NOTCH1 fusions in pediatric T-cell lymphoblastic lymphoma: A high-risk subgroup with CCL17 (TARC) levels as diagnostic biomarker

Abstract

Twenty percent of children with T-cell lymphoblastic lymphoma (T-LBL) will relapse and have an extremely poor outcome. Currently, we can identify a genetically low-risk subgroup in pediatric T-LBL, yet these high-risk patients who need intensified or alternative treatment options remain undetected. Therefore, there is an urgent need to recognize these high-risk T-LBL patients through identification of molecular characteristics and biomarkers. By using RNA sequencing which was performed in 29/49 T-LBL patients who were diagnosed in the Princess Maxima Center for Pediatric Oncology between 2018 and 2023, we discovered a previously unknown high-risk biological subgroup of children with T-LBL. This subgroup is characterized by NOTCH1 gene fusions, found in 21% of our T-LBL cohort (6/29). All patients presented with a large mediastinal mass, pleural/pericardial effusions, and absence of blasts in the bone marrow, blood, and central nervous system. Blood CCL17 (C-C Motif Chemokine Ligand 17, TARC) levels were measured at diagnosis in 26/29 patients, and all six patients with NOTCH1 gene fusions patients exclusively expressed highly elevated blood CCL17 levels, defining a novel and previously not known clinically relevant biomarker for T-cell lymphoblastic lymphoma. Four out of these six patients relapsed during therapy, a fifth developed a therapy-related acute myeloid leukemia during maintenance therapy. These data indicate that T-LBL patients with a NOTCH1 fusion have a high risk of relapse which can be easily identified using a blood CCL17 screening at diagnosis. Further molecular characterization through NOTCH1 gene fusion analysis offers these patients the opportunity for treatment intensification or new treatment strategies.

Figure 1

NOTCH1‐rearrangements in T‐cell lymphoblastic lymphoma. (A) Schematic representation of three different NOTCH1 fusions with different fusion partners. The in‐frame IKZF2::NOTCH1 fusion generates a chimeric protein in which the N‐terminal DNA binding domain of IKZF2 is fused to the C‐terminal intracellular domains of NOTCH1. Fusions transcripts with miR142HG and TRBJ use an alternative translation start site in exon 28 (Met1727). (B) Western blot analysis using Val1744 antibody (Cell Signaling Inc.) shows that miR142HG::NOTCH1 and the TRBJ::NOTCH1 fusions lead to a larger NICD protein, likely representing uncleaved NICD with translation initiation at Met1727. Simultaneous beta‐actin staining was performed for loading comparisons.

Figure 2

Gene expression differences in NOTCH1‐rearranged, mutated and wildtype T‐cell lymphoblastic lymphoma. (A, B) Volcano plots showing differentially expressed genes between NOTCH1‐rearranged and NOTCH1‐WT samples (n = 1288; A) and between NOTCH1‐mutated and NOTCH1 WT samples (n = 101; B) (C) Expression analysis of the 200 most significantly upregulated and downregulated genes (from a total of 1288 genes) in NOTCH1‐rearranged compared to NOTCH1‐WT samples, revealed that NOTCH1‐rearranged samples cluster separately from NOTCH1 WT and NOTCH1‐mutated samples using Euclidean distance as a measure of similarity. The relapse sample of TLBL042 was used because of better quality. Range of 0–10 showing the log2‐transformed TPM values. Significance was determined using false discovery rate (FDR)‐adjusted p‐values.

Figure 3

Gene set enrichment analysis (GSEA) results of the NOTCH signaling pathway. (A) Enrichment plot for the NOTCH signaling pathway in the NOTCH1‐rearranged versus wild‐type (WT) samples, showing the profile of the running enrichment score and positions of genes associated with this pathway on the rank‐ordered gene list. (B) Z‐scores of log2‐transformed TPM values of genes associated with the NOTCH signaling pathway within the complete T‐LBL cohort. Mean z‐scores are depicted for NOTCH1‐rearranged, NOTCH1‐mutated, and WT samples. Genes are ranked based on their position in the rank‐ordered gene list used for GSEA between NOTCH1‐rearranged and WT samples.

Figure 4

CCL17 (TARC) in NOTCH1‐rearranged patients. (A) CCL17 levels in pg/mL per patient, showing highly elevated CCL17 in blood of NOTCH1‐rearranged T‐LBL patients but none of the other patients. 10,000 pg/mL is the maximum measurable CCL17 level with used assay. Orange line in (A–C) represents maximum normal CCL17 level (1300 pg/mL) based on what has been described in Hodgkin lymphoma. Patients that had a relapse are indicated with an asterisk. Patient TLBL049 developed a therapy‐related acute myeloid leukemia (double asterisk). (B) For three NOTCH1‐rearranged patients, blood CCL17 levels could be determined for a time point of remission after diagnosis, revealing normalized CCL17 levels in all three cases. (C) For four NOTCH1‐rearranged patients blood CCL17 levels were determined at time point of relapse and remission after relapse (second remission), revealing increased levels in three relapses that again normalized in second remission. (D) Staining for TARC using anti‐CCL17 antibody for four NOTCH1‐rearranged patients showing that T‐LBL cells do not express high levels of CCL17 based on immunohistochemistry.

Figure 5

Cumulative incidence plot of events reveals a significant higher cumulative incidence of relapse in NOTCH1‐rearranged cases compared to the rest of the T‐LBL cohort (3 years) (p < 0.001). The p‐value is estimated using Gray's test. Four relapses occurred in the NOTCH1‐rearranged cohort. No relapses occurred in the cohort. In both cohorts, one other event occurred, which was a therapy‐related‐AML in the NOTCH1‐rearranged cohort and death due to pancreatitis complicated by a septic shock in the other cohort.