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Cox et al. J Transl Genet Genom 2021;5:80-8 https://dx.doi.org/10.20517/jtgg.2021.06 Page 82

In parallel to advancements in CART cell therapy, genome sequencing also has a long history. The idea of
[16]
somatic changes in the genome as a cause of cancer originated in the 1900s . It was not until the 1970s that
we began to link these changes to specific genes.

[17]
The first discussions of the human genome sequencing project began in the 1980s , but the sequencing
didn’t begin until a decade later and wasn’t completed until early 2000s. Both the time needed to sequence
and the cost of sequencing per genome have improved dramatically, and millions of genomes have been
sequenced since then . The thousand genomes project was completed in 2015 and played an important
[18]
role in defining normal, non-deleterious genetic variation by providing a database of variant frequency [19,20] .
The progression of laboratory techniques and public availability of genetic data not only allows us to better
understand the development of cancer, but it also enlightens our therapeutic intervention. These
technological advancements allow us to use genetics to characterize resistance and toxicity of CART cell
therapy [Figure 1]. In this manuscript, we will review lessons learned from multi-omics studies to assess the
response and toxicities after CART cell therapy (summarized in Table 1).

T CELL MOLECULAR STATE TO UNCOVER MECHANISMS OF RESISTANCE
Increasing evidence from correlative studies of clinical trials suggests that T cell fitness is an important
determinant of CART cell response . In order to generate CART cells, T cells undergo ex vivo activation,
[21]
ex vivo expansion, and transduction or transfection to express the CAR transgene. After a period of 7-14
days, cell manufacturing is completed and CART cells are cryopreserved for their future use. The
components that govern T cell fitness at the end of their expansion and manufacturing process are unclear.
Critical T cell factors include expansion condition, stimulatory and co-stimulatory molecules used in the
CAR design, T cell subsets, CART cell activation threshold, and the susceptibility to T cell exhaustion
during the engineering process . The optimal level of T cell activation in vivo to achieve adequate
[21]
expansion and anti-tumor activity is unknown. It is also unknown how much T cell receptor stimulation
contributes to CART cell activation, expansion, and response. The advancement of sequencing and
computational tools allows us to uncover the answers to some of these questions using high-throughput
data.

In an analysis of baseline CART19 products [Axicabtagene ciloleucel (Axi-Cel), CD28 co-stimulated
CART19], transcriptome sequencing was used to reveal that non-responders to CART cell therapy had an
increased population of exhausted CD8 T cells compared to a population of memory T cells in patients with
complete response . This is consistent with prior studies indicating a correlation between a baseline
[22]
memory T cell phenotype with increased response after CART cell therapy . The outcomes of these
[21]
patients were also associated with cell-free DNA sequencing on day 7 after CART infusion. The patients
who had a 5-fold decrease in tumor-related, cell-free DNA correlated with improved anti-tumor response at
3 months post infusion .
[22]

Another strategy is to study the molecular status of the tumor and the T cells simultaneously, in an attempt
to identify a genetic signature or phenotype of CART cells and/or the tumor that would result in a specific
outcome. A classification tool called LymphGen was created that inputs tumor biopsy results and uses a
probabilistic model to output classification as one or more of the seven genetic subtypes of diffuse large B
cell lymphoma (DLBCL) . These subtypes differ in oncogenic signaling, genetic signature, TME, and
[23]
survival rates . This tool could lead to more precise CART cell therapies targeting a specific subtype of
[23]
DLBCL.