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Background T cell receptor (TCR)-T cell therapy has shown clinical activity in a variety of cancers including melanoma, synovial cell sarcoma, and human papillomavirus (HPV)-associated cancers. 1–3 Despite responses in an assortment of malignancies, damaging mutations to human leukocyte antigen (HLA) class I alleles have been identified in non-responding tumors.3 4 To date, these alterations have been identified through examination of solid tumor biopsies. Practical limitations of tumor biopsy acquisition pose a challenge to studying the frequency and timing of these resistance mechanisms.5 6 Conversely, liquid biopsy offers a minimally invasive and easily repeatable method for real-time monitoring of tumors difficult or inaccessible to biopsy.7 8 This study aimed to determine if HLA-A*02:01 damaging mutations previously identified in tumor biopsies from 2 patients treated in a phase I trial of E7 TCR-T cell therapy for HPV-associated cancers could also be detected with liquid biopsy.3 Methods Archived plasma specimens collected before and after treatment from each of the 2 patients (Patients 4 and 5) were studied. Cell-free (cf)DNA was extracted using the QIAamp Circulating Nucleic Acid kit. Whole exome sequencing (WES) was performed on the Illumina NextSeq200. Somatic damaging mutations were identified by comparing patient-matched germline sequencing to WES of cfDNA using manual curation and the mutation callers mutect, mutect2, vardict and strelka.Results Patient 4 had primary resistance to E7 TCR-T cell therapy due to a nonsense mutation at amino acid position 51 in the α1 helix of HLA-A*02:01 as determined by a tumor biopsy on day +40 ( figure 1, left).3 Liquid biopsy detected this mutation in samples collected at each timepoint: pre-treatment (day -10), early post-treatment (day +10) and progression (day +40). Patient 5 had acquired resistance following a deep partial response with elimination of over 80 tumors. A progressing tumor specimen from day +349 showed a damaging mutation at amino acid position 141 in the peptide binding region of the α2 helix of HLA-A*02:01 (figure 1, right).3 Liquid biopsy did not detect this mutation pre-treatment (day -12) and at day +150 but did detect it at day +284.Conclusions These findings highlight the potential for liquid biopsy to detect both primary and acquired resistance to TCR-T cell therapy. This method holds promise for prospectively predicting response and monitoring for the development of resistance to TCR-based therapeutics.Ethics Approval The trial was registered on clinicaltrials.gov, trial number NCT02858310. This study was approved by the NIH IRB; approval number 16C0154.References Robbins PF, Kassim SH, Tran TLN, Crystal JS, Morgan RA, Feldman SA, et al. A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: long-term follow-up and correlates with response. Clin Cancer Res Off J Am Assoc Cancer Res. 2015 Mar 1;21(5):1019–27.D’Angelo SP, Araujo DM, Abdul Razak AR, Agulnik M, Attia S, Blay JY, et al. Afamitresgene autoleucel for advanced synovial sarcoma and myxoid round cell liposarcoma (SPEARHEAD-1): an international, open-label, phase 2 trial. Lancet Lond Engl. 2024 Apr 13;403(10435):1460–71.Nagarsheth NB, Norberg SM, Sinkoe AL, Adhikary S, Meyer TJ, Lack JB, et al. TCR-engineered T cells targeting E7 for patients with metastatic HPV-associated epithelial cancers. Nat Med. 2021 Mar;27(3):419–25.Norberg SM, Hinrichs CS. Engineered T cell therapy for viral and non-viral epithelial cancers. Cancer Cell. 2023 Jan 9;41(1):58–69.Boeddinghaus I, Johnson SRD. Serial biopsies/fine-needle aspirates and their assessment. Methods Mol Med. 2006;120:29–41.Hirahata T, Ul Quraish R, Quraish AU, Ul Quraish S, Naz M, Razzaq MA. Liquid biopsy: a distinctive approach to the diagnosis and prognosis of cancer. Cancer Inform. 2022;21:11769351221076062.Herberts C, Annala M, Sipola J, Ng SWS, Chen XE, Nurminen A, et al. Deep whole-genome ctDNA chronology of treatment-resistant prostate cancer. Nature. 2022 Aug;608(7921):199–208.Scaini MC, Catoni C, Poggiana C, Pigozzo J, Piccin L, Leone K, et al. A multiparameter liquid biopsy approach allows to track melanoma dynamics and identify early treatment resistance. NPJ Precis Oncol. 2024 Mar 28;8(1):78.Abstract 38 Figure 1Detection of damaging mutations in HLA-A*o2:01 by liquid biopsy. Illustration depicting somatic damaging mutations in HLA-A*02:01 molecule from treatment resistant tumor for patient 4 (left) and patient 5 (right)