Subclonal Mutations of TP53 Are Common in Untreated Follicular Lymphoma and Mutation Status Is Predictive of PFS When CHOP Is Combined with 131-Iodine Tositumomab but Not with Rituximab: An Analysis of SWOG S0016
Authors: Burack Richard , Li Hongli , Spence Janice M , Adlowitz Diana , Rimsza Lisa M. , Braziel Rita M. , Spier Catherine M. , Kaminski Mark S , Leonard John P. , Fisher Richard I. , Leblanc Michael L. , Smith Sonali M. , Friedberg Jonathan W. 
Published: October 2019

Abstract
Background: TP53 is reported to be mutated in about 20% of transformed and/or relapsed follicular lymphoma (FL) specimens but in fewer than 5% of pre-treatment FL specimens. The disparity in the reported prevalence of TP53 mutations at diagnosis versus transformation/relapse suggests that TP53-mutated subpopulations, which are too small for detection, might be present at the time of diagnosis in tumors destined to relapse. To test this hypothesis, we developed a more sensitive assay for TP53 mutations and applied the assay to SWOG S0016, a phase III randomized intergroup trial of CHOP chemotherapy plus rituximab (R-CHOP) compared with CHOP chemotherapy plus 131-iodine tositumomab (radio-immunotherapy (RIT)-CHOP) for previously untreated follicular non-Hodgkin lymphoma.
Methods: Formalin Fixed Paraffin Embedded (FFPE) biopsies were available for 213/571 enrollees. 147 samples fulfilled inclusion criteria: 1) sufficient DNA for 3 PCR-reactions each at 250 ng gDNA (~40,000 diploid equivalents; 5'UTR and all exons of TP53 were amplified in 3 multiplexed reactions to obtain consistently sized amplicons); 2) appropriate size distribution of DNA products after library preparation (~300 bp). To maintain specificity with high read-coverage, each PCR included an amplicon of eUCR41 (eukaryotic ultra-conserved region) to provide specimen-specific metrics of DNA-damage and sequencing noise. Sequence reads (Ilumina 300 nt paired end) were merged, mapped to reference (>10,000x coverage), and single nucleotide variants (SNV) were called using LoFreq based on case and base-change error thresholds (from the eUCR41 data). Pathogenicity of each TP53 sequence variant was based on a consensus of the Seshat and IARC databases. Variant Allele Frequencies (VAF) of passenger SNVs (5'UTR of BCl2) allowed normalization to the minimum tumor fraction.
Results: A total of 73 SNVs within TP53 were detected in 147 FFPE specimens (80 RIT-CHOP; 67 R-CHOP); the SNVs in 34 patients (23%; 17-30% 95% CI) created missense or nonsense mutations which met criteria for pathogenicity. The VAF for these SNVs ranged from 0.008 to 0.7 (median 0.02) and 82% had a VAF of less than 0.1. Among the 34 patients with a pathogenic TP53 mutation, no relationship was observed between the VAF and time to progression. The group of 34 patients with a pathogenic TP53 mutation had a shortened Progression Free Survival (PFS) (HR=1.68; p=0.035) and Overall Survival (OS) (HR=2.02; p=0.045). However, the relationship between TP53 mutations and PFS depended on treatment: for the R-CHOP arm, the pathogenic TP53 mutations were not associated with PFS (10-year PFS 44% vs 42%); in contrast, for the RIT-CHOP arm, pathogenic TP53 mutations were associated with a shortened PFS (10 year PFS 27% vs 65%; HR=3.2; p=0.001) (see figure). Furthermore, among patients with no detectable pathogenic TP53 mutation, RIT-CHOP was associated with a longer PFS than R-CHOP (10 year PFS 65% vs 44%; HR=0.58; p=0.046).
Conclusions: Using specimens from SWOG S0016, we find that pathogenic TP53 mutations are present in 23% of untreated, advanced stage FL specimens, a considerably higher fraction than reports based on less sensitive methods. Long term follow-up of S0016 has shown that the RIT-CHOP arm had a small but statistically significant improvement in the 10-year PFS compared to R-CHOP (Shadman 2018). We now show that the interaction of RIT and TP53-status substantially affects PFS. First, in the cohort without a detectable TP53 mutation, a longer PFS was observed with RIT-CHOP than R-CHOP. Second, in the cohort with a detectable TP53 mutation, a shorter PFS was observed with RIT-CHOP than R-CHOP. This observation is consistent with the well-defined role of TP53 as the mediator of the apoptotic response to ionizing radiation. Therefore, an assay which detects subclonal populations with TP53 mutations defines two cohorts for which the inclusion of radio-immunotherapy had substantial and opposite effects on the risk of progression. Finally, this identification of subclonal TP53 mutations as a predictive biomarker highlights that preserving specimens from clinical trials to mine with new technologies can be key to the advancement of precision medicine.
We wish to recognize the late Dr. Oliver Press for his central role in S0016 and his many contributions to SWOG.
Support: NIH/NCI grants U10CA180888, U10CA180819, U10CA180821, R21CA198072 (RB); and in part by GlaxoSmithKline.
Figure. Figure.

Disclosures
Rimsza: NanoString: Other: Inventor on the patent for the Lymph2Cx assay. Leonard:Juno: Consultancy; ADC Therapeutics: Consultancy; Pfizer: Consultancy; Gilead: Consultancy; Sutro: Consultancy; Biotest: Consultancy; United Therapeutics: Consultancy; BMS: Consultancy; Bayer: Consultancy; Genentech/Roche: Consultancy; Celgene: Consultancy; MEI Pharma: Consultancy; AstraZeneca: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy. Smith:BMS: Consultancy; Portola: Honoraria. Friedberg:Bayer: Honoraria.