ct-DNA is useful to diagnose mutations at codon 641 of exon 16 of EZH2, which is a biomarker for relapse in patients with Diffuse Large B-cell Lymphoma treated with the R-CHOP scheme

Jose Díaz-Chavez Instituto Nacional de Cancerología (INCan) Olga Gutiérrez-Hernández Instituto Nacional de Cancerología (INCan) Lucia Taja-Chayeb Instituto Nacional de Cancerología (INCan) Sindy Gutiérrez-Chavarría Instituto Nacional de Cancerología (INCan) Alejandro Aviles-Salas Instituto Nacional de Cancerología (INCan) Myrna Candelaria (  candelariahmgloria@gmail.com ) Instituto Nacional de Cancerología (INCan)


Background
Diffuse Large B Cell Lymphoma (DLBCL) constitutes the most common of all aggressive types of lymphomas [1]. It is a clinically and molecularly heterogeneous malignant lymphoproliferative disease [2,3]. Traditionally, it has been classi ed into morphological variants, molecular subtypes, and distinct disease entities. Among no-otherwise-speci ed (NOS) cases, an accepted grouping is either germinal center (GC) subtype and or non-germinal center (non-GC) subtype. GC subtype has a signi cantly better prognosis. However, within the GC-subtype, some patients show Myc rearrangement with co-expression of BCL2 or BCL6, de ned as double or triple hit lymphomas [2)] which have a more aggressive clinical behavior. Recently, the presence of EZH2 mutations has also been implicated in the prognosis of DLBCL [4][5][6].
The epigenetic regulator EZH2 is a subunit of the polycomb repressive complex 2 (PRC2), methylates H3K27, resulting in transcriptional silencing [7][8]. The overexpression of EZH2 has been identi ed as a driver in lymphomagenesis [9]. In addition, the mutation at Y646 amino acid in the EZH2 gene is recurrently and signi cantly mutated in up to 40 % of B-cell lymphomas [10], and particularly in approximately 13-22 % of DLBCL [4,[11][12]. The mutations of Tyr641 (Y641F, Y641N, Y641S, and Y641H) are de cient catalysts of unmodi ed H3K27 methylation relative to the wild type (wt) enzyme. However, these mutants are superior to the wt enzyme in catalyzing further methylation to mono, and especially the demethylated H3K27 peptides [13,14]. The nal effect of these mutations is gene silencing.
Traditionally, mutations are documented in tumor samples. However, with the development of new techniques, the identi cation of circulating tumor DNA (ctDNA) is available and useful to monitor tumorspeci c molecules in the blood, with a sensitivity approaching 1 x 10 6 cells [15] and also with high tumor speci city. Also, the access to serial blood samples allows monitoring these tumor-speci c changes and follow-up during treatment [16]. This quantitative approach has been used as a marker for the identi cation of tumor biology, and to predict long-term outcome [17].
Although ctDNA promises as a monitoring tool, the standardization of the collection and processing is necessary to improve DNA preservation and facilitate accurate testing and interpretation of the results.

Discussion
The detection of somatic mutations directly from ctDNA is an attractive alternative, because ctDNA is a non-invasive, real time, tumor-speci c biomarker, an alternative source of tumor DNA for genotyping purposes. However, the molecular aberrations within lymphomas are heterogeneous and different methods are employed for such purposes. In this cohort, the addition of ddPCR analysis to Sanger sequencing increased the sensitivity from 52 to 95 %, with a PPV and of 100 % and 92 %, respectively.
In healthy subjects the cell-free DNA (cfDNA) derives from the apoptosis of hematopoietic cells. In lymphoma patients, the total amount of cfDNA has a median concentration of 30 ng/mL of plasma [23][24]. The normal cfDNA requires to be discriminated from ctDNA, and the test used for ctDNA detection and quanti cation requires to suppress the technical and biological noise in order to achieve the required sensitivity and speci city.
The ctDNA concentrations vary among the lymphoma subtypes, with higher values in Diffuse large B cell lymphoma, Hodgkin lymphoma and mantle cell lymphoma, and lower levels in low-grade B lymphomas, as follicular lymphoma. [16,[25][26][27][28]. In this cohort, the median ctDNA concentrations were within the reported range. Some studies [29][30][31] have concluded that higher levels of ctDNA are associated with different prognostic markers, as advanced clinical stage, poor risk prognostic categories evaluated with IPI score, or survival. However, in this cohort we only found an association between ctDNA and elevated levels of LDH, none other prognostic parameter, as clinical stage, or IPI correlated with ctDNA concentrations. This difference may be related to the fact that we had very few patients with low clinical stages by low grade, according to IPI score, when compared with other authors. In the same direction, we could not demonstrate a correlation with ctDNA levels and response to treatment, or survival since we collected only a sample at diagnosis; however other authors [30][31][32][33], have indeed demonstrated a correlation between the ctDNA levels and a worse response to treatment, or with survival [30,[34][35], evaluating the kinetics of ctDNA.
It has been proposed, that the liquid biopsy can inform about the whole intratumor heterogeneity. The concordance of results between the analysis in tumor samples, in comparison with ctDNA may vary with the proportion of a mutation within tumoral tissue and in ctDNA. In this study, the analysis by Sanger sequencing had a very low Se (52 %). However, the addition of a more sensible technique, as ddPCR increased the detection of EZH2 mutations in all patients analyzed by this technique.
Ultra-deep generation sequencing (NGS) methodologies can identify a range of genetic alterations. For example, The Cancer Personalized Pro le by Deep Sequencing (CAPP-Seq) is considered a diseasespeci c selector, covering a set of exonic and intronic regions of known recurrent mutations for a speci c cancer setting [25,36]. The CAPP-Seq relies on an updatable gene panel containing lymphoma genes, as well as somatic mutations; unfortunately, none commercial kits are available. Also, ddPCR assays are used to detect mutations but may not be representative of a fraction of ctDNA, unless a targeted mutation is known to be trunk in all lymphoma cases. In this sense, genotyping of ctDNA by CAPP-Seq allows the recovery of 100 % of the tumor con rmed actionable mutations of DLBCL, as EZH2, MYD88, CD79B [23,32].
Camus et al. [37] documented the usefulness of ddPCR to quantify recurrent and potentially somatic mutations in ctDNA from 88 patients with DLBCL, including EZH2 Y641 mutations. In addition, this author found a 100 % concordance for somatic mutation detection between ddPCR and NGS. In our study, the Se of this approach was 95 %, with a 100 % Sp, and as has been described [37], no falsepositive cases have been documented with this method. Dubois et al. [38] documented initially 22 %, and thereafter up to 24 % frequency of EZH2 Y641 mutations in GC-DLBCL, which is slightly higher than initially reported by Morin et al. (n=18/83, 21.7%) [39]. In our study, we found a similar frequency of EZH2 Y641 mutations (n=20/98, 20.4 %) in the same population.
Different authors have evaluated the clinical impact of EZH2 mutations in DLBCL in tumor samples [11,16,29,[40][41]. However, recently, only Nagy et al. [42] has used liquid biopsy to evaluate the clinical role of EZH2 mutations by ddPCR, but in patients with follicular lymphoma, and correlated the variant allele frequency with the volume of metabolically active tumor sites observed on 18F-uorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. To our knowledge, this is the rst study analyzing the EZH2 mutation using ctDNA to evaluate the frequency and the negative impact on PFS in Diffuse large B cell lymphoma. Furthermore, it will be interesting to determine other actionable mutations of DLBCL, as MYD88 and CD79B together EZH2 mutations in ctDNA, and analyze their impact in response to therapy and other clinical variables.
A personalized approach to cancer diagnosis implies integral tumor pro ling for each patient, which might be possible by tracking plasma ctDNA tumor-related mutations. The purpose of studying biopsy specimens may be the selection of a personalized anticancer therapy, relevant to the mutational pro le of the speci c tumor. But, on the other hand, application of the plasma ctDNA analysis allows for the monitoring of disease dynamics and the prescribed therapy effectiveness in order to detect any residual tumor after resection, relapse, or even metastasis within a particular patient [43].

Conclusions
In conclusion, our data support the implementation in the clinic of the analysis of recurrent somatic mutations like EZH2 in ctDNA to diagnose early detection of molecular relapse, guide salvage therapy based on molecular targets, and identify molecular resistance mechanisms.

Methods
We did a prospective cohort, non interventional study, with the aim to analyze the feasibility to detect the presence of exon 16 EZH2 mutations in ctDNA, and also evaluated the clinical impact of these mutations in terms of response, relapse and survival in a cohort of patients with DLBCL. We included consecutive patients diagnosed with DLBCL, who were attended at the National Cancer Institute (Mexico City, Mexico) between January 2017 till December 2019. The last follow-up was on July 31st 2021. The inclusion criteria were: age older than 18 years, histopathological diagnosis of DLBCL, without previous treatment and candidate to be treated with RCHOP. We excluded patients with hepatitis B or C or HIV, as well as those receiving any other treatment regimen.
All patients were treated with 6 cycles of RCHOP regimen: IV rituximab, 375 mg/m2 on day 1; IV cyclophosphamide, 750 mg/m2 on day 1; IV doxorubicin, 50 mg/m2 on day 1; IV vincristine, 1.4 mg/m2, with capping at 2 mg, on day 1; and oral prednisone, 100 mg daily on days 1-5. 18 Fluoro-deoxyglucose PET-CT positron emission tomography combined with computer tomography (PET-CT) was done at diagnosis, and at the end of treatment.
Analyzed outcomes were clinical response after chemotherapy, risk of relapse, Progression-free survival (PFS), and overall survival (OS). All patients signed informed consent. initial denaturation at 95°C for 5 min and a nal extension at 72°C for 5 min; denaturation at 95°C for 30 seconds, annealing for 30 seconds at 58°C and extension was done at 72°C for 30 seconds for 40 cycles. Ampli cation was veri ed by gel electrophoresis.
The PCR products were sequenced in at least two independent ampli cation reactions to analyze the presence of mutations in Exon 16 of EZH2, using the Reverse primer: 5´-CCAATCAAACCCACAGACTTAC-3'(Integrated DNA Technologies; Standard desalted puri cation synthesis) PCR amplicons were puri ed using isopropanol precipitation. According to the manufacturer's instructions, the puri ed DNA was diluted and cycle-sequenced using the ABI BigDye Terminator kit v3.1 (ABI, Foster City, CA, USA). Sequencing reactions were electrophoresed in an ABI3500 genetic analyzer. Electropherograms were analyzed, and the sequences obtained were compared with the EZH2 reference sequence (GenBank NG_032043.1).
Those samples of ctDNA that showed a discordant result after Sanger sequencing were analyzed by ddPCR. The tumor DNA was considered the standard of reference.
The reaction mixture for ddPCR contained 10 μg of ctDNA, 250 nmol/L forward and reverse primers, 250 nmol/L FAM-labeled wt probe, 250 nmol/L HEX-labeled Y641N, 250 nmol/L FAM-labeled Y641S, Y641H, Y641F probe, and 11 μl of 2 × ddPCR Supermix for Probes (BioRad Laboratories, Pleasanton, CA, USA). Distilled water was added to achieve a nal volume of 22 μl. The reaction mixture was then partitioned into nanoliter-sized droplets using QX200 Droplet Generator TM (BioRad Laboratories), in which the target DNA was randomly distributed into the droplets. Then, the droplets were transferred to a 96-well plate for PCR reaction in a thermal cycler (Biorad). The PCR program was initiated and held at 95°C for 10 min, followed by 39 cycles at 94°C for 30 sec 58°C for 1 min, and 98°C for 10 min. The PCR product from each well was then subjected to the QX200 Droplet Reader (BioRad Technologies), which analyzed the uorescence of each droplet individually using a two-color detection system. Custom software (QuantaSoft; BioRad Technologies) was used to count the number of droplets within each gate.

Statistical analysis.
A descriptive analysis was done for demographic and clinical characteristics. Median and its interquartile range (IQR) was used as a measure of dispersion. Clinical and histological variables were compared between wt and mutated cases by Chi-squared test, and T-student's test, as required. Response was evaluated by Lugano criteria (18). Progression-free survival (PFS) was de ned, from the date of diagnosis, until relapse, progression, or the last visit. Overall survival (OS) was de ned from the diagnosis date until death or last visit.
ctDNA concentrations were measured and compared by bivariate analysis with the following clinical prognostic factors: LDH levels, clinical stage, IPI score, response to treatment, and presence of relapse.
Results of EZH2 mutations in the tumor sample and ctDNA were compared, considering the tumor sample as the standard of reference. Sensitivity (Se), speci city (Sp), positive predictive value (PPV), as well as negative predictive value (NPV) were calculated. Se was calculated with true positive/ (true positive + false negative). Sp was calculated with: True negative / (true negative + false positive). PPV was: True positive/ (true positive+ false positive), and NVP was obtained with: true negative/ (false negative + true negative).
The Kaplan-Meier method was used to construct survival curves, and the Log-rank test was used for comparisons. The survival curves compared the mutated and wt cases.
The proportionality assumptions and interaction terms were checked in the nal models. The SPSS version 23 software (IBM, Corp. Armonk, NY) was used for computations.

Declarations
Ethical approval and consent to participate: This trial was approved by IRB (register number CEI/966/15).
All patients signed informed consent.
Competing interest.
All authors declare none con ict of interest.

Funding.
This research was supported by the pharmacogenetics Laboratory from the Instituto Nacional de Cancerología.
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript." Competing Interests.
The authors have no relevant nantial or non-naltial interests to disclose Author's contribution.     Progression free survival and risk of relapse curves, according to EZH2 mutation status.
Progression free survival in tumor (a) and ctDNA (b), and risk of relapse, when analyzing tumor (c) and ctDNA by ddPCR (d) Blue line= EZH2 wildtype, Red line= EZH2 Mutated