Whole genome sequencing reveals potentially complex genomic features in lymphoma patients who have failed CD19 CAR-T cell therapy

A total of 49 LBCL patients were included in this review: 40 were new-onset DLBCL, 8 were converted follicular lymphoma (FL), and 1 was transformed chronic lymphoblastic leukemia (CLL).

The median age of patients was 65 years (range: 44-79), 11 women (22.
4%), and the median number of previous lines of treatment was 2 (range: 1-6).

Forty patients (81.
6%) were treated
with a platinum-containing regimen.
Eleven patients (22.
4%) received high-dose melphalan-based conditioning and salvage therapy
with autologous hematopoietic stem cell transplantation.
Thirty-four patients (69.
4%) received bridging therapy
prior to apheresis and CAR-19 infusion.
After CAR-19 treatment, 4 patients (8.
2%) developed grade 3 or above cytokine release syndrome (CRS), and 15 patients (30.
6%) developed grade 3 or above immune effector cell-associated neurotoxic syndrome (ICANS).

One patient died of CAR-19 toxicity within one week of infusion, and the disease response was unknown
.
The median overall survival (OS) for all patients was 11.
6 months and progression-free survival (PFS) was 8 months
.
The median follow-up of persistent responders was 17.
3 months
.
Eighteen patients (36.
7%) showed progression-free remission (Table 1).

This study explored the clinical features associated with poor prognosis after CAR-19 treatment and found that tumor metabolic volume (MTV), sex, age, and number of previous treatment lines before treatment were associated with a higher risk of recurrence (P=0.
048; P=0.
029; P=0.
008; P=0.
016) (Figure 1).

In patients with new-onset DLBCL, age and number of prior treatment lines were associated with a higher risk of recurrence (P=0.
004, P=0.
02).

In addition, MTV (P=0.
048) and bridging therapy (P=0.
012) were significantly associated
with shorter OS.

Figure 1

The study found no difference
in total mutation load between progressors and persistent responders.
TP53 is the most common driver mutation gene, with 42% of patients with R/R LBCL containing at least one mutation
.
But TP53 and any other individual driver mutant gene could not predict adverse outcomes
after CAR-19 treatment.

Previous studies have suggested that CD19 loss is one of the mechanisms of
CAR-19 resistance.
In this study, the genome-level study found that 3 patients had CD19 monoallele copy number loss, 2 patients showed CD19 mutations, and 4 of these 5 patients achieved a durable CAR-19 response
.
In addition, flow cytometry measured the expression of CD19 before treatment in 38 patients, of which 8 (10.
5%) had missing or decreased CD19 protein expression, and 6 of these 8 patients had disease progression, but the difference between CD19 protein expression level and poor prognosis was not statistically significant (P=0.
46), and there was no effect on OS (P=0.
61).

Finally, transcriptome studies of CD19 expression levels in 16 patients by RNA sequencing (RNA-seq) did not find any effect
on PFS or OS.

In this study, researchers identified 12 gene mutation signatures involved in R/R lymphoma patients (Figure 2), of which 8 have been included in COSMIC v.
2 and have been reported in newly diagnosed DLBCL patients, including: SBS1, SBS2, SBS5, SBS8, SBS9, SBS13, SBS17b, and SBS18
。 The other 4 were due to exposure to different chemotherapy (SBS-MM1: melphalone; E_SBS37: oxaliplatin; SBS31: cisplatin/carboplatin; SBS35: cisplatin), 2 of which are not yet included in
COSMIC.
Studies have shown that when aggressive lymphoma recurs, the mutagen-related burden increases and the clonal dominance of individual surviving cells can also drive its progression
.

Figure 2

Different mutation characteristics and patient response to CAR-19 were studied
.
The results showed a significantly shorter PFS in patients with APOBEC mutant activity (SBS2 and SBS13), and disease progression occurred in 12 of 13 patients (92%) (all patients: P=0.
0023; new-onset DLBCL: P=0.
045) (Figure 3).

Other mutational signature tests found that SBS18 was associated with disease progression after CAR-19 treatment (9/11 [81%]) (all patients: P=0.
0396; new-onset DLBCL: P=0.
045) (Figure 3).

SBS signaling reflects damage to the genome by oxygen radical stress
.
Both APOBEC and SBS18 are associated
with OS shortening.

Figure 3

TP53 single allele or double allele deletion is common (59.
2%), but it does not affect the survival of
patients after CAR-19.
3p21.
31 (RHOA) deletion was the only strong predictor of poor prognosis after CAR-19 (all patients: P=0.
0.
0013; new-onset DLBCL: P<0.
0001) (Figure 4).
<b11> After CAR-19 infusion, progression
occurred in 10 of 11 patients (91%).
RHOA deletion was also associated with OS shortening (P=0.
023).

Figure 4

WGS identifies detailed SV and complex variations
.
4011 SVs were found in 51 WGS samples (median R/R sample: 60; Range: 9-277).

In this study, 5 major complex SVs that could affect patient survival were observed, including: bimicrosomes (6 cases), chromosome fragmentation (23 cases), chromosome weaving (18 cases), and template insertion (11 cases).

Chromosomal fragmentation was associated with shorter PFS after CAR-19 treatment (P = 0.
026), with early progression in 18 of 22 patients (81.
8%), but not related to OS
.
All patients with new-onset DLBCL with bimicrobodies (4/4) had rapid disease progression and all patients died after CAR-19 treatment (PFS: P=0.
017 [Figure 5]; OS：P=0.
0011）
。

Figure 5

In addition, the results suggest that established prognostic markers in newly diagnosed DLBCL have a lower
ability to predict efficacy for CAR-19 treatment.
In patients treated with CAR-19, different SBS characteristics, copy number variation (CNV), and complex SV were independent predictors of
PFS and OS.