NOTCH1 Mutations Are Associated with Therapy-Resistance in Patients with B-Cell Lymphoma Treated with CD20xCD3 Bispecific Antibodies
Authors: Kyvsgaard Emil Ramsø , Grauslund Morten , Sjö Lene , Melchior Linea , Grantzau Trine , Gjerdrum Lise Mette , Trab Trine , Andersen Lærke Sloth , Gang Anne Ortved , Breinholt Marie Fredslund , Møller Michael Boe , Christensen Jacob Haaber , Larsen Thomas Stauffer , Clausen Michael Roost , Hasselbalch Riley Anna Caroline , Niemann Carsten Utoft , Gronbaek Kirsten , Hutchings Martin , Husby Simon 
Published: December 2024

Introduction:
CD20xCD3 bispecific antibody therapy (BsAb) have been approved for treatment of relapsed/refractory diffuse large B cell lymphomas (DLBCL) and follicular lymphoma (FL) with ≥2 prior lines of treatment. Approximately 50% of patients do not achieve long lasting remission when treated with single agent CD20xCD3 BsAb. Mechanisms of treatment resistance to these novel T-cell engaging antibodies are unclear. Previously, tumor mutations in TP53 have been associated with poor response to both immunochemotherapy (i.e. R-CHOP) and CD19 CAR-T cell therapy in patients with B-cell lymphoma. Genetic tumor aberrations have not yet been examined thoroughly in patients with long follow-up treated with CD20xCD3 BsAb.
Methods:
We conducted a retrospective study including patients with R/R B cell lymphoma treated at Rigshospitalet Copenhagen and Vejle University Hospital, both in Denmark from 2017-2023 as part of phase 1/2 clinical trials with CD20/CD3-targeted BsAb. Pre-treatment formalin-fixed and paraffin-embedded (FFPE) archival specimens were collected from 106 patients, of which 56 had sufficient tumor involvement and DNA quantity for clinical grade diagnostic next-generation sequencing (NGS). For analysis of clonal development under CD20xCD3 BsAb exposure, we collected paired relapse biopsies from 17 patients.A custom NGS lymphoma gene-panel covering 59 genes recurrently mutated in lymphoid malignancies were used with a median coverage &gt;10,000x for all samples.
Results:
We examined pre-treatment tumors from 56 patients with B-cell NHL who received CD20xCD3 BsAbs from 2017-2023. Median age was 70 years, 60.7% were male, and the median number of prior lines of therapy was three. Median follow-up time was 24.2 months.
The most frequently altered genes in pre-treatment tumors were CREBBP (47%), KMT2D (47%), TP53 (30%), TNFRSF14 (23%), BCL2 (25%), and GNA13 (18%).
In a univariate Cox model examining mutations in pre-CD20xCD3 BsAb treatment biopsies, NOTCH1 mutations were associated with significantly inferior progression-free survival (PFS) (crude hazard ratio (HR): 3.46, p = 0.026), while TP53 mutations did not impact overall response rate (ORR) or survival.
Examining CRS grade ≥2, we found that mutations in GNA13 (n =10) and CARD11 (n=9) were negatively associated with CRS grade ≥2 (p = 0.004 and p = 0.006). When adjusting for multiple testing, both mutations remained significant (q = 0.060 and q = 0.067). Mutations in CARD11 or GNA13 were not associated with inferior overall response rate (ORR) or PFS.
The most common mutations at relapse after CD20xCD3 exposure were CREBBP (41%), STAT6 (24%), BCL2 (24%), NOTCH1 (24%), KMT2D (18%) and BCL2 (18%).
Analyzing clonal evolution during CD20xCD3 BsAb, we identified gross expansion of NOTCH1 mutated clones in 29% of the paired relapse biopsies (these NOTCH1 mutations were undetectable in pre-treatment biopsies). Of the four new NOTCH1 clones identified at relapse, three were in exon 34. All exon 34 clones were detected in germinal center B cell like DLBCL samples. All patients with NOTCH1-mutated tumors (either in biopsy before or after CD20xCD3 BsAb; total 8 patients [14%] of the cohort) died within 6 months of lymphoma progression after initial detection of NOTCH1 aberration. Surprisingly, three patients were observed to have diminishing TP53 clone size at post-CD20xCD3 BsAb relapse.
Conclusions: NOTCH1 mutations predict poor survival and are a resistance mechanism to CD20xCD3 BsAb, while TP53 mutations do not seem to have an adverse effect on response or survival. We suggest investigation of NOTCH1 mutations in patients before undergoing treatment with CD20 targeted T-cell engaging therapies.