A New Post-Transplant Monitoring Paradigm: Combined Pharmacogenetic and Torque Teno Viral Load Testing to Determine Individual Sensitivity to Tacrolimus
Authors: Chadha Abhi , Alhadid Yazan , Mamroth Max , Wang Zhiwei , Nejad Dariush , Rumpler Marc , McCloskey Christopher , Vlassov Alexandre , Chikova Anna 
Published: December 2024

Tacrolimus is a commonly prescribed immunosuppressant for GVHD prophylaxis and treatment in HSCT patients. Effective tacrolimus management balances the risk of GVHD from insufficient dosing against the risk of toxicity and other complications associated with excessive drug concentrations, which can be further complicated by individual variations in therapeutic response.
Tacrolimus is metabolized by the CYP3A5 enzyme, where reductions in CYP3A5 activity slow the rate of metabolism resulting in rapid increases in blood concentration and a decreased clearance for a given dose of the drug. Identifying individual CYP3A5 polymorphisms may therefore inform proper tacrolimus dosing. Three CYP3A5 alleles (*3, *6 and *7) are associated with full or partial loss of protein activity, depending on zygosity. Both the Dutch Pharmacogenetic Working Group (DPWG) and the Clinical Pharmacogenetics Implementation Consortium (CPIC) agree on recommendations for tacrolimus dosing based on CYP3A5 genotyping for the *3, *6 and *7 alleles. Zhu et al. (2020) observed a similar association between CYP3A5 genotypes and tacrolimus kinetics in the blood of HSCT patients, and suggested use of this marker could support faster, more efficient dose optimization. However, the investigators reported that CYP3A5 typing alone was insufficient for long-term treatment success and indicated that additional biomarkers should be leveraged to inform immunosuppression management post-transplant. One such biomarker is the human Alpha Torquetenovirus (TTV), a non-pathogenic virus that is highly prevalent across all major populations and age groups. Multiple studies conducted among more than 1,000 transplant recipients, including HSCT recipients, have observed significant associations between TTV titers and individual immunosuppression levels. Monitoring TTV levels post-transplant may therefore offer clinicians a pathway to assess individual sensitivity to immunosuppressive treatment and provide data that further inform patient management decisions.
We developed two qPCR based tests: one that identifies patients' CYP3A5 genotype and another that provides a precise measurement of TTV in patient blood to support post-transplant monitoring.
Our new test for CYP3A5 *3, *6 and *7 genotyping uses a 6-plex qPCR assay format and exhibits a high level of reliability. We conducted analytical performance testing by assessing accuracy, sensitivity, precision and robustness of the assay, known as TacroType, using multiple sources of human DNA. Accuracy was assessed for each possible genotype at all three alleles using well-characterized reference samples. TacroType consistently demonstrated accurate and robust performance within a broad range of DNA input. Precision studies indicated reproducible assay results across operators, instruments and reagent lots. Assay performance was further demonstrated using EDTA and ACD blood and buccal swabs prepared by a variety of DNA extraction methods.
Our new, quantitative TTV test utilizes multiplex real-time PCR as well. Despite the highly polymorphic nature of the TTV genome, the test accurately measures the 27 most common groups of TTV with high sensitivity and precision, as demonstrated in analytical testing. We confirmed our ability to measure TTV in EDTA blood samples from 40 donors. We also confirmed the specificity of our assay against select human Anelloviruses (TTMV, TTMDV), characterized by highest sequence similarity to TTV. No cross-reactivity was detected with TTMV and TTMDV in concentrations equivalent of 280 million copies per 1 mL of blood. The assay displayed satisfactory linearity in TTV quantification and a single-copy level of sensitivity for all 27 target variants.
Combining pharmacogenetic data with post-transplant monitoring for GVHD management may help to address individual variabilities in treatment sensitivity among HSCT patients.