Droplet Digital PCR Monitoring of TP53 Mutated Circulating Tumor DNA Levels in Lymphoma Patients
Authors: Long Xiaolu , Gu Jia , Lou Yaoyao , Xu Qian , Cai Haodong , Zhang Wei , Shen Kefeng , Xiao Min , Zhang Yicheng , Chen Liting , Zhou Jianfeng 
Published: October 2019

Abstract
INTRODUCTION: Circulating tumor DNA (ctDNA), a portion of circulating cell-free DNA (cfDNA), is released from tumor cells into the circulatory system, which contains mutations corresponding to the patient's tumour alleles. Detection of ctDNA may noninvasively signal the presence of minimal residual disease (MRD) and predict prognosis. In recent years, droplet digital PCR (ddPCR) has emerged as a sensitive and effective tool for detection of point mutations in cfDNA. In this work, by application of ddPCR, we monitored the mutated TP53 ctDNA levels in serial plasma samples of lymphoma patients with identified TP53 hotspot mutations in their tumor biopsies. These results may give clues about the prognostic implications of different mutation locations and therapeutic strategies.
METHODS: Lymphoma-focused next-generation sequencing were performed in tumor biopsies from over 200 lymphoma patients. A total of 134 sequential plasma samples from 32 lymphoma patients with TP53 hotspot mutations were subsequently monitored. cfDNA was extracted from a median sample volume of 4.8 ml (range 3.5-7.0 ml) of plasma. Specific MGB probes were designed for each TP53 mutation site and were then validated by tumor biopsies as well as healthy plasma samples as positive and negative controls respectively. Patient samples were considered to be mutation-positive if the mutant concentration in the sample was higher than the 95% confidence interval of the assay-specific false positive rate. The ctDNA sequential samples were quantitatively tracked using the ddPCR system QX200 (Bio-Rad Laboratories).
RESULTS: 30 different TP53 mutations in 134 plasma samples from 32 lymphoma patients were monitored. All the mutations are hotspot in tumors according to COSMIC database, and are predicted to make damaging effect on TP53 protein by SIFT prediction. The average TP53 mutation frequency in tumor biopsies is 40.41% (2.30~92.21%). A total number of 30 specific MGB probes were designed for each TP53 mutation site. Their average false positive rate is 0.0004 copies/ul (0~0.002 copies/ul).
In our results, 6 out of 20 (30%) patients with mutations in Loop3 and LSH motifs within the DNA binding domain of TP53 had a clearance of ctDNA, while 6 of 12 (50%) patients with mutations outside of the Loop3 and LSH motifs had a clearance of ctDNA. Literatures reported that patients with non-detectable ctDNA in plasma have negative minimal residual disease (MRD). So these results suggested that Loop3 and LSH motifs, which were reported to interact with DNA groove directly, are more critical than other structures.
To investigate the association between different Ann Arbor stages and prognosis in TP53 mutated patients, we divided all the patients according to their stages. We found that patients with earlier Ann Arbor stages were more likely to have their ctDNA cleared (stage I/II: 83.33%; stage III/IV: 28%).
In addition, we also analyzed the prognostic implication of cytogenetic abnormality. We found that, in patients with del (17p) as well as TP53 mutation, 7 of 8 had persistent detectable TP53 mutated ctDNA, only 1 of 8 (12.5%) had a clearance of ctDNA, While among the patients carrying TP53 mutations but not accompanied del (17p), 11 of 24 (45.83%) patients had a clearance of ctDNA. These results suggested that it is more difficult to achieve remission in patients with no wide-type TP53 allele compared to those have one wide-type TP53 allele.
We further evaluated CAR-T cell immunotherapy and chemotherapy alone to 30 refractory recurrent TP53 mutated lymphoma patients. After CAR-T cell immunotherapy, 11 of 21 (52.38%) patients have non-detectable TP53 mutated ctDNA, while only 2 of 9 (22.22%) patients who received chemotherapy alone have non-detectable ctDNA. It suggested that CAR-T cell immunotherapy is more effective than chemotherapy alone to clear TP53 mutated ctDNA.
CONCLUSIONS: Circulating tumor DNA is a promising biomarker to noninvasively monitor tumor load and quantify MRD. By applying droplet digital PCR, ctDNA could be measured and monitored sensitively, specifically and fast. In this work, we designed and validated 30 TP53 probes and tracked 134 sequential plasma from 32 patients. We found that it is easier to clear ctDNA for earlier stages of lymphoma patients with TP53 mutations. CAR-T cell immunotherapy is more effective than chemotherapy alone to continuously clear ctDNA.
DISCLOSURE: No relevant conflicts of interest to declare.

Disclosures
No relevant conflicts of interest to declare.