ALBERT

Description: BACKGROUND: Primary mediastinal B-cell lymphoma (PMBCL) are a subtype of diffuse large B cell lymphoma (DLBCL), that represent 2-4% of all non-Hodgkin lymphoma (NHL) and 6-12% of DLBCL. They affect young adults with a median age at diagnosis of 35 years old and are more frequent in females (2:1). Approximately 80% of patients have stage I or II disease at the time of diagnosis, with B symptoms, bulky mass and superior vena cava syndrome (SVCS) in 50 %. There is no standard initial therapy regimen, anthracyclines with Rituximab are the most used. Local consolidation radiotherapy (RT) is reserved for bulky disease and to complete partial remissions (PR) after chemotherapy. AIMS: To analyze prognostic and epidemiological factors, treatment administered and response and survival rates of patients diagnosed with PMBCL in our center. METHODS: Retrospective cohort study between September 2003 and June 2014. The treatment received was 6 to 8 cycles of CHOP-like chemotherapy (Cyclophosphamide, Adriamycin, Vincristine, Prednisone ± methotrexate / Intrathecal triple therapy MTX- TIT ), with or without Rituximab, and subsequent radiotherapy if necessary. The following results were evaluated by univariate analysis: overall survival (OS), disease-free survival (DFS) and incidence of relapse after diagnosis. DFS was defined as the interval of time from complete remission (CR) to relapse or last visit. Response to treatment was assessed by PET-CT. RESULTS: We observed 14 patients diagnosed with PMBCL in our hospital with a median age of 33 years (r, 21-58 years) and female predominance (Š:‰ 10:4). At diagnosis 42.8 % had B symptoms and 42.8% elevated lactic dehydrogenase. Fifty percent of patients presented with SVCS. Central nervous system and bone marrow infiltration was not observed. Early-stage disease at diagnosis (I 30% and II 53%) was observed in 83%, with IPI 1 in 78.5% of patients. The number of cycles received was 6 in 78.5 %, 8 cycles in 14.2 %, and only 1 cycle of CHOP in one patient. Altogether 71.4 % received Rituximab and 71.4 % received MTX-TIT. Ten patients (71.4%) received RT (median 36 Gy) after chemotherapy, 9 of which had initial bulky disease. We observed an overall response rate of 92.8 % after chemotherapy (57.1 % CR, 35.7% PR). After a median follow-up of 60 months (r, 4.4 to 130.8 months) 12 patients had responded to treatment, were alive, without relapse and in complete response, and one patient is currently receiving the 6º cycle of CHOP pending reevaluation (awaiting last MTX-TIT and RT consolidation) and obtained CR after the third cycle of R-CHOP-MTX-TIT. Median overall survival was not reached and median DFS was of 54.8 months. Only one patient died (mortality 7.1 %) due to influenza A in the context of postchemotherapy aplasia after the first cycle of CHOP. CONCLUSIONS: We observed prognostic and epidemiological factors similar to those described in literature, although in our series, CHOP -like chemotherapy ( ± MTX- TIT ) with or without Rituximab and RT has shown improved survival rates and 100% of CR. Consolidation radiotherapy was successfully used to complete treatment in patients that only achieved PR after chemotherapy. Rescue chemotherapy followed by autologous hematopoietic cell transplantation was not required. It is difficult to draw conclusions on the impact of the therapeutic regimen received, we believe multicenter analysis with larger numbers of patients are necessary. Disclosures No relevant conflicts of interest to declare.

Description: We recently reported that Tyk2 was essential for IFN-a-induced B lymphocyte growth inhibition, although Stat1 is not required for this IFN-a-mediated inhibition. This means that other signaling molecules besides Stat1, and which are activated by Tyk2, are thought to transduce the IFN-a signal inhibiting B lymphocyte growth. We performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Rack-1, originally described as a receptor for activated C kinase beta, associated with Tyk2. Receptor for activated C kinase (Rack)-1 is a protein kinase C interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase C gamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alpha/beta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudo-kinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N-terminus and one in the middle portion (a.a.138–203) of Rack-1. In addition, not only Tyk2 but other Jak kinases associate with Rack-1, and each Jak activation causes the phosphorylation of Tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated from cytoplasm or membrane toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.

Description: Background: Pulmonary Hypertension (PH) is a well-known complication of the advanced phase of Myeloproliferative Diseases (MPD) such as Polycythemia Vera (PV), Essential Thrombocytopenia (ET) and Myelofibrosis (MF). Although initially attributed to increased pulmonary vascular resistance (group 1 PH of the WHO classification), this entity was classified in 2009 in group 5 PH (unknown mechanism and miscellaneous PH). Our aim was to describe the prevalence and possible causes of PH in a series of patients with MF. Methods: We studied a series of patients with Primary MF or Secondary MF to other MPD with cardiac ultrasound, right heart catheterization and scintigraphy after intraarterial infusion of Tc99-labeled albumin macroaggregates. Results: We studied 11 consecutive patients with MF (7 male, mean age 58 years, 4 with MF post-PV, 4 post-TE and 3 Primary MF) during the period 2009-2014. All of them had mutations of JAK-2 gene, fibrosis in bone marrow biopsy and visceromegaly (all with intermediate-2 or higher IPSS). Median NTproBNP levels were 4597 pg/ml (range 175-5700). Echocardiogram showed high systolic pulmonary pressure in most cases, with a mean of 54 ± 17 mmHg (range 35-80). Right heart catheterization showed high cardiac output (HCO) in all patients (table 1). After ruling out other causes of HCO, a scintigraphy was performed after administration of Tc99-labeled albumin macroaggregates in descending thoracic aorta. In every case, a percentage of the labeled macroaggregates (6.1 ± 2.0% of the radioactivity) were plugged in the pulmonary capillary bed, what is diagnostic of the presence of microfistulas in infradiaphragmatic territory. In two of these patients, scintigraphies were performed at diagnosis and after been treated with the anti-JAK drug Ruxolitinib (Novartis Pharma). A favourable impact of this drug was obtained in the two cases, probably due to a reduction of spleen volume. Conclusion: Most patients with MF show pulmonary hypertension associated with high cardiac output caused by microfistulas, without significant increase in pulmonary resistance. This finding has important clinical implications, because pulmonary vasodilators (once recommended) should be contraindicated, since they could cause worsening of the clinical picture. Ruxolitinib could resolve PH in MF and a prospective study in this sense could be indicated. Table 1. Patient 1 2 3 4 5 6 7 8 9 10 11 Pulmonary Artery Pres. (S/D/Mean) 58/38/ 42 69/41/ 50 48/29/ 40 84/26/ 45 57/17/ 30 22/17 / 19 51/16 / 28 22/9 / 13 25/8 / 14 71/23 / 39 30/7 / 15 Pulmonary wedge pres. 28 37 27 13 9 7 13 7 8 12 6 Cardiac output (l/min) 12.5 8.0 11 7.1 8 7.3 8.7 6.7 7.45 6 7.8 Cardiac index (l/m/m2) 5.5 4.0 6.6 3.6 5 4 4.8 4.0 4.5 3.9 5.1 Pulmonary vascular resistance (Wood U.) 1.2 1.4 1.2 4.5 2.6 3 1.7 0.9 0.8 4.5 1.1 Disclosures Ojeda: Alexion Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Fores:Alexion Pharmaceuticals: Consultancy, Speakers Bureau.

Description: Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. Using traditional embryoid body methods, iPS cell-derived erythroid cells predominantly produce ε-globin and γ-globin without β-globin expression. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production, which is associated with emergence of CD34+ hematopoietic stem/progenitor cells (HSPCs) (2014 ASH). In the current study, we extend this work to evaluate erythroid cell generation using iPS cell lines generated from various sources including patients with sickle cell disease (SCD). To test our two hypotheses; (1) erythroid progenitor (EP)-derived iPS cells more efficiently differentiate to erythroid cells, and (2) stromal cell (ST)-derived iPS cells more efficiently emerge hemangioblast-like immature HSPCs which results in greater erythroid cell generation, we generated several clones of iPS cells which were derived from (1) EPs (6 clones) which were differentiated from peripheral blood mononuclear cells and (2) bone marrow STs (5 clones) in SCD patients. Transgene-free iPS cells were generated and characterized according to Merling et al. (Blood. 2013). These iPS cells and controls (2 clones of fibroblast (FB)-derived iPS cells and H1 ES cells) were used to generate ES/iPS sacs for 15 days. After a 2 day culture of ES/iPS sac-derived spherical cells on OP9 feeder cells, the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002). Following ES/iPS sac generation, 3.5-4.8 fold greater amounts of CD34+CD45+ HSPCs emerged in both EP- and ST-derived iPS sacs, compared to FB-derived iPS sacs (p

Description: Jak-Stat pathway is essential to transduce cytokine signalings. We previously reported that tyrosine kinase 2 (Tyk2), one of the Jak kinases, was essential but Stat1 was dispensable in IFN-alpha-induced B lymphocyte apoptosis. This indicates the existence of other signaling molecules besides Stat1 downstream of activated Tyk2. Therefore, in order to find Tyk2-activated signaling molecules which transduce IFN-alpha signal and induce B lymphocyte apoptosis, we performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Siva-1, which had been originally cloned as a CD27 binding protein. Siva-1 has a death domain (DD) homology region and induces apoptosis in various cells through binding CD27, which belongs to the tumor necrosis factor receptor (TNFR) family. We found that Tyk2 interacts with and phosphorylates Siva-1. Two regions of Siva-1, the N-terminus and the middle portion of the protein containing a death domain homology region, contributed to binding to Tyk2, and two tyrosines of Siva-1, Tyr53 and Tyr162, were phosphorylated by Tyk2. Because Siva-1 is so far thought to be a proapoptotic protein, we assessed whether Tyk2 had any effects on Siva-1-mediated apoptosis through the Tyk2-Siva-1 interaction. First, we established BaF3 cell (IL-3 dependent murine proB cell) lines stably expressing either wild-type Tyk2 (BaF3/WT Tyk2) or constitutively activated Tyk2 which Valine678 was replaced by Phenylalanine (BaF3/V678F Tyk2). In BaF3/V678F Tyk2 cells, Tyk2 and Stats are constitutively activated, and they grow in the absence of IL-3. We transiently transfected either GFP-Siva-1 or control GFP vector into BaF3/parent cells, BaF3/WT Tyk2 cells or BaF3/V678F Tyk2 cells. Thirty-six hours after transfection, we measured annexin V positive cells by flow cytometry on GFP-positive cells. Approximately 24% of both BaF3/parent cells and BaF3/WT Tyk2 cells transfected with GFP-Siva-1 fell into apoptosis, whereas only less than 7% of the cells transfected control vector showed apoptosis. The frequency of this Siva-1-induced apoptosis was increased in BaF3/V678F Tyk2 cells (over 43% vs

Description: Wilms tumor protein (WT1) is a transcription factor selectively over expressed in several types of leukemia and solid tumors, making it a promising potential target antigen for immunotherapy. Several open clinical trials use native or altered peptide sequences derived from the WT1 protein in order to overcome the weak immunogenicity of the self-antigen. Here we report a new strategy to circumvent tolerance by designing peptides that incorporate non-natural amino acids into the native sequence of WT1 peptides. Starting from the nonamer sequences WT1 187–195 and WT1 235–243, eight peptides containing natural amino acids and nine peptides in which different chemical modifications (fluorination, photo-reactive azido groups or benzophenone groups) were introduced at major histocompatibility complex (MHC) and T cell receptor binding positions, were synthesized. The new non-natural peptides could stabilize MHC class I molecules better than the native sequences and were also able to elicit strong specific T-cell responses. Photo-reactive peptides were additionally modified with biotin handles to allow streptavidin-biotin pull down and western blot analysis of kinetics of binding and catabolism. Upon UV irradiation, these peptides covalently bound to MHC molecules on the live cells; clearance of the peptide-MHC covalent complex occurred over 24 hours, consistent with the T2 thermo-stabilization data for the same peptide. Further catabolic studies may elucidate the important or novel cellular proteins involved in antigenic peptide processing and cross presentation and should aid in vaccine development. We are investigating whether covalent interaction with the MHC may lead to alterations in immune responses as well. T cells stimulated with one of the synthetic peptides (WT1J-W4WF) cross-reacted with the native WT1J sequence and were able to kill WT1 positive HLA-A0201 matched acute lymphoblastic leukemia cell lines. In conclusion, this study shows that peptides with non-natural amino acids can be successfully incorporated into T cell epitopes to provide increased immunogenicity and novel biological information.

Description: Sickle cell disease (SCD) is caused by a 20A〉T mutation in the β-globin gene, and can be cured by therapeutic β-globin gene addition into hematopoietic stem cells (HSCs) with lentiviral transduction. However, this method relies upon random integration, leaving the SCD mutation intact and potentially inducing insertional mutagenesis. Genome editing technologies have the potential to correct the SCD mutation without integration, producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. In this study, we investigated CRISPR/Cas9-based gene correction for SCD CD34+ cells. Plerixafor-mobilized SCD CD34+ cells were transfected by electroporation using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver SCD mutation-specific guide RNA at 200mg/ml, SpCas9 mRNA at 200mg/ml or protein at 120mg/ml, and single strand donor DNA with a normal β-globin sequence at 80, 120, or 200mg/ml. We chose Cas9 mRNA and single strand donor DNA due to the ease of clinical grade large-scale production and to avoid the need for viral vector manufacturing. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. Cell viability was reduced to 76-87% after electroporation, compared to 90% in the control. We observed high-efficiency genome editing (29-34% gene correction and 49-58% indels) with Cas9 mRNA, showing donor DNA concentration dependence, and editing levels were comparable to Cas9 protein (39% correction and 43% indels). 15-23% Biallelic and 17-26% monoallelic gene correction were detected at the clonal level by colony assay. After erythroid differentiation, up to 54% normal β-globin production was observed with Cas9 mRNA (Figure), comparable to Cas9 protein (67%), while βs-globin amounts were markedly reduced under both conditions (6-10%). Similar correction efficiencies were obtained from two additional SCD patients' CD34+ cells at DNA levels (28-35%) and protein levels (33-56%). These data demonstrate that Cas9 mRNA and single strand donor DNA allow for efficient gene correction in SCD CD34+ cells, exceeding the therapeutic threshold of 20% in SCD. We then evaluated off-target effects on the δ-globin gene, which was reported as a major off-target site in β-globin gene editing due to high homology; however, almost no off-target effects (0.6-1.3% indels) were detected. Interestingly, gene conversion in the 9T〉C polymorphism (11bp upstream of SCD mutation) on the β-globin gene was observed, and this conversion always occurred with SCD gene correction (26-33% of SCD gene correction), suggesting that gene conversion is strongly affected by distance from the target site. In addition, we evaluated genome editing among subpopulations of CD34+ cells from 3 healthy donors under the same conditions (normal β-globin to SCD mutation). We observed similar editing efficiencies (conversion and indels) among more immature (CD34+CD133+CD90+) and relatively differentiated populations (CD34+CD133+CD90-, CD34+CD133-, and CD34-) as well as among cells at different phases of the cell cycle (G0/G1, S, and G2/M), suggesting that similar gene correction efficiencies are obtained in all CD34+ cell populations, including the HSC population. We have begun efforts to evaluate gene-corrected SCD CD34+ cell engraftment in the mouse xenograft model, as similarly corrected X-CGD CD34+ cells were engrafted in immunodeficient mice. To examine the effects of indels in the β-globin gene, we next evaluated Hb production from genome-edited SCD CD34+ cells (2 patients) without donor DNA. Editing without donor DNA resulted in 63-70% indels (compared to 26-29% correction and 46-53% indels with donor DNA) and increased non-adult Hb production (small amounts of fetal Hb and significant amounts of a Hb variant), which will require further investigation to characterize. In summary, we observed efficient gene correction in SCD CD34+ cells with a simple Cas9 mRNA, single strand donor DNA, and guide RNA method, resulting in ~30% gene correction and ~50% indels. After erythroid differentiation, the majority of Hb detected was adult Hb; we detected up to 54% normal β-globin production with a marked reduction of βs-globin to ~10%. Evaluation of engraftment potential is required for gene-corrected CD34+ cells, but these methods would be clinically applicable for gene correction in SCD. Figure. Figure. Disclosures Li: MaxCyte, Inc.: Employment. Allen:MaxCyte, Inc.: Employment. Peshwa:MaxCyte, Inc.: Employment.

Description: Elevated fetal hemoglobin (HbF, α2γ2) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved. We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p

Description: Background: Nivolumab, an immune checkpoint inhibitor, has been tested in patients with classical Hodgkin lymphoma (cHL) who failed standard treatment options and has demonstrated remarkable activity with acceptable safety profile in clinical trials. After the impressive results of nivolumab phase I study, a significant number of patients were granted early access to nivolumab through a Name Patient Program (NPP) or compassionate use in Spain. Demonstrating that results of nivolumab use in real-life are similar to those in clinical trials is of major clinical relevance. Objective: The aim of this retrospective study was to analyze the efficacy and safety profile of nivolumab for the treatment of relapsed/refractory (RR) cHL in a real-life context. Methods: We retrospectively collected data from 34 GELTAMO centers. Eligible patients included RR cHL patients treated with at least one cycle of nivolumab. The primary end-point was to describe the overall response rate (ORR). Secondary objectives were to assess the complete response rate (CR), safety of nivolumab, and clinical outcomes (overall survival [OS], and progression free survival [PFS]). Results: Between September 2015 and May 2018, 74 patients with RR cHL received nivolumab monotherapy dosed at 3mg/kg once every 2 weeks (97%). The median age was 38 years (range 17-78). Patients have received a median of 4 (1-15) prior therapy lines; all but 2 were previously treated with brentuximab vedotin (97%), and 38 (51%) of them underwent a hematopoietic stem cell transplantation (HSCT) (n=33 autologous, autoHSCT, and n=5 allogeneic, alloHCST). Median number of nivolumab cycles was 8 (1-65). Ten (14%) patients are still on treatment. Reasons for nivolumab discontinuation were disease progression in 23/64 (36%), referral to HSCT in 27/64 (42%), adverse events (AE) in 8/64 (13%), patient or physician's decision in 5/64 (8%), and unknown in 1/64 (1%). Treatment related AE were reported in 42/69 (61%). Half of them (21, 30%) were probably immune related AE: grade 1-2, 67% (cutaneous n=5, hepatitis n=3, hypothyroidism n=3, gastrointestinal n=3, suprarenal insufficiency n=1); grade 3-4, 24% (pneumonitis n=2, hepatitis n=1, encephalitis n=1, hypothyroidism n=1); grade 5, 3% (pneumonitis n=1, Stevens-Johnson syndrome + hepatitis + nephritis n=1). ORR was 58% (CR 21/72 patients, partial response [PR] 21/72). Stable disease (SD) was achieved in 9 patients (13%). After an initial response (4 PR and 3 SD), 7 patients developed lymphoma progression. A total of 40 (54%) patients finally underwent HSCT, 4 autoHSCT and 36 alloHCST. AlloHSCT was performed after a median of 63 days (41-115) and 8 patients received prior salvage therapy. Complications after alloHSCT consisted of non-infectious fever requiring steroid treatment in 13 (36%), acute graft-versus-host disease in 19 (53%) (2 of them grade 3-4, 1 death), hepatic venocclusive disease in 2 (6%, 1 death), and non-infectious pulmonary complications in 2 (6%). Five (14%) patients died due to transplant complications. At the last follow-up, all autoHSCT patients and 23/36 alloHSCT were in CR. The 2-year OS for the whole series (n=74) was 54% (median not reached). After a median follow-up of survivors patients of 12.5 months (1-31), 29 (39%) were alive in CR. Conclusions: Our real-life experience confirms the efficacy of nivolumab in very heavily pretreated cHL patients with an ORR of 58%. The safety profile of our cohort is comparable with that previously reported in clinical trials with manageable side effects and low treatment related mortality. In our study the percentage of patients who bridged to transplantation was significantly higher to that previously reported indicating this preference for Spanish physicians. AlloHSCT post-nivolumab showed encouraging results and toxicity seemed comparable to that previously described with other treatment regimens. Authors thank Bristol Myers Squibb for its support in this study. Disclosures Martinez: BMS: Research Funding; Takeda: Consultancy. García-Sanz:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Hospira: Research Funding; Pharmacyclics: Research Funding; Spanish Government: Research Funding; Gilead: Research Funding; Amgen Inc.: Research Funding; Incyte: Consultancy.

Description: Background: Whole genome amplification (WGA) has become an invaluable method for working with small amounts of starting DNA and for preserving limited samples of precious stock material. Next-Generation Sequencing (NGS) techniques can benefit from WGA, but due to their high sensitivity, WGA reliability needs to be certified to ensure an unbiased and accurate amplification of whole genomes. Myelodysplastic Syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. We have performed whole exome sequencing (WES) and targeted deep sequencing in tumoral samples from MDS patients. With the aim to determine if Multiple Displacement Amplification-based WGA can be applied to perform NGS in these type of samples and to obtain valuable results, targeted deep sequencing was performed on both fresh-DNA and WGA-DNA from the same patients. Mehtods: Whole bone marrow samples from four MDS patients were included in the study. WGA was performed in tumoral DNA samples with REPLI-g (Qiagen). WES libraries were generated in tumoral-control paired samples using the SureSelect Human Exome Kit 51Mb v4 (Agilent) and sequenced on an Illumina HiSeq2000. Targeted sequencing libraries were prepared for fresh-DNA and WGA-DNA following the manufacturer specifications for TruSight Myeloid Sequencing Panel protocol (Illumina), and then sequenced on one single run on an Illumina MiSeq. WES sequencing data was analyzed using an in-house pipeline, as previously reported. Targeted sequencing data analysis was performed with theMiSeq Reporter Software (Illumina). Filtering was performed in all cases by eliminating sequencing and mapping errors and by discarding intronic or synonym variants, variants located at highly variable regions or with low coverage, as well as know polymorphisms. Additional filtering was performed by visualization on Integrative Genome Viewer Software v.2.3.72. Results: Regarding targeted sequencing, fresh-DNA samples generated 6 million reads (SD = 1.9 million), with 98.5 % (SD = 0.8) of the mapped reads on-target and a mean target coverage of 12148.8 (SD = 3872.9). WGA-DNA samples yielded about 5.2 million reads (SD = 1.5 million), with 98.3 % (SD = 0.4) of the mapped reads on-target and a mean target coverage of 10447.5x (SD = 2946.3). A mean of 77% of total bases displayed a Q score ≥30, which did not differ between fresh and WGA-DNA. Comparison of all filtered variants within the four pairs revealed a high level of discordance between fresh/WGA samples (Figure 1A). A mean of 86% of the detected variants, considering both fresh and WGA-DNA, were detected at a low frequency (