Background:MYC rearrangements (MYC-R), genetic hallmark of Burkitt lymphoma (BL), have been identified in 5-15% of DLBCL and 30-60% of high-grade B-cell lymphomas (HGL). MYC-R in BL always involve an immunoglobulin (IG) gen (IGH, IGK, IGL), whereas in DLBCL and HGL non-IG partners are frequent. The prognostic significance of the MYC partner gene in non-BL is controversial. Aims: We aimed to describe the histobiological features of the aggressive B-cell lymphomas (aBCL) with MYC-R identified in our center and explore their clinical and outcome differences within the context of MYC partner gene (IGH vs non-IGH). Methods: We retrospectively collected data from all adult patients with a MYC-R aBCL diagnosed in our center between 2010-2021. Diagnostic samples were reviewed by 2 expert hematopathologists. Fluorescence in situ hibridation (FISH) studies were performed either on paraffin tissue sections or bone marrow smears using Vysis LSI MYC, BCL2, BCL6 Dual color break apart probes and Vysis IGH/MYC/CEP 8 Tri-Color Dual Fusion probes (Abbott Molecular, USA). The study was approved by the local ethics committee. Results: We identified 61 cases of MYC-R aBCL. Tissue sections were available for MYC-IGH FISH analysis in 57/61 cases, MYC partner was evaluable in 52 patients. The morphologic distribution was: 34 (64.5%) cases with diffuse large B-cell histology, 3 (5.8%) with intermediate-cell or high-grade features and 15 (29%) with Burkitt histology. According to the current WHO classification (Table 1a) 28/34 large-cell morphology cases corresponded to double/triple hit (DH/TH) HGL because of additional BCL2 and/or BCL6-R and 6/34 were DLBCL NOS. 2/3 intermediate-cell cases were DH/THL and 1/3 was a HGL NOS. 14/15 cases with Burkitt histology were diagnosed as BL and one case was reclassified into DHL due to concurrent BCL6-R. Excluding BL, 38 cases (6 DLCBL NOS and 32 HGL) were selected for further analysis. Among this subset MYC-IGH-R was detected in 17/38 (44.7%) cases: 14 DH/THL and 3 DLBCL NOS (Table 1a). Noteworthy, 14/31 (45.2%) of all DH/THL and 3/6 (50%) of LBDCG NOS had an IGH partner. Considering cell of origin (COO) by Hans algorithm, MYC-IGH fusion was detected in 40% of Germinal center-derived (GC) lymphomas and in 62.5% of non-GC. Comparing clinico-biological data and outcome depending on MYC partner (Table 1b) our results show that patients with MYC-IGH fusion were significantly younger (p=0.019) and more likely to have multiple (≥2) extranodal (EN) sites (p=0.023). No other variable was significant. Referring to treatment, 33 patients (92%) received R-CHOP or R-EPOCH regimens, with no differences between groups. First line complete remission (CR) rates were 52% in both MYC-IGH and MYC-non-IGH groups. With a median follow up of 62 months (37-114), no significant association was observed between MYC-R partner and duration of response (p=0.784), event free survival (EFS) (p=0.933) or overall survival (OS) (p=0.706). At last follow-up 15 patients remain alive in CR. Image:Summary/Conclusion: Excluding BL, most patients with MYC-IGH rearrangement had diffuse large B-cell histology (93.8%) and were classified as DH/THL (82.3%). Among all DH/THL cases, 14/31 (45.2%) had IGH partner. MYC-IGH patients were younger and had more EN site involvement as MYC-non-IGH, but no differences in EFS and OS were noted. We are working to incorporate molecular techniques for a more precise analysis of BCL2, BCL6 and MYC translocations and their partners that permit a better understanding of the behavior of patients with aBCL.

EZH2 is mutated in nearly 25% of follicular lymphoma (FL) cases. Little is known about how EZH2 affects patients' response to therapy. In this context, the aim of this study was to retrospectively analyze the frequency of mutations in EZH2 at diagnosis in tissue and ctDNA in patients with FL and to assess the patients' outcomes after receiving immunochemotherapy, depending on the EZH2 mutation status. Among the 154 patients included in the study, 27% had mutated EZH2 (46% with high-grade and 26% with low-grade FL). Of the mutated tissue samples, the mutation in ctDNA was identified in 44% of cases. EZH2 mutation in ctDNA was not identified in any patient unmutated in the tissue.Unmutated patients who received R-CHOP had significantly more relapses than patients who received R-Bendamustine (16/49 vs. 2/23, p = 0.040). Furthermore, our results show that patients with mutated EZH2 treated with R-CHOP vs. those treated with R-Bendamustine present a lower incidence of relapse (10% vs. 42% p = 0.09 at 4 years), a higher PFS (92% vs. 40% p = 0.039 at 4 years), and higher OS (100% vs. 78% p = 0.039 at 4 years). Based on these data, RCHOP could be a more suitable regimen for mutated patients, and R-bendamustine for unmutated patients. These findings could mean the first-time identification of a useful biomarker to guide upfront therapy in FL.

Background: DNA is standardly preferred to cDNA for the analysis of FLT3- internal tandem duplication (ITD). Although its screening is mandatory at diagnosis in clinical routine, its use as minimal residual disease (MRD) marker after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains controversial due to the poor sensitivity of its detection method and its apparent instability during the disease. Aims: To compare FLT3-ITD mutation analysis in DNA and cDNA samples at diagnosis and to prove the usefulness of its expression measurement as MRD marker after allo-HSCT. Methods: Forty-six diagnosis samples from 46 patients and 80 samples from 34 patients who underwent an allo-HSCT (34 pre-HSCT samples, 34 at day 30 after infusion and 12 before relapse) were analyzed. DNA was purified using a Maxwell RSC Blood DNA Kit (Promega), RNA was isolated using TRIzol reagent (Invitrogen) and cDNA synthesis was performed using the First Strand cDNA Synthesis kit (Roche). FLT3-ITD was analyzed in both DNA and cDNA samples by fragment analysis using an ABI3130xl DNA sequencer (Applied Biosystems). Fragment analysis was analyzed through Peak Scanner Software (Thermo Fisher). Allelic ratio (AR) quantification was performed by dividing the area under the curve of the mutant allele by the area under the curve of the wild type allele. Every clone was analyzed as an event, comparing its DNA ratio with its cDNA ratio. Results: Median AR of the 58 FLT3-ITD clones at diagnosis were 0.54 [0-9.47] in DNA samples and 0.63 [0.012-13] in cDNA samples, (Wilcoxon test, p<0.001, Figure A). In six patients, FLT3-ITD AR was <0.5 in DNA and ≥0.5 in cDNA, changing their prognosis based on ELN algorithm. One of them was not candidate for chemotherapy and of the five remaining, four were refractory or relapsed after intensive treatment. In terms of sensitivity, cDNA was better than DNA, quantifying higher AR values in most cases, optimizing the detection of minor clones and the prognostic classification. In respect of HSCT samples, seven patients were positive for FLT3-ITD mutation pre-HSCT in cDNA and two in DNA. Of the seven patients which were positive by cDNA, four relapsed after HSCT. The three patients who did not relapse had FLT3-ITD AR lower than 0.05. As for the two patients who tested positive in the DNA samples, only the one who also tested positive for cDNA relapsed. Despite the small sample size, these results reveal that the analysis of pre-HSCT cDNA samples could have a predictive value for relapse. No patient was positive on day 30 after HSCT, suggesting that it may not be useful, whether the DNA or cDNA sample is studied. However, the analysis at this time point could be crucial in those patients who may relapse early. On respect of the last sample before relapse (a median of 22 days before relapse, range 7-85), of the 12 cases, three were positive for FLT3-ITD mutation in DNA samples and nine were positive in cDNA samples. Great differences were observed between AR of both type of samples (Wilcoxon test, p<0.001, median 0 [0-0.04] vs. 0.15 [0-0.57], Figure B). In three patients FLT3-ITD cDNA measurement did not anticipate the relapse, due to the loss of the mutation or because the day on which the sample was analyzed was too early to detect the mutation, issues that can be solved by complementing with other MRD markers. Image:Summary/Conclusion: In conclusion, cDNA fragment analysis of FLT3-ITD by capillary electrophoresis is an easy-to-implement technique that could be a great alternative approach in AML patients at diagnosis and in allo-HSCT monitoring.

Background: Monitoring of CD19-targeted chimeric antigen receptor (CAR)-T cell levels is essential for an optimal follow-up of patients. In this regard, methodologies such as multiparametric flow cytometry (MFC) or qPCR are commonly performed. In addition, the use of new approaches, such as digital PCR (dPCR), has improved the analysis of cell kinetics. Nevertheless, the role of CAR circulating cell-free DNA (ccfDNA) dynamics during CAR-T cell treatment is not clear. Aims: To evaluate the usefulness of CAR ccfDNA measurement by dPCR in patients treated with CD19-CAR-T cell therapy and its correlation with CAR-T cell levels measured by MFC and dPCR. Methods: Forty patients diagnosed with a B-cell lymphoma who underwent CAR-T therapy (28 Axicabtagen Ciloleucel and 12 Tisagenlecleucel) between May 2019 and May 2021 were included in the analysis. One hundred and five peripheral blood (PB) samples were collected in EDTA tubes at days +1, +7, +14, +30 (10, 31, 33, and 31 samples, respectively) after CAR-T administration. Plasma was obtained from 20 mL of PB by centrifugation at 2500g for 30 minutes at 4°C. DNA was purified from PB samples using the Maxwell 16 Blood DNA Purification Kit (Promega) and ccfDNA was extracted from 5 mL of plasma, using QIAamp® Circulating Nucleic Acid (Qiagen). dPCR was carried out in DNA and ccfDNA samples, using specific primers and probes for FMC63 and a reference gene. MFC was performed on a DxFLEX cytometer (Beckman Coulter), using CD19 (20-291) protein-FITC (ACRO Biosystems). Continuous variables were expressed as median and range. Correlations between CAR copies in plasma determined by dPCR and CAR-T cells measured in PB by MFC and dPCR were analyzed using Pearson’s test. Comparison between number of copies and clinical variables (cytokine release syndrome -CRS-, immune effector-cell associated neurotoxicity syndrome -ICANS-, and relapse) was analyzed using Mann-Whitney U test. All statistical analyses were performed using GraphPad Prism 8.0.1. Results: Median values of CAR copies of ccfDNA per milliliter measured by dPCR were 39, 38, 25 and 2 at days +1, +7, +14 and +30. Similarly, median reference gene values were 3079, 1799, 2588 and 2183 (Figure 1). Thus, we validate previous observations of an initial peak concentration followed by a constant decrease in plasma CAR copies kinetics. Moderate correlation was found between CAR copies/mL of plasma and CAR-T cells in PB determined by MFC and dPCR (R=0.49, p<0.001, Figure 2A; R=0.51, p<0.001, Figure 2B, respectively). So, although a part of plasma CAR copies variation is explained by changes in whole blood CAR content, there might be other influencing factors. Regarding the association between CAR copies in plasma at +7 and clinical variables, an association was only found in the case of development of CRS<2 and CRS grade ≥2 (28 [0-220] vs 168 [11-1479], p<0.01, Figure 3). Further experiments are needed to evaluate whether this reflects the overall CAR-T cell kinetics. Image:Summary/Conclusion: The analysis of multiple variables is important for an optimal monitoring of patients undergoing CAR-T cell therapy. In this sense, ccfDNA dynamics may play an important role during the follow-up and even have relevant clinical implications as we have demonstrated in the present study. However, more studies are necessary for validating our data.

Background: CD19-targeted chimeric antigen receptor (CAR)-T cell is a treatment for B-cell lymphoma patients. CAR-T cell monitoring is important to ensure a correct follow-up, being multiparametric flow cytometry (MFC) the actual gold standard technique. However, other molecular techniques such as digital PCR (dPCR) could add complementary information that could be valuable to predict CAR-T response. Both available commercial antiCD19 CAR-T cell therapies, Tisagenlecleucel (tisa-cel) (Kymriah®) and Axicabtagen Ciloleucel (axi-cel) (Yescarta®) share the single variable fragment domain (FMC63), which makes possible the measurement by dPCR. Aims: To evaluate the usefulness of digital PCR for monitoring CAR-T cell levels in comparison to multiparametric flow cytometer. Methods: Forty-five patients diagnosed with diffuse large B-cell lymphoma (36), transformed-follicular lymphoma (7) and mediastinal primary large B-cell lymphoma (2), treated consecutively with anti-CD19 CAR-T cell therapy between June 2019 and May 2021 were included in this study. One hundred and forty-two peripheral blood (PB) samples were collected at days +7, +14, +30, +90 (40, 39, 40 and 23 samples, respectively) after infusion. DNA was purified using the Maxwell® RSC Whole Blood DNA Kit (Promega, USA). dPCR assays were performed on the QIAcuity One platform (QIAgen, Germany). MFC analysis was performed on a DxFLEX cytometer (Beckman Coulter), using CD19 (20-291) protein-FITC (ACRO Biosystems). To assess the sensitivity of molecular methodologies, serial dilutions from 100% to 0.001% of CAR-T were analyzed by dPCR. Correlation of CAR-T cell detection between MFC and dPCR was calculated using Pearson’s test. Results: A high correlation between dPCR and MFC was found (r = 0.90), demonstrating the usefulness of dPCR to quantify absolute number of CAR copies (Figure 1A and 1B). dPCR improved the detection capacity of MFC, detecting CAR-T in 15 samples that were negative by MFC because of low CAR-T cell presence in PB (Figure 1C), as MFC measures CAR-T events in total PB the low content of CAR-T could reduce MFC sensitivity. Median number of CAR-T cells detected by dPCR was greater than that quantified by MFC in each days of the follow-up (Figure 1D). Different CAR-T cell products (Yescarta and Kymriah) differ in its expansion rates, therefore tisa-cel expand before axi-cel (near day 7th and day 14th, respectively) (Figure 1C). Image:Summary/Conclusion:FMC63 quantification through dPCR was effective for CAR-T measurement, even improving MFC sensitivity, especially when CAR-T counting in PB is low in late monitoring days. dPCR monitoring could complement data given by MFC and being a very sensible gold standard technique in late monitoring as its sensitivity is very superior to MFC in those follow-up days.

Background: Cytomegalovirus (CMV) is the most common viral infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Without prophylaxis, 80% of CMV-seropositive patients experience CMV infection after allo-HSCT. Cytokines and chemokines are the first line of defence against viral infections and recent studies have shown that gene polymorphisms result in inter-individual differences in cytokine production. Aims: To determine the genotype of Cytokines and chemokines in the donor (D) and recipient (R) and their association with the CMV reactivation of patients receiving an allo-HSCT. Methods: Eighty-five patients who received allo-HSCT from an HLA-identical sibling donor from 2000 to 2015 were included. CMV DNAemia was evaluated until approximately day 100 after allo-HSCT. CMV reactivation was defined as the detection of CMV DNAemia ≥100 copies/ml in plasma. All of patients were classified according to epidemiological risk factors of clinical interest. Fifty genes were selected for their potential role in the pathogenesis of CMV: C-C Motif Chemokine Ligand (CCL) and Receptor (CCR), C-X-C Motif Chemokine Ligand (CXCL) and Receptor (CXCR), Forkhead Box P3 (FOXP3), CD48 Molecule (CD48), Interferon (IFN), Interleukin (IL), Killer Cell Immunoglobulin Like Receptor (KIR), Transforming Growth Factor Beta (TGFB), tumor necrosis factor alpha (TNF) and Lymphotoxin Alpha (LTA). Genomic DNA was purified from 170 PB (D and R) using Maxwell® RSC Blood DNA Kit (Promega, USA). Libraries were performed using an enrichment-capture gene panels according to the manufacturer's protocol. Paired-end 2x101 bp was performed using the Illumina HiSeq platform (Illumina, USA). Variants located in coding region, splicing sites and intronic polymorphisms were analyzed. Synonymous variants were excluded. During the variant calling process, the values of Depth, variant allele frequency (VAF) and the minor allele frequency (MAF) in European population are obtained and used to variants filtration (Depth ≥ 30, VAF ≥ 0.4 and MAF≥ 10%). Differences among groups were evaluated by Chi-Squared and Fischer Exact Test. Results: CMV reactivation was observed in 51/85 (60%) patients. Initial episodes of CMV DNAemia occurred at a median of 48 days (2–151) after transplant. Clinical variables are not associated with CMV reactivation: age, gender, stem cells, hematological disease, conditioning regimen, serology status and prior autologous transplant. Twenty-four patients (47.1%) had only one episode of CMV reactivation, 12 (23.5%) had two episodes and 15 (29.3%) had more than two episodes after allo-HSCT. Using filters discussing previously, 221 polymorphisms were detected in D and R. Although 209 variants studied had no apparent impact on the features of CMV reactivation, but we found that 12 variants in 7 different genes were associated significantly with the development or protection of CMV reactivation (Table 1). Our data showed that determined polymorphism in TNF-α, IL12A, TGBF2, IL1RN and CD48 play an important role to protection against CMV. On other hand, KIR3DL1 and CXCL12 represent examples of how receptor polymorphism can influence CMV development.Summary/Conclusion: The data presented suggest that screening of patients and donor pre-transplantation could help to predict the individual risk of the development of CMV infection. These results might also enable the identification of patients at high risk of CMV reactivation, enabling pre-emptive therapy or attempts to cure the infection by administrating antiviral therapy or CMV-specific T lymphocytes.