ALBERT

Description: Introduction. Genetic studies in patients with Ph-negative myeloproliferative neoplasms (MPNs) are essential to establish a correct diagnosis and to optimize their management. Recently, it has been demonstrated that it is possible to detect molecular alterations present in solid tumors and hematologic neoplasms by the analysis of circulating tumor DNA in plasma samples, which is known as liquid biopsy. It has been reported that most of the circulating cell-free DNA (cfDNA) has its origin in immature hematopoietic and bone marrow cells; however, there is limited information about liquid biopsy applications in MPNs. Objective. To analyze the molecular profile of circulating tumor DNA in patients with MPNs. Patients and methods. Peripheral blood samples from 75 patients with MPNs were collected at the time of diagnosis: 21 polycythemia vera (PV), 42 essential thrombocythemia (ET), 10 primary myelofibrosis (PMF) and two non-classifiable MPNs. Cellular DNA was extracted from the granulocytic fraction isolated by density gradient centrifugation and cfDNA was obtained from 1-3ml of plasma (MagMAX Cell-Free DNA Isolation Kit, Thermo Fisher Scientific). cfDNA purity was ascertained by capillary electrophoresis (4200 TapeStation system, Agilent). Molecular characterization was performed in paired samples of granulocytes DNA and cfDNA by next generation sequencing (NGS). Libraries were prepared using a custom panel that covered the whole codifying region of 25 myeloid-associated genes (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina technology (Miseq, Nextseq) with a 3000x minimum coverage. Results. The amount of total cfDNA/mL in plasma was significantly higher in PMF (mean 97 ng/ml) than in PV and ET (mean 18 and 23g/ml, respectively) (p = 0.003, Kruskal-Wallis). Overall, 144 mutations in driver (JAK2, CALR, MPL) and non-driver genes were detected in the granulocytic fraction with similar frequencies to what has been described for PV, ET and PMF. The most frequently mutated non-driver genes where ASXL1 (18.7%), TET2 (17.3%), DNMT3A (6.7%), SRSF2 (6.7%) and IDH2 (5.3%). Sequencing of cfDNA showed a total of 146 mutations. All mutations detected in the granulocytic fraction were also detected in the paired cfDNA sample (100% concordance); two additional mutations in MPL and ASXL1 were detected in plasma in one case. The median variant allele frequency (VAF) present in cfDNA was 29% (range 0.86 - 91.73%), which is far superior to what has been described in solid neoplasms or lymphomas (median 0.41%, range 0.03% - 97.6%). A strong correlation was observed between the VAFs of granulocytic DNA and cfDNA (r = 0.875, p 〈 0.001, Spearman) (Figure 1). The mutation VAFs detected in cfDNA were significantly higher than VAFs detected in granulocytes (p 〈 0.001, Wilcoxon). In particular, MPL mutations presented 2.5 higher VAF in cfDNA than in granulocytes (p = 0.018, Wilcoxon). This finding was confirmed and quantified by digital PCR. Interestingly, in one PMF patient the p.W515L MPL driver mutation was originally only detectable by NGS in cfDNA, but not in granulocytes. This mutation was identified by ultra-sensitive digital PCR in both cfDNA (VAF 2.30%) and granulocytes (VAF 0.16%). Conclusions. The analysis of circulating tumor DNA allows the characterization of the molecular abnormalities of patients with Ph negative myeloproliferative neoplasms. The sensitivity for mutation detection in driver and non-driver genes was equal or even superior to that obtained when studying the isolated granulocytic population. Disclosures Salar: Roche: Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy. Besses:Gilead: Research Funding. Bellosillo:TermoFisher Scientific: Consultancy, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

Description: Background Adult T cell acute lymphoblastic leukemia (T-ALL) is a rare disease that affects less than 10 individuals in one million. It has been less studied than its cognate pediatric malignancy, which is more prevalent. A higher percentage of the adult patients relapse, compared to children. It is thus essential to study the mechanisms of relapse of adult T-ALL cases. Results We profile whole-genome somatic mutations of 19 primary T-ALLs from adult patients and the corresponding relapse malignancies and analyze their evolution upon treatment in comparison with 238 pediatric and young adult ALL cases. We compare the mutational processes and driver mutations active in primary and relapse adult T-ALLs with those of pediatric patients. A precise estimation of clock-like mutations in leukemic cells shows that the emergence of the relapse clone occurs several months before the diagnosis of the primary T-ALL. Specifically, through the doubling time of the leukemic population, we find that in at least 14 out of the 19 patients, the population of relapse leukemia present at the moment of diagnosis comprises more than one but fewer than 108 blasts. Using simulations, we show that in all patients the relapse appears to be driven by genetic mutations. Conclusions The early appearance of a population of leukemic cells with genetic mechanisms of resistance across adult T-ALL cases constitutes a challenge for treatment. Improving early detection of the malignancy is thus key to prevent its relapse.

Description: Background: Polycythemia vera (PV), Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPNs) with median age at diagnosis of ~56-70 years old. However, around 10%-15% of cases are diagnosed during young adulthood and there are scanty data about their molecular profile and its implications in clinical outcomes. Objective: To analyze the clinical and molecular characteristics of young adult patients (≤45y.o.) with MPNs (Y-MPN) and to correlate them with clinical features and outcomes. Material and Methods: This is a retrospective single-center study including MPN patients diagnosed below the age of 45 years. Molecular characterization was performed using DNA from granulocytes at diagnosis or before the start of cytoreductive therapy. JAK2V617F was assessed by quantitative allele-specific PCR and CALR mutations by fragment analysis of exon 9. Further molecular profiling was performed by next generation sequencing (NGS) with a custom panel of 25 myeloid-associated genes (ASXL1, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, PRPF8, RUNX1, SETPB1, SF3B1, S2HB3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2) using Illumina technology. Pathogenic mutations in genes previously related with poor outcomes (ASXL1, EZH2, IDH1, IDH2, SRSF2 and U2AF1) were named as Mutations of Adverse Significance (MAS). Molecular alterations were correlated with diagnosis, progression to PV post ET (PVpET), progression to MF post PV/ET (MFpPV/ET), start of cytoreduction and major thrombotic events (MTE). Time to progression (TTP) and overall survival (OS) were calculated from diagnosis to progression (ELN criteria) and to last visit. Results: From 646 MPN followed in our clinic, 109 (17%) cases were Y-MPN; females 72 (66.1%). At diagnosis the median age was 35 y.o. (9-45). 23 patients (21%) were PV, 91% carried JAK2V617F, 4% (1) carried an exon-12 JAK2 mutation and 1 was JAK2V617F and exon-12 negative. 84 cases (77.1%) were ET, 53.5% (45) JAK2V617F, 25% (21) CALR, (52% type-1 mutation, see table) and 21.4% (18) triple negative (TN). There was 1 PMF with CALR type-1 mutation. No MPL canonical mutations were found. ET was predominantly diagnosed in females (M/F: 26/58). Regarding clinical variables, we found a high proportion of ET-JAK2V617F with high LDH values, higher platelet count for CALR-ET and ET-TN patients (967 and 978 vs 728 for ET-JAK2V617F, p=0.03) and higher frequency of MTE at or before diagnosis for JAKV617F cases (p=0.001). The mean follow-up was 152 months (SD +/-10.4); 16 progressions were registered (PFS 305 months); 8 patients to MFpPV/ET and 8 ET-JAK2V617F to PVpET. An increase in the VAF of JAK2V617F was observed at the time of progression either to PVpET or to MFpET/PV (see table). Seven MTE were registered during this time, 3 in JAK2V617F, 2 in CALR type-1, 1 in exon-12 and 1 in a TN case. 38 (34.8%) cases started cytoreduction, with median time to cytoreductive therapy of 251 (172-330) months; JAK2V617F cases started cytoreduction more often (p=0.04) than patients with other genotypes. No progression to AML nor deaths were recorded. The NGS panel was performed in 102 (93.5%) cases. Pathogenic mutations in non-driver genes were found in 41.2% (42) of cases, being TET2 (7%), ASXL1 (6%) and DNMT3A (5%) the most frequently mutated genes. Also, in 28.4% (29) variants of unknown significance (VUS) were found, involving TET2 (6%), SETBP1 (4%), SH2B3 (5%), and JAK2 (4%) among others. The mutations in SH2B3 (1 pathogenic, 5 VUS) were more frequent in JAK2V617F patients and those in DNMT3A were more common in PV patients. The presence of mutations in non-driver genes (pathogenic or VUS) did not correlate with MTE before or after diagnosis, the start of cytoreduction nor clonal progression. Regarding the 19 TN cases, in 7 (36.8%) one or more non-canonical pathogenic variants implicating MPL, JAK2 and TET2 genes were found. Finally, 8 patients (7.8%) harbored a MAS, of which 3 progressed to MF (2 CALR to MF and 1 ET-JAK2V617F to PVpET); TTP was similar to the rest of the cohort. Conclusions: Our data show that 41% of Y-MPN patients harbor pathogenic mutations in non-driver genes. There was no correlation between their presence and clonal progression, major thrombotic events or overall survival. Mutations of adverse significance did not predict major clinical outcomes. Monitoring of JAK2V617F allele-burden can help to predict progression to MFpPV/ET or PVpET. Disclosures Andrade-Campos: Sanofi-Genzyme: Consultancy, Speakers Bureau; Takeda-Shire: Speakers Bureau; Celgene-BMS: Consultancy. Fernández:Roche: Consultancy, Speakers Bureau. Salar:Janssen: Speakers Bureau; Roche: Speakers Bureau; Celgene: Speakers Bureau. Bellosillo:Qiagen: Consultancy, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau.