Engineered Nanobody-Bearing Extracellular Vesicles Enable Precision Trop2 Knockdown in Resistant Breast Cancer

Background: Trophoblast cell surface antigen 2 (TROP2) promotes breast cancer (BC) development, invasion and metastasis, with promising role as a biomarker and therapeutic target in triple negative BC. Due to the dynamic tumor evolution during disease progression, a significant discordance in tumor cell profiles is frequently observed among primary tissues and distant metastases. In this regard, analyses of circulating tumor cells (CTCs) in the peripheral blood (PB) can inform on the expression of biomarkers in real-time. In the current study we assessed in parallel the expression of TROP2 on CTCs and matched primary tumors and metastatic sites from patients with triple negative BC. Methodology: PB was collected from 54 patients and CTCs were enriched by ficoll-density gradient centrifugation. Cytospins were immunofluorescently stained using antibodies for cytokeratins (Clones: AE1/AE3 & C11), CD45 and TROP-2 (Enzo Life Sciences); TROP2 expression on CTCs was defined as high, low or negative, by using the high TROP2-expressing MDA.MB.231 triple negative BC cell line as internal control. Matched primary (n=51) and metastatic (n=7) tumor tissue samples were evaluated for TROP-2 expression by immunocytochemistry (IHC); H-score was calculated as follows: (3 × % cells with strong intensity staining) + (2 × % cells with moderate intensity staining) + (1 × % cells with mild intensity staining), ranging from 0 to 300, and the following expression categories were defined: H-score 0 to <100: TROP2 low; H-score 100-200: TROP2 medium; H-score >200-300: TROP2 high. Results: CTCs (CK+/CD45- cells) were identified in 12 out of 54 patients evaluated (total CTC counts: n=80; mean CTC counts per patient: n=6.7). TROP2-expressing CTCs were detected in 75% of CTC-positive patients and represented the 95% of total CTCs. Specifically, high and low TROP2-expressing CTCs were identified in 66.7% and 41.7% of patients, representing the 81.3% and 13.7% of total CTCs, respectively. Differential TROP2 expression levels (high, medium and low) were also observed in both primary and metastatic tumors, showing a great intra-tumoral heterogeneity. High TROP2 expression was identified in 58.8% and 57.1% of primary and metastatic tissues, respectively. When matched primary and metastatic tissues were analyzed, a decrease in TROP2 expression was observed [median H-Score: 172.5 (range: 11-300) versus 87.5 (range: 5-150) in primary and metastatic tissue, respectively, p=0.068)]. CTC detection in the PB was not associated with TROP2 expression levels in primary or metastatic tissue (CTCs were identified in 13.3% and 50% of patients with high TROP2-expressing primary and metastatic tumors, respectively). Finally, there was no concordance in TROP2 expression pattern among CTCs and the respective tumor (high, low and negative TROP2-expressing CTCs were identified in 50%, 25% and 25% of patients with high TROP2-expressing primary tumors, respectively; high and low TROP2 -expressing CTCs were evident in all patients with high TROP2-expressing metastatic tumors). Conclusions: Herein we demonstrate for the first time a significant discrepancy in TROP2 expression among CTCs, primary and metastatic tumor tissue samples in triple negative BC. A lower TROP2 expression was observed in metastatic as compared to primary tissue, while no concordance was demonstrated among CTCs and the respective tumors. The results suggest the dynamic change in TROP2 expression status among different disease sites, thus highlighting the value of using liquid biopsy as a tool for real-time biomarker assessment in triple negative BC. Citation Format: Dimitrios Mavroudis, Eleni Lagoudaki, Sofia Gounaki, Sofia Hatziavraam, Charalampos Fotsitzoudis, Kleita Michaelidou, Sofia Agelaki, Maria A Papadaki. Comparative analysis of TROP2 expression in tumor tissues and circulating tumor cells (CTCs) in the peripheral blood of patients with triple negative breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-05-27.

5031 Background: Trophoblast cell surface antigen 2 (TROP-2) is a tumor associated antigen overexpressed in several malignancies including breast and urothelial cancer. The TROP-2 antibody-drug conjugate (ADC) Sacituzumab govitecan is approved for treatment of metastatic breast cancer. The expression of TROP-2 in GCT is unknown. We present immunohistochemistry results of TROP-2 expression in GCT. Methods: Patients who underwent resection for GCT at Indiana University were included. Sixty formalin-fixed paraffin-embedded (FFPE) GCT samples were available. FFPE slides were selected from differing GCT histology and surgical sites including primary tumor, retroperitoneal lymph node, and distant metastases. Immunohistochemical (IHC) staining for TROP-2 (clone 1, mouse monoclonal, Enzo Life Sciences) was conducted and scored by intensity on a 0-3 scale by an experienced pathologist. Results: Samples from 60 individual specimens were available for IHC analysis. TROP2 expression was detected in 29 (48%) of these samples. Intensity expression differed from pure seminoma, mixed non-seminoma (NSGCT), teratoma, yolk sac tumor, and choriocarcinoma samples. Both primary and metastatic samples had TROP-2 expression of varying degrees. Conclusions: TROP-2 expression varies across histology in GCT. Seminoma appears to have the lowest expression of TROP-2. Higher TROP-2 expression was noted in choriocarcinoma and yolk sac tumor samples indicating potential as a target in these histologic subtypes in future clinical trials. Detectable TROP-2 by histology. Sample histology (N) Total detectable TROP-2 expression (%) 3+ 2+ 1+ Seminoma (20) 3 (15) 2 1 NSGCT (19) 12 (63) 9 (6*) 2* 1* Teratoma (9**) 6 (66) 3* 3* Yolk sac (9) 5 (56) 2 3 Choriocarcinoma (3) 3 (100) 1 1 1 *In epithelial elements of teratoma. **Three negative samples with only small fragments of teratoma and false negative may be present.

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.

The prognostic and predictive role of trophoblast cell-surface antigen-2 (Trop-2) overexpression in human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer is currently unknown. We retrospectively analyzed Trop-2 expression and its correlation with clinicopathologic features and pathological complete response (pCR) in HER2-positive early breast cancer (EBC) patients treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab in the PHERGain study. Trop-2 expression at baseline was determined in formalin-fixed, paraffin-embedded primary tumor biopsies by immunohistochemistry and was first classified into expressing (Trop-2-positive) or not-expressing (Trop-2-negative) tumors. Then, it was classified by histochemical score (H-score) according to its intensity into low (0-9), intermediate (10-49), and high (≥ 50). The association between clinicopathologic features, pCR, and Trop-2 expression was performed with Fisher's exact test. Forty-one patients with tissue evaluable for Trop-2 expression were included, with 28 (68.3%) Trop-2-positive tumors. Overall, 17 (41.46%), 14 (34.15%), and 10 (24.40%) tumors were classified as low, intermediate, and high, respectively. Trop-2 expression was significantly associated with decreased pCR rates (50.0% vs. 92.3%; odds ratio [OR] 0.05; 95% CI, 0.002-0.360]; p adjusted = 0.01) but was not correlated with any clinicopathologic features (p ≥ 0.05). Tumors with the highest Trop-2H-score were less likely to obtain a pCR (OR 0.03; 95% CI, 0.001-0.290, p adjusted < 0.01). This association was confirmed in univariate and multivariate regression analyses. These findings suggest a potential role of Trop-2 expression as a biomarker of resistance to neoadjuvant chemotherapy plus dual HER2 blockade and may become a strategic target for future combinations in HER2-positive EBC patients.

Trophoblast cell surface antigen 2 (TROP2) is the target of sacituzumab govitecan (SG), an antibody-drug conjugate that was recently approved for previously treated triple negative breast cancer and urothelial carcinomas. To comprehensively determine TROP2 expression in normal and neoplastic tissues, a tissue microarray containing 18,563 samples from 150 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. TROP2 positivity was found in most normal epithelial cell types and in 109 of 150 tumor categories, including 92 of 95 epithelial tumor categories. Particularly high rates of TROP2 positivity and highest expression levels were seen in squamous cell carcinomas of various origins and various categories of urothelial, breast, prostate, pancreatic, and ovarian cancers (&amp;gt;95% positive). High TROP2 expression was linked to advanced stage (p=0.0069) and nodal metastasis (p&amp;lt;0.0001) in colorectal cancer as well as to nodal metastasis in gastric adenocarcinoma (p=0.0246) and papillary thyroid cancer (p=0.0013). Low TROP2 expression was linked to advanced stage in urothelial carcinoma (p&amp;lt;0.0001), high pT (p=0.0024) and high grade (p&amp;lt;0.0001) in breast cancer, as well as with high grade (p=0.0005) and pT stage (p=0.0009) in papillary renal cell carcinomas. Associations between TROP2 expression and clinicopathological features were not found in clear cell renal cell carcinomas, high grade serous ovarian carcinomas, pancreatic adenocarcinomas and in endometroid endometrium carcinomas. In summary, TROP2 is abundantly expressed in a broad range of epithelial neoplasms. Both TROP2 upregulation and downregulation can be associated with cancer progression in a tumor type dependent manner. As anti-TROP2 cancer drugs have demonstrated efficiency and induce tolerable side effects they may be applicable to a broad range of tumor entities in the future. Citation Format: David Dum, Claudia Hube-Magg, Ronald Simon, Elena Bady, Tim Mandelkow, Niclas C. Blessin, Maximilian Lennartz, Guido Sauter, Sarah Minner, Andreas M. Luebke. Trophoblast cell surface antigen 2 (TROP2) expression in human tumors: A tissue microarray study on 18,563 tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1861.

e12558 Background: Trophoblast cell-surface antigen-2 (Trop-2) is a transmembrane calcium signal transducer highly expressed in multiple tumor types including breast cancer. Trop-2 overexpression is associated with poor survival. Given the approval of sacituzumab govitecan in advanced triple-negative breast cancer (TNBC), Trop-2 emerges as an important target for antibody-drug conjugates. Limited data exist about correlations of Trop-2 expression with clinicopathological characteristics and outcome in early TNBC. Methods: We included 127 patients with early TNBC, treated with primary surgery at UZLeuven from 2005-2017. Trop-2 expression was determined with IHC (ab227689, Abcam) on whole slide tumor sections from resection specimens and assessed as continuous (H-score 0-300) and categorical (high 201-300, medium 100-200 and low &lt; 100) variables. Stromal tumor infiltrating lymphocytes (sTIL; low, intermediate and high), mitotic score and androgen receptor (AR) expression (1% or 10%-cutoff) were scored. We assessed associations between Trop-2 expression and age, BMI, BRCA status, tumor grade and size, lymphovascular invasion (LVI), DCIS, nodal status, sTILs, AR, mitotic index and outcome (Invasive disease free survival and breast cancer specific survival). Results: The median age at diagnosis was 51y (range 26-80y) and the median follow-up 8.5y. Low, medium and high Trop-2 expression was seen in 50%, 37% and 13% of cases. AR was positive in 32% of cases (10%-cutoff). Higher Trop-2 expression was correlated with age (ρ = 0.192, p = 0.03) and inversely correlated with mitosis/mm² (ρ = -0.2, p = 0.02). Patients with high Trop-2-expression were older compared to patients with low Trop-2-expression (median 58 vs 47 years, p = 0.02). LVI was more frequent in Trop-2-high (65%) compared to TROP-2-medium (15%) or -low (16%) (p &lt; 0.001). Median mitosis/mm² was higher in Trop-2-low (14) compared to Trop-2-medium (10) and -high (8) (p = 0.004). Patients in Trop-2-high subgroup had more nodal involvement (53%) compared to Trop-2-medium (23%) and -low (21%) (p = 0.03). There was no correlation between Trop-2 expression and sTILs, AR or outcome. Conclusions: In this patient cohort with TNBC treated with upfront surgery, higher Trop-2 expression was correlated with older age, with more LVI and nodal involvement, while mitosis/mm² were higher in tumors with low Trop-2 expression. Trop-2 expression was not correlated with sTILs, AR or outcome. Limited numbers of events warrant caution in interpretation.

The trophoblast cell-surface antigen 2 (Trop2) is markedly overexpressed in breast cancers, with a particularly high incidence in triple-negative breast cancer. The therapeutic relevance of Trop2 expression is underscored by the approval of an antibody-drug conjugate for triple-negative breast cancer treatment. However, there is no a predictive technique for accurate whole-body mapping of Trop2 expression in patients. In this study, we developed a novel Trop2-specific molecular probe, [99mTc]Tc-MY6349, and evaluated its safety and feasibility for detecting Trop2 expression in breast cancer using SPECT/CT imaging. Methods: Trop2 expression in different breast cancer cell lines was assessed via immunofluorescence and flow cytometry. The Trop2-specific nanobody MY6349 was site-specifically labeled with 99mTc via a C-terminal GGGC tag, and its binding affinity to the Trop2 receptor was tested invitro. The invivo tumor uptake and distribution of [99mTc]Tc-MY6349 were examined through SPECT imaging and biodistribution studies. Furthermore, a pilot clinical study of [99mTc]Tc-MY6349 SPECT/CT was conducted in 8 patients with breast cancer, and the results were compared with [18F]FDG PET/CT. Results: [99mTc]Tc-MY6349 achieved a greater than 95% radiochemical purity after purification. In vitro and invivo experiments demonstrated the binding specificity of [99mTc]Tc-MY6349 to the Trop2 receptor. In vivo imaging and biodistribution studies revealed a significant correlation between tumor uptake and Trop2 expression levels. In the pilot clinical study, SPECT imaging with [99mTc]Tc-MY6349 successfully detected Trop2-positive tumors 15 min after tracer injection. Delayed imaging showed reduced uptake in normal organs but sustained retention of [99mTc]Tc-MY6349 in tumors. Importantly, [99mTc]Tc-MY6349 SPECT/CT imaging highlighted Trop2 expression heterogeneity and visualized primary and metastatic lesions with a favorable tumor-to-background ratio in breast cancer. Conclusion: [99mTc]Tc-MY6349 was successfully prepared and exhibited a high binding affinity and Trop2 specificity. The pilot clinical study validated the safety and feasibility of [99mTc]Tc-MY6349 SPECT/CT for detecting Trop2 expression invivo in patients with breast cancer. This imaging modality could complement existing methods, aiding in the guidance of Trop2-targeted therapies and advancing personalized treatment while also promoting the application of SPECT/CT nuclear medicine imaging technology.

This study aimed to evaluate the prognostic and predictive significance of Claudin-6 and trophoblast cell surface antigen-2 (Trop-2) expression in serous ovarian carcinoma, assessing their influence on treatment efficacy and clinical outcomes. A retrospective cohort of 73 patients diagnosed with serous ovarian carcinoma was analyzed. All patients underwent standard cytoreductive surgery, with or without neoadjuvant or adjuvant chemotherapy. Immunohistochemistry was used to assess the expression levels of Claudin-6 and Trop-2. Survival outcomes were evaluated using Kaplan-Meier and Cox proportional hazards models, with overall survival (OS) as the primary endpoint. High Trop-2 expression was observed in 67.1% of the patients, while 46.6% exhibited high Claudin-6 expression. Both markers were more common in older, postmenopausal patients, those with larger tumors, and those with distant metastasis. However, no significant associations were found with clinical factors (P > .05). Survival analysis demonstrated that high Trop-2 and Claudin-6 expression were associated with shorter OS and progression-free survival (PFS). Patients with high Trop-2 expression exhibited a median OS of 38 months and PFS of 32 months, whereas those with low Trop-2 expression had a median OS of 62 months and PFS of 60 months (OS: P = .039, PFS: P = .020). Similarly, high Claudin-6 expression was associated with a median OS of 32 months and PFS of 21 months, compared to an OS of 60 months and PFS of 56 months in patients with low Claudin-6 expression (OS: P = .005, PFS: P = .003). Both univariate and multivariate analyses confirmed that advanced age, and high Trop-2, and high Claudin-6 expression were significant predictors of poor OS and PFS (P < .05). These findings underscore the prognostic significance of Claudin-6 and Trop-2 in ovarian cancer, with elevated expression correlating with poorer survival outcomes. These markers may serve as independent prognostic factors, and their targeting through antibody-drug conjugates offers a potential therapeutic strategy to improve survival outcomes and overcome treatment resistance.

Purpose: TROP2 is overexpressed in many tumor types and is being actively pursued as a target. Sacituzumab govitecan (SG), a humanized anti-TROP2 antibody conjugated with SN-38, was approved for treatment of metastatic triple negative breast cancer, with the greatest efficacy in patients with medium or high TROP2 levels. We sought to enhance efficacy of TROP2-targeted therapies by pharmacological regulation of TROP2 expression. Methods: TROP2 levels were assessed by immunohistochemistry (IHC) in two sets of breast tumors: a set of surgical samples and a tissue microarray. TROP2 mRNA expression was assessed in surgical samples and breast cancer patient-derived xenografts (PDXs) with RNAseq and assessed in the TCGA. In cell lines, expression of TROP2, E-cadherin (E-cad), and Schlafen family member 11 (SLFN11) were assessed by immunoblotting and qPCR following drug treatment or cell line manipulation. Epithelial-mesenchymal transition was evaluated by cell migration, cell invasion, and anchorage-independent growth assays. Antitumor efficacy of drug combination was assessed by cell survival, cell colony formation, and apoptosis assays. Results: By IHC, TROP2 was expressed in only 40% of metaplastic breast cancers (MpBC), but nearly all non-MpBC tumors. TCGA database evaluation further showed higher TROP2 levels in non-MpBC tumors than metaplastic tumors. In breast cancer surgical specimens, breast cancer PDXs, and the TCGA, there was a strong correlation between TROP2 and E-cad expression. In vitro, we demonstrated that downregulating transcriptional factor zinc finger E-box binding homeobox 1 (ZEB1) led to mesenchymal-epithelial transition with upregulation of both E-cad and TROP2 expression in breast cancer cells, leading to increased sensitivity to SG treatment. Screening of epigenetic modulators identified DNA methyltransferase inhibitor decitabine as an enhancer of TROP2 and E-cad expression in PDX cell lines of metaplastic cancer origin and mesenchymal subtype breast cancer cell lines. Decitabine increased TROP2 expression by decreasing TROP2 promoter methylation. Decitabine was significantly synergistic with SG, and enhanced apoptosis. Similarly, overexpression of TROP2 (by plasmid) in cell lines enhanced activity of SG. Furthermore, decitabine increased expression of SLFN11, a putative biomarker of SN38 sensitivity, and was synergistic with SG in TROP2 expressing, SLFN11 low breast cancer cell lines. Conclusion: TROP2 is expressed in most breast cancers, but is expressed less frequently in MpBC, an aggressive subtype unresponsive to traditional therapies. Epigenetic modulator decitabine upregulates TROP2 and SLFN11 expression and enhances antitumor efficacy of SG. Combinatorial treatment of TROP2 ADCs with epigenetic modulators of TROP2 represent a novel therapeutic strategy for tumors with low TROP2 or SLFN11 expression. Citation Format: Ming Zhao, Timothy P. DiPeri, Gabriela Raso, Yasmeen Q. Rizvi, Xiaofeng Zheng, Kurt Evans, Argun Akcakanat, Fei Yang, Debu Tripathy, Ecaterina Ileana Dumbrava, Senthil Damodaran, Funda Meric-Bernstam. Epigenetically upregulating TROP2 enhances therapeutic efficacy of TROP2 ADC sacitizumab govitecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1791.

Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² &amp;gt; 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-11.

4579 Background: Patients (pts) with mUC have an estimated 5-year overall survival (OS) rate of 14%. Trop-2 is a transmembrane glycoprotein with elevated expression in many cancers, including UC. SG is a Trop-2–directed antibody-drug conjugate with accelerated FDA approval for pts with LA unresectable or mUC who previously received platinum (PT) and a checkpoint inhibitor (CPI). SG has demonstrated activity in 3 phase 2 TROPHY-U-01 mUC cohorts: Cohort 1 (C1), 28% objective response rate (ORR); Cohort 2 (C2), 32% ORR; and Cohort 3 (C3), 41% ORR. Here, we assess efficacy outcomes in C1-3 by Trop-2 archival tumor expression. Methods: Pts (≥18 years) with previously treated (PT [C3], CPI [C2], or both [C1]) LA or mUC received SG (10 mg/kg IV) on D1 and D8 of 21-D cycles; C3 pts also received pembrolizumab (200 mg) on D1 of 21-D cycles. The primary endpoint was ORR by independent review. Archival tumor samples collected at enrollment were assessed for Trop-2 protein expression using SP295 anti–Trop-2 antibody by IHC with assessment by histological scores (H-scores; scale, 0-300) and % of membrane-positive tumor cells (Roche Tissue Services). Trop-2 association with clinical endpoints was evaluated using unstratified Cox proportional hazards models for survival data and logistic regression for ORR. Results: At data cutoff, 192 pts were enrolled in C1-3; 144 pts (75%) had samples evaluable for Trop-2 prevalence and 139 pts (72%) were evaluable for efficacy analysis by Trop-2 expression (5 pts not assigned to any cohort were excluded). Baseline characteristics for pts with evaluable samples were consistent with the overall population. Median (IQR) Trop-2 H-score and % of membrane-positive tumor cells for evaluable pt samples were 215 (180-247) and 92% (75-98), respectively; these readouts were highly correlated (ρ=0.82, P&lt;0.001). ORRs for C1 samples with below (n=42) and above (n=45) median Trop-2 H-scores were 24% and 29% ( P=0.59), respectively; median progression-free survival (PFS) was 3.4 and 6.7 months (HR=0.765, P=0.262), respectively; and median OS was 9.9 and 10.9 months (HR=0.978, P=0.927), respectively. Median PFS for C1 samples with below and above median Trop-2 membrane positivity were 3.9 and 6.2 months (HR=0.835, P=0.443), respectively. Analyses of efficacy endpoints for C2 and C3 pt samples by Trop-2 expression were consistent with C1 results. Conclusions: In this analysis of archival tumor samples from pts with pretreated LA or mUC, Trop-2 protein was highly expressed across cohorts, supporting prior Trop-2 expression analyses in UC. Similar outcomes were observed between C1 pts with samples that were below and above median Trop-2 H-score with most pronounced numerical differences observed for PFS. These results suggest that SG activity may be independent of Trop-2 expression in UC, but additional studies are needed to confirm these results. Clinical trial information: NCT03547973 .

Background: Trop2 (trophoblast cell surface antigen) is a transmembrane calcium transducer which has been associated with tumor growth, aggressiveness and metastasis. Compared to normal tissue, Trop2 is expressed at much higher levels in many epithelial tumors, which makes it an excellent target for ADCs (antibody-drug conjugates). Sacituzumab govitecan-hziy, an ADC that combines Trop-2 antibody with a SN-38 payload has been recently approved for treatment of refractory metastatic triple-negative breast cancer, and several other TROP2 targeted therapies are in development. To better understand Trop 2 expression in breast tumor that may help to identify patients who could benefit most from Trop2-targeted therapy, we aimed to screen Trop2 protein in multiple types of breast tumor. Design: Using chromogenic immunohistochemistry (IHC), Trop2 protein was examined in 42 formalin fixed paraffin embedded (FFPE) specimens from surgically resected breast tumors, including 21 TNBC, 16 HR+, 4 HER2+ (3 HR+/HER2+ and 1 HR-/HER2+), and 1 sarcoma. Sections were stained on an automated staining system (BOND-MAX; Leica Microsystems) using anti-TROP2 antibody (clone ERP20043, Abcam, cat# 214488). Membrane staining was assessed. The percentage of positivity (0% to 100%) and the staining intensity (0 = no staining, 1+ = weak staining, 2+ = moderate staining, and 3+ = strong staining) were evaluated and multiplied to generate H-score (0-300). Results: Among of 41 breast carcinoma cases, 38 (92.6%) cases expressed Trop2 protein in tumor cells, with a median H-score of 162.5, including 18/21 TNBC, 16/16 HR+, 4/4 HER2+ cases. Trop2 was expressed in several breast cancer histopathology subtypes, including 32 invasive ductal carcinomas (H score ranged from 29 - 290), 2 invasive lobular carcinomas (H score 140 and 145), 1 invasive papillary carcinoma (H score 70), 1 invasive micropapillary carcinoma (H score 210), 1 invasive squamous cell carcinoma (H score 245) and 1 metaplastic carcinoma with mesenchymal differentiation (cartilaginous sarcomatoid type) (H score 17). Three breast carcinomas were negative for Trop2 (H score 0). All three cases are metaplastic carcinomas, including two spindle cell carcinomas and one with mesenchymal differentiation, which are TNBC. Only two of 5 (40%) metaplastic carcinomas tested expressed Trop2 (p = 0.0034). One breast sarcoma was tested and had no TROP2 expression. Conclusion: Trop2 is commonly expressed in breast cancer. However, metaplastic carcinoma of breast has shown a trend of Trop2 protein loss. Further validation on Trop2 loss in metaplastic carcinoma might help with identifying breast cancer patients less likely to benefit from Trop2-targeted therapy. Citation Format: Fei Yang, Yasmeen Rizvi, Erkan Yuca, Ming Zhao, Kurt Evans, Xiaofeng Zheng, Aysegul Sahin, Funda Meric-Bernstam. Loss of Trop2 protein in metaplastic carcinoma of breast by IHC [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-37.

e12558 Background: Human trophoblastic cell surface antigen 2 (Trop2) is a transmembrane protein overexpressed in breast cancer tissues. However, it is present at minimal levels in adult somatic tissues, making it an ideal target for treatment with antibody-drug conjugates (ADCs). Sacituzumab govitecan, an anti-Trop2 ADC, has been approved for the treatment of triple-negative breast cancer and urothelial cancer. Recent studies have demonstrated that human cytotoxic T lymphocytes can recognize Trop2. Accordingly, we aimed to assess the role of Trop2 in the tumor immune activity of different subtypes of breast cancer, and laying the foundation for further studies on the efficacy of the combination of Trop2 ADC and immune checkpoint inhibitors in breast cancer. Methods: We retrospectively retrieved tissues from 152 invasive breast carcinoma of no special type and 111 benign breast tumor tissues and undergoing surgical resection at our institution from 2018 to 2023. Immunohistochemistry was used to detect Trop2 and PD-L1 expression and lymphatic invasion in patients, and their correlations with clinicopathological factors were analyzed. Trop2 expression was evaluated using the H-score. The median as the cut-off value, a score of 0–240 indicated low expression, and 240–300 indicated high expression. Cases with CPS ≥ 1% were considered positive for PD-L1. Results: The expression was Trop2 in breast cancer tissues than in benign tumor tissues (P&lt;0.0001). Immune infiltration analysis suggested that Trop2 expression was correlated with immune cell infiltration and the abundance (P&lt;0.001). Kaplan–Meier plotter analysis demonstrated that high Trop2 expression was related to poor prognosis in patients with triple-negative breast cancer (overall survival: HR=1.53, P=0.029; relapse-free survival: HR=1.26, P=0.045). In contrast, Trop2 expression was not significantly associated with prognosis in other breast cancer subtypes. However, there was no statistically significant correlation found between PD-L1 expression and Trop-2 expression in breast cancer. Conclusions: The findings suggest that Trop2 correlates with poor prognosis and immune cell infiltration in breast cancer. This indicates the role of Trop2 as a potential prognostic biomarker, laying the foundation for further research focusing on the immune regulatory role of Trop2. [Table: see text]

Background: Trophoblast cell surface antigen (TROP2), is expressed on many tumors including breast cancer, thus TROP2 antibody drug conjugates are being pursued as a therapeutic strategy. Datopotamab deruxtecan (Dato-DXd) is composed of a humanized anti-TROP2 IgG1 monoclonal antibody attached to a highly potent topoisomerase I inhibitor payload (an exatecan derivative DXd) via a stable tetrapeptide-based cleavable linker. Dato-DXd has shown promise in the treatment of TROP2 expressing lung and breast cancers. However, the role of TROP2 as a predictor of therapeutic sensitivity is yet to be elucidated. We sought to determine efficacy of Dato-DXd in breast cancer patient-derived xenografts (PDXs) as well as explore biomarkers of response and combinations with PARP inhibitors. Methods: Membrane expression of TROP2 was assessed in 31 breast cancer PDXs and matching patient tumors by immunohistochemistry (IHC). Schlafen family member 11 (SLFN11) nuclear expression was also assessed by IHC. The antitumor efficacy of two doses (1 and 10 mg/kg, q3wk, IV) of Dato-DXd and the isotype control-DXd (IgG-DXd) were tested against 9 breast cancer PDXs derived from residual tumors after neoadjuvant chemotherapy. The PDXs represented a range of TROP2 expression levels, including 3 TROP2 negative/low PDXs. The antitumor activity of Dato-DXd in combination with PARP inhibition (olaparib) was assessed in 3 PDXs with intermediate Dato-DXd activity. Tumor volumes were measured twice weekly; antitumor activity was assessed by treatment-to-control ratio (T/C) and event-free survival (EFS-2). T/C ratio was calculated as (Vt,21/Vt,0)/(Vc,21/Vc,0), where t = treatment, c = control (no treatment or IgG-DXd), and V = tumor volume (mm3). Stable disease (SD) and response (R) were based on day 21 (-30- to 20% and &amp;lt;-30%, respectively). An event was defined as tumor doubling. Results: TROP2 H-score in PDXs correlated with TROP2 expression in matched patients (r= 0.7264, p&amp;lt;0.0001), however expression was lower in PDXs (p= 0.04). Dato-DXd caused R/SD in 2 of 9 models at 1 mg/kg, and in 4 of 9 models at 10 mg/kg. All models that regressed with Dato-DXd expressed TROP2. TROP2 expression was associated with higher antitumor activity compared to IgG-DXd based on T/C ratio (r= -0.7448, p= 0.0213) and EFS-2 (r= 0.9318, p= 0.0068) at 10 mg/kg but not at 1 mg/kg. Three models with low TROP2 expression had &amp;gt;50% SLFN11 positivity in the nucleus by IHC; 2 of 3 had doubling of EFS-2 both by Dato-DXd and IgG-DXd. The combination of Dato-DXd and olaparib had improved activity over single agents in 2 of 3 models. Additional comparative predictive and pharmacodynamic biomarker studies will be presented. Conclusion: Dato-DXd is a promising therapy for breast cancer patients including those resistant to standard chemotherapy. Additional biomarkers may better integrate DXd sensitivity into patient selection. Citation Format: Erkan Yuca, Kurt Evans, Argun Akcakanat, Gabriela Raso, Yasmeen Q. Rizvi, Fei Yang, Lauren Byers, Senthil Damodaran, Okajima Daisuke, Funda Meric-Bernstam. Anti-tumor activity and biomarker analysis for datopotamab deruxtecan in breast cancer PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1768.

BackgroundSacituzumab govitecan is an antibody–drug conjugate that delivers SN-38, an active metabolite of irinotecan, to the target molecule, trophoblast cell-surface antigen 2 (Trop-2). It is a promising drug for triple-negative breast cancer and is anticipated to be effective for luminal breast cancer. The efficacy of the agent relies on the expression of Trop-2 rather than its intracellular function. However, conditions that alter the Trop-2 expression have not been well investigated.MethodsWe tested a range of clinically related treatments for their effect on Trop-2 expression in cultured breast cancer cell lines.ResultsThe expression level of Trop-2 differed among cell lines, independent of their subtypes, and was highly variable on treatment with kinase inhibitors, tamoxifen, irradiation, and chemotherapeutic agents including irinotecan. While inhibitors of AKT, RSK, and p38 MAPK suppressed the Trop-2 expression, tamoxifen treatment significantly increased Trop-2 expression in luminal cancer cell lines. Notably, luminal cancer cells with acquired resistance to tamoxifen also exhibited higher levels of Trop-2. We identified transcription factor EB (TFEB) as a possible mechanism underlying tamoxifen-induced elevation of Trop-2 expression. Tamoxifen triggers dephosphorylation of TFEB, an active form of TFEB, and the effect of tamoxifen on Trop-2 was prevented by depletion of TFEB. A luciferase reporter assay showed that Trop-2 induction by TFEB was dependent on a tandem E-box motif within the Trop-2 promoter region.ConclusionsOverall, these results suggest that the effectiveness of sacituzumab govitecan could be altered by concomitant treatment and that tamoxifen could be a favorable agent for combined therapy.