CD19 Isoforms Enabling Resistance to CART-19 Immunotherapy Are Expressed in B-ALL Patients at Initial Diagnosis

Abstract

B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer and the prognosis of children with relapsed or therapy refractory disease remains a challenge. Treatment with chimeric antigen receptor-modified T cells targeting the CD19 antigen (CART-19 therapy) has been presented as a promising approach toward improving the outcome of relapsed or refractory disease. However, 10%-20% of the patients suffer another relapse. Epitope-loss under therapy pressure has been suggested as a mechanism of tumor cells to escape the recognition from CART-19 therapy. In this work, we analyzed the expression of CD19 isoforms in a cohort of 14 children with CD19 B-ALL and 6 nonleukemia donors. We showed that an alternatively spliced CD19 mRNA isoform lacking exon 2, and therefore the CART-19 epitope, but not isoforms lacking the transmembrane and cytosolic domains are expressed in leukemic blasts at diagnosis in children and in the bone marrow of nonleukemia donors. Furthermore, we clarified the sequence of a further isoform lacking the epitope recognized by CART-19 therapy and disclosed the presence of new isoforms. In comparison with the children, we showed that alternatively spliced CD19 mRNA isoforms affecting exon 2 are also expressed in 6 adult patients with CD19 B-ALL. On top of that, one of the adults expressed an isoform lacking the CD19 transmembrane and cytosolic domains. In conclusion, we proved that some of the CD19 isoforms contributing to CART-19 escape already preexist at diagnosis and could evolve as a dominant clone during CART-19 therapy suggesting the application of combined treatment approaches.

FIGURE 1

CD19 isoforms lacking the CART-19 epitope exist before any therapy in children and adults. Expression of CD19 isoforms was analyzed by RT-PCR with a primer pair amplifying exon1–5 in 3 pediatric leukemia patients (A), 6 controls (B), and 6 adult leukemia patients (D). The numbers of the pediatric patients (P), controls (C), and adult patients (A) according to Tables 1–3 are shown. bm indicates bone marrow; pb, peripheral blood. The full-length (fl)-, ex2part-, and Δex2-CD19 isoforms are indicated. Expression of the CD19 ex2part-isoform, the Δex2-isoform, and CD19 ex8–9 in pediatric patients and controls (C) and adult patients (E) was analyzed by qRT-PCR. The percentage of the expression of the isoforms relative to CD19 ex8–9 is shown. Expression analysis was done in triplicates.

FIGURE 2

The CD19 isoform lacking the membrane and cytosolic domains is not expressed at the time of diagnosis in children. Expression of CD19 isoforms was analyzed by RT-PCR with a primer pair amplifying exon4–8 in 14 pediatric leukemia patients (A), 6 controls (B), and 6 adult leukemia patients (C). The full-length (fl) plus a new isoform of 800 bp are indicated. The position of the expected ∆ex5–6 isoform is also indicated. The size of 500 bp is indicated for adult patients. The numbers of the pediatric patients (P), controls (C), and adult patients (A) according to Tables 1–3 are shown. bm indicates bone marrow; pb, peripheral blood.

FIGURE 3

CD19 isoforms lacking the CART-19 epitope are specifically expressed in leukemic blasts. A, Expression of the CD 19 ex2part-isoform, the ∆ex2-isoform, and CD19 ex8–9 in 3 pediatric (P) and 2 adult patients (A) after cell sorting was analyzed by qRT-PCR. The percentage of the expression of the isoforms relative to CD19 ex8–9 is shown. Expression analysis was done in triplicates. Expression of CD19 isoforms was analyzed by polymerase chain reaction with a primer pair amplifying exon4–8 in 3 pediatric leukemia patients (B) and 2 adult leukemia patients (C). The full-length (fl) plus a new isoform of 800 bp are indicated. The position of the expected ∆ex5–6 isoform is also indicated. The size of 500 bp is indicated for adult patients. The numbers of the pediatric patients (P) and adult patients (A) according to Tables 1 and 3 are shown. bm indicates bone marrow.

FIGURE 4

The CD19 ex2part-isoform leads to a truncated protein. A, Alignment of the CD19 ex2part-isoform sequence against the wild-type CD19 (NM_001770.5). B, Protein sequence of ex2part and wild-type exon2 (ex2). C, UCSC browser showing the CD19 gene structure, acetylation of H3K27 (H3K27ac), and DNase 1 hypersensitive peaks.