Cell-Free DNA Genotyping Analysis in Diffuse Large B-Cell Lymphoma; The Correlation between TP53 Gene Mutation and Cereblon Gene Mutation
Authors: Uryu Hideki , Mishima Yuko , Mishima Yuji , Yokoyama Masahiro , Nishimura Noriko , Kusano Yoshiharu , Okabe Takashi , Inoue Norihito , Fukuta Takanori , Shirouchi Yuko , Hatake Kiyohiko , Terui Yasuhito 
Published: November 2019

Introduction: Recently cell-free DNA (cfDNA) genotyping analysis has been utilized as a non-invasive procedure for detecting tumor specific genes or clarifying gene mutations instead of tumor sample biopsies. In diffuse large B-cell lymphoma (DLBCL), cfDNA analysis has been proceeding and the relationships between somatic gene mutation and the disease status have been considered. We retrospectively analyzed somatic gene mutations in DLBCL and examined the correlation between mutant genes and clinical outcomes by using serum cfDNA samples.
Patients and Methods: 50 patients newly diagnosed with DLBCL (including 4 patients mixed with follicular components) in our institute between March 2016 and March 2017 were enrolled in this study. All cases were sub-divided into germinal center B-cell (GCB) or non-germinal center B-cell (NGC) through immuno-staining, as CD10, bcl-6, and MUM-1 as Hans algorithm with biopsied tumor specimen. The stage at diagnosis and evaluation of treatment effects were assessed by PET-CT scan and bone marrow aspiration. All patients were treated by R-CHOP-like regimens with or without radiation. The serum samples from the patients were obtained before treatment and the cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 121 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. We compared the validation of the sequence results dependent on the characteristics and prognoses of the patients. Connections between each gene mutation and clinical features were assessed using Fisher's exact test. Mann-Whitney U test was used for evaluating factors associated with the number of gene mutations. Survivals were estimated by the Kaplan-Meier method and differences were compared using the log-rank test. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research.
Results: Median age was 65.4 years old (range 33-83), 27 patients were male and 23 were female. The stage of Ⅲ or Ⅳ were 17 (34%), IPI high or high intermediate were 17 (34%), and 20 (40%) had LDH>ULN. 27 patients (54%) were GCB and 23 (46%) were NGC. With a median follow up was 26.6 months (range 6-35). Factors affecting both shorter overall survival (OS) and progression-free survival (PFS) were LDH>ULN (P=0.037 and P<0.001), NGC (P=0.012 and P=0.002), IPI≧3 (P=0.023 and P<0.001) in univariate analysis. According to the cfDNA gene mutation analysis, the mutations of HDAC4 (88%), KDM3B (86%), HDAC6 (86%), KDM1A (84%), CREBBP (80%), JMJD1C (78%), and EGR1 (62%) were frequently detected in all DLBCL patients. CREBBP, PCLO, and KDM5A tended to be relatively abundant in NGC (P=0.084, 0.087, and 0.085, respectively). No other mutations had deviation in either GCB or NGC and correlated with other prognostic factors. 7 patients had TP53 mutation, 6 were TP53 p.N178H and 1 was TP53 p.P58L. All cases with TP53 p.N178H mutation were accompanied by more than 27 types of other gene mutations. This was significantly abundant compared to patients without TP53 p.N178H (mean of 38 types vs 16 types, P<0.001). Patients with 15 or more types of gene mutations tended toward poor PFS (2-year PFS of 89.5% vs 77.2%, P=0.092). Additionally, 7 patients had cereblon (CRBN) p.F101S mutation and 5 of 7 were concurrent with TP53 p.N178H mutation.
Discussion and Conclusion: This study showed serum cfDNA could be used as an alternative resource for analysis by tumor specimen. Our results indicated that TP53 mutation occurred in latter period undergoing multistep genetic variation and it showed the tendency that multistep mutations were related to poor prognosis. Interestingly, relative rate in TP53 mutation accompanied by CRBN mutation was high. In some reports, it was described that deletion p53 caused drug resistance including immunomodulator which targets CRBN in multiple myeloma cases. Zijun Y et al mentioned CRBN expression correlated with favorable prognosis in DLBCL with wild type TP53. Further studies are warranted to confirm the relationships between TP53 and CRBN mutations and its prognosis of DLBCL.

Disclosures
Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Celgene K.K.: Honoraria; Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Hatake:Takeda Pharmaceutical Co.,Ltd.: Honoraria; Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding. Terui:Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria; Bristol-Myers Squibb K.K.: Research Funding.