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77 An NGS assay to identify HLA loss of heterozygosity for future CEA and MSLN logic-gated CAR-T solid tumor protocols designed for reduced on-target, off-tumor toxicity
  1. Scott Kopetz1,
  2. Maria Pia Morelli1,
  3. Julian Molina2,
  4. Diane Simeone3,
  5. J Randolph Hecht4,
  6. Kedar Kirtane5,
  7. Mitesh Borad6,
  8. Theodore Welling3,
  9. Edward Garon4,
  10. Armen Mardiros7,
  11. Xueyin Wang7,
  12. Eric Ng7,
  13. Tyler Danek8,
  14. Shannon Gallagher8,
  15. Ariane Lozac’hmeur8,
  16. Karl Beutner8,
  17. John Welch7,
  18. David Maloney9,
  19. William Go7 and
  20. Sandip Patel10
  1. 1The University of Texas MD Anderson Cancer Center, Houston, TX, USA
  2. 2Mayo Clinic, Rochestser, MN, USA
  3. 3New York University Langone Health, New York, NY, USA
  4. 4University of California at Los Angeles, Los Angeles, CA, USA
  5. 5H. Lee Moffitt Cancer Center, Tampa, FL, USA
  6. 6Mayo Clinic Cancer Center, Scottsdale, AZ, USA
  7. 7A2 Biotherapeutics, Inc., Agoura Hills, CA, USA
  8. 8Tempus, Chicago, IL, USA
  9. 9Fred Hutchinson Cancer Research Center, Seattle, WA, USA
  10. 10University of California San Diego, La Jolla, CA, USA


Background Chimeric antigen receptor (CAR) T-cell therapy has shown clinical efficacy in hematologic cancers, but success is limited in solid tumors due to a lack of tumor-specific targets that distinguish cancer from normal cells and an immunosuppressive tumor microenvironment.1 Integrating synthetic biology and comprehensive molecular profiling of tumors may provide active and tolerable approaches to CAR T-cell therapy in patients with solid tumors.

Human leukocyte antigen (HLA) loss of heterozygosity (LOH) in tumors offers a definitive tumor vs normal discriminator target for CAR T-cell therapy.2 The Tmod platform3,4 is a modular logic-gated CAR T system comprising different versions including a carcinoembryonic antigen (CEA)- or mesothelin (MSLN)-targeting CAR activator and a separate blocker receptor targeting HLA-A*02 or other HLA alleles to protect normal cells. Compared with existing immunohistochemistry (IHC) tests, Tempus xT-Onco is a standard-of-care next-generation sequencing (NGS) assay5 that detects somatic alterations including HLA LOH and generates whole transcriptome RNA data (eg, CEA or MSLN expression) and a tumor immune infiltration profile, which can effectively identify patients appropriate for Tmod CAR T-cell therapy.

Methods HLA LOH in solid tumors was assessed with paired germline and somatic DNA sequencing. Common driver mutations, microsatellite instability status, and tumor mutational burden were examined in HLA-A LOH or HLA-A intact cohorts. Tumor expression of CEA and MSLN was evaluated via RNA sequencing and compared with immunohistochemistry (IHC) results.

Results A total of 21,053 tumor samples in the Tempus database were compared with their matched-normal samples. HLA-A LOH was detected in 16% of 10,867 advanced solid tumors (table 1) and similar LOH frequencies were observed among common HLA-A alleles. Clinical factors and molecular biomarkers were similar between HLA-A LOH and HLA-A intact cohorts. High CEA expression was seen in IHC-positive patients.

Conclusions The frequency of HLA-A LOH in solid tumors in the Tempus database is similar to that reported in the Cancer Genome Atlas.6 Tempus xT-Onco reliably detects HLA LOH and quantifies CEA and MSLN expression. Based on these data, patients with solid tumors are now being prospectively screened for HLA LOH using xT-Onco in an ongoing tissue banking study (BASECAMP-1, NCT04981119), preparing for future interventional protocols.


  1. Martinez M, Moon EK. CAR T cells for solid tumors: new strategies for finding, infiltrating, and surviving in the tumor microenvironment. Review. Front Immunol. 2019;10:128.

  2. Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A. 2021;118(12):e2022410118

  3. Hamburger A, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol. 2020;128:298–310.

  4. DiAndreth B, Hamburger AE, Xu H, Kamb A. The Tmod cellular logic gate as a solution for tumor-selective immunotherapy. Clin Immunol. 2022;241:109030.

  5. Beaubier N, Tell R, Lau D, et al. Clinical validation of the tempus xT next-generation targeted oncology sequencing assay. Oncotarget. 2019;10(24):2384–2396.

  6. The Cancer Genome Atlas (TCGA) Research Network [Internet]. Accessed June 2022.

Abstract 77 Table 1

Solid tumor samples (n) and HLA-A LOH frequency (%)

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