<i> <i>NOTCH1</i> </i> gene Fusions Occur Frequently in Refractory or Relapsed (R/R) T-Cell Lymphomablastic Lymphoma

Background : T-cell lymphoblastic lymphoma (T-LBL) developed from immature precursor T cells, occurs mostly in children and adolescents. It is generally confined to a tissue lesion and may progress rapidly or relapse if not properly treated. Previous T-LBL genetic studies have shown high frequency of NOTCH1/FBXW7 and PI3K-AKT signaling gene mutations somewhat similar to T-ALL. Additionally, recent reports with limited cases suggested NOTCH1 gene fusion (NOTCH1r) was another kind of exclusive oncogenic driver event of T-LBL accompanied with higher relapse rate. However, large scale verification, further discovery of mechanisms, novel molecular features, and prognostic biomarkers are still urgently needed. Method: Retrospective analyses were performed for target sequencing of 203 T-LBL cases tested in our lab between April 2019 and July 2024. Among those, 90 patients were refractory or relapsed (R/R) including those relapsed after HSCT, and they were admitted to Beijing GoBroad Boren hospital for further therapy. NOTCH1 gene fusions and hotspot gene mutations were analyzed and annotated with an in-house workflow. Clinical and molecular features of these R/R T-LBL cases were then further characterized and analyzed. We also performed survival analyses for the NOTCH1r patients to consolidate the recent clinical findings. Results: NOTCH1 r were frequently observed with a much higher incidence of 25.6% (23/90) in our R/R as compared to 10.6% (12/113) in other patients without detailed clinical information (p &amp;lt; 0.01). The median age of the 9 female (39.1%) and 14 male (60.9%) NOTCH1r R/R T-LBL patients was 15.0 (IQR: 9.0-31.0) years, while the median age of the 12 female (17.9%) and 55 male (82.1%) non-NOTCH1r R/R cases was 12.5 (IQR: 9.3-14.0) years. Female seem to have a higher NOTCH1r incidence than male (p &amp;lt; 0.01), while the disease onset age between NOTCH1r and non-NOTCH1r group was not significantly different (p = 0.08). The bone marrow involvement was rare in NOTCH1r R/R cases (1/11, 9.21%) than that of non-NOTCH1r (28/42, 66.7%, p &amp;lt; 0.01), similar to previous report. A total of five NOTCH1 fusion partner genes, i.e., IKZF2 (9/23, 39.1%), TRB (5/23, 21.7%), TSPOAP1-AS1(4/23, 17.4%), IKZF1(3/23, 13.0%), and TRA (2/23, 8.7%) were discovered. All the fusions of NOTCH1 with these genes were previously reported as driver event in sporadic cases. The hotspot IKZF2/1 genes fused to NOTCH1 with a breakpoint in either exon 27 or 28 (NM_017617.5), result in a chimeric transcription factor protein retaining the IKZF2/1's DNA-binding domain and the NOTCH1's transmembrane and intracellular subunit. The other three genes fused to NOTCH1 with wide-range breakpoints located between intron 24 and exon 34, which may play a divergent pathogenic mechanism. Only 1 NOTCH1r patient (4.3%) harbored a NOTCH1/FBXW7 gene mutation, while 32 of 67 (47.8%) non-NOTCH1r patients harbored NOTCH1/FBXW7 gene mutation, indicating the mutual exclusivity of the two types (p &amp;lt; 0.01). Additionally, 9 of 23 NOTCH1r patients (39.1%) carried PI3K-AKT pathway gene mutations, which was much higher than 7.5% of non-NOTCH1r patients (5/67, p &amp;lt; 0.01). Median follow-up time for all the 23 R/R NOTCH1r patient was 24.5 (95% CI：15.9-24.7) months. The one year overall survival (OS) rate was 94.7% (95% CI: 85.2-100%), and the mOS was 30.4 (95% CI: 30.4-NA) months. The one year cumulative incidence of relapse rate (CIR) was 29.9% (95% CI: 11.0-49.4%), which tends to be much higher than the overall five year CIR of 12.2% as show in the EURO-LB02 trial study, further consolidating the recent findings of NOTCH1r's T-LBL. Conclusion: NOTCH1 gene fusion with divergent partner genes occurred frequently in R/R T-LBL. These oncogenic driver events were mutually exclusive with NOTCH1/FBXW7 gene mutations. However, these fusions accompanied frequently with PI3K-AKT pathway alteration, which were also regarded as worse prognostic factors. The one year CIR of 29.9% was much higher than the reported overall five year CIR of 12.2%.