEAEs) were reported in 71.3%, the most common were neutrophil count decreased (62.5%), white blood cell count decreased (48.8%) and anemia (21.3%); 6.3% discontinued SG because of TEAEs. SG demonstrated substantial clinical activity in heavily pretreated Chinese patients with mTNBC. The observed safety profile was generally manageable.

TROP2, androgen receptor, and PD-L1 status in histological subtypes of high-grade metaplastic breast carcinomas.

High-grade metaplastic breast carcinoma (HG-MBC) is a rare subtype of invasive breast carcinoma, mostly triple-negative. Metaplastic carcinomas are less responsive to neoadjuvant chemotherapy and are associated with a worse outcome than invasive carcinomas of no special type. Clinicopathological characteristics and immunophenotype were retrospectively assessed in a series of 65 patients diagnosed with HG-MBC between 2005 and 2017 at the Curie Institute (antibody panel: oestrogen receptor [ER], progesterone receptor [PR], androgen receptor [AR], human epidermal growth factor receptor 2 [HER2], programmed death ligand-1 [PD-L1], and trophoblast cell surface antigen 2 [TROP2]). The median age at diagnosis was 59.5 years. Six (9%) patients had metastatic disease at diagnosis. Among the nonmetastatic patients receiving neoadjuvant therapy, 26% (5/19) achieved pathological complete response. Most tumours were pT1/pT2 (77%) and 12% were pN+. Histological subtypes (mixed, squamous, mesenchymal, and spindle cell) were 40%, 35.5%, 15.5%, and 9%, respectively. Tumour-infiltrating lymphocytes were low or moderate except when squamous differentiation was present. Most tumours were triple-negative (92%). AR and TROP2 were positive in 34% and 85% of the cases, respectively. PD-L1 was positive in tumour cells in 18% (cutoff: 1% of positive tumour cells) of the cases and in tumour-infiltrating immune cells in 40% (cutoff: 1% of tumour area) of the cases. Notably, spindle cell and mesenchymal metaplastic breast carcinomas were mostly PDL1-negative. Lastly, 21 (32.3%) cases were HER2-low, all being HER2 1+, with no HER2 2+. Metaplastic breast carcinoma could benefit from tailored therapeutic strategies adapted to the phenotypic specificities of histological subtypes.

A literature review of the promising future of TROP2: a potential drug therapy target.

Previous studies have demonstrated that the oncogene trophoblast cell surface antigen 2 (TROP2) has great application prospects as a therapeutic target. However, few literature reviews have systematically summarized and evaluated its role in cancer therapy. This study aims to summarize the molecular structure, functions, signal transduction pathways, and prognostic value of TROP2, and explore therapeutic agents that target TROP2. A total of 1,376 published literatures from PubMed and 614 published literatures from EMBASE were retrieved by searching "TROP2" or "Trophoblast cell surface antigen 2". The search was conducted on December 12, 2020, and updated on November 20, 2022. The cBioportal and GEPIA (Gene Expression Profiling Interactive Analysis) databases were used to analyze the expression, mutation, and prognostic value of TROP2 in different types of cancer. TROP2 is overexpressed in different tumor tissues and plays roles in cell proliferation, invasion, migration, apoptosis, and treatment resistance by binding to or interacting with several molecules. As a therapeutic target, TROP2 is particularly suitable for antibody-based therapies. Monoclonal antibodies, bispecific antibodies, antibody-drug conjugates (ADCs), virus-like particles, and antibody drugs in combination with traditional chemotherapy, immunotherapy, radioimmunotherapy, photoimmunotherapy, and nanoparticles that target TROP2 have thus far been rapidly developed. For example, sacituzumab govitecan (IMMU-132), a TROP2-targeting ADC, was granted accelerated approval for the treatment of metastatic triple-negative breast cancer (TNBC). Anti-TROP2 antibody-conjugated nanoparticles (ST-NPs) are a promising vehicle for delivering doxorubicin in targeted TNBC therapy. The availability of TROP2-targeting ADCs makes TROP2 an accessible and promising therapeutic target for advanced metastatic cancers. The present review describes the important role of TROP2 in tumorigenesis and its potential applications as a promising biomarker and therapeutic target that is capable of reversing resistance.

Antibody Drug Conjugates in Lung Cancer

An antibody-drug conjugate (ADC) comprises a monoclonal antibody that is specific to a tumor cell protein, bound to a cytotoxic agent, known as the payload. The use of ADCs is already common practice in several cancers, thanks to their efficacy and potentially more manageable toxicity profile, resulting from the release of the cytostatic payload directly in the tumors. Currently, early-phase trials of ADCs in non-small cell lung cancer are rapidly gaining ground, with promising results targeting HER2 (human epidermal growth factor 2), HER3, TROP2 (trophoblast cell surface antigen 2), MET, CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5), and PTK7 (tyrosine protein kinase-like 7). Unfortunately, in small cell lung cancer, trials targeting the ubiquitous DLL3 (delta-like ligand 3) protein have failed to show clinically relevant results, despite significant toxicity.

Antibody-drug conjugates targeting TROP-2: Clinical development in metastatic breast cancer

Antibody drug conjugates (ADCs) combine the potent cytotoxicity of chemotherapy with the antigen -specific targeted approach of antibodies into one single molecule. Trophoblast cell surface antigen 2 (TROP-2) is a transmembrane glycoprotein involved in calcium signal transduction and is expressed in multiple tumor types. TROP-2 expression is higher in HER2-negative breast tumors (HR+/HR-) and is associated with worse survival. Sacituzumab govitecan (SG) is a first-in-class TROP-2-directed ADC with an anti-TROP-2 antibody conjugated to SN-38, a topoisomerase inhibitor via a hydrolysable linker. This hydrolysable linker permits intracellular and extracellular release of the membrane permeable payload enabling the “bystander effect” contributing to the efficacy of this agent. There was significant improvement in progression free survival (PFS) and overall survival (OS) with SG versus chemotherapy in pretreated metastatic triple negative breast cancer (TNBC), resulting in regulatory approval. Common adverse events (AE) reported were neutropenia and diarrhea. SG also demonstrated clinical activity versus chemotherapy in a phase III trial of HR+/HER2-metastatic breast cancer (MBC) and is under evaluation in first-line metastatic and early stage TNBC as well. Datopotamab deruxtecan (Dato-DXd) is a TROP-2 ADC that differs from SG in that it has a cleavable tetrapeptide linker and a more potent topoisomerase inhibitor payload. This construct is highly stable in circulation with a longer half-life than SG, and undergoes cleavage in presence of intracellular lysosomal proteases. Dato-DXd demonstrated preliminary efficacy in unselected metastatic TNBC, with common AEs of low-grade nausea and stomatitis. Dato-DXd is being investigated in phase III studies in metastatic TNBC and HR+/HER2- MBC. These novel TROP-2 ADCs have the potential to deliver enhanced efficacy with reduced toxicity in MBC and possibly in early stage breast cancer (EBC).

Antibody-Drug Conjugate Sacituzumab Govitecan Drives Efficient Tissue Penetration and Rapid Intracellular Drug Release.

Antibody-drug conjugates (ADC) are a rapidly growing class of targeted cancer treatments, but the field has experienced significant challenges from their complex design. This study examined the multiscale distribution of sacituzumab govitecan (SG; Trodelvy), a recently clinically approved ADC, to clarify the mechanism(s) of efficacy given its unique design strategy. We employed a multiscale quantitative pharmacokinetic approach, including near-infrared fluorescence imaging, single-cell flow cytometry measurements, payload distribution via γH2AX pharmacodynamic staining, and a novel dual-labeled fluorescent technique to track the ADC and payload in a high trophoblast cell-surface antigen 2 expression xenograft model of gastric cancer (NCI-N87). We found that rapid release of the SN-38 payload from the hydrolysable linker inside cells imparts more DNA damage in vitro and in vivo than an ADC with a more stable enzyme cleavable linker. With SG, little to no extracellular payload release in the tumor was observed using a dual-labeled fluorescence technique, although bystander effects were detected. The high dosing regimen allowed the clinical dose to reach the majority of cancer cells, which has been linked to improved efficacy. In addition, the impact of multiple doses (day 1 and day 8) of a 21-day cycle was found to further improve tissue penetration despite not changing tumor uptake [percent injected dose per gram (%ID/g)] of the ADC. These results show increased ADC efficacy with SG can be attributed to efficient tumor penetration and intracellular linker cleavage after ADC internalization. This quantitative approach to study multiscale delivery can be used to inform the design of next-generation ADCs and prodrugs for other targets.

Trophoblast Cell Surface Antigen 2 (TROP2) as a Predictive Bio-Marker for the Therapeutic Efficacy of Sacituzumab Govitecan in Adenocarcinoma of the Esophagus.

Simple SummaryThe trophoblast cell surface antigen 2 (TROP2) is a protein produced by many carcinomas. Sacituzumab govitecan (SG) is a drug consisting of an antibody that binds to TROP2 on the tumor cell when TROP2 is present and is taken up into the cell interior after binding. The antibody is coupled with a cytotoxic substance (SN38) that is released inside the cell after uptake. As a result, a lethal dose of SN38 acts specifically in the tumor cell while having a small systemic effect, reducing the extent of side effects. We can show here that TROP2 is formed in nearly 90% of esophageal adenocarcinomas (EAC) and that sacituzumab govitecan is also effective in EAC. We can show that efficacy is dependent on the presence of TROP2 on the cancer cell - complete absence of TROP2 is associated with poor response rate to SG. We therefore advocate determining TROP2 at the protein level prior to therapy. Suitable immunohistochemical antibodies for routine testing exist.Introduction: The Trophoblast cell surface antigen 2 (TROP2) is expressed in many carcinomas and may represent a target for treatment. Sacituzumab govitecan (SG) is a TROP2–directed antibody-drug conjugate (ADC). Nearly nothing is known about the biological effectiveness of SG in esophageal adenocarcinoma (EAC). Material and Methods: We determined the TROP2 expression in nearly 600 human EAC. In addition, we used the EAC cell lines (ESO-26, OACM5.1C, and FLO-1) and a xenograft mouse model to investigate this relationship. Results: Of 598 human EACs analyzed, 88% showed varying degrees of TROP2 positivity. High TROP2 positive ESO-26 and low TROP2 positive OACM5.1C showed high sensitivity to SG in contrast to negative FLO-1. In vivo, the ESO-26 tumor shows a significantly better response to SG than the TROP2-negative FLO-1 tumor. ESO-26 vital tumor cells show similar TROP2 expression on all carcinoma cells as before therapy initiation, FLO-1 is persistently negative. Discussion: Our data suggest that sacituzumab govitecan is a new therapy option in esophageal adenocarcinoma and the TROP2 expression in irinotecan-naïve EAC correlates with the extent of treatment response by sacituzumab govitecan. TROP2 is emerging as a predictive biomarker in completely TROP2-negative tumors. This should be considered in future clinical trials.

Trophoblast cell-surface antigen 2 (TROP2) expression in triple-negative breast cancer

BackgroundTrophoblast cell-surface antigen 2 (TROP2) is related to tumor proliferation enhancement and poor prognosis. An antibody targeting TROP2 was developed to treat metastatic triple-negative breast cancer (TNBC) which has a limited treatment modality. To characterize the TROP2 expressing tumors in TNBC, we analyzed TROP2 expression in three cohorts; (1) primary tumor without neoadjuvant chemotherapy, (2) primary tumor with neoadjuvant chemotherapy, and (3) metastatic tumor.MethodsA total of 807 TNBC cases were evaluated for TROP2 immunohistochemical expression. We evaluated the TROP2 H-score distribution in the three cohorts. Tumors were divided into two groups based on TROP2 expression (high vs. low). We analyzed the relationship between clinicopathologic features and markers, including epidermal growth factor receptor, cytokeratin 5/6, p53, and Ki-67, and prognostic significance at high vs. low TROP2 expression.ResultsThere was no difference in TROP2 H-score distribution between the three cohorts. Moderate-to-strong membranous expression of TROP2 in at least 10% of tumor cells was present in 662 cases (82.0%) in Cohort 1, 59 cases (89.4%) in Cohort 2, and 23 cases (88.5%) in Cohort 3. There was no significant difference in clinicopathologic features between high vs. low TROP2 in all cohorts. TROP2 H-score was an independent poor prognostic factor for overall survival in Cohort 3.ConclusionsTNBC showed similar TROP2 expression regardless of neoadjuvant treatment or primary tumor/metastasis. Although the prognostic significance of TROP2 expression in metastatic TNBC has been revealed, further evaluation of the predictive value of TROP2 expression for targeted therapy is needed.

3D microfluidics-assisted modeling of glucose transport in placental malaria

The human placenta is a critical organ, mediating the exchange of nutrients, oxygen, and waste products between fetus and mother. Placental malaria (PM) resulted from Plasmodium falciparum infections causes up to 200 thousand newborn deaths annually, mainly due to low birth weight, as well as 10 thousand mother deaths. In this work, a placenta-on-a-chip model is developed to mimic the nutrient exchange between the fetus and mother under the influence of PM. In this model, trophoblasts cells (facing infected or uninfected blood simulating maternal blood and termed “trophoblast side”) and human umbilical vein endothelial cells (facing uninfected blood simulating fetal blood and termed “endothelial” side) are cultured on the opposite sides of an extracellular matrix gel in a compartmental microfluidic system, forming a physiological barrier between the co-flow tubular structure to mimic a simplified maternal–fetal interface in placental villi. The influences of infected erythrocytes (IEs) sequestration through cytoadhesion to chondroitin sulfate A (CSA) expressed on the surface of trophoblast cells, a critical feature of PM, on glucose transfer efficiency across the placental barrier was studied. To create glucose gradients across the barrier, uninfected erythrocyte or IE suspension with a higher glucose concentration was introduced into the “trophoblast side” and a culture medium with lower glucose concentration was introduced into the “endothelial side”. The glucose levels in the endothelial channel in response to CSA-adherent erythrocytes infected with CS2 line of parasites in trophoblast channel under flow conditions was monitored. Uninfected erythrocytes served as a negative control. The results demonstrated that CSA-binding IEs added resistance to the simulated placental barrier for glucose perfusion and decreased the glucose transfer across this barrier. The results of this study can be used for better understanding of PM pathology and development of models useful in studying potential treatment of PM.

83P Patterns of trophoblast cell surface antigen 2 (TROP2) and epithelial cell adhesion molecule (EPCAM) expression in human tumors: A tissue microarray study on 14,766 tumors

Trophoblast cell surface antigen 2 (TROP2) and epithelial cell adhesion molecule (EPCAM) are cell surface receptors that play a role in growth signal transduction and cell adhesion. Antibody drug conjugates against both proteins are approved or under development.