Adlowitz, Diana G.

Abstract Although TP53 is commonly mutated in transformed follicular lymphoma, mutations are reported in <5% of pretreatment follicular lymphoma (FL) specimens. We assayed archival follicular B-cell non-Hodgkin lymphoma specimens from a completed clinical trial, Southwest Oncology Group S0016, a phase 3 randomized intergroup trial of CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone) chemotherapy plus R-CHOP (rituximab-CHOP) compared with CHOP chemotherapy plus 131-iodine tositumomab (radioimmunotherapy [RIT]-CHOP). Subclonal TP53 mutations (median allele frequency 0.02) were found in 25% of diagnostic FL specimens and in 27% of a separate validation cohort. In the R-CHOP arm, pathogenic TP53 mutations were not associated with progression-free survival (PFS) (10-year PFS 43% vs 44%). In contrast, among patients with no detectable pathogenic TP53 mutation, RIT-CHOP was associated with a longer PFS than with R-CHOP (10-year PFS 67% vs 44%; hazard ratio = 0.49; P = .008). No relationship was detected between PFS and the extent of activation-induced cytidine deaminase (AICDA)–mediated heterogeneity. In summary, subclonal TP53 mutations are common in FL and are a distinct phenomenon from AICDA-mediated genetic heterogeneity. The absence of a detectable subclonal mutation in TP53 defined a population that particularly benefited from RIT.

Background: Activation induced cytidine deaminase (AICDA or AID) causes somatic hypermutation (SHM) of the immunoglobulin heavy chain variable genes (IGHV). It has been widely hypothesized that aberrant targeting of AICDA creates additional driver mutations and intra-clonal heterogeneity which enables clonal evolution and thus disease progression. This hypothesis predicts that greater AICDA-mediated intraclonal heterogeneity will be associated with shortened progression free survival (PFS). To definitively test this hypothesis that AICDA-mediated heterogeneity is linked to risk of progression, we evaluated available samples from the largest US-based phase 3 trial of untreated follicular lymphoma patients treated with CHOP-based therapies, SWOG trial S0016. Methods: We have previously shown that ultradeep sequencing of two genes, the expressed IGHV and the 5' UTR of the translocated BCL2 allele, provide unrelated measures of AICDA-mediated genetic heterogeneity (Spence, J Immunol. 2014). Furthermore, the number of AICDA-mediated mutations in the 5' UTR of BCL2, but not in IGHV, correlates with the number of AICDA-mediated mutations at other known AICDA sites throughout the genome (such as PIM1, BCL6, RHOA, etc). Genomic DNA was prepared from all available paraffin-embedded specimens. Ultra-deep, amplicon-based sequencing (~10,000x) with internal controls to establish specimen-specific signal-to-noise parameters allowed sensitive and specific detection of sequence variants at 0.2% (FDR 1%). Results: For subjects with (152) and without (419) available specimens, the median duration of follow-up (11.3 years), the number of patients who progressed (60%), and other patient characteristics (demographics, histology, stage, FLIPI) were indistinguishable. Sequencing data that fulfilled strict quality metrics for low frequency variants were obtained for >75% of the specimens (114 for BCL2; 120 for IGHV); further analysis was restricted to these subjects. The extent of intraclonal variation within both the IGHV and the 5' UTR of BCL2 was highly variable. For BCL2, a median of 7 mutations/case were detected (range 0-61); the mutations were highly skewed to the RCYW motif (p<0.001), the preferred target of AICDA. These mutations defined a median of 5 subclones/case (range 1-22). The clonal IGHV was somatically mutated (<0.98 germline identity) in 94% of samples (median 0.89). For IGHV, a median of 113 subclones/case were detected (range 0-1531). Consistent with our prior study, no correlation was seen between the number of mutations detected in the IGHV and BCL2 or the number of subclones defined by IGHV and BCL2. Neither the number of mutations in nor the number of subclones defined by BCL2 or IGHV were prognostic. Cox regression showed no relationship between PFS and mutation number or subclone count based on either IGHV or BCL2 (hazard ratios are indistinguishable from 1; parameters were treated both as a continuous variables and dichotomized as quartiles). Furthermore, receiver operator curves did not suggest a cut point for any of these measures that would predict disease progression at any time (areas under the curve are indistinguishable from 0.5). Conclusions: Several measures of AICDA-mediated mutation and resulting subclonal heterogeneity were not found to be prognostic in previously untreated, advanced stage FL treated with CHOP-based regimens. In contrast, AICDA-mediated mutations have been reported to accrue during transformation from low grade FL to large cell lymphoma. This paradox calls for a more refined model to explain the role of AICDA in the progression of FL. Support: NIH/NCI grants U10CA180888, U10CA180819, U10CA180821, R21CA198072(WRB); and in part by GlaxoSmithKline. Disclosures Rimsza: NanoSting: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution]. Leonard:Bayer Corporation: Consultancy; AstraZeneca: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Sandoz: Consultancy; ADC Therapeutics: Consultancy; Miltenyi: Consultancy; BeiGene: Consultancy; Sutro Biopharma: Consultancy; Celgene: Consultancy; Nordic Nanovector: Consultancy; Gilead: Consultancy; Karyopharm Therapeutics: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Bayer Corporation: Consultancy; Celgene: Consultancy; Epizyme, Inc: Consultancy; Merck: Consultancy; MorphoSys: Consultancy; AstraZeneca: Consultancy; Epizyme, Inc: Consultancy; Akcea Therapeutics: Consultancy; Miltenyi: Consultancy; ADC Therapeutics: Consultancy; BeiGene: Consultancy; Karyopharm Therapeutics: Consultancy; Merck: Consultancy; Sandoz: Consultancy; Gilead: Consultancy; Akcea Therapeutics: Consultancy; MorphoSys: Consultancy; Nordic Nanovector: Consultancy; Sutro Biopharma: Consultancy. Fisher:Celgene: Consultancy; Barclays: Honoraria; AstraZeneca: Consultancy; prIME: Honoraria. Smith:Portola Pharmaceuticals: Research Funding. Friedberg:Bayer: Honoraria, Other: Data & Safety Monitoring Committee; Acerta: Other: Data & Safety Monitoring Committee.

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.