Double-hit alterations of TP53 identify ultra high-risk disease in previously treated, MYD88 mutated waldenstrom macroglobulinemia. Free

Background: BTK-inhibitors and chemotherapy (CT) with rituximab are widely used frontline treatments in Waldenström Macroglobulinemia (WM). Higher rates of TP53 alterations (TP53^ALT) were detected in previously treated versus treatment naïve WM patients in the ASPEN study. Most previously treated patients in the ASPEN study received alkylators as well as nucleoside analogues raising concern about prior treatment exposures and acquisition of TP53^ALT. We therefore sought to clarify prior treatment exposures and risk of acquiring TP53^ALT, as well as delineate types of TP53^ALT that could contribute to high-risk disease in previously treated WM patients.

Methods: Whole-exome sequencing was performed in CD19⁺ sorted bone marrow (BM) mononuclear cells and CD19-depleted mononuclear cells from peripheral blood (germline control) for 247 previously treated WM patients. The mean read coverage was 625 and 115 for tumor and germline, respectively. TP53^ALT included either TP53 mutations (TP53^MUT) or del17p. Prior CT exposure was defined by previous treatment with an alkylating agent (AA) or nucleoside analogue (NA). Logistic regression analysis evaluated the association between TP53^ALTand CT exposure.

Results: We restricted our analysis to 164 patients who had a >5% MYD88 VAF to ensure adequate tumor content. Their median age was 64.9 years, and 61% were male. The median time from diagnosis and first therapy were 6.0 and 4.3 years, respectively. Patients received a median of 1.5 (range 1-9) prior therapies, and 50% had previous CT exposure. At the time of study biopsy, 57.9% were progressing. CXCR4mutations were identified in 75/164 (45.7%) patients. The median follow-up from study BM biopsy was 9.2 years (95% CI 8.1-10). TP53^ALT were identified in 19/164 patients (11.6%). Of these, TP53 double-hits were observed in 10/19 (52.6%), comprising cases with TP53 mutations plus del17p (n=6), UPD17 (n=3), and compound heterozygosity (n=1). Single events were found in 9/19 (47.4%) of TP53^ALT patients comprising single TP53^Mut (n=5) or del17p (n=4). TP53^ALT were more common in CT-vs. non-CT- exposed patients (15.9% vs. 7.3%; p=0.088). Double-hit TP53^ALT were more common in patients who received both AA and NA (18.8%) versus either an AA or NA (6.1%) or no CT (3.6%); p=0.069 for three-way comparison. Multivariate analysis adjusting for age, CXCR4 mutation status, progression status at study biopsy, and number of prior lines of therapy showed an association between prior CT exposure and acquisition of TP53^ALT (OR 2.8, p=0.10).

Importantly, more somatic variants (median 44 vs. 31, p=0.013) and copy number events (median 4 vs. 1, p<0.001) occurred in TP53^ALT versus TP53^WT patients. CT-exposed patients had more somatic variants than CT-unexposed patients (median 35.5 vs. 30, p=0.022). Among these, patients who received both an AA and NA showed a higher number of somatic variants (median 44.5 vs. 34.5, p=0.065) and copy number events (median 3 vs. 1, p=0.075) versus those who received AA or NA alone. After adjusting for age and prior lines of therapy, TP53^ALT status and prior CT exposure associated with increased risk for acquiring somatic variants (p<0.001).

OS was significantly worse for TP53^ALT versus TP53^WT WM patients (9-year OS: 45% vs. 74%; p=0.019). Stratification of OS by number of TP53 events showed that inferior survival outcome was driven by double versus single hits. The 9-year OS for double versus single hits was 19% vs. 88%; p=0.098. Conversely, those with single-hit TP53^ALT showed no significant difference versus TP53 wild-type patients (9-year OS: 88% vs. 75%; p=0.98). Boruta analysis showed that double hit TP53^ALT and age were the most relevant predictors for OS. A multivariate Cox regression confirmed sex (HR: 2.01, p=0.043), age (HR: 1.08, p<0.001), and double-hit TP53^ALT (HR: 3.6, p=0.002) significantly impacted OS, whereas single-hit TP53 ^ALT was not significant (p=0.73).

Conclusions: Prior CT exposure is associated with increased acquisition of TP53^ALT (including TP53^Mut and del17p), as well as other somatic variants and copy number alterations when compared to CT-unexposed patients. Double-hit TP53^ALT was a major predictor of poor survival thereby identifying an ultra-high risk disease population. Our studies further inform risks of upfront CT use and provide support for the routine assessment of TP53 and del17p in WM patients, and the investigation of novel treatment approaches for patients with ultra-high risk TP53^ALT.