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.

CAR-T cell therapy is approved for the treatment of adults with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL). However, 40-60% of patients relapse after therapy and characterization of relapse has been poorly reported. To describe relapse characteristics in biopsy samples in patients with DLBCL receiving CAR-T cell therapy in our center. All consecutive patients diagnosed with R/R DLBCL who were infused with commercial CAR-T cells from June 2019 to December 2020 were included. CAR-T cell expansion in PB was monitored by multiparameter flow cytometry (FC) through detection of labeled CD19 in T cells (Human CD19 Protein®). PET-CT scanner evaluation was performed on days +30, +90 and +180. In cases where PET-CT detected progression or indeterminate response, core-needle biopsy of the accessible lesions was performed. Study of biopsy included: histological analysis; FC immune phenotyping including CAR-T cell presence and detection of exhausted CD3+ populations (LAG3, TIM3, PD1). DNA was purified from biopsy using GeneRead DNA FFPE Kit (Qiagen). Mutations of CD19 (exon2-5) were analyzed by Sanger sequencing. 30 patients with R/R lymphoma were treated: 21 with axi-cel 9 with tisa-cel. Median age at infusion was 57 years (r: 22-79); 16 (53%) were female. Median follow up-was 9.5 months (r: 3-18). PFS at 6 months was 42% and OS 72%. CAR-T cell expansion in PB was detected in 29 patients (97%). Complete and overall response rate at day 30 (CR and ORR) were 39% and 75%, respectively (28 evaluable patients), and 41% and 55% at day 100, respectively (27 evaluable patients). At day 180, 10 out of 14 evaluable patients (38.5%) showed CR. A total of 15 patients experienced relapse or progression. Biopsy of the accessible lesions was performed in 10 (66%) (Table 1). Relapsed was confirmed in all cases except in one in whom sample was insufficient. Three patients who were CD19+ by IHQ on the pre-infusion biopsy showed CD19- at relapse. CAR-T cell in PB at relapse was detected in all except one patient that did not expand CAR-T after 2 infusions and 60% of patients presented CAR-T in the biopsy. TME (tumor microenvironment) TCD3+ lymphocytes were >20% in 80% of the cases and no patient had <5%. In tumor sample, FC failed to identify cellularity in 37% of patients; in the rest of samples, percentage of exhausted TCD3 presented an average of 80% (range 64-84). CD19 study revealed mutation in one patient (p.V279L) who presented a CD19+ relapse. In our series of relapsed lymphomas after CAR-T anti-CD19 treatment, CAR-T cell was present in all patients in PB but only 60% in tissue. CD19 loss and mutations in DNA are possible relapse mechanisms. Exhausted TCD3 lymphocytes were abundant in TME, which would support its relevant role preventing a good function of the carts in situ. More complex biopsies analyses are necessary to better understand relapse mechanisms. No conflicts of interests pertinent to the abstract.

Background: Postpartum haemorrhage (PPH) is an unpredictable obstetric emergency that requires a multidisciplinary approach. Pelvic arterial embolization (PAE) is considered as a second-line treatment, although the published results have not been reviewed systematically since 2007.Objectives: To evaluate success and complication rates of PAE to treat PPH in the study hospital between 2009 and 2015, and to perform a systematic review of the literature on the reported efficacy and safety of PAE for the management of PPH.Search strategy: A systematic review of articles on PAE in English or Spanish was conducted using Medline and the Cochrane Library.Selection criteria: All published articles assessing success and complication rates of PAE in cases of PPH. The search was restricted to articles published in English or Spanish between 2000 and 2015, with at least 25 cases.Data collection and analysis: Obstetric variables, maternal haemodynamic state, pre-/postembolization management, technique-related variables, post-PAE evolution and complications were recorded in the case series study. Study characteristics, success rates and PAE-related complication rates were recorded in the systematic review.Main results: The case series included 29 patients. The majority of these patients were primiparous, with singleton term pregnancies and spontaneous labour. Caesarean section was performed in 62.1% of patients undergoing PAE for PPH. PAE was successful in 89.6% [95% confidence interval (CI) 78.3-100] of cases. Twenty studies were included in the systematic review, providing data from 1739 patients. PAE was successful in 89.4% (95% CI 87.9-90.9) of cases. The mortality rate was 0.9%, and other major complications were uncommon (1.8%).Conclusions: PAE was found to be a minimally invasive, highly successful and safe technique for the management of PPH. It should be considered in PPH refractory to initial treatment. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Allogeneic transplantation is the only curative option for patients with high risk leukemias or MDS. Only one third of them have an HLA identical sibling donor and around 60-70% will find an unrelated donor; that's why haploidentical stem cell transplantation (HAPLO-HSCT) offers a therapeutic option to most of these patients. Myeloablative conditioning (MAC) used to obtain better disease control than reduced intensity conditioning regimens (RIC), but with higher toxicity, rendering long term similar results.