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Introduction: Prostate cancer therapy involves the use of androgen deprivation therapy, chemotherapy, targeted therapy and immunotherapy. Chemotherapy involves the use of anti-tubulins (docetaxel, cabazitaxel), platinum salts, and topoisomerase inhibitors (TOPO1, TOPO2A). There is no biomarker of chemotherapy that is routinely used. We examined 87 prostate cancer samples using targeted proteomics for biomarkers of response or resistance to chemotherapy agents. Biomarkers of resistance include ERCC1 (Platinum), TUBB3 (taxanes), ALDH1A1 (cyclophosphamide), while response biomarkers include TOPO1 (irinotecan, topotecan), TOPO2A (doxorubicin, epirubicin), and hENT1 (Gemcitabine). We also measured markers for several antibody-drug conjugates targets (Her2, Her3, Trop2) in our clinical proteomics (CLIA) platform and several ADC/CAR-T proteins (PSMA, STEAP1, Nectin4, Claudin 18.2. in our research platform Methods: Tumor areas from Formalin-fixed, paraffin-embedded (FFPE) tumor tissues from clinical samples of prostate cancer received at our CLIA certified laboratory were microdissected and a quantitative proteomic analysis of 72 biomarkers were conducted using selected reaction monitoring mass spectrometry (SRM-MS). Discussion:[WL1] Androgen receptor was detected in majority of the samples (83%) with a 19x range of distribution (329 amol/ug - 24063 amol/µg). Majority of prostate cancer samples expressed a range of resistance markers for anti-tubulin inhibitors (TUBB3: 69% detection with a 14x range) indicating that the high expressors is likely to be resistant to docetaxel/cabazitaxel based regimens. ERCC1, a marker for resistance to platinum-based agents, was not detected in 26% of cases, potentially enabling a cisplatin/carboplatin-based regimen in 1/4th of prostate cancer patients. Cyclophosphamide could be effective in 10% of cases where ALDH1A1 was not detected. TOPO1, a marker for irinotecan-based therapy was observed in 94% of cases with a range of ~6x (488 - 2760 amol/µg). Other chemotherapy agents that are not routinely used in prostate cancer was observed in select groups. These include doxorubicin biomarker TOPO2A in 34% of cases with 10x range, gemcitabine biomarker hENT1 in 36% of cases (4x range), temozolomide biomarker MGMT (ND in 12% of cases). ADC biomarker analysis revealed HER2 which was observed in 59% of the cases with range of expression from 301 - 1255 amol/µg including a significant population of low HER2 (84%, Citation Format: Sheeno P. Thyparambil, Wei-Li Liao, Robert Heaton, Amanda Strasbaugh, Marya Melkie, Negin Ghafourian, Xuefeng B. Ling. Quantitative proteomics of antibody-drug conjugates and chemotherapy targets in prostate 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 2160.

Introduction: Trop2 is overexpressed in many cancers and currently Trop2 ADC is approved in TNBC. In ADC drug design, it is imperative to assess not only the levels of the receptor but also the payload biomarkers. We have developed a multiplex mass spectrometry method to quantitate 72 actionable proteins from FFPE samples that requires minimal tissue (2-3 sections). This panel includes chemotherapy, targeted therapy and immunotherapy agents. The test is run in our CAP, CLIA, and NYSDOH approved laboratory. For this study, we examined a subset of samples run in the clinical lab for the levels of Trop2 (target biomarker) and payload biomarkers (Topo1, TUBB3). Topo1 is a chemosensitive marker for irinotecan, while TUBB3 is a chemoresistance marker for tubulin inhibitors. Methods: FFPE tissue sections from 1140 clinical samples from a variety of cancers were examined. These include breast (n=318), colorectal (n=228), ovarian (n=199), GBM (n=69), NSCLC (n=169), HNSCC (n=91), and Gastric cancer (n=66). Two sections (10 µ) of FFPE tissue were cut on DIRECTOR slides and only the tumor areas were laser microdissected for downstream analysis which resulted in tryptic peptides. 1µg of peptides (~4000 cells) along with heavy peptides was injected into a triple quad mass spec and 72 biomarkers were quantitated concurrently. Results: Trop2 showed a wide range of expression in various cancers. Almost all (95%)breast cancer samples expressed Trop2 which exhibited a wide range (93x; 157 - 14650 amol/µg). Topo1 and TUBB3 were expressed in 93% and 60% of samples respectively. 1/4th of NSCLC does not express Trop2 and there is a 106x difference in expression of Trop2 in NSCLC. Topo1 was expressed in almost all samples while TUBB3 was expressed in 80% of NSCLC. Majority of ovarian cancer samples (85%), HNSCC (89%), Gastric cancer (88%) samples expressed Trop2 with a 113x, 134x, 47x difference in expression. Chemosensitive biomarker Topo1 was expressed in almost all ovarian (96%), HNSCC (91%) and Gastric (99%) cancer samples. Chemoresistant marker TUBB3 was expressed in 66% of ovarian cancer, 44% of HNSCC and 45% of gastric cancer samples. In contrast to above cancers, only 10% of Glioblastoma samples express Trop2 and only 3/4th of GBMs express Topo1. Discussion: In a randomly selected group of cancers, we have found Trop2 is expressed in majority of Breast, Ovarian, Lung, HNSCC and gastric cancers and minimal expression in GBM. Given the range of expression of anti-tubulin resistance marker in many solid tumors, a payload biomarker guided clinical trial is highly recommended in ADCs that employ anti-tubulin inhibitors. In contrast, wide expression of chemosensitive biomarker for Topo1 payload makes it a promising candidate for many solid tumors. Further studies are warranted to determine the level of target and payload biomarkers that will be required for a clinical response. Citation Format: Sheeno P. Thyparambil, Wei-Li Liao, Robert Heaton, Guolin Zhang, Amanda Strasbaugh, Marya Melkie, Xuefeng B. Ling. Clinical survey of Trop2 antibody drug conjugate target and payload biomarkers in multiple cancer indications using multiplex mass spectrometry [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 4099.