Proteomics Analysis Identifies Candidate Biomarkers for Prediction of Post-CAR-T Cytopenias
Authors: Madhukar Rashmi , Flora Christopher , Olesnavich Mary , Sandford Erin , Rozwadowski Michelle , Li Gen , Geer Marcus Johannes , Yanik Gregory , Ghosh Monalisa , Frame David , Agrusa Jennifer Elise , Bonifant Challice L. , Jain Tania , Choi Sung W. , Tewari Muneesh 
Published: December 2024

Introduction: CAR-T cell therapy demonstrates significant promise and is increasingly being adopted in the management of hematological malignancies. A subset of patients receiving CAR-T cell therapy remain cytopenic for a prolonged duration post-treatment, placing them at an increased risk for severe infections, bleeding, and reduced quality of life. Identifying early biomarkers of post-CAR-T cell cytopenia could provide insights into its underlying mechanisms and potential therapeutic targets.

Methods: All complete blood count measurements after CAR-T therapy (for up to one year) were obtained from the electronic health records and were used to identify patients who developed early (14-30 days post-infusion), prolonged (30-90 days post infusion) and late (&gt;90 days post-infusion) post-CAR-T cytopenias. Cytopenia and neutropenia were defined as having Common Terminology Criteria for Adverse Events (CTCAE) grade 3 or more decline in cell counts of any one cell line or absolute neutrophil counts respectively. In a subset of patients (n=27), proximity ligation-based proteomics screening (Olink Proteomics, Waltham, MA, USA) was used to measure 453 proteins in plasma samples from patients at baseline and 5-11 days after CAR-T infusion. To identify early biomarkers of post CAR-T cytopenia differential expression analysis was used to compare the proteomes of those who went on to develop post-CAR-T cytopenia and those who did not.

Results: 79 patients were included with a median age of 62 years (range: 6-79); 30% (24/79) of our study population was female. Early, prolonged, and late cytopenia were seen in 46/79 (58.2%), 34/76 (44.7%), and 35/65. (53.8%) patients respectively. Early, prolonged and late neutropenia were seen in 37/79 (46.8%), 31/79 (39.2%), and 20/65 (30.8%) patients respectively. Those with early cytopenia were more likely to have prolonged (X-squared = 23.6, p &lt; 0.001) and late cytopenia (X-squared = 3.325, p = 0.06).

Pre CAR-T infusion biomarkers: In the subset of patients (n = 27) whose plasma specimens underwent proteomics analysis, 20 proteins were differentially abundant between those who went on to develop early cytopenia (decline in any one cell line) vs. those who did not (with a fold change (FC) &gt; 2 and false discovery rate (FDR) of &lt; 5%). The list included an activator of the Wnt pathway, and 2 proteins involved in fibrinolysis. Nine proteins were differentially abundant when patients who specifically developed prolonged neutropenia were compared to patients who did not (with FC &gt; 2 and FDR &lt; 5%). These included a danger associated molecular pattern molecule (DAMP) and an activator of the Wnt signaling pathway.

Post CAR-T infusion biomarkers: Plasma samples collected between 5 to 11 days after CAR-T infusion were used to discover biomarkers measurable after CAR-T cell infusion. Two proteins, including a hematopoietic cytokine, were differentially abundant (FC &gt; 2 and FDR of &lt; 5%) between those patients who went along to develop late cytopenia (decline in any one cell line) compared to those who did not. Three proteins were differentially abundant when comparing proteomes of the patients who went on to specifically develop late neutropenia to those who did not. (FC &gt; 2 and FDR of &lt; 5%) These included two proteins involved in innate immune regulation, as well as a hematopoietic cytokine and a Wnt signaling antagonist.

Conclusions: Our results nominate candidate biomarkers for prediction of post-CAR-T cytopenias, which are suited for further investigation in larger validation studies. Our findings also point to dysregulation of innate immunity and Wnt signaling as lines of investigation for future mechanistic studies of post-CAR-T cytopenias.