Articles de revues : « Targeted resequencing panel »

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Hildebrand, Michael S., Candace T. Myers, Gemma L. Carvill, Brigid M. Regan, John A. Damiano, Saul A. Mullen, Mark R. Newton et al. « A targeted resequencing gene panel for focal epilepsy ». Neurology 86, n^o 17 (30 mars 2016) : 1605–12. http://dx.doi.org/10.1212/wnl.0000000000002608.

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Kim, Eun-Hye, Sunghoon Lee, Jongsun Park, Kyusang Lee, Jong Bhak et Byung Chul Kim. « New Lung Cancer Panel for High-Throughput Targeted Resequencing ». Genomics & ; Informatics 12, n^o 2 (2014) : 50. http://dx.doi.org/10.5808/gi.2014.12.2.50.

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Walter, Christiane, Zhenyu Xu, Martin Zimmermann, Dirk Reinhardt et Nils Von Neuhoff. « Amplicon Based Panel Targeted Resequencing Identified ZRSR2 As a Potential New Favorable Marker in Pediatric AML ». Blood 128, n^o 22 (2 décembre 2016) : 2905. http://dx.doi.org/10.1182/blood.v128.22.2905.2905.

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Abstract Introduction: Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing (NGS) as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time. The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) to identify variants in 54 genes. Methods Patients: In total 150 samples derived from pediatric patients diagnosed with AML at the time of initial diagnosis or relapse were analysed regarding their mutational profile. All patients treated according to the AML-BFM therapy protocols (n=103) were chosen to determine the potential impact in prognosis. Sequencing and analysis of variants Sequencing with the TruSight Myeloid panel (Illumina) was performed on a MiseqDX. The sequencing panel is designed to identify somatic mutations associated with myeloid malignancies in 54 genes. To define variants, we excluded intronic, synonymous and variants with an allele frequency below 5% and a read depth below 50 reads. False positive variants were excluded by including healthy donors and reference samples. Variants were detected and analysed using Variant studio (Illumina) and Sophia DDM (Sophia Genetics). Almost all variants were detectable in both software, although great insertions and deletions were detectable only by Sophia DDM. Results In the cohort of 150 patients, we detected 408 mutations in the 54 genes included in the panel (fig. 1). 26% of the patients showed more than 4, 24% 3, 24% 2, 17% 1 and 9% of the patients showed 0 mutations. Four and more mutations occurred mostly in AML FAB M1 (n=17) and patients with a complex karyotype (n=6). Treatment related AML show less mutations compared to primary AML. Within the group of patients treated according the 1st line AML-BFM protocol (n=103), CEBPA, FLT3, KIT, NRAS, KRAS, NPM1 or WT1 mutations did not have prognostic relevance. Interestingly, we were able to detect mutations in ZRSR2 in 21 patients in total (SNVs in 6, InDels in 9 and splice acceptor variants in 6 patients). 15 patients were part of the group of patients who were treated according to the 1st line AML-BFM protocol. ZRSR2 encodes an essential splicing factor and the encoded protein associates with the U2 auxiliary factor heterodimer and may play a role in network interactions during spliceosome assembly [RefSeq 2008]. The presence of a ZRSR2 mutation seems to be associated with better EFS and lower cumulative incidence of relapse, respectively (fig.2). Even if patients with favourable cytogenetics were excluded, patients with mutated ZRSR2 might have better EFS (fig.2). Conclusions: Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days. This will enable a faster and possible more precise characterisation of pediatric AML, either for risk group stratification or the addition of more specific treatment options. Due to the limited number of patients, the results concerning the prognostic relevance of ZRSR2 need to be confirmed in a larger patient group. Table patient characteristics Table. patient characteristics Figure 1 Number of variants detected in 54 genes Figure 1. Number of variants detected in 54 genes Figure 2 Event-free survival (EFS) and cumulative incidence for relapse for patients showing no mutation (blue) or mutations (red) in ZRSR2. Figure 2. Event-free survival (EFS) and cumulative incidence for relapse for patients showing no mutation (blue) or mutations (red) in ZRSR2. Disclosures Reinhardt: Jazz Pharma: Other: Travel Accomodation; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

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Ciezarek, A., Antonia G. P. Ford, Graham J. Etherington, Nasser Kasozi, Milan Malinsky, Tarang K. Mehta, Luca Penso-Dolfin et al. « Whole genome resequencing data enables a targeted SNP panel for conservation and aquaculture of Oreochromis cichlid fishes ». Aquaculture 548 (février 2022) : 737637. http://dx.doi.org/10.1016/j.aquaculture.2021.737637.

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Galatolo, Daniele, Giovanna De Michele, Gabriella Silvestri, Vincenzo Leuzzi, Carlo Casali, Olimpia Musumeci, Antonella Antenora et al. « NGS in Hereditary Ataxia : When Rare Becomes Frequent ». International Journal of Molecular Sciences 22, n^o 16 (6 août 2021) : 8490. http://dx.doi.org/10.3390/ijms22168490.

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The term hereditary ataxia (HA) refers to a heterogeneous group of neurological disorders with multiple genetic etiologies and a wide spectrum of ataxia-dominated phenotypes. Massive gene analysis in next-generation sequencing has entered the HA scenario, broadening our genetic and clinical knowledge of these conditions. In this study, we employed a targeted resequencing panel (TRP) in a large and highly heterogeneous cohort of 377 patients with a clinical diagnosis of HA, but no molecular diagnosis on routine genetic tests. We obtained a positive result (genetic diagnosis) in 33.2% of the patients, a rate significantly higher than those reported in similar studies employing TRP (average 19.4%), and in line with those performed using exome sequencing (ES, average 34.6%). Moreover, 15.6% of the patients had an uncertain molecular diagnosis. STUB1, PRKCG, and SPG7 were the most common causative genes. A comparison with published literature data showed that our panel would have identified 97% of the positive cases reported in previous TRP-based studies and 92% of those diagnosed by ES. Proper use of multigene panels, when combined with detailed phenotypic data, seems to be even more efficient than ES in clinical practice.

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Bailey, Nathanael G. « Visualization of the Effect of Assay Size on the Error Profile of Tumor Mutational Burden Measurement ». Genes 13, n^o 3 (26 février 2022) : 432. http://dx.doi.org/10.3390/genes13030432.

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Tumor mutational burden (TMB) refers to the number of somatic mutations in a tumor per megabase and is a biomarker for response to immune checkpoint inhibitor therapy. Immune checkpoint inhibitors are currently approved for tumors with TMB greater than or equal to 10 mutations/megabase. Many laboratories are currently reporting TMB values based upon targeted resequencing panels with limited genomic coverage. Due to sampling variation, this leads to significant uncertainty in the assay’s TMB result, particularly at relatively low TMB levels near the 10 mutation per megabase therapeutic threshold. In order to allow clinicians and laboratorians to explore this uncertainty, we built a novel web application that allows a user to view the potential error of a TMB result given the sequencing panel size. This application also allows the user to explore the effect of incorporating knowledge of a specific tumor type’s typical TMB distribution on the error profile of the TMB result.

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Das, Reena, Manu Jamwal, Anu Aggarwal, Prashant Sharma, Man Updesh Singh Sachdeva, Deepak Bansal, Sreejesh Sreedharanunni et al. « Spectrum of Genetic Defects and Phenotype-Genotype Correlation in Dyserythropoietic Anemias : Bench to Bedside Approach in the Indian Scenario ». Blood 134, Supplement_1 (13 novembre 2019) : 950. http://dx.doi.org/10.1182/blood-2019-126453.

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Introduction Congenital dyserythropoietic anemias (CDA) are rare inherited red cell disorders characterized by ineffective erythropoiesis and inappropriate reticulocytopenia. CDAs are usually difficult to diagnose due to variable phenotypes and overlapping bone marrow (BM) morphology with other disorders. Numerous implicated causal genes make Sanger sequencing a less likely approach and hence, the use of targeted resequencing can expedite molecular diagnosis. This study aimed at determining the genetic spectrum of CDAs and translating the results into patient care. Methods Twenty nine patients with clinical and laboratory evidence suggestive of CDA and 1 patient suggestive of CDA with thrombocytopenia by BM morphology were studied. Various biochemical and molecular tests were done to exclude common hemolytic anemias. Common SEC23B: p.Tyr462Cys variant in our patients with CDA was screened by Sanger sequencing. DNA libraries were prepared using TruSight One Sequencing Panel and TruSeq Custom Amplicon Panel and sequenced on Illumina platform. After data analysis variants were classified and the most likely disease-causing variants were validated by Sanger sequencing followed by pedigree analysis. Results Out of 27 patients of suspected CDA, SEC23B: p.Tyr462Cys variant was found in 10 patients. Rest of the remaining 17 patients were subjected to targeted resequencing. Data analysis revealed novel potentially pathogenic variants in compound heterozygosity in SEC23B in 4 patients and 1 patient had a heterozygous variant in SEC23B. There could be the possibility of intronic or large indel in her. The variants were distributed throughout the SEC23B gene. Notably, in 7 patients with suspected CDA, the final molecular diagnosis were hemolytic anemias. Of them, 4 showed likely pathogenic variants in PKLR gene and 1 each had probably causal variant in MTRR, SPTB and PIEZO1 genes. In the patient's with pyruvate kinase deficiency, screening by enzyme assays were normal. Except for the patient with MTRR gene defect all 6 had transfusion dependent anemia and BM showed dyserythropoiesis. One patient each of GATA1 gene variant (novel) and a known pathogenic variant p.Glu325Lys in KLF1 gene (CDA type IV) was detected. Of 17 cases subjected to targeted resequencing the diagnosis was achieved in ~76% (13/17) of cases. The phenotypes correlated with the genetic defects found in the SEC23B gene. The homozygous and compound heterozygous defects in this gene cause CDA type II. As anticipated GATA1 gene defect (p.Val205Leu) was found in a patient of X-linked thrombocytopenia with dyserythropoietic anemia. Patient with KLF1 had high levels of fetal hemoglobin along with features of dyserythropoiesis in BM compatible with the phenotype of variant p.Glu325Lys causing CDA type IV. Phenotype-genotype correlation was discrepant in 7 cases of CDA. In 4 cases pyruvate kinase deficiency (PKLR) was found and each case of hereditary xerocytosis (PIEZO1), membrane defect (SPTB) and MTRR defect was found. Conclusion(s) CDA showed a highly varied etiology. Our experience demonstrates a high diagnostic yield (~76%) of targeted resequencing for molecular diagnosis of suspected CDAs. Discrepancy was noted in 41% (7/17) cases with suspected CDA which were diagnosed as hemolytic anemia after molecular analysis. Establishing the correct diagnosis of pyruvate kinase deficiency led to an evidence-based decision of splenectomy that eliminated transfusion dependence. In the patient with MTRR defect change in therapy was suggested. Prenatal diagnosis was done for 2 families, where in 1 of the family both the SEC23B variants were novel and in compound heterozygosity. This study highlights the importance of genetic testing in patients under frequent blood transfusions and suspected CDAs, to provide accurate diagnosis and therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

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Bartels, Stephan, Elisa Schipper, Hans Heinrich Kreipe et Ulrich Lehmann. « Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing ». PLOS ONE 10, n^o 7 (29 juillet 2015) : e0133930. http://dx.doi.org/10.1371/journal.pone.0133930.

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Roy, Noémi B. A., Edward A. Wilson, Shirley Henderson, Katherine Wray, Christian Babbs, Steven Okoli, Wale Atoyebi et al. « A novel 33‐Gene targeted resequencing panel provides accurate, clinical‐grade diagnosis and improves patient management for rare inherited anaemias ». British Journal of Haematology 175, n^o 2 (19 juillet 2016) : 318–30. http://dx.doi.org/10.1111/bjh.14221.

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Jin, Wang, Zhao Lei, Sun Xu, Zhou Fachen, Zhang Yixiang, Zhao Shilei, Guo Tao et al. « Genetic Mutation Analysis in Small Cell Lung Cancer by a Novel NGS-Based Targeted Resequencing Gene Panel and Relation with Clinical Features ». BioMed Research International 2021 (5 avril 2021) : 1–8. http://dx.doi.org/10.1155/2021/3609028.

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Background. Small cell lung cancer (SCLC) is an aggressive and invasive malignancy that presents at advanced clinical stage with no more effective treatments. Development of a method for its early detection would be useful, also new therapeutic target need to be discovered; however, there is a lack of information about its oncogenic driver gene mutations. Objectives. We aim to identify the SCLC-related genomic variants that associate with clinical staging and serum protein biomarkers observed in other types of lung cancer. Methods. We screened formalin-fixed paraffin-embedded (FFPE) biopsy tissues of 32 Chinese SCLC patients using the 303 oncogenic driver gene panel generated by Tiling PCR amplification sequencing (tPAS) and analyzed the patients’ corresponding serum protein levels of CYFRA21-1 CEA, NSE, and SCCA. Results. In total, we found 147 SCLC-related mutant genes, among these, three important genes (TP53, RB1, KMT2D) as well as five novel genes LRRK2, BRCA1, PTCH1, ARID2, and APC that altogether occurred in 90% of patients. Furthermore, increased mutations to 6 genes (WT1, NOTCH1, EPHA3, KDM6A, SETD2, ACVR1B) significantly associated with higher serum NSE levels ( P = 0.0016 ) and higher clinical stages II + III compared to stage I ( P = 0.06 ). Conclusions. Our panel is relatively reliable in detecting the oncogenic mutations of Chinese SCLC patients. Based on our findings, it may be possible to combine SCLC-related mutations and serum NSE for a simple detection of clinical staging.

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Vandamme, T., M. Beyens, G. Boons, A. Schepers, K. Kamp, K. Biermann, P. Pauwels et al. « Hotspot DAXX, PTCH2 and CYFIP2 mutations in pancreatic neuroendocrine neoplasms ». Endocrine-Related Cancer 26, n^o 1 (janvier 2019) : 1–12. http://dx.doi.org/10.1530/erc-18-0120.

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Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs). However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor. This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing. Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel. Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance). Found variants were validated by Sanger sequencing. Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476. Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation. Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging. Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes. Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2. This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations. The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX. In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN.

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Claerhout, Sofie, Paulien Verstraete, Liesbeth Warnez, Simon Vanpaemel, Maarten Larmuseau et Ronny Decorte. « CSYseq : The first Y-chromosome sequencing tool typing a large number of Y-SNPs and Y-STRs to unravel worldwide human population genetics ». PLOS Genetics 17, n^o 9 (7 septembre 2021) : e1009758. http://dx.doi.org/10.1371/journal.pgen.1009758.

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Male-specific Y-chromosome (chrY) polymorphisms are interesting components of the DNA for population genetics. While single nucleotide polymorphisms (Y-SNPs) indicate distant evolutionary ancestry, short tandem repeats (Y-STRs) are able to identify close familial kinships. Detailed chrY analysis provides thus both biogeographical background information as paternal lineage identification. The rapid advancement of high-throughput massive parallel sequencing (MPS) technology in the past decade has revolutionized genetic research. Using MPS, single-base information of both Y-SNPs as Y-STRs can be analyzed in a single assay typing multiple samples at once. In this study, we present the first extensive chrY-specific targeted resequencing panel, the ‘CSYseq’, which simultaneously identifies slow mutating Y-SNPs as evolution markers and rapid mutating Y-STRs as patrilineage markers. The panel was validated by paired-end sequencing of 130 males, distributed over 65 deep-rooted pedigrees covering 1,279 generations. The CSYseq successfully targets 15,611 Y-SNPs including 9,014 phylogenetic informative Y-SNPs to identify 1,443 human evolutionary Y-subhaplogroup lineages worldwide. In addition, the CSYseq properly targets 202 Y-STRs, including 81 slow, 68 moderate, 27 fast and 26 rapid mutating Y-STRs to individualize close paternal relatives. The targeted chrY markers cover a high average number of reads (Y-SNP = 717, Y-STR = 150), easy interpretation, powerful discrimination capacity and chrY specificity. The CSYseq is interesting for research on different time scales: to identify evolutionary ancestry, to find distant family and to discriminate closely related males. Therefore, this panel serves as a unique tool valuable for a wide range of genetic-genealogical applications in interdisciplinary research within evolutionary, population, molecular, medical and forensic genetics.

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Herold, Sylvia, Thoralf Stange, Matthias Kuhn, Ingo Roeder, Christoph Röllig, Gerhard Ehninger et Christian Thiede. « Targeted Resequencing of MLL-PTD Positive AML Patients Reveals a High Prevalence of Co-Ocurring Mutations in Epigenetic Regulator Genes ». Blood 124, n^o 21 (6 décembre 2014) : 1035. http://dx.doi.org/10.1182/blood.v124.21.1035.1035.

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Abstract Background Partial tandem duplication mutations of the Mixed Lineage Leukemia gene (MLL-PTD) can be found in about 10% of patients with AML, especially in patients with normal karyotype AML. The mutation generates a self-fusion within the N-terminal part of MLL and has been shown to be leukemogenic in mouse models. In patients, the presence of the mutation is associated with poor prognosis. Little is known on the molecular profile of patients with MLL-PTD and on the cooperating mutations. In order to identify accompanying molecular alterations, we performed whole exome sequencing (WES) of eight AML patients harbouring MLL-PTD mutations. Based on the observed alterations we then designed a custom amplicon panel and performed targeted resequencing in a cohort of 90 MLL-PTD mutated AML patients. Materials and Methods All patients included in this analysis were treated in prospective treatment protocols of the Study Alliance Leukemia (SAL). To enrich for malignant cells and to obtain germline reference material (T-cells), FACS sorting was performed on viable cells banked at diagnosis. After whole genome amplification of the primary DNA, whole exomes were enriched (TruSeq chemistry; Illumina), and paired-end sequenced using Illumina HiSeq2000 2x100 bp runs. Resulting data were mapped against human genome (Hg19). Only somatic single nucleotide variants (SNVs) were included in the final analysis. Based on the SNVs identified by whole exome sequencing (WES), a custom amplicon panel (TruSeq Custom Amplicon, TSCA, Illumina) for targeted resequencing was designed. The assay included either the entire coding region or mutational hot spots of 56 genes (Fig.1). In total, 700 targets were amplified in a single reaction for each patient and paired end sequenced on a MiSeq NGS system (Illumina). Paired end reads were BWA mapped against targeted regions and data analysis was done using the Sequence Pilot software package (JSI Medical Systems) with a 20% variant allele frequency (VAF) mutation calling cutoff. Only non-synonymous variants not specified as SNP in the db137 database and predicted as deleterious (Provean) were included in the final analysis. All variations were confirmed by Sanger sequencing. Results WES of eight MLL-PTD (7/8 FLT3-ITD negativ) patients revealed a total 490 SNVs (range 13-254 per patient). Most frequently mutated genes were DNMT3A, IDH1/2 and TET2. Somatic mutations were also found in genes rarely mutated in AML, such as ATM, GNAS, TET1 and EP300. Based on the WES-data, 90 MLL-PTD patients were screend for a panel of 56 genes using the TSCA assay, which revealed in total of 169 mutations. 18 genes were not found to be mutated and in 8 patients, no co-occurring mutations were identified. Due bad assay performance EP300, EZH1, JAK3, MLL2, MLL3 and NOTCH1 were excluded from the data analysis. Here again, the most frequently mutated genes were DNMT3A (34.4%), IDH1 (20.0%), IDH2R140 (18.9%), IDH2R172 (7.9%), TET2 (16.7%) and FLT3 (11.3%). Mutations were less frequently found in RUNX1 (8.9%) and ASXL1, SMC1A, U2AF1 (5.6% each) (Fig. 1). In addition to these known genes, most prevalent mutations were found in ATM (8.9%) as well as DNMT3B and TET1 (4.4% each). Overall, we oberserved a low frequency of mutations in typical class 1 genes such as NRAS, KRAS and FLT3, which was lower than reported in the TCGA data set. Conclusions This analysis in a large set of patients with MLL-PTD mutations did not reveal any new and specific individual mutation present in patients with this alteration. Instead, our finding of a very high prevalence of alterations in epigenetic regulator genes, found in more than 85% of patients with MLL-PTD, strongly argues for a particular disease biology in these patients. These findings might also implicate that treatment based on demethylating agents or histone-deacetylase inhibitors might be especially attractive in patients with MLL-PTD. Figure 1: Figure 1:. Distribution of mutations in MLL-PTD patients The assay included either the entire coding region or mutational hot spots of the following 56 genes; ASXL1, ATM, BCOR, BRAF, CBL, DDR1, DNMT1, DNMT3A, DNMT3B, EIF4A2, EP300, ETV6, EZH1, EZH2, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KDM4A, KDM5A, KDM5C, KDM6A, KIT, KRAS, MET, MLL, MLL2, MLL3, NOTCH1, NOTCH4, NPM1, NRAS, PDGFRA, PDGFRB, PHF6, PTEN, PTPN11, RAD21, RUNX1, SF3A1, SF3B4, SMC1A, SMC3, SMC4, TET1, TET2, TP53, U2AF1 and WT1. Disclosures Thiede: AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

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Hirabayashi, Kyoko, Daniela Tiaki Uehara, Hidetoshi Abe, Atsushi Ishii, Keiji Moriyama, Shinichi Hirose et Johji Inazawa. « Copy number variation analysis in 83 children with early-onset developmental and epileptic encephalopathy after targeted resequencing of a 109-epilepsy gene panel ». Journal of Human Genetics 64, n^o 11 (30 août 2019) : 1097–106. http://dx.doi.org/10.1038/s10038-019-0661-x.

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Roy, Noemi, Melanie Proven, Irene Roberts, Hannah Tamary, Dorine W. Swinkels, Aguilar-Martinez Patricia, Paola Bianchi et al. « Towards an External Quality Assessment for Next Generation Sequencing in the Diagnosis of Rare Inherited Anaemias ». Blood 132, Supplement 1 (29 novembre 2018) : 4936. http://dx.doi.org/10.1182/blood-2018-99-111609.

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Abstract The diagnosis of patients with inherited anemias is increasingly made in conjunction with high-throughput 'next-generation' sequencing (NGS) analysis, largely using targeted resequencing panels validated for clinical use in diagnostic labs. While there is a joint UK NEQAS and European Molecular Quality Network pilot scheme for Molecular Genetics to assess NGS quality control, this is not disease-specific. Patients with inherited anaemias can have multiple mutations with complex genotype phenotype interactions therefore a scheme assessing interpretation of these results could be of value. Likewise, guidelines for variant reporting (eg ACMG- American College of Medical Genetics and Genomics) provide excellent advice on how to interpret the likely pathogenicity of genetic variants, but no disease-specific guidance exists to assist in the clinical interpretation of NGS findings for individuals with rare inherited anemias. The European Hematology Association Scientific Working Group on Red Cells and Iron carried out a survey among its members to investigate variability in current practice and determine the need for, and feasibility of, a formal external quality assessment (EQA) scheme. Surveys and two clinical vignettes with sample variant call files (VCFs) were distributed among 14 participating labs from 9 countries; 13/14 labs used a targeted panel and 1/14 lab (8%) used a 366-gene virtual panel derived from whole exome sequencing data. Accreditation: 12/14 labs had ISO (International Organisation for Standardisation) accreditation for their NGS; 2/12,used local accreditation schemes. The number of genes per targeted panel ranged from 18 to 215 genes (median 64), covering: congenital dyserythropoietic anemias, Diamond-Blackfan anemia, sideroblastic anemia, red cell membrane and enzyme disorders. Some panels included other related bone marrow failure or iron metabolism disorders. 7/13 labs with targeted panels sequenced only exons, with variable padding into introns, while 6/13 routinely sequenced 3' and 5' untranslated regions. Capture methods were variable between labs and 11/14 labs used Illumina platforms for sequencing and 3/14 Ion Torrent. Sanger sequencing was used for confirmation of NGS variants in 12/14 labs, but used for gap-filling of uncovered regions in 10/14 labs. Reporting of variants followed ACMG guidelines in 10/14 labs, ACGS (Association for Clinical Genomics Science) guidelines in 2/14 and no published guidelines in 2/14. Reporting of Tier 3 (variants of uncertain significance): 8/14 labs reported strict adherence to ACMG guidelines, including only Class 4 and Class 5 variants in clinical reports, while 6/14 labs admitted looser adherence and reporting of Class 3 variants depending on circumstances. The number of samples analysed per year was highly variable between labs (10-600, median 60). Two mini-EQA VCFs were sent with clinical vignettes, for which 100% of labs correctly identified a case of autosomal recessive sideroblastic anaemia due to compound heterozygous mutations in SLC25A38, and 100% correctly identified a case negative by NGS. Class 3 variants were not reported at all in 50% of clinical reports, reported in the main body of the report in 20% and in a separate table in 30% of labs. In conclusion, we have identified common approaches to NGS sequencing in 14 diagnostic laboratories but highlighted variability in accreditation, use of Sanger sequencing and adherence to ACMG guidelines. The feasibility of carrying out an EQA scheme has been established and work will continue with UK NEQAS to formally create such a scheme, with the aim of ensuring improved patient care through the use of objective quality assessments. Disclosures Colombatti: ADDMEDICA: Consultancy; BlueBirdBio: Consultancy; Global Blood Therapeutics: Consultancy; NOVARTIS: Consultancy. Viprakasit:Protagonist Therapeutics: Consultancy, Research Funding; Agios: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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Jayaraman, Pushkala, Timothy Mosbruger, Taishan Hu, Nikolaos G. Tairis, Chao Wu, Peter M. Clark, Monica D’Arcy et al. « AnthOligo : automating the design of oligonucleotides for capture/enrichment technologies ». Bioinformatics 36, n^o 15 (2 juin 2020) : 4353–56. http://dx.doi.org/10.1093/bioinformatics/btaa552.

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Abstract Summary A number of methods have been devised to address the need for targeted genomic resequencing. One of these methods, region-specific extraction (RSE) is characterized by the capture of long DNA fragments (15–20 kb) by magnetic beads, after enzymatic extension of oligonucleotides hybridized to selected genomic regions. Facilitating the selection of the most appropriate capture oligos for targeting a region of interest, satisfying the properties of temperature (Tm) and entropy (ΔG), while minimizing the formation of primer-dimers in a pooled experiment, is therefore necessary. Manual design and selection of oligos becomes very challenging, complicated by factors such as length of the target region and number of targeted regions. Here we describe, AnthOligo, a web-based application developed to optimally automate the process of generation of oligo sequences used to target and capture the continuum of large and complex genomic regions. Apart from generating oligos for RSE, this program may have wider applications in the design of customizable internal oligos to be used as baits for gene panel analysis or even probes for large-scale comparative genomic hybridization array processes. AnthOligo was tested by capturing the Major Histocompatibility Complex (MHC) of a random sample. The application provides users with a simple interface to upload an input file in BED format and customize parameters for each task. The task of probe design in AnthOligo commences when a user uploads an input file and concludes with the generation of a result-set containing an optimal set of region-specific oligos. AnthOligo is currently available as a public web application with URL: http://antholigo.chop.edu. Supplementary information Supplementary data are available at Bioinformatics online.

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Xu, Liya, Mary E. Kim, Ashley Polski, Rishvanth K. Prabakar, Lishuang Shen, Chen-Ching Peng, Mark W. Reid et al. « Establishing the Clinical Utility of ctDNA Analysis for Diagnosis, Prognosis, and Treatment Monitoring of Retinoblastoma : The Aqueous Humor Liquid Biopsy ». Cancers 13, n^o 6 (13 mars 2021) : 1282. http://dx.doi.org/10.3390/cancers13061282.

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Because direct tumor biopsy is prohibited for retinoblastoma (RB), eye-specific molecular biomarkers are not used in clinical practice for RB. Recently, we demonstrated that the aqueous humor (AH) is a rich liquid biopsy source of cell-free tumor DNA. Herein, we detail clinically-relevant molecular biomarkers from the first year of prospective validation data. Seven eyes from 6 RB patients who had AH sampled at diagnosis and throughout therapy with ≥12 months of follow-up were included. Cell-free DNA (cfDNA) from each sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing for pathogenic variants using a RB1 and MYCN custom hybridization panel. Tumoral genomic information was detected in 100% of diagnostic AH samples. Of the seven diagnostic AH samples, 5/7 were positive for RB SCNAs. Mutational analysis identified RB1 variants in 5/7 AH samples, including the 2 samples in which no SCNAs were detected. Two eyes failed therapy and required enucleation; both had poor prognostic biomarkers (chromosome 6p gain or MYCN amplification) present in the AH at the time of diagnosis. In the context of previously established pre-analytical, analytical, and clinical validity, this provides evidence for larger, prospective studies to further establish the clinical utility of the AH liquid biopsy and its applications to precision oncology for RB.

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Rao, Pavitra N., Swapna Uplekar, Sriti Kayal, Prashant K. Mallick, Nabamita Bandyopadhyay, Sonal Kale, Om P. Singh et al. « A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India ». Journal of Clinical Microbiology 54, n^o 6 (23 mars 2016) : 1500–1511. http://dx.doi.org/10.1128/jcm.00235-16.

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A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of sixPlasmodium falciparumgenes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverseP. falciparumreference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

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Das, Reena, Manu Jamwal, Prashant Sharma, Deepak Bansal, Amita Trehan, Pankaj Malhotra et Arindam Maitra. « Genetic Spectrum of Inherited/Congenital Hemolytic Anemias in Indian Patients ». Blood 138, Supplement 1 (5 novembre 2021) : 4151. http://dx.doi.org/10.1182/blood-2021-154452.

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Abstract Introduction Hemolytic anemias are a group of disorders caused by the premature destruction of red blood cells with reticulocytosis. Common causes of inherited/congenital hemolysis are hemoglobinopathies and thalassemia syndromes, red blood cell membrane, and enzyme disorders. Most of the common causes (thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary spherocytosis, etc.) are diagnosed based on laboratory testing; however, for remaining causes laboratory tests are either inaccessible or cumbersome. We follow a stepwise diagnostic pipeline and red cell morphology is helpful with membrane disorders. Phenotypes vary from severe hemolysis (transfusion-dependent) to mild/asymptomatic patients. Undiagnosed haemolytic anemias are taken up for multi-gene panel-based targeted resequencing which is rapid, accurate, and cost-effective. The use of these panels expedites the diagnoses of inherited hemolytic anemias and is eventually helpful for evidence-based genetic counseling. Objectives This study aimed to determine the genetic defects in inherited/congenital hemolytic anemias which remained unexplained after routine laboratory tests. Methods Seventy-five families were enrolled based on the clinical and laboratory features of inherited/congenital hemolytic anemias. Common causes of inherited hemolysis are G6PD deficiency, hemoglobinopathies and thalassemia syndromes, autoimmune hemolytic anemias, hereditary spherocytosis, and pyruvate kinase (PK) deficiency were excluded on the basis of biochemical and molecular tests. DNA extraction was done QIAamp DNA Blood Mini Kit. Quantity and quality of DNA were verified using NanoDrop and Qubit Fluorometer respectively. DNA libraries were prepared using Amplicon custom panels for genes implicated in hemolytic anemias and sequenced on Illumina MiSeq Sequencer. Alignment and variant calling were done in Illumina Local run Manager and Variant annotation was done in Basespace VariantInterpretor. Sanger sequencing was done as orthogonal validation in the index case. Predictive testing was performed for the family members. Results After targeted resequencing of the total 75 index cases, 19 patients were found to have red blood cell enzymopathies, 15 patients had stomatocytosis, 13 had membranopathies and three patients had unstable hemoglobins. In 8 patients cause was not established either only heterozygous variant was found for autosomal recessive or due to the lack of samples of family members for screening. Seventeen cases remained unexplained even after next-generation sequencing. Out of 19 patients, unexpected PK deficiency was found in 12 patients and G6PD deficiency was found in 3 patients; despite the enzyme assay being normal in these cases. We also found 2 patients with glucose-6-phosphate isomerase deficiency. One case each with hexokinase deficiency and glutathione synthetase deficiency was found. Among 15 patients with stomatocytosis, 8 had Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5/ABCG8). These 8 patients showed the presence of stomatocytosis along with giant platelets on peripheral smear evaluation. Of the remaining 7 cases , 2 were found to have overhydrated hereditary stomatocytosis (RHAG) and dehydrated Stomatocytosis/xerocytosis was found in 5 (PIEZO1/KCNN4). We also found 13 cases of hemolytic anemia to have a genetic defect in red blood cell membrane protein-coding genes. Of these 5 had probably pathogenic variants in the ANK1 gene, 5 had a pathogenic variant in SPTA1, 2 had SPTB 2, and 1 patient SLC4A1. We also encountered 3 cases of unstable hemoglobins where no abnormality was noted in Hb-HPLC patterns. A total of seven patients underwent splenectomy and are transfusion free. Conclusions Our cohort of 75 families of hemolytic anemia of unexplained etiology showed a highly heterogeneous genetic spectrum. Of the total cases, the confirmed diagnosis was achieved in 67% of the patients. This approach of using a multi-gene panel is cost-effective and can provide a rapid and accurate diagnosis. Unexpected PK deficiency, G6PD deficiency, and unstable hemoglobins suggest that such cases can be missed. Providing accurate diagnosis in such cases provides evidence-based counseling and saves the families from inappropriate treatments. Disclosures No relevant conflicts of interest to declare.

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Paracchini, Lara, Laura Mannarino, Luca Beltrame, Fabio Landoni, Robert Fruscio, Tommaso Grassi, Maria Luisa Dalessandro, Maurizio D’Incalci et Sergio Marchini. « Targeted Mutational Analysis of Circulating Tumor DNA to Decipher Temporal Heterogeneity of High-Grade Serous Ovarian Cancer ». Cancers 14, n^o 15 (29 juillet 2022) : 3697. http://dx.doi.org/10.3390/cancers14153697.

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We have previously demonstrated that longitudinal untargeted analysis of plasma samples withdrawn from patients with high-grade serous ovarian cancer (HGS-EOC) can intercept the presence of molecular recurrence (TRm) earlier than the diagnosis of clinical recurrence (TRc). This finding opens a clinical important temporal window to acquire through plasma sample analysis a real-time picture of those emerging molecular lesions that will drive and sustain the growth of relapsed disease and ultimately will confer resistance. In this proof of principle study, the same genomic libraries obtained at the diagnosis (T0), TRm and TRc were further analyzed by targeted resequencing approach to sequence the coding region of a panel of 65 genes to provide longitudinal analysis of clonal evolution as a novel strategy to support clinical decisions for the second-line treatment. Experiments were performed on plasma and tumor tissues withdrawn on a selection of previously analyzed cohorts of cases (i.e., 33 matched primary and synchronous lesions and 43 plasma samples from 18 patients). At T0, the median concordance of mutations shared by each tumor tissue biopsy and its matched plasma sample was 2.27%. This finding confirms the limit of a single tumor biopsy to be representative of the entire disease, while plasma analysis can recapitulate most of the main molecular lesions of the disease. A comparable scenario was observed during longitudinal analysis, where, with the exception of the TP53 gene and germline mutations in BRCA1/2 genes, no other gene shared the same locus specific gene mutation across T0, TRm and TRc time points. This high level of temporal heterogeneity has important implications for planning second-line treatment. For example, in three out of 13 cases, plasma ctDNA analysis at TRm or TRc reported acquired novel variants in the TP53BP1 gene not present at T0. In particular, patient 21564, potentially eligible for PARP-inhibitor (PARPi) treatment at the time of diagnosis (BRCA1 c.5182delA mutation), would unlikely respond to these drugs in second-line therapy due to the presence of eight distinct TP53BP1 variants in plasma samples collected TRc. This study demonstrates that liquid biopsy provides a real-time molecular picture to intercept those actionable genetic vulnerabilities or drug resistance mechanisms that could be used to plan a more rational second-line treatment.

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Bonora, Elena, Federica Isidori, Isotta Bozzarelli, Marialuisa Lugaresi, Deborah Malvi, Henna Söderström, Chiara Bolognesi et al. « PS02.046 : UNRAVELING TUMOR HETEROGENEITY OF ESOPHAGEAL ADENOCARCINOMA (EAC) THROUGH HIGH-THROUGHPUT OF SORTED TUMOR CELL POPULATIONS ». Diseases of the Esophagus 31, Supplement_1 (1 septembre 2018) : 133. http://dx.doi.org/10.1093/dote/doy089.ps02.046.

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Abstract Background In EAC clinical, histological, immune-histochemical and genetic patterns were documented, which support the existence of biologically different sub populations. We studied genetic intra/inter tumor heterogeneity of EAC with a new technology based on sorting of specific cell populations. Methods Formalin embedded material obtained from 16 EAC surgical specimens, classified according to the presence/absence of intestinal metaplasia in esophagus (BIM) and stomach (GIM) in BIM + GIM- (Barret's like), BIM-GIM- (cardio-pyloric like), BIM—GIM + (gastric like) types, was processed using a high-throughput cell sorting technology. Stromal and tumor cell populations were sorted based on antibodies against vimentin/pan-cytokeratin and on DNA content. Targeted resequencing on DNA extracted from the sorted cells was performed for 63 cancer-related genes (OncoSeek panel). Results In 11/16 (68.75%) of cases a mutation in TP53 was detected, and in 2 we observed CDKN2A (TP53 regulator) mutations. In Barret's like and Gastric like sub types only mutations of TP53 or TP53regulator genes were present. In pyloric like type TP53 and/or other different mutations of whom 2 in HNF1A were detected (Figure 1). Conclusion Selective sorting led to new patterns of EAC tumor mutational status and heterogeneity, among others somatic mutations in HNF1A not previously found. Parallel analysis of unsorted samples failed to detect these specific mutations. The cardio-pyloric like sub type contains mutational patterns different than those of Barret's and gastric like types. Further research on additional cases is necessary to confirm these findings. Disclosure All authors have declared no conflicts of interest.

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Hardigan, Michael A., F. Parker E. Laimbeer, Linsey Newton, Emily Crisovan, John P. Hamilton, Brieanne Vaillancourt, Krystle Wiegert-Rininger et al. « Genome diversity of tuber-bearing Solanum uncovers complex evolutionary history and targets of domestication in the cultivated potato ». Proceedings of the National Academy of Sciences 114, n^o 46 (30 octobre 2017) : E9999—E10008. http://dx.doi.org/10.1073/pnas.1714380114.

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Cultivated potatoes (Solanum tuberosum L.), domesticated from wild Solanum species native to the Andes of southern Peru, possess a diverse gene pool representing more than 100 tuber-bearing relatives (Solanum section Petota). A diversity panel of wild species, landraces, and cultivars was sequenced to assess genetic variation within tuber-bearing Solanum and the impact of domestication on genome diversity and identify key loci selected for cultivation in North and South America. Sequence diversity of diploid and tetraploid S. tuberosum exceeded any crop resequencing study to date, in part due to expanded wild introgressions following polyploidy that captured alleles outside of their geographic origin. We identified 2,622 genes as under selection, with only 14–16% shared by North American and Andean cultivars, showing that a limited gene set drove early improvement of cultivated potato, while adaptation of upland (S. tuberosum group Andigena) and lowland (S. tuberosum groups Chilotanum and Tuberosum) populations targeted distinct loci. Signatures of selection were uncovered in genes controlling carbohydrate metabolism, glycoalkaloid biosynthesis, the shikimate pathway, the cell cycle, and circadian rhythm. Reduced sexual fertility that accompanied the shift to asexual reproduction in cultivars was reflected by signatures of selection in genes regulating pollen development/gametogenesis. Exploration of haplotype diversity at potato’s maturity locus (StCDF1) revealed introgression of truncated alleles from wild species, particularly S. microdontum in long-day–adapted cultivars. This study uncovers a historic role of wild Solanum species in the diversification of long-day–adapted tetraploid potatoes, showing that extant natural populations represent an essential source of untapped adaptive potential.

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Oultram, Jackson M. J., Joseph L. Pegler, Greg M. Symons, Timothy A. Bowser, Andrew L. Eamens, Christopher P. L. Grof et Darren J. Korbie. « Genetic Variants Associated with Long-Terminal Repeats Can Diagnostically Classify Cannabis Varieties ». International Journal of Molecular Sciences 23, n^o 23 (22 novembre 2022) : 14531. http://dx.doi.org/10.3390/ijms232314531.

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Cannabis sativa (Cannabis) has recently been legalized in multiple countries globally for either its recreational or medicinal use. This, in turn, has led to a marked increase in the number of Cannabis varieties available for use in either market. However, little information currently exists on the genetic distinction between adopted varieties. Such fundamental knowledge is of considerable value and underpins the accelerated development of both a nascent pharmaceutical industry and the commercial recreational market. Therefore, in this study, we sought to assess genetic diversity across 10 Cannabis varieties by undertaking a reduced representation shotgun sequencing approach on 83 individual plants to identify variations which could be used to resolve the genetic structure of the assessed population. Such an approach also allowed for the identification of the genetic features putatively associated with the production of secondary metabolites in Cannabis. Initial analysis identified 3608 variants across the assessed population with phylogenetic analysis of this data subsequently enabling the confident grouping of each variety into distinct subpopulations. Within our dataset, the most diagnostically informative single nucleotide polymorphisms (SNPs) were determined to be associated with the long-terminal repeat (LTRs) class of retroelements, with 172 such SNPs used to fully resolve the genetic structure of the assessed population. These 172 SNPs could be used to design a targeted resequencing panel, which we propose could be used to rapidly screen different Cannabis plants to determine genetic relationships, as well as to provide a more robust, scientific classification of Cannabis varieties as the field moves into the pharmaceutical sphere.

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Rothenberg-Thurley, Maja, Binje Vick, Stephanie Schneider, Bianka Ksienzyk, Philipp A. Greif, Michael Fiegl, Marion Subklewe et al. « Genetic Profiling By Targeted, Deep Resequencing Confirms That a Murine Xenograft Model Of Acute Myeloid Leukemia (AML) Recapitulates The Mutational Landscape Of The Human Disease and Provides Evidence For Clonal Heterogeneity and Clonal Evolution ». Blood 122, n^o 21 (15 novembre 2013) : 49. http://dx.doi.org/10.1182/blood.v122.21.49.49.

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Abstract Background A growing number of recurrent gene mutations have been identified in AML through novel sequencing techniques. To uncover the functional consequences of these mutations and identify novel therapeutic targets, adequate model systems are needed. Such models should recapitulate the mutational landscape and genetic heterogeneity of AML as closely as possible. In this respect, analyses of primary tumor cells are superior to established permanent cell lines. Xenografts of primary AML blasts in immunodeficient mice thus may represent a valuable platform for functional analyses on a wide spectrum of AML subtypes. However, it has not been determined whether the mutational architecture of AML xenografts faithfully represents the human disease they originate from. Methods Fresh bone marrow or peripheral blood samples were obtained from adult AML patients. One to ten million cells were injected intravenously or intrafemorally into recipient NOD-scid IL2R-gammanull(NSG) mice. Engraftment was monitored by serial immunophenotyping using anti-human CD33 and anti-human CD45 antibodies. Mice showing >35% human cells in the peripheral blood or signs of disease were sacrificed, and xenotransplanted cells re-isolated from femurs and spleens. Using genomic DNA from xenografts and paired primary human specimens, we profiled mutations in 44 genes recurrently mutated in human hematologic malignancies by targeted, deep amplicon resequencing (Agilent Haloplex / Illumina MiSeq). Results To date, 4 pairs of primary AML specimens and matched xenografts have been genetically characterized by targeted resequencing. We obtained between 480k and 1100k paired-end reads (2x250bp) per sample, resulting in >30x coverage for >98.7% of the target sequence. The median coverage for individual target regions ranged from 116-fold (CEBPA) to ∼5200-fold (FLT3). In each patient specimen, 2 to 5 known AML-associated driver mutations were identified in our panel of 44 genes. The Figure shows variant allele frequencies (VAFs) for mutations and known germline polymorphisms (SNPs; dbSNP database v137), in the patient samples and matched xenografts. All mutations that were present in the primary patient samples with a VAF of >10% were also found in the matched xenograft. None of the xenografts acquired new mutations that were undetectable in the original patient specimen. However, each sample pair showed evidence for clonal diversity and clonal evolution. In patient AML361, NPM1, DNMT3A and BCOR mutations were detected at a VAF slightly below .5, consistent with heterozygous mutations present in most cells in the primary specimen. Additionally, a FLT3-internal tandem duplication (ITD) was present at a lower VAF, likely representing a subclonal mutation. In the corresponding xenograft, the FLT3-ITD was observed in a significantly larger fraction of cells, suggesting that the FLT3-mutated clone had a relative growth advantage. Similarly, patient AML393 carried a subclone with mutated BCOR that became the dominant clone in the xenograft. Conversely, in patients AML372 and AML373 we found subclonal NRAS mutations (VAF, 6% and 7%, respectively) that were undetectable in the matched xenografts, indicating that the NRAS-mutated subclones did not engraft. We are currently studying parallel lines of xenografts generated from the same patient, followed by serial re-passaging in NSG mice, to better characterize growth patterns of such patient-derived subclones in our model. Updated results will be presented at the meeting. Genotypes for known germline SNPs were fully concordant between the human specimens and matched xenografts in 3/4 pairs. The fourth patient (AML393), who had AML relapse after an allogeneic stem cell transplant, showed several SNPs with a low VAF that were not identified in the corresponding xenograft. These SNPs most likely originate from residual donor hematopoiesis in the patient, indicating that non-malignant cells of donor origin did not engraft in the NSG mouse. Conclusion Xenografts of primary AML blasts in NSG mice recapitulate the patterns of gene mutations observed in AML patients, and thus provide an opportunity to study the biology of various genetic AML subgroups. Deep, targeted amplicon resequencing can sensitively detect subclonal driver mutations, and in conjunction with the xenograft model can be used to study clonal diversity and clonal evolution in AML. Disclosures: Greif: Illumina: Honoraria.

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Clifford, Ruth M., Pauline Robbe, Susanne Weller, Adele T. Timbs, Michalis Titsias, Adam Burns, Maite Cabes et al. « Towards Response Prediction Using Integrated Genomics in Chronic Lymphocytic Leukaemia : Results on 250 First-Line FCR Treated Patients from UK Clinical Trials ». Blood 124, n^o 21 (6 décembre 2014) : 1942. http://dx.doi.org/10.1182/blood.v124.21.1942.1942.

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Abstract Background: Major progress has been made in understanding disease biology and therapeutic options for patients with chronic lymphocytic leukaemia (CLL). Recurrent mutations have been discovered using next generation sequencing, but with the exception of TP53 disruption their potential impact on response to treatment is unknown. In order to address this question, we characterised the genomic landscape of 250 first-line chemo-immunotherapy treated CLL patients within UK clinical trials using targeted resequencing and whole-genome SNP array. Methods: We studied patients from two UK-based Phase II randomised controlled trials (AdMIRe and ARCTIC) receiving FCR-based treatment in a first-line treatment setting. A TruSeq Custom Amplicon panel (TSCA, Illumina) was designed targeting 10 genes recurrently mutated in CLL based on recent publications.Average sequencing depth was 2260X. The cumulated length of targets sequenced was 7.87 kb from 330 amplicons covering 160 exons. Alignment and variant calling included a combination of three pipelines to confidently detect SNVs, indels and low level frequency mutations. SNP array testing was performed using HumanOmni2.5-8 BeadChips, (Illumina) and data analysed using Nexus 6.1 Discovery Edition, Biodiscovery. We performed targeted resequencing and genome-wide SNP arrays using selected samples’ germline material to confirm somatic mutations (n=40). Univariate and multivariate analyses using minimal residual disease (MRD) as the outcome measure were performed for 220 of the 250 patients. Results: Pathogenic mutations were identified in 165 (66%) patients, totalling 268 mutations in 10 genes. ATM was the most frequently mutated gene affecting 67 patients (29%) followed by SF3B1 (n=56, 24%), NOTCH1 (n= 32, 14%), TP53 (n= 21, 9%), BIRC3 (n= 17, 7%) and XPO1 (n=14, 6%). Less frequently recurrent mutations were seen in SAMHD1 (n=8, 3%), MYD88 (n= 4, 2%), MED12 (n=7, 3%) and ZFPM2 (n=5, 2%). Integrating sequencing and array results increased the patients with one or more CLL driver mutation from 66% to 94%. As previously reported del17p and TP53 mutations are co-occurring and associate with MRD positivity in all cases (n=15, p=0.0002). We report on minor TP53 subclones in 11 patients (VAF 1-5%), 8 of whom have MRD data available and were also associated with MRD positivity. Deletions of 11q were present in 44 patients. These lesions always included ATM but not always BIRC3. Bialleleic disruption was present in ATM for 27 patients (significantly associated with MRD positivity) and in BIRC3 for 4 patients. Rather surprisingly, trisomy 12 (n=33) and NOTCH1 mutations (n=28) were associated with MRD negativity (p=0.006 and 0.097, respectively). Analysing clonal and subclonal mutations per gene revealed the majority of mutations in SF3B1 and BIRC3 were subclonal (65% and 87% respectively). In contrast almost all SAMHD1 and MYD88 mutations were clonally distributed. There was an association between NOTCH1 subclonal mutations and MRD negativity, compared to clonal mutations, but this difference was not seen in the remaining mutated genes. From our copy number data, the presence of subclones was associated with MRD positivity (p=0.05). Combining important lesions in a multiple logistic regression analysis to predict MRD positivity, bialleleic ATM disruption, together with TP53 disruption, were the strongest predictors, followed by SAMHD1, whereas BIRC3 monoalleleic mutations were a medium predictor for MRD negativity. Conclusion: This is the first integrated genome-wide analysis of the distribution and associations of CLL drivers, using targeted deep resequencing and whole genome SNP arrays in an FCR-based first-line treatment setting. We have shown subclonal and clonal mutation profiles in all patients. For patients with two or more CLL-associated mutations we have begun to unravel clonal hierarchies. We have developed a comprehensive model using MRD as an outcome measure and have found bialleleic ATM mutations and SAMHD1 disruption to strongly predict for MRD positivity. Using MRD status as a robust proxy for PFS not only enables us to confirm results of previous studies, but is advantageous also in considerably reducing the timeframe for results. Indeed, we suggest that MRD status should be assessed routinely in future studies to complement modern integrated genomics approaches. Disclosures Hillmen: Pharmacyclics, Janssen, Gilead, Roche: Honoraria, Research Funding.

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Jamwal, Manu, Anu Aggarwal, Prashant Sharma, Deepak Bansal, Pankaj Malhotra, Arindam Maitra et Reena Das. « The Spectrum of Genetic Defects in Indian Patients with Rare Congenital Anemias : Next Generation Sequencing Based Approach ». Blood 132, Supplement 1 (29 novembre 2018) : 2328. http://dx.doi.org/10.1182/blood-2018-99-116092.

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Abstract Introduction Diagnosis of inherited anemias is based on automated red cell indices, morphology and reticulocyte count in an appropriate clinical presentation and a stepwise diagnostic algorithm needs to be followed. Major inherited causes of hemolysis include the hemoglobinopathies, membranopathies and enzymopathies. In contrast, ineffective erythropoiesis, a term that refers to a disturbance in proliferation, differentiation and maturation of erythroblasts, includes megaloblastic anemia, myelodysplastic syndromes, thalassemias, and congenital dyserythropoietic anemias (CDA). Phenotypes vary from severe transfusion-dependent hemolytic anemia (HA) to fully compensated hemolysis. Patients are encountered where the entire spectrum of diagnostic tests cannot identify the etiology. Causal genes implicated are numerous, making a gene-by-gene approach time consuming, expensive and laborious. Hence, the use of targeted resequencing can expedite molecular diagnosis, genetic counseling and prenatal diagnosis if indicated. Objectives This study aimed at determining the spectrum of mutations in uncommon HA and CDA. Methods Sixty-one patients (HA=44, CDA=17) with clinical and laboratory evidence suggestive of HA/CDA were enrolled after excluding common causes of anemias. Various biochemical and molecular tests were used to exclude glucose-6-phosphate dehydrogenase (G6PD) deficiency, hemoglobinopathies, autoimmune HA, hereditary spherocytosis and pyruvate kinase (PK) deficiency. Peripheral blood genomic DNA was extracted using QIAamp DNA Blood Midi Kit, quantified on NanoDrop 2000 spectrophotometer and Qubit® 2.0 Fluorometer. Common G6PD, PKLR variants, SEC23B mutation were excluded by molecular tests. Since the genetic defects in Indian patients with unexplained HA/CDA are not available, we first tested 16 patients with a comprehensive commercially available sequencing panel that covers 4,813 clinically-relevant genes. We next sought to streamline testing and designed a customized panel of 55 genes (Illumina). Variants were annotated and classified and the most likely disease-causing variants were validated by Sanger sequencing in the patient and available affected and unaffected family members. Results Out of 61 patient's sequenced, unexpected pyruvate kinase (PK) deficiency were found in 11 patients. PK enzyme activity assay was within normal limits in all these cases. Three patients with G6PD deficiency, 2 patients with glucose-6-phosphate isomerase deficiency and 1 case with hexokinase deficiency were found. Of 5 patients with stomatocytes on peripheral blood film, 3 had Mediterranean stomatocytosis/ macrothrombocytopenia (ABCG5/ABCG8) and 2 were found to have overhydrated hereditary stomatocytosis (RHAG). We also found 6 cases of HA to have a mutation in erythrocyte membrane protein-coding genes. Heterozygous nonsense mutation in the ANK1 gene was found in 2 cases and compound heterozygous missense mutations in ANK1 were seen in 1. Missense mutations were also found in SPTB gene in other 3 cases. Xerocytosis or dehydrated hereditary stomatocytosis (PEIZO1) was found in 3 patients. Seventeen cases for suspected CDA were studied and SEC23B mutations were present in 7. In one patient only heterozygous mutation was found. There could be the possibility of intronic mutation or long deletion/insertion in her. Notably, 6 patients with CDA showed mutations in PKLR (n=3), 1 each had mutations in MTRR, SPTB and PIEZO1 genes. We also found a mutation in GATA1 gene in a patient of CDA with thrombocytopenia. Conclusion(s) HA/CDA showed highly varied etiology. Our experience demonstrates the high diagnostic yield (>75%) of targeted resequencing for molecular diagnosis of unexplained anemias. It is cost effective and can expedite the diagnosis where conventional testing is not helpful. Timely detection of the etiology was helpful in genetic counseling. It not only offered therapeutic benefits to our patients but may help us in future antenatal diagnosis if required. Therapeutic implications include performing splenectomy which can ameliorate the anemia in selected subgroups of patients (HS, PK deficiency, CDA type II). Disclosures No relevant conflicts of interest to declare.

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Wang, Han, Kathy Chan, Po Yi Lee, Alex WK Leung, Chi Kong Li et Kam Tong Leung. « Integrative Drug and Genomic Profiling Identify Therapeutic Vulnerabilities and Inform Precision Medicine for Pediatric Acute Myeloid Leukemia ». Blood 138, Supplement 1 (5 novembre 2021) : 2297. http://dx.doi.org/10.1182/blood-2021-147693.

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Abstract Background/Aims: Despite advances in chemotherapy-based treatment protocols, the outcomes of children with acute myeloid leukemia (AML) remain suboptimal. Implementation of targeted therapy based solely on genomics is challenging due to the complex mutational patterns and scarcity of pharmacologic agents for most lesions. In addition, pediatric and adult AML are genetically and biologically distinct, which poses a major hurdle for extrapolation of new agents approved for adult AML to the pediatric population. This study aims to adopt a functional approach that directly measure the response of patient-derived leukemic cells to targeted agents, and to establish the drug sensitivity pattern and identify candidates of immediate clinical relevance for precision usage in high-risk pediatric AML. Methods: A high-throughput drug screening, comprising 39 targeted agents (2 in Phase I, 10 in Phase II, 5 in Phase III, 22 FDA-approved) and 6 conventional chemotherapeutics, was performed on 30 pediatric AML samples collected at diagnosis or relapse using a serum-free, cytokine-supported culture system. A counter-screen of active drugs on cord blood hematopoietic stem cells was accomplished to reveal leukemia-selective activities. The robustness of the drug testing platform for predicting in vivo activities was validated in xenograft models. Genomic profiling was complementarily performed to identify the genetic markers and underlying mechanisms of drug sensitivity. Patients with refractory AML were treated with targeted agents based on drug profiling results, and assessed for clinical responses. Results: Unsupervised clustering revealed 5 distinct clusters of drug response: highly active compounds (IC50 <15 nM, 5 drugs); generally active compounds (IC50 <250 nM, 11 drugs); compounds with bimodal activities (wide IC50 ranges, 3 drugs); generally inactive compounds (16 drugs); and inactive compounds (IC50>2000 nM, 10 drugs). Targeted agents, including Bcl-2, HDAC, proteasome, HSP and survivin inhibitors, had substantially higher potency and selectivity over standard chemotherapeutic agents. New agents approved for adult AML were essentially inactive in pediatric AML. Drug sensitivity ex vivo accurately predicted in vivo single-agent and combinatorial activities with cytarabine in cell line- and patient-derived xenografts. Targeted resequencing of a 141-gene panel revealed novel mutations of prognostic relevance, such as KMT2C, in pediatric AML and their vulnerability to targeted agents. Whole-genome RNA-sequencing identified distinct gene expression signatures shaping the response to individual drugs. Administration of venetoclax to a child with refractory AML resulted in rapid blast clearance and achieved long-term remission. Complementary genomic profiling on serial specimens dictated the dynamic drug responses during disease evolution. Conclusions: Our study establishes a reliable drug testing platform and a pediatric-specific drug response profile of AML, which enables an evidence-based selection of targeted agents for patients without treatment options and endows therapies increasingly precise and personalized. The study also generates a valuable gene-drug-clinical dataset that could be leveraged to address the fundamental and translational biology of pediatric AML. It will ultimately impact the future design of clinical trials and protocols for managing this life-threatening malignancy. Disclosures No relevant conflicts of interest to declare.

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Maxwell, Kara Noelle, Daniel De Sloover, Lyndsey Emery, Bradley Wubbenhorst, Kurt P. D'Andrea, Jessica Long, Rebecca Mueller et al. « The mutational spectrum of breast and ovarian tumors from BRCA1 and BRCA2 mutation carriers. » Journal of Clinical Oncology 31, n^o 15_suppl (20 mai 2013) : 1510. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.1510.

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1510 Background: Individuals who carry one mutated copy of the BRCA1 or BRCA2 genes have elevated lifetime risks of breast and ovarian cancer. A number of studies have investigated the somatic mutational spectra of breast and ovarian tumors; however, BRCA1/2mutated tumors are underrepresented. Methods: Sixty-eight formalin-fixed paraffin embedded samples from BRCA1/2patients have been identified. Massively parallel sequencing using 48 gene capture is in process, whole exome sequencing of tumor and matched germline DNA is planned. Data are analyzed using a custom bioinformatics pipeline. Results: In analysis of data from the first 26 breast (4 BRCA1, 6 BRCA2) and ovarian (8 BRCA1, 8 BRCA2) tumors, the majority (23/26, 88%) had 0-2 variants in 48 cancer genes. Known deleterious TP53 mutations were the only variants identified in 2/4 BRCA1 and 2/6 BRCA2 breast tumors. Of those remaining, 2 BRCA1 and 1 BRCA2 breast tumors had no identified deleterious mutations. Two BRCA2 breast tumors with no TP53 mutations had known deleterious mutations in a single gene each - FGFR2 and PI3KCA. One BRCA2 breast tumor with no TP53 mutation had a variant of uncertain significance in FLT3. Finally, one BRCA2 breast tumor had a very high mutational rate, with one deleterious TP53 mutation and 7 other small deletion and single nucleotide variants. For the ovarian tumors, 15/16 BRCA1 and BRCA2 tumors had known deleterious TP53 mutations; the ovarian tumor with no TP53 mutation had no other variants. TP53 mutations were the sole identified mutations in 8 ovarian tumors. One ovarian tumor carried a known JAK3 activating mutation and 4 ovarian tumors carried one variant of uncertain significance in a single gene - SMO, PDGFRA, GNA11 and NRAS. Finally, two ovarian tumors were found to have high mutational rates. Conclusions: Using a targeted resequencing panel, we confirmed the high rate of TP53 mutations in BRCA1/2 breast tumors and observed a higher than expected rate in BRCA1/2 ovarian tumors. Importantly, we have identified mutations in other known driver genes using FFPE samples, allowing generalizability to other sites. These analyses may uncover novel mutations that could be exploited in the development of targeted therapeutic agents for BRCA1/2 carriers.

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Konstandin, Nikola P., Klaus H. Metzeler, Annika Dufour, Philipp A. Greif, Tobias Herold, Bianka Ksienzyk, Stephanie Schneider et al. « Genetic Characterization of Patients with Monoallelic and Biallelic CEBPA Mutations Using a Targeted Sequencing Approach Reveals Differences in the Spectrum of Cooperating Mutations ». Blood 124, n^o 21 (6 décembre 2014) : 2385. http://dx.doi.org/10.1182/blood.v124.21.2385.2385.

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Abstract Mutations in the CCAAT/enhancer binding protein alpha (CEBPA) are detected in about 10% of patients with cytogenetically normal acute myeloid leukemia (CN-AML). CEBPA mutation can either affect both CEBPA alleles (biallelic, biCEBPA) or only one allele (monoallelic, moCEBPA). We and others have shown that only patients with biCEBPA mutations have favorable outcomes when compared to other CN-AML patients (Dufour et al, JCO 2009; Green et al, JCO, 2010). Interestingly, biCEBPA mutations are rarely associated with other known prognostic mutations (e.g. FLT3-ITD, NPM1). In this study we aimed to characterize the mutational spectrum of CN-AML patients with mo- and biCEBPA mutations using a targeted amplicon sequencing approach. We analyzed 45 biCEBPA patients and 35 moCEBPA patients. 55 of these patients (26 biCEBPA and 29 moCEBPA) were enrolled in a multicenter trial of the German AML Cooperative Group (AMLCG-1999). Our amplicon resequencing panel included 42 genes which are known to be frequently mutated in AML (Haloplex, target region 62 kilobases). Out of these 42 genes we identified 23 different mutated genes in the biCEBPA subgroup and a total of 28 different mutated genes in the moCEBPA cohort. The mean number of mutated genes per moCEBPA patient was significantly larger (4.37±1.6) than in biCEBPA patients (2.96±1.22) (P<0.05). The two groups also differed remarkably with regard to the genes that were mutated. In the moCEBPA group FLT3 (46%), NPM1 (46%), TET2 (37%) and DNMT3A (26%) were the most frequently mutated genes, whereas the biCEBPA group showed frequent mutations in TET2 (40%), GATA2 (36%) and FLT3 (18%). Thus there was a strong association of NPM1 (P<0.0001), FLT3 (P=0.01) and IDH2 (P=0.04) mutations with the moCEBPA group. GATA2 mutations were significantly associated with biCEBPA mutations (P=0.0003). NPM1 and biCEBPA mutations were mutually exclusive. In this large and well characterized CEBPA-mutated patient cohort we identified distinct mutational landscapes in patients with moCEBPA and biCEBPA mutated CN-AML. The lower number of mutated genes within the biCEBPA group suggests that biallelic CEBPA mutations may act as a strong driver. In almost all cases, patients with biallelic mutations of CEBPA have a C-terminal mutation in one allele of CEBPA and an N-terminal mutation in the other allele. These date provide further insight into the genetic background of CEBPA mutated CN-AML. We are currently analyzing the prognostic impact of the associated mutations. Disclosures No relevant conflicts of interest to declare.

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Mehrvarz Sarshekeh, Amir, Michael J. Overman, Bryan K. Kee, David R. Fogelman, Arvind Dasari, Kanwal Pratap Singh Raghav, Eduardo Vilar Sanchez et al. « Demographic, tumor characteristics, and outcomes associated with SMAD4 mutation in colorectal cancer. » Journal of Clinical Oncology 34, n^o 4_suppl (1 février 2016) : 565. http://dx.doi.org/10.1200/jco.2016.34.4_suppl.565.

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565 Background: SMAD4 regulates signaling in TGF-β pathway as inactivation of SMAD4 renders resistance of cells to TGF-β1-induced growth inhibition. Sporadic mutation of SMAD4 has been reported to be present in 8.6-25% of colorectal cancers (CRC), but the clinicopathologic features and outcomes associated with this mutation have not been described. Methods: Data for patients (pts) with metastatic or locally advanced unresectable CRC who received treatment at MD Anderson Cancer Center whose tumors underwent genotyping for SMAD4 mutation were reviewed and clinicopathologic characteristics, and survival outcomes were evaluated. Tumors were sequenced using a hotspot panel (Ion Torrent, Life Technology) predicted to cover 80% of the reported mutations in SMAD4, and further targeted resequencing that included full-length SMAD4 was performed on mutated ones by HiSeq (Illumina) with full exome coverage to an average depth of 800. Results: Among 616 pts (47.4% females) with CRC, 11.2% of pts (n = 69) had SMAD4 mutation by hotspot testing. SMAD4 mutation was associated with colon cancer versus rectal cancer (OR = 2.1, p = 0.01) and female gender (15% vs 8%, OR = 2.12, p = 0.004). There was no association between the presence of SMAD4 mutation and age, stage at the presentation, tobacco history, colonic location, presence of distant metastasis, histology, nor tumor grade. When compared to pts with wild-type SMAD4, those with SMAD4 mutation had shorter survival from date of testing (median survival of 20.2 months versus 14 months, respectively; log-rank, HR = 1.4, p = 0.014). Full-length sequencing was performed on mutated tumors and it confirmed that missense mutations in R361 and P356 in the MH2 domain were the most common, which are predictive of disruption of both homo- and hetero-oligomerization required for activation. Conclusions: This study is the largest retrospective study to date characterizing SMAD4 mutation in metastatic CRC, and demonstrates a prognostic role for this subgroup of CRC. The prevalence and spectrum of SMAD4 mutations is consistent with previous studies and data from TCGA. Further studies are required to evaluate the implication of the dysregulated TGF- β pathway on response to therapy.

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Sachdev, Pallavi, Omid Hamid, Kevin Kim, Axel Hauschild, Steven O'Day, Corina Andresen, Yasuhiro Funahashi, Tadashi Kadowaki, James P. O'Brien et Keith Flaherty. « Analysis of serum biomarkers and tumor genetic alterations from a phase II study of lenvatinib in patients with advanced BRAF wild-type melanoma. » Journal of Clinical Oncology 31, n^o 15_suppl (20 mai 2013) : 9058. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.9058.

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9058 Background: Lenvatinib is an oral receptor tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1-4, RET, KIT, and PDGFRβ. Phase I and II studies of lenvatinib demonstrate that a subset of advanced melanoma patients (pts) appears to derive benefit from lenvatinib treatment (tx), prompting a need to identify predictive biomarkers for response to lenvatinib. Methods: Serum and archival tumor samples were collected from 93 enrolled pts in the BRAF wild-type (wt) cohort of the lenvatinib phase II study of advanced melanoma. Concentrations of 50 factors were measured in pre- and post-tx serum samples; 58 archival tumor tissues were obtained and gene mutation (mut) (n=53) and gene expression profiling (GEP) (n=39) analyses were performed. For mut analysis, targeted resequencing was performed on a select panel of genes using the Ion PGM Sequencer and mut validation studies were performed. For GEP analysis, the nCounterAnalysis System was used to measure the expression level of 330 genes. Results: Clinical benefit rate (32%) and objective response rate (9%) by independent radiologic review were observed in the BRAF wt cohort. In serum biomarker analysis, lenvatinib tx resulted in a decrease in soluble VEGFR2 and increase in VEGF and PlGF levels consistent with target engagement. Correlation with clinical outcome demonstrated that baseline (BL) levels of Ang-2, IL8, PlGF, and PDGFBB associated with OS, but only pts with low BL Ang-2 levels also demonstrated improved response. High BL levels of FLT3LG and eotaxin associated with longer OS and improved response. In GEP analysis, expression levels of a set of genes including TARBP2, ZNF544, and NF1 (targets identified in phase I and preclinical studies) associated with OS. In gene mut analysis, NRAS mut showed a trend towards longer OS. Pts with NRAS mut along with wt PIK3CA status demonstrated longer OS. Conclusions: Lenvatinib demonstrated limited response in BRAF wt advanced melanoma pts. A subset of pts may derive extended clinical benefit. NRAS mut/PIK3CA wt status and low BL Ang-2 levels appear to associate with improved clinical outcome in this pt population and require further study to assess their predictive value. Clinical trial information: NCT01136967.

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Hirsch, Pierre, Ruoping Tang, Nassera Abermil, Pascale Flandrin, Hannah Moatti, Mohamad Mohty, Ollivier Legrand, Luc Douay, Chrystele bilhou Nabera et Francois Delhommeau. « Clono-Specific Evaluation of Minimal Residual Disease in Acute Myeloid Leukemia ». Blood 128, n^o 22 (2 décembre 2016) : 1208. http://dx.doi.org/10.1182/blood.v128.22.1208.1208.

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Abstract Background: The genetic landscape of adult acute myeloid leukemias (AML) has been recently unravelled. This makes achievable the determination of a comprehensive profile of driver lesions for virtually all patients at diagnosis. Recent studies using multi-target minimal residual disease (MRD) strategies with around 1% sensitivity indicate that the clearance of all molecular events after chemotherapy is associated with better survival. To improve the clono-specificity and the sensitivity of this approach, after a precise determination of AML clonal composition, we combined cytogenetic, FISH, and high sensitivity deep sequencing technologies to monitor the MRD in a series of 69 patients. Methods: Forty-five consecutive patients reflecting the genetic diversity of AML were prospectively included and 24 patients were retrospectively studied. All patients received an anthracycline + cytarabine based regimen. The clonal architecture was established at diagnosis based on NGS-targeted resequencing (122 gene panel) and cytogenetic data. Lesions were next investigated in complete remission (CR). Based on the initial clonal composition, targeted resequencing panels were designed to improve the sensitivity by the use of unique molecular barcodes (Haloplex High Sensitivity, or HS-NGS assay). Cytogenetic events were evaluated by FISH. Results: In the 69 patients, a median of 4 genetic or chromosomal events were identified per patient (range 0-10). One patient had no evaluable target allowing MRD evaluation in 68/69 patients. To determine the threshold of detection of the HS NGS assay, we analyzed the frequency of mutant reads in multiple samples expected to be wild type for 31 given SNVs and 2 indels. A consensus threshold of detection was set at a variant allele frequency (VAF) of 0.2% for all lesions. In CR samples, early initiating events frequently persisted after treatment, especially mutations in DNMT3A, TET2, ASXL1, EZH2, IDH1, TP53, SRSF2, and U2AF1. Mutations in FLT3, NRAS, KIT, NPM1, CEBPA, WT1, IDH2 and BCOR were the most frequently cleared events. Seven patients did not reach CR after one course, and two had no available material after one course. In the 59 remaining patients, we tested whether the global response level of all targets was associated with prognosis. We used the median VAF of the first events of all clonal architectures to separate good responder from poor responders (i.e. VAF = 1.66%). At 2 years, there was a trend to lower leukemia free survival (LFS) probability in poor responders (31.7+/-9.9% vs 51.7+/-9.8%, p=0.08) with no translation in overall survival (OS). We next investigated if the persistence of two or more detectable markers was associated with prognosis. The 58 patients with more than one evaluable event were consequently separated in two groups: patients with 0 or 1 marker above the detection threshold after treatment (n=31), and patients with 2 or more detectable lesions (n=27). At 2 years, DFS was 64.9+/-9.3 % in patients with 0 or 1 detectable marker vs 19.8+/-8.7% in patients with 2 or more detectable markers (p=0.001). OS probability was higher in patients with 0 or 1 detectable marker 84+/-6.6% vs 57.1+/-10.5% (p=0.023). When focusing on the 40 patients with intermediate cytogenetics, persistence of 2 or more markers was associated with lower LFS (57+/-11.8% vs 19.4+/-10.5 p=0.0048) and with a trend to lower OS (85+/-8% vs 61+/-11.9% p=0.07). Similar results were observed when restricting the analyses to the 42 prospectively included patients (At 2 years: LFS 73+/-10% vs 24+/-10%, p=0.0026 and OS 90.2+/-6.6% vs 62.8+/-11.5%, p=0.036). In 50 patients with 3 or more identified events, the persistence of 3 or more markers after one course was associated with a very high risk of relapse (DFS 23.5+/-10.3 % vs 75.8 +/-7.5% at one year, p<0.0001; median DFS at 7 months in the non-responder group and not reached after two years in the responder group), and lower OS probability (84.8+/-6.2 vs 45.2+/-13.5% at 2 years, p=0.026) Conclusion Our study shows the high prognostic value of a personalized multi-target clono-specific MRD evaluation that can be used in nearly all AML patients. Detection of two or more events in more than 0.4% cells after one course is associated with lower survival, in particular in intermediate cytogenetic patients. Larger studies are needed to confirm the results and to evaluate if this strategy could be useful to guide treatment decisions. Disclosures No relevant conflicts of interest to declare.

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Mack, Elisabeth, Danny Langer, André Marquardt, Alfred Ultsch, Michael G. Kiehl, Andreas Neubauer et Cornelia A. Brendel. « Comprehensive Genetic Diagnostics of Acute Myeloid Leukemia By Next Generation Sequencing ». Blood 128, n^o 22 (2 décembre 2016) : 1665. http://dx.doi.org/10.1182/blood.v128.22.1665.1665.

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Abstract Background Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults with a poor overall prognosis. Although the disease has been extensively characterized on the molecular level, this knowledge is translating only slowly into the clinic, particularly with regard to novel therapeutic concepts. Presumably, this striking imbalance substantially is due to the long time required to complete genetic analyses so that results are not available when treatment has to be initiated. Specifically, cytogenetic examinations to determine the karyotype of the malignant blasts, which has been the most important parameter for risk stratification for more than thirty years, take up to two weeks. Next generation sequencing (NGS) technology essentially catalyzed efforts to dissect the genomic landscape of AML, leading to the identification of a large variety of AML driver genes and distinct molecular risk groups. However, these emerging molecular classes of AML do not cover all patients, implying that karyotyping is not dispensable for AML diagnostics at this point. Here we present an integrated approach to AML diagnostics that incorporates these complementary genetic examinations - focused mutational screening of AML-related genes and karyotyping - in one NGS assay. Methods We combined targeted resequencing of DNA and RNA using commercially available panels (TruSigth Myeloid, Illumina and FusionPlex Heme, ArcherDx) to detect AML-associated short sequence variants and gene fusions with low coverage whole genome sequencing for copy number variation analysis. Sequencing was performed on an Illumina MiSeq instrument with a read length of 2x150 bp and a coverage of 3.75 M reads for the TruSight Myeloid panel, 2.25 M reads for the FusionPlex panel and 1.5 M reads for the whole genome library. Variants and fusions were called using the manufacturers' analysis software and a previously published algorithm to identify ITDs (ITD-seek, Au et al., 2016). CNV analysis was performed by comparing read distribution in an AML whole genome library to in silico randomly sampled reads from the reference genome using an in house-developed algorithm. Results Initial testing of our approach on leukemia cell lines and peripheral blood leukocytes from healthy donors revealed sensitivities of 2% and 1-25% for the detection of DNA variants and fusions, respectively. Applying stringent filter criteria, we recovered 75% of verified COSMIC variants and 100% of known fusions in undiluted AML samples without false positives. Chromosomal gains and losses were detected with high confidence with a sensitivity of 10%. We were able to reliably distinguish between normal and complex karyotypes, although NGS-karyotyping based on known fusions and CNV-analysis missed some details of highly aberrant karyotypes such as derivative chromosomes and chromosomal translocations that did not involve genes included in the FusionPlex panel. Our preliminary experience on our method in a diagnostic setting confirms high correlation with reference laboratory results and no relevant differences with regard to treatment decisions. Moreover, we find that NGS considerably accelerates genetic diagnostics of AML as the entire workflow from sample to report including three parallel library preparations, sequencing and data analysis can be completed within 5 days. Operational costs amount approximately 1,700 USD (1,500 EUR) per sample with the low throughput equipment used in this work, which is in the range of expenses for currently established AML diagnostics. Conclusions NGS allows for comprehensive translocation and mutation screening, however, some technical and bioinformatics optimization is required to achieve consistently high sensitivity and specificity for all target genes. CNV analysis of low coverage whole genome sequencing data adds valuable information on numerical chromosomal aberrations, thus allowing construction of a virtual karyotype to substitute for difficult and time-consuming cytogenetics. In summary, we present a reliable, fast and cost-effective strategy to combine molecular and cytogenetics for AML diagnostics in a single NGS run in order to pave the way for a more differentiated clinical management of AML patients in the near future. Disclosures Kiehl: Roche: Consultancy, Other: Travel grants, Speakers Bureau.

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Metzeler, Klaus H., Nadine Sandhöfer, Tanja Hinrichsen, Evelyn Zellmeier, Bianka Ksienzyk, Annika Dufour, Stephanie Schneider et al. « Analysis of Cooperating Genetic Events in MLLT3-MLL Rearranged Acute Myeloid Leukemia (AML) by Targeted Next-Generation Sequencing of 16 Leukemia-Related Genes Reveals Frequent Mutations Affecting Growth Factor Signalling Pathways and Provides Evidence for Clonal Heterogeneity ». Blood 120, n^o 21 (16 novembre 2012) : 1379. http://dx.doi.org/10.1182/blood.v120.21.1379.1379.

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Abstract Abstract 1379 Background: A large number of gene mutations have been recently detected in AML using novel sequencing technologies. We established a rapid, amplicon-based resequencing assay that allows efficient analysis of 16 of the most commonly mutated genes in AML and used it to study a cohort of AML patients (pts) carrying a translocation t(9;11)(p22;q23) (MLLT3-MLL; MLL-AF9). This genetic subgroup, accounting for ∼1% of adult AML, is associated with young age, treatment-related disease, FAB M4/M5 morphology, and an intermediate prognosis. There is limited information on the cooperating genetic lesions in adult AML with t(9;11). Importantly, several widely used murine AML models are based on MLLT3-MLL fusion transcript expression. Thus, a better understanding of the genetic basis of human MLLT3-MLL-rearranged AML is necessary to understand how well these animal models reflect their human counterpart and whether findings from MLLT3-MLL-induced disease are generalizable to other genetic subsets. Patients and Methods: We studied 33 bone marrow samples from adult AML pts with t(9;11)(p22;q23) (age range, 20–71 years; median, 44 years; 21 de novo and 12 therapy-related AML). Mutations in ASXL1, CBL, DNMT3A, FLT3, IDH1, IDH2, KIT, KRAS, NRAS, NPM1, RUNX1, SF3B1, SRSF2, TET2, U2AF1 and WT1 were analyzed from 250ng of genomic DNA using a multiplexed, amplicon-based next-generation sequencing approach (Illumina TruSeq Custom Amplicon assay and MiSeq sequencer). KRAS mutations were independently verified using PCR followed by 454 sequencing (Roche), and NRAS and FLT3 mutations by PCR and melting curve analysis or Sanger sequencing. Results: Per patient, we obtained between 96k and 235k paired-end reads (2×150bp) mapping to the regions of interest, resulting in median coverage depths of the target genes ranging from 180-fold (SRSF2) to >2500-fold (KRAS). Overall, mutations affecting growth factor signalling pathways were detected in 73% of MLLT3-MLL rearranged AML (24/33; Figure): Fourteen pts (42%) carried KRAS mutations mostly affecting the known hotspot codons 12, 13 and 61, 6 pts (18%) had NRAS mutations (mainly at codons 12 or 13), 5 pts had FLT3 mutations (4 tyrosine kinase domain mutations and 1 internal tandem duplication), and 2 pts had mutated CBL. The frequency of RAS gene mutations did not differ significantly between de novo AML and pts with treatment-related disease (P=.26). More than one RAS mutation was found in 7 pts, including pts with 2 (n=3) or 3 (n=1) distinct KRAS mutations, 2 pts with mutations in both NRAS and KRAS, and one patient with 2 NRAS mutations. Interestingly, in some of these pts, one mutation was present in a relatively large proportion of sequencing reads (e.g., patient UPN12 showing a KRAS p.Q61H mutation in 36% of reads, consistent with a heterozygous mutation present in the majority of cells in the specimen), while other coexisting mutations affected a much smaller proportion of reads (in patient UPN12, two different KRAS exon 2 mutations in 5% and 2% of reads, respectively). These results suggest the presence of different subclones within the AML blast population, each carrying a different KRAS mutation. Analyses of follow-up samples are underway to assess changes of clonal architecture over time. Other gene mutations were rarely found in this cytogenetic subgroup of AML: In our 33 pts, we detected 2 ASXL1 mutations, 1 mutation each in TET2, SRSF2 and U2AF1, and no mutations in the other 8 genes we studied. Conclusion: Targeted resequencing using a multiplexed amplicon-based assay is a sensitive and rapid method to screen for mutations in a panel of genes commonly involved in AML pathogenesis. To our knowledge, our report is the first comprehensive analysis of cooperating gene mutations in adult AML with t(9;11)(p22;q23). We demonstrate that MLLT3-MLL-rearranged AML is characterized by frequent mutations in genes involved in growth factor signalling (particularly KRAS and NRAS, mutated in 40% and 18%, respectively, of our MLL-MLLT3 AML cohort compared to only about 5% of unselected AML pts), in the absence of other common AML-related gene mutations. Our results complement recent studies reporting RAS mutations in 45% of infant MLL-rearranged ALL, and functional data from mouse models showing that RAS mutations cooperate with the MLLT3-MLL fusion during leukemogenesis. Finally, our results provide evidence for clonal heterogeneity within MLLT3-MLL rearranged human AML. Disclosures: No relevant conflicts of interest to declare.

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Hamblin, Angela, Adam Burns, Christopher Tham, Ruth Clifford, Pauline Robbe, Adele Timbs, Joanne Mason et al. « Development and Evaluation of the Clinical Utility of a Next Generation Sequencing (NGS) Tool for Myeloid Disorders ». Blood 124, n^o 21 (6 décembre 2014) : 2373. http://dx.doi.org/10.1182/blood.v124.21.2373.2373.

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Abstract Background Historically diagnosis and prognosis of myeloid disorders including acute myeloid leukemia (AML) have been determined using a combination of morphology, immunophenotype, cytogenetic and more recently single gene, if not single mutation, analysis. The introduction of NGS technology has resulted in an explosion in the quantity of mutation data available. However, the feasibility and utility of NGS technology with regards to decision-making in routine clinical practice of myeloid disorders is currently unknown. We therefore developed an advanced NGS tool for simultaneous assessment of multiple myeloid candidate genes from low amounts of input DNA and present clinical utility analysis below. Methods We designed a targeted resequencing assay using a TruSeq Custom Amplicon panel with the MiSeq platform (both Illumina) consisting of 341 amplicons (~56 kb) designed around exons of genes frequently mutated in myeloid malignancies (ASXL1, ATRX, CBL, CBLB, CBLC, CEBPA, CSF3R, DNMT3a, ETV6, EZH2, FLT3, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PDGFRA, PHF6, PTEN, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1 & ZRSR2). Filtering, variant calling and annotation were performed using Basespace and Variant Studio (Illumina) with additional indel detection achieved using Pindel. A cohort of samples previously characterised with conventional techniques was used for validation and the lower limit of detection established using qPCR. Post-validation, DNA from 152 diagnostic blood or bone marrow samples from patients with confirmed or suspected myeloid disorders; both AML (n=46) and disorders with the potential to transform to AML i.e. myelodysplasia (confirmed n=54, suspected n=10) and myeloproliferative neoplasms (n=42), were analysed using this assay. To gather clinical utility data we developed a reporting algorithm to feed back information to clinicians; only those variants with a variant allele frequency (VAF) of >10% and described as acquired in publically available databases were reported with the exception of novel mutations predicted to result in a truncated protein. Further utility data was obtained using published mutation algorithms to determine the proportion of patients in whom mutation data altered prognosis. Results In the validation cohort, initial concordance for detection of clinically significant mutations was 88% rising to 100% once Pindel was used to identify FLT3 ITDs. The lower limit of detection was 3% VAF, and mean amplicon coverage was 390 reads. Using our reporting algorithm 66% of patients in the post-validation cohort had a suspected pathogenic mutation relevant to a myeloid disorder, rising to 74% in patients with confirmed diagnoses. The median number of reported variants per sample for all diagnoses was one (range 0-6). When mutation data for patients with AML with intermediate risk cytogenetics was analysed using the algorithm of Patel et al (N Engl J Med. 2012;366:1079-1089), 4/22 (18%) moved into another risk category. A further two patients had double CEBPA mutations, improving their prognosis. Identification of complex mutations in KIT exon 8 in 2/6 patients with core binding factor AML resulted in more intensive MRD monitoring due to the increased risk of relapse. Interpretation of mutation data for patients with confirmed myelodysplasia using the work of Bejar et al (N Engl J Med. 2011;364:2496-2506) revealed 13/54 (24%) had a high risk mutation independently associated with poor overall survival. 2/8 (25%) patients with chronic myelomonocytic leukemia and 1/12 (8.3%) patients with primary myelofibrosis had high risk ASXL1 exon 12 mutations, independently associated with a poor prognosis. Among suspected diagnoses confirmatory mutations were found in 2/19 (11%), while the absence of mutations reduced the probability of myeloid disease in 11/19 (58%), in some cases sparing elderly patients invasive bone marrow sampling. A further 20 patients had clinically relevant mutations. Conclusions The NGS Myeloid Gene Panel provided extra information to clinicians in 57/152 patients (38%) helping inform diagnosis, individualize disease monitoring schedules and support treatment decisions. The targeted panel approach requires rigorous validation and standardisation in particular of bio-informatics pipelines, but can be adapted to incorporate new genes as their relevance is described and will become central to treatment decisions. Disclosures No relevant conflicts of interest to declare.

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Soverini, Simona, Caterina De Benedittis, Manuela Mancini, Michela Rondoni, Cristina Papayannidis, Antonella Padella, Giorgina Specchia et al. « Genome-Wide Molecular Portrait of Aggressive Systemic Mastocytosis and Mast Cell Leukemia Depicted By Whole Exome Sequencing and Copy Number Variation Analysis ». Blood 126, n^o 23 (3 décembre 2015) : 4085. http://dx.doi.org/10.1182/blood.v126.23.4085.4085.

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Abstract Background and Aims: The term Systemic Mastocytosis (SM) identifies a poorly understood group of rare and clinically heterogenous myeloproliferative neoplasms characterized by abnormal growth and activation of mast cells (MCs) and their precursors in the bone marrow and in various tissues and organs. Based on phenotype and extent of organ infiltration/dysfunction, a spectrum of disease variants can be recognized ranging from indolent SM (ISM) to aggressive SM (ASM) and mast cell leukemia (MCL). The fact that in all cases, including ISM who have a (near) normal life expectancy, neoplastic MCs display the same D816V KIT gene mutation points to additional mechanisms and molecular defects as responsible for ASM and MCL. So far, however, this issue has mainly been addressed with targeted resequencing studies of candidate gene panels. We thus decided to undertake an integrated molecular characterization study of ASM and MCL to identify novel, functionally relevant molecular lesions and/or clinically actionable signaling pathways. Methods: A discovery panel including 6 patients with ASM and 6 patients with MCL was studied using whole exome sequencing (WES) and copy number variation (CNV) analysis. WES (80x) was performed on a Hiseq 2500 (Illumina). CNV was done using Cytoscan HD Arrays (Affymetrix). Paired normal/MC DNA was analyzed in all but 2 archival MCL cases for whom germline DNA was not available. A validation panel of 30 ISM, 5 smoldering SM and 20 additional ASM was also included in this study. Results: In the discovery panel, WES identified a total of 1554 point mutations, small insertions and deletions. Seven hundred and eighty-five were non-silent mutations in 698 genes, with an average of 51 (range, 30-186) non-silent mutations per patient. Non-silent mutations included 354 missense mutations, 188 nonsense mutations, 145 frameshift insertions/deletions, 98 non-frameshift insertions/deletions. C to T transitions were by far the most frequent. Orthogonal validation estimated the accuracy of mutation calls at >95%. Interrogation of the COSMIC and OMIM databases revealed 42 known cancer genes. Among the missense mutations, 87 were predicted to have a high probability of being deleterious by Condel. MCL cases were found not to harbour a higher mutation load as compared to ASM cases. High resolution CN analysis showed that focal amplifications/deletions/loss-of-heterozygosity (LOH) were prevalent over arm-level alterations (found in 3 patients only). Genes were selected for further assessment when recurrently mutated in ≥2 patients or concurrently identified in WES and CNV analyses or previously associated with leukemogenesis or cancer pathogenesis. Among these, genes already reported to be affected by mutations in SM included TET2, NRAS, ASXL1, CBL, IDH1, SRSF2, SF3B1, RUNX1. We also identified genetic alterations in genes not previously implicated in SM pathogenesis including TP53BP1, RUNX3, NCOR2, CDC27, CCND3, EI24, MLL3, ARID1B, ARID3B, ARID4A, SETD1A, SETD1B, KDM1B, PRDM1, ATM, WRN. A long tail of infrequently mutated genes dominated, resulting in significant intertumoural heterogeneity. However, when genes were assigned to functional pathways to discern patterns of mutations across different patients, we found that PI3K/Akt and MAPK pathways, calcium pathway, chromatin modification, DNA methylation, and DNA damage repair were consistently affected (Figure 1). Further assessment of the mutation frequency of selected genes within each pathway and functional validation at the protein level are currently ongoing in the validation panel. Preliminary findings on a tumor suppressor selected among those identified by WES show transcript and/or protein downmodulation due to inactivating mutations, transcriptional silencing or enhanced degradation in 17/20 ASM. Detailed results will be presented at the meeting. Conclusions: WES and CNV analyses of ASM and MCL revealed a complex landscape, not unexpected when considering the clinical heterogeneity of these patients. Nonetheless, key pathways were found to be recurrently altered. Further investigation of selected candidate genes and pathways is warranted and will cast light on the cooperative genetic (and epigenetic?) events underlying the more aggressive forms of SM - paving the way to a better prognostic stratification and more effective treatment. <>This study was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

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Herold, Tobias, Klaus H. Metzeler, Sebastian Vosberg, Stephanie Schneider, Max Hubmann, Evelyn Zellmeier, Bianka Ksienzyk et al. « Acute Myeloid Leukemia With Isolated Trisomy 13 Is a Genetically Homogenous Entity With a High Frequency Of Mutations In Genes Encoding Components Of The Splicing Machinery and Extremely Poor Prognosis ». Blood 122, n^o 21 (15 novembre 2013) : 608. http://dx.doi.org/10.1182/blood.v122.21.608.608.

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Abstract Acute myeloid leukemia (AML) with isolated trisomy 13 (AML+13) is rare and frequently associated with FAB M0 morphology. The clinical course is not well characterized but according to the ELN classification of intermediate prognosis. Eighty to one-hundred percent of patients (pts) with AML+13 carry mutations in the RUNX1 gene. Over-expression of FLT3 (located on chromosome 13 [chr 13]) due to the additional gene copy on the third chr 13 was proposed as a mechanism of leukemogenesis in AML+13 (gene dosage hypothesis). We set out to characterize the clinical course of AML+13 pts and elucidate their molecular background using whole exome sequencing, targeted resequencing and gene expression profiling. We identified 23 pts with AML+13 enrolled in a multicenter trial of the German AML Cooperative Group (AMLCG-1999) and compared this group to 386 pts without +13 who were classified in the ELN Intermediate-II genetic category. All pts received intensive induction chemotherapy. There was no significant difference in age, white blood cell or platelet count between the two groups. However, LDH levels were significantly (p=.01) lower in the AML+13 group while bone marrow blast percentage was significantly higher (p=.04). Twelve AML+13 pts (52%) reached complete remission, but all relapsed. Relapse-free and overall survival were inferior in the AML+13 group compared to other ELN Intermediate-II pts (median RFS, 9 vs 15 months, p=.01; median OS, 7 vs. 13 months, p=.03). Remission samples from two AML+13 pts were available as normal control for exome sequencing. Using SureSelect human all exon target enrichment (Agilent) followed by 80bp paired-end sequencing on an Illumina GAIIx platform, we were able to identify non-synonymous leukemia-specific mutations affecting, among others, RUNX1, ASXL1, PTPN11 and CEBPZ. Genes identified by exome sequencing and a panel of genes recurringly mutated in AML were studied by targeted amplicon resequencing in all AML+13 pts with available material (16/23; Figure). As described before, a high incidence of RUNX1 mutations (75%) was identified. In addition, we detected mutations in spliceosome components in 14/16 (88%) of AML+13 pts, including SRSF2 codon 95 mutations in 13/16 pts (81%). One patient without SRSF2 mutation showed a mutation in SF3B1. Moreover, recurring mutations were found in ASXL1 (44%) and BCOR (25%), and were associated with RUNX1 and SRSF2 mutations. Interestingly, both pts without mutations in the splicing machinery had mutations in DNMT3A, which were also mutually exclusive with mutations in RUNX1 or ASXL1. Two pts carried mutations in CEBPZ suggesting that CEBPZ is a novel recurringly mutated gene in AML.FigureMutation frequencies in 16 patients with AML+13Figure. Mutation frequencies in 16 patients with AML+13 To further characterize this genetically homogenous subgroup, we compared gene expression profiles of 9 pts with AML+13 with 509 AML pts without +13. We identified 678 (up-regulated 492; down-regulated 186) probe sets as significantly deregulated. Only 59 (8.7%) of these probe sets were localized on chr 13, but of those, 55 were up-regulated and only 4 were down-regulated. Up-regulated probe sets on chr 13 included FOXO1, FLT3 and RB1. The strongest down-regulated probe set on chr 13 belonged to the tumor suppressor gene SPRY2, which is a negative regulator of receptor tyrosine kinases. Gene set enrichment analysis showed significant deregulation of gene sets associated with regulation of transcription and nuclear transport. In summary, our study is the first to show that AML+13 is significantly associated with inferior OS and RFS compared to other intermediate-risk cytogenetic abnormalities in a homogeneously treated cohort. Furthermore, we present evidence that AML+13 leukemias are a genetically quite homogenous subgroup. AML+13 is not only associated with a high rate of RUNX1 mutations but also with mutations in SRFS2, ASXL1 and BCOR. The incidence of mutations in SRSF2 in AML+13 is the highest of any AML subgroup reported so far. In addition, our gene expression data show a homogenous expression profile associated with AML+13. The striking association of a few recurring mutations in AML+13 suggests a biological relationship with synergistic lesions during leukemogenesis. While mutations in RUNX1, ASXL1 and up-regulation of FLT3 were previously reported as markers of poor prognosis in AML, the combination of these lesions might be responsible for the extremely poor outcome of AML+13. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

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Rossi, Davide, Simone Ferrero, Alessio Bruscaggin, Paola Ghione, Alice Di Rocco, Valeria Spina, Vittorio Stefoni et al. « A Molecular Model for the Prediction of Progression Free Survival in Young Mantle Cell Lymphoma Patients Treated with Cytarabine-Based High Dose Sequential Chemotherapy and Autologous Stem Cell Transplantation : Results from the MCL0208 Phase III Trial from Fondazione Italiana Linfomi (FIL) ». Blood 126, n^o 23 (3 décembre 2015) : 336. http://dx.doi.org/10.1182/blood.v126.23.336.336.

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Abstract Background. Recent studies have described the landscape of recurrently mutated genes in mantle cell lymphoma (MCL), including genes involved in DNA damage response/cell cycle (ATM, TP53, CCND1), epigenetic regulation (KMT2D also known as MLL2, WHSC1), and cell signaling (BIRC3, TRAF2, NOTCH1). However, with the exception of TP53 abnormalities, little is known about the clinical relevance of recurrent mutations in MCL. Thus, we performed deep sequencing analysis of a MCL gene panel in the prospective series of patients enrolled in the ongoing FIL-MCL0208 phase III trial (EudraCTNumber: 2009-012807-25). Patients and Methods. The study included untreated, advanced stage <65 years MCL patients that: i) were eligible into MCL0208 trial (R-CHOP followed by high-dose cytarabine, autologous stem cell transplantation, and randomization between lenalidomide maintenance vs observation); ii) were provided with baseline tumor material. A targeted resequencing gene panel including coding exons and splice sites of genes recurrently mutated in MCL (ATM, BIRC3, CCND1, KMT2D, TP53, TRAF2, WHSC1, NOTCH1) has been specifically designed. The gene panel allows the recovery of at least one mutation in >70% of cases. The gene panel was analyzed in tumor DNA from baseline bone marrow CD19+ purified MCL cells and, for comparative purposes to filter out polymorphisms, in the paired normal genomic DNA (available in 55% of cases) using a TruSeq Custom Amplicon target enrichment system followed by deep next generation sequencing (Illumina, median depth of coverage 2356x). Variants represented in >10% of the alleles were called with VarScan2 with the somatic function when the paired germline DNA was available. For patients lacking germline DNA, a bioinformatic pipeline including a number of stringent filters was applied to protect against the misclassification of polymorphisms as somatic variants. Primary endpoint of the analysis was progression free survival (PFS). Results. Out of the enrolled patients, 151 are currently evaluable for mutations and clinical outcome (median age: 57 years, range 35-66; males 75%). Among prognostic factors, the MIPI was intermediate or high-risk in 49% of patients, the Ki67 ≥30% in 39%, and blastoid histology occurred in 8%. At the first planned interim analysis, median follow-up of alive patients was 26 months. At 2-years, 79% of patients were progression free and 91% alive (Cortelazzo et al EHA 2015). Overall, at least one mutation was detected in 106/151 cases (70%), including mutations of ATM in 42% of cases, CCND1 in 14%, WHSC1 in 13%, KMT2D in 12%, TP53 in 7%, NOTCH1 in 6%, BIRC3 in 5% and TRAF2 in 1% (Figure 1A). By univariate analysis, mutations of TP53 (2-years PFS 48% vs 82%; p<0.0001) and KMT2D (2-years PFS 67% vs 81%; p=0.004) associated with a significantly shorter PFS. By multivariate analysis, mutations of TP53 (HR: 4.39) and KMT2D (HR: 2.38) associated with an increase of the hazard of progression after adjusting for the MIPI. The hierarchical order of relevance in predicting PFS among molecular lesions was established by recursive partitioning analysis. TP53 mutations were the most predictive variable in the survival tree, followed by KMT2D mutations, thus providing the rationale to utilize these molecular features in the development of a model to predict PFS. By this approach, three MCL subgroups were hierarchically classified (Figure 1B). The high-risk category accounted for 8% of the cohort and included patients harboring TP53 mutations independent of the co-occurrence of KMT2D mutations (2-year PFS: 48%). The intermediate risk category accounted for 9% of the cohort and included patients harboring KMT2D mutations in the absence of TP53 mutations (2-year PFS: 74%). The low-risk category accounted for 83% of the cohort and comprised patients lacking both TP53 and KMT2D mutations (2-year PFS: 83%). The low number of events so far recorded prevented any analysis on overall survival. Conclusions. Though limited by the short follow-up, our data show that: i) the combination of two genetic biomarkers (i.e. TP53 and KMT2D mutations) allows to predict the benefit that young MCL patients can gain from a cytarabine-based high dose sequential chemotherapy followed by autologous stem cell transplantation; ii) intensive chemotherapy does not overcome the negative prognostic impact of TP53 mutations; and iii) KMT2D mutations may represent a novel genetic biomarker in MCL patients. Figure 1. Figure 1. Disclosures Santoro: Celgene: Research Funding.

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Rothenberg-Thurley, Maja, Stephanie Schneider, Tobias Herold, Nikola P. Konstandin, Annika Dufour, Kathrin Bräundl, Bianka Ksienzyk et al. « Targeted, Deep Sequencing of Adult AML Patients Treated on the AMLCG-2008 Trial Detects Clonal Heterogeneity in 52% of Patients at Initial Diagnosis and Reveals Patterns of Clonal Evolution ». Blood 124, n^o 21 (6 décembre 2014) : 697. http://dx.doi.org/10.1182/blood.v124.21.697.697.

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Abstract Background: Recurrent mutations in >100 different genes have been described in AML, but the clinical relevance of most of these alterations has not been defined. Moreover, high-throughput sequencing techniques revealed that AML patients (pts) may harbor multiple, genetically related disease subclones. It is unclear whether clonal heterogeneity at diagnosis also associates with clinical characteristics or outcomes. To address these questions, we set out to characterize a relatively large, uniformly treated patient cohort for mutations in known and putative AML driver genes. Patients and Methods: We studied pretreatment blood or bone marrow specimens from adult AML pts who received high-dose cytarabine-based induction chemotherapy within the German multicenter AMLCG-2008 trial. Sequence variants (single nucleotide variants and insertions/deletions up to approx. 150bp) in 70 genes known to be mutated in AML or other hematologic neoplasms were analyzed by multiplexed amplicon resequencing (Agilent Haloplex; target region, 321 kilobases). Sequencing was performed on an Illumina MiSeq instrument using 2x250bp paired-end reads. A variant allele frequency (VAF) threshold of 2% was set for mutation detection, corresponding to heterozygous mutations present in 4% of cells in a specimen. Variants were classified as known/putative driver mutations, variants of unknown significance, or known germline polymorphisms based on published data (including dbSNP, the Catalogue Of Somatic Mutations In Cancer [COSMIC] and The Cancer Genome Atlas [TCGA]). In patients with more than one single nucleotide variant, the chi square test was used assess if the observed VAFs, adjusted for ploidy, were compatible with the presence of a single clone. Results: Material for genetic analyses was available for 280 of the 396 participants (71%) enrolled on the AMLC-2008 trial. To date, analyses have been completed for 248 pts (130 male, 118 female; median age, 54y; range 19-81y). Updated results for the entire cohort will be presented at the meeting. Mean coverage of target regions was >600-fold, and on average, 98.2% of target bases were covered >30-fold. We detected a total of 914 mutations in 46 genes, including 37 genes mutated in >1 patient (Fig. A). Nine genes (NPM1, FLT3, DNMT3A, NRAS, WT1, IDH2, RUNX1, TET2 and ASXL1) were mutated in >10% of patients (red dashed line in Fig. A). We found a median of 4 mutations per patient (range: 0-10). Of note, only 1 patient had no detectable mutation and no abnormality on cytogenetic analysis. Patients with Intermediate-risk cytogenetics according to the MRC classification harbored a higher number of driver gene mutations (median, 4) compared to patients with MRC Favorable (median, 2 mutations) or Unfavorable (median, 3 mutations) cytogenetics (P<.001). When analyzing patterns of co-occurring and mutually exclusive mutations, we confirmed well-known associations (e.g., between CEBPA and GATA2 mutations) and identified novel pairs of mutations that frequently occur in combination and, to our knowledge, have not yet been reported in AML (e.g., ASXL1/STAG2, SRSF2/STAG2). These findings may guide functional studies on the molecular mechanisms of leukemogenesis. We found evidence for clonal heterogeneity in 129 (52%) of 248 pts, based on the presence of mutations with significantly (P<.001) different VAFs within the same sample. Our analyses reveal differences in allele frequencies between different AML driver genes. Mutations can be grouped into "early" events that often are present in the founding clone, and "late" events which frequently appear to be restricted to subclones (Fig. B). Conclusion: Targeted sequencing allowed detection of mutations affecting a panel of known and putative AML driver genes in clinical specimens with high sensitivity. Our data from the AMLCG-2008 patient cohort reveal novel patterns of cooperating gene mutations, and show that the presence of subclonal driver mutations is a frequent event in AML pts. Differentiating between "founding clone" mutations, and subclonal mutations that typically occur later in the disease has implications for choosing targeted therapies aimed at disease eradication. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Lee, Seung-Shin, Jae-Sook Ahn, Taehyung Kim, Hyeoung Joon Kim, Yeo-Kyeoung Kim, Seo-Yeon Ahn, Sung-Hoon Jung et al. « RUNX1 Mutation in Cytogenetically Normal Acute Myeloid Leukemia : Clinical Implications, Co-Mutation Analysis ». Blood 128, n^o 22 (2 décembre 2016) : 5253. http://dx.doi.org/10.1182/blood.v128.22.5253.5253.

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Abstract Background and Objectives Acute Myeloid Leukemia (AML) is a cytogenetically and molecularly heterogeneous disease. In the recent decades, many genetic mutations and their clinical significances in AML have been identified with the development of new genomics technology. Based on these advances, new 2 entities were added to the WHO 2008 classification : AML with mutated NPM1 and AML with mutated CEBPA. Likewise, AML with RUNX1 mutation are now considered as a new provisional entity in the next update of WHO classification. In this work, we characterized patients with cytogenetically normal AML according to RUNX1 mutational status and analyzed several co-mutations by next generation sequencing. Patients and Methods A total of 419 patients were included in the present study who met the following eligibility criteria: 1) age ≥ 15 years; 2) a diagnosis of AML with normal karyotype confirmed by conventional cytogenetic analysis. Analysis of genetic mutations were performed using targeted resequencing by Illumina Hiseq 2000 (Sureselect custom probe set targeting 94 myeloid gene panel including RUNX1 mutation). Samples for the confirmation of first complete response were also analyzed in 163 patients. The majority of patients (97%) received '3+7' standard induction chemotherapy. Median age was 53(range 15-84). Results Overall, most common mutations for this cohort were NPM1(33.9%), DNMT3A(30.3%), NRAS(20.2%), IDH2(15.0%), FLT3(12.2%), CEBPA(11.1%). RUNX1 mutations were found in 22 of 419 (5.4%) patients. 7 of 13 available samples in complete remission still had RUNX1 mutation. The patients with RUNX1 mutations were older than those with wild-type RUNX1. (p=0.006) and RUNX1 mutation had a trend of male preponderance. The WBC count and blast percentage of peripheral blood and bone marrow were not different according to RUNX1 mutational status. The complete response rate was significantly lower in RUNX1 mutated group compared with wild-type group. (57% vs. 84%, p=0.005) In univariable survival analysis, RUNX1 mutations were significantly associated with inferior event-free survival (EFS) (p<0.001), relapse-free survival (RFS) (p=0.009) and overall survival (OS) (p=0.002). However, in multivariable analysis, RUNX1 mutation was not an independent prognostic factor for inferior EFS (hazard ratio(HR) 1.48, p=0.286), RFS (HR 2.15, p=0.057) OS (HR 1.14, p=0.716). Co-mutation analysis revealed that ASXL1 (26%,p=0.001), KRAS (26%, p=0.009), BCOR (16%, p=0.032) were correlated with RUNX1 mutation. None of the patients with RUNX1 mutation had NPM1 mutation and only one patient had CEBPA mutation. Conclusion In cytogenetically normal AML, RUNX1 mutation is observed in 5.4% and is mutually exclusive of the NPM1 and CEBPA mutation. Older age and lower complete response rate is correlated with RUNX1 mutation. In univariable survival analysis, RUNX1 mutation is associated with poor clinical outcomes. Disclosures No relevant conflicts of interest to declare.

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Di Trani, Martina, Ettore Rizzo, Silvia Locatelli, Fabrizio Marino, Vanessa Cristaldi, Valeria Spina, Alessio Bruscaggin et al. « Longitudinal Assessment of Circulating Tumor Mutational Burden Using a Next-Generation Sequencing Cancer Gene Panel : A Potential Biomarker of Response to Programmed Cell Death 1 (PD-1) Blockade in Patients with Relapsed/Refractory Classical Hodgkin Lymphoma ». Blood 134, Supplement_1 (13 novembre 2019) : 131. http://dx.doi.org/10.1182/blood-2019-131096.

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Introduction: The programmed cell death 1 (PD-1) monoclonal antibodies (MoAbs) nivolumab and pembrolizumab induce response rates exceeding 70% in relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL). The lack of response to PD-1 MoAbs, and the relapse occurring in most patients who had responded to PD-1 blockade suggest that tools to identify the determinants of response/resistance to PD-1 MoAbs are urgently required. We hypothesized that the characterization of the mutational profile of circulating tumor DNA (ctDNA) could represent a valuable tool to track clonal evolution-driven resistance to checkpoint inhibitors. Patients and Methods: 21 R/R cHL (median age, 32 years; range, 19-51) who had received a median of 5 (range, 3-7) chemotherapy lines, including autologous stem cell transplantation (77%) and brentuximab vedotin (100%), were treated with PD-1 MoAbs. Blood samples were profiled by CAPP-Seq strategy. We analyzed ctDNA and paired DNA from peripheral blood mononuclear cells (PBMCs), as source of germline DNA to filter out polymorphism and sequencing errors. A targeted resequencing panel optimized to include the coding exons and splice sites of 133 genes (320 Kb) that are recurrently mutated in B-cell lymphomas was used. Libraries were prepared from ctDNA and germline gDNA according to the CAPP-seq targeted enrichment strategy (Nimblegen-Roche) and subjected to ultra-deep-next generation sequencing (NGS) using the Nextseq 500 platform (Illumina). The sequencing was performed to obtain a depth of coverage >2000x in >80% of the target region in all samples, which allowed a sensitivity of 3x10-3. A stringent and completely automated bioinformatic pipeline was applied to call non-synonymous somatic mutations, using the somatic function of VarScan2. Results: After a median of 26 (range, 9-63) cycles of PD-1 inhibitors best response was complete remission (CR) for 9 patients (42%), partial remission (PR) for 6 (29%) and progressive disease (PD) for 6 (29%). Patients achieving PR experienced a disease control lasting for 4.5 to 24 months and subsequently underwent PD. Plasma and PBMC samples were collected at baseline, every five cycles of therapy, and end-of-therapy (EOT). At baseline, 18 of 21 patients could be successfully genotyped, whereas three were not. Evaluable patients showed a mean (±SD) number of mutated genes and mutations per patient of 7.3±5.1 (range, 2-22) and 9.9±8.4 (range, 2-37), respectively. Genes recurrently affected by non-synonymous somatic mutations in >20% of R/R cHL included STAT6 (45%), SOCS1 (40%), ITPKB (35%), GNA13 (35%), TP53 (20%), TNFAIP3 (15%). At baseline, no association of distinct DNA mutations with resistance to PD-1 inhibitors could be demonstrated. Signaling pathways targeted by DNA mutations included JAK-STAT, NF-κB, PI3K-AKT, cytokine, NOTCH, immune evasion. The concentration of ctDNA reported as haploid genome equivalent per ml (hGE/ml) was 592.2 (range, 2-2,746), with values of hGE/ml detected in PD patients being significantly higher as compared to CR patients (P=.0437). As compared to cycle 0, the hGE/ml of ctDNA at cycle 5 showed a significant reduction (592.2 vs. 67, P<.0008) which was followed by further hGE/ml decline in CR patients (to 14 P=.05) and further hGE/ml increase in PD patients (to 1,300 P=.1). At cycle 5, all CR/PR patients showed complete disappearance of baseline mutations, which were replaced by completely novel clones. In all CR/PR patients, this pattern of "clonal reshaping" was repeatedly detected over time. In striking contrast, at cycle 5, PD patients showed the persistence of baseline mutations. In all PD patients, this pattern of "clonal persistence", was repeatedly detected over time. In 4 patients, resistance to PD-1 inhibitors was associated with the appearance of a TP53 mutated clone. Although, a formal correlation of circulating DNA mutations with standard FDG-PET imaging was outside the objective of this study, both the "clonal reshaping" and "clonal persistence" patterns could be demonstrated to correlate with the results of FDG-PET. Conclusions: Analysis of ctDNA allows detecting tumor-specific mutations in R/R cHL. The longitudinal tracking of circulating DNA mutations in these patients identifies two different patterns of clonal evolution associated with sensitivity (clonal reshaping) or resistance (clonal persistence) to checkpoint blockade. Disclosures Santoro: Eisai: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Lilly: Speakers Bureau; Sandoz: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Arqule: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; AstraZeneca: Speakers Bureau; Celgene: Speakers Bureau; Servier: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; BMS: Speakers Bureau; Roche: Speakers Bureau; Abb-Vie: Speakers Bureau; Amgen: Speakers Bureau; BMS: Consultancy; Bayer: Consultancy, Speakers Bureau; MSD: Speakers Bureau. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Carlo-Stella:ADC Therapeutics: Consultancy, Other: Travel, accommodations, Research Funding; Sanofi: Consultancy, Research Funding; Celgene: Research Funding; Janssen Oncology: Honoraria; MSD: Honoraria; Servier: Consultancy, Honoraria, Other: Travel, accommodations; Amgen: Honoraria; Boehringer Ingelheim: Consultancy; Novartis: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Other: Travel, accommodations, Research Funding; BMS: Honoraria; Janssen: Other: Travel, accommodations; Takeda: Other: Travel, accommodations; Rhizen Pharmaceuticals: Research Funding; AstraZeneca: Honoraria; Genenta Science srl: Consultancy.

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Varettoni, Marzia, Silvia Zibellini, Ettore Rizzo, Luca Malcovati, Virginia Valeria Ferretti, Matteo Giovanni Della Porta, Anna Gallì, Luca Arcaini, Irene Defrancesco et Mario Cazzola. « Targeted Next Generation Sequencing Identifies Novel Genetic Mutations in Patients with Waldenstrom's Macroglobulinemia/Lymphoplasmacytic Lymphoma or IgM-Monoclonal Gammopathies of Undetermined Significance ». Blood 128, n^o 22 (2 décembre 2016) : 2928. http://dx.doi.org/10.1182/blood.v128.22.2928.2928.

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Abstract Background. TheMYD88 (L265P) somatic mutation is present in more than 90% of patients (pts) with Waldenström's Macroglobulinemia (WM)/lymphoplasmacitic lymphoma (LPL). The second most common mutations are nonsense (NS) or frameshift (FS) mutations in the CXCR4 gene, detectable in approximately 25-30% of WM pts by Sanger sequencing. Limited data are available about other genetic mutations in WM/LPL and its precursor condition IgM-monoclonal gammopathy of undetermined significance (IgM-MGUS). Pts and methods. Using targeted next generation sequencing (NGS), we evaluated the prevalence of somatic mutations of 11 genes selected on the basis of evidences available from the literature (MYD88, CXCR4, ARID1A, KMT2D, TP53, NOTCH2, PRDM1, CD79b, TRAF3,TNFAIP3, MYDBBP1A) in 119 pts, classified as WM/LPL (n=63) or IgM-MGUS (n=56) according to International Consensus Criteria. Median age of pts (67 males, 52 females) was 65 years (range: 38-82). Samples were collected at diagnosis (n=101), after diagnosis but before any treatment (n=9) or at progression after therapy (n=9). Paired tumor and germline DNA extracted respectively from CD19-selected and CD19-depleted bone marrow (BM) mononuclear cells was available in all pts. Mean resequencing depth across gene panel was 1009x. Only mutations tagged as oncogenic or possibly oncogenic based on information derived from the literature and on in silico prediction effect were considered in the analysis. For MYD88 (L265P) and CXCR4 mutations, results obtained with NGS were compared with those obtained respectively with allele-specific PCR (AS-PCR) and Sanger sequencing. Results. Overall, we found 151 mutations in 88 pts (74%). The median number of mutations was significantly higher in WM/LPL as compared with IgM-MGUS and in pts previously treated as compared with untreated ones (median 2 versus 1, P < 0.001 for both comparisons). MYD88 mutations were found in 80/119 pts (67%), with a median allele burden of 34.2% (range: 2.5-93.3%). The prevalence of MYD88 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (86% versus 46%, P <0.001). MYD88 mutations other than classical L265P (n=76) were found in 4 pts and were represented by V217F (n=2), S219C (n=1), M232T (n=1). Fifteen pts who were MYD88 (L265P) wild-type by NGS were found to be mutated by AS-PCR (K coefficient of concordance between NGS and AS-PCR: 70%, P < 0.001). CXCR4 mutations were found in 19/119 pts (16%), with a median allele burden of 34% (range: 4.2-84%). The prevalence of CXCR4 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (24% versus 7%, P < 0.02). The K coefficient of concordance between NGS and Sanger was 83% (P < 0.001), with 2 pts mutated only by NGS and 2 pts mutated only by Sanger. Somatic mutations were also found in KMT2D (formerly known as MLL2) (16% of pts), TP53 (8%), NOTCH2 (7%), PRDM1 (4%), ARID1A (3%), CD79b (2%), and TRAF3 (1%). No mutations were found in MYBBP1A and TNFAI3. Overall, the prevalence of these mutations was significantly lower in pts wild-type either for MYD88 or CXCR4 as compared with those with MYD88 and/or CXCR4 mutations (15% versus 41%, P = 0.04). The prevalence of KMT2D mutations was significantly higher in WM/LPL as compared with MGUS (25% versus 5%), while for the other genes the distribution was not statistically different according to diagnosis. With a median follow-up of 20 months (range: 0-264), we did not find a statistically significant correlation between genetic mutations and pts' outcome in terms of overall survival or time to first treatment. Conclusions. In this cohort of pts with WM/LPL and IgM-MGUS studied with NGS we could demonstrate that: i) NGS identifies MYD88 mutations other than L265P in a small proportion of pts; ii) the prevalence of CXCR4 mutations by Sanger is confirmed by NGS, despite the higher sensitivity of the latter method; iii) the subgroup of pts wild type either for MYD88 or CXCR4 shows a low incidence of other genetic mutations; iv) 25% of pts with WM/LPL were found to carry KMT2D mutations, a prevalence similar to that reported in marginal zone lymphoma; v) genetic mutations are more common in WM/LPL than in IgM-MGUS in agreement with the hypothesis that multiple genetic hits are required for progression from a pre-benign condition to a neoplastic disease; vi) due to the indolent nature of these disorders, longer follow-up is probably needed to see the prognostic impact of these mutations, if any. Disclosures No relevant conflicts of interest to declare.

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Palomo, Laura, Blanca Xicoy, Montse Arnan, Marta Cabezon, Rosa Coll, Vera Ademà, Francisco Fuster et al. « Molecular Genetic Profiling in Chronic Myelomonocytic Leukemia with Low Risk Cytogenetic Features ». Blood 126, n^o 23 (3 décembre 2015) : 2883. http://dx.doi.org/10.1182/blood.v126.23.2883.2883.

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Abstract Background: Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease that can vary from a myelodysplastic (MD) predominant profile to a myeloproliferative (MP) one. CMML has a variable course, with a median overall survival of 20 months and 15-30% of progression to acute myeloid leukemia (AML). Cytogenetic abnormalities are present in 20-40% of cases and recurrent gene mutations have been reported in several genes. Patients with low risk cytogenetic features (normal karyotype and isolated -Y) account for approximately 80% of CMML patients and often fall into the low risk categories of CMML prognostic scores. Our hypothesis is that mutational study can contribute to diagnosis and prognostic stratification in this subset of patients. Methods: A retrospective study was performed on 57 patients with CMML. Cases with normal karyotype (n=53), isolated -Y (n=2) and no metaphases (n=2) were selected. DNA was extracted from BM (n=52) and PB (n=5) samples at diagnosis. Targeted deep-sequencing was performed in a panel of 83 myeloid-related genes. Libraries were prepared with 1μg of genomic DNA using the KAPA Library Preparation Kit (Kapa Biosystems) and then enriched using the SeqCap EZ capture chemistry (Nimblegen, Roche). Libraries were sequenced with 150 bp paired-end reads on an Illumina MiSeq. Herein we present the results of 43 cases which were preliminarily analyzed using the Illumina MiSeq Reporter and Variant Studio softwares. High-probability oncogenic mutations were called by eliminating sequencing and mapping errors and known SNPs based on the available databases. An in-house bioinformatics pipeline will be designed to analyze the whole series of patients. A preliminar statistical analysis was performed with SPSS. Fisher's exact test was used to compare variables between patient subsets. Complete study, including the correlation of the sequencing findings with the clinical data, will be presented in the meeting. Results: Median age at diagnosis was 70 years (range 48-87) and there was a 2:1 male predominance. Median follow up of patients was 23 months (range 1-116) during which 23% (11/43) of cases progressed to AML. Morphological WHO subtypes were CMML-1 in 36 (84%) cases and CMML-2 in 7 (16%). According to the FAB criteria 34 (79%) cases were classified as CMML-MD and 9 (21%) as CMML-MP. According to the CMML-specific scoring system (CPSS) 28/43 (65%) patients belonged to the low-risk category, 10/43 (23%) to the intermediate-1 and 4/43 (12%) to the intermediate-2. The mean depth of the targeted resequencing per base per sample was 810-fold. After excluding sequencing and mapping errors a mean of 293 single nucleotide variants (snv) and insertions/deletions (indels) were called per sample. After filtering non-silent variants and excluding known polymorphisms a mean of 6 variants per sample were called as high-probability somatic changes. Distribution of detected variants across the patients can be seen in Figure 1. Most frequently affected genes were TET2 (70%), ASXL1 (47%) and SRSF2 (35%); followed by RUNX1 (23%), NRAS (16%), CBL (12%), EZH2 (12%), SETBP1 (12%) and ZRSR2 (12%). Variants detected in 5-10% of patients included IDH2, CRBBP, SH2B3, UMODL1, DNMT3A, JAK2, PTPN11, SF3B1 and U2AF1 genes. Statistical analysis revealed that some variants correlated with CMML subtypes: SH2B3 (P=0.010) and STEBP1 (P=0.024) associated with CMML-2; JAK2 (0.007), NRAS (P=0.026) and EZH2 (P=0.05) associated with CMML-MP. Variants in NRAS also correlated with progression to AML (P=0.04) and patients in intermediate groups of CPSS associated with JAK2 (P=0.008) and EZH2 (P=0.011) variants. Conclusions: Genetic profiling using targeted deep-sequencing is a highly promising approach for CMML diagnosis and varies according to the cytological subtypes. With the correlation of the results with the clinical data of patients, we expect to determine if targeted molecular profiling can contribute to prognostic stratification of patients with CMML and low risk cytogenetic features. For the moment, we have already found a correlation with progression to AML. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519; PI 11/02010); RTICC, FEDER (RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Josep Carreras, Obra Social "La Caixa" and Celgene Spain. Figure 1. Distribution of the affected genes across the 43 studied patients with CMML Figure 1. Distribution of the affected genes across the 43 studied patients with CMML Disclosures Sole: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Cabezon, Marta, Joan Bargay, Blanca Xicoy, Laura Palomo, Sílvia Marcé, Ramón Guàrdia, Salut Brunet et al. « Mutational Studies Using Next Generation Sequencing in High Risk Myelodysplastic Syndromes and Secondary Acute Myeloid Leukemia Patients Treated with Azacitidine (High risk MDS 2009 protocol from CETLAM Group) ». Blood 126, n^o 23 (3 décembre 2015) : 2905. http://dx.doi.org/10.1182/blood.v126.23.2905.2905.

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Abstract INTRODUCTION: Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms originated in hematopoietic stem cells, characterized by citopenias, dysplasia in one or more cell lines, ineffective hematopoiesis and an increased risk of progression to acute myeloid leukemia (AML). Treatment of MDS depends on subtype and prognostic category. DNA methyltranferase inhibitors are approved for high risk MDS. Over the past decade, the application of new high-throughput technologies to the study of MDS has led to the identification of several recurrently mutated genes. These include genes producing proteins involved in RNA splicing, DNA methylation, chromatin modification, transcription, DNA repair control, cohesin function, RAS pathway, and DNA replication. There is a significant overlap between the genes mutated commonly in MDS with those found in AML. Mutation status is not widely used to select treatment in MDS. The aim of this study is to define the mutational status of MDS and secondary AML (sAML) patients at diagnosis that have been treated with azacitidine (AZA) to see if it could help to discriminate which patients will respond from those who will not. MATERIAL AND METHODS: A prospective study was performed on 36 patients with MDS and sAML treated with AZA. Genomic DNA was obtained from bone marrow at diagnosis. SeqCap EZ and KAPA Library Preparation Kit (Roche) reagents have been used to enrich DNA of 83 genes implicated in myeloid neoplasm. The customized panel has been analyzed in MiSeq Illumina platform with 150bp paired-end reads. Samples were preliminary analyzed using Illumina MiSeq Reporter and Variant Studio softwares. Data from response to treatment and survival have been collected from all patients. RESULTS:The mean depth of the targeted resequencing per base was 685-fold. After filtering all the variations obtained for quality, biological consequence and discard the known SNPs, we have obtained 162 variations, including 145 single nucleotide variants (SNV) and 17 insertions/deletions. All patients harbored at least 1 alteration with a mean of 4.5 variants per sample. The average of alterations detected in each cytological category can be observed in Table 1.Table 1.Average abnormalities detected by cytological category.Nº patientsAverage of alterations detected for patient (range)sAML104,8 (1-8)RAEB-274,9 (2-8)RAEB-1123,7 (1-6)RCDM54,4 (3-7)RCDM-RS16RARs11The most frequent altered genes have been TP53, TET2 and DNMT3A. The numbers of variations detected for each gene are represented in Table 2.Complete results, including correlation with treatment response will be presented in the meeting.Table 2.Number of variations in each gene.GeneNº of variations foundNº of diferent variationsNº of patients with variationsFrequency of variationsTP5322191952,8%TET214101027,8%DNMT3A88822,2%CREBBP75719,4%SRSF271719,4%ASXL165616,7%U2AF162616,7%EP30053513,9%STAG255513,9%CUX144411,1%ETV643411,1%MLL (KMT2A)43411,1%RUNX14438,3%BCOR3338,3%CDH133338,3%CTNNA13238,3%EZH23338,3%GCAT3338,3%MLL2 (KMT2D)3338,3%NF13338,3%PDGFRB3338,3%SH2B33338,3%TGM23238,3%UMODL13338,3%CEBPA2125,6%CSF3R2225,6%GATA22125,6%PHLPP12225,6%RAD212225,6%SF3B12125,6%SUZ122225,6%TIMM502125,6%Others*1112,8%*ABL1, BCORL1, CALR, CDH3, IDH2, KRAS, LUC7L2, NPM1, NRAS, PHF6, SF3A1, SFPQ, SMC3, TERT, WT1, ZRSR2. CONCLUSIONS: Targeted deep-sequencing technique is a good tool to study mutational profile in MDS and sAML. SNV are the most frequent type of alteration found in our cohort. The patients with sAML and RAEB-2 present more variations than patients with RAEB-1. The rest of groups are less representing to be evaluated. The most affected genes match with those described in the literature, with some exceptions that need to be studied in more detail. We expect to predict in advance which patients are going to respond when we study the correlation of mutational analysis with treatment response. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Josep Carreras, Obra Social "La Caixa" and Celgene Spain. Diana Domínguez for her technical assistance Disclosures Valcarcel: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Oliveira, Giacomo, Gabriele Bucci, Cristina Toffalori, Carolina Caserta, Lara Crucitti, Barbara Camisa, Raffaella Greco et al. « Clinical and Biological Features Associated with Engraftment of Acute Myeloid Leukemia Patient-Derived Xenografts ». Blood 128, n^o 22 (2 décembre 2016) : 2858. http://dx.doi.org/10.1182/blood.v128.22.2858.2858.

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Abstract Background: Patient-derived xenografts (PDXs) are key models for interrogating the biology of tumor cells that poorly survive in vitro. In particular, over the last decade, immunodeficient mouse models have been extensively used to assess the in vivo growth potential of human leukemia, to provide insights into its biology, and to perform preclinical validation of therapies. Still, only a fraction of the cases of acute myeloid leukemia (AML) are able to engraft into mice, and the biological and clinical correlates of the ability to generate PDXs are unknown. Methods: Primary AML harvested from 52 patients at diagnosis (n=37, 71%), at relapse after treatments (n=15, 29%), or both (n=6) were purified and infused into non-irradiated NOD-SCID γ-chain null (NSG) mice. Upon leukemia engraftment, assessed by multiparametric flow cytometry, mice were sacrificed and leukemic cells were isolated, characterized, and reinfused in serial recipients, in up to four serial passages. Gene expression profile was analyzed using Illumina microarray, and deregulated genes and processes identified by pairwise LIMMA analysis and classified using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) curated databases. The mutational asset of infused AML was assessed through targeted resequencing, using a custom panel comprising 192 targets and based on the Agilent Haloplex HS technology. Results: Twenty-six out of 52 primary AML samples (50%) generated xenografts. Engraftment and growth kinetics of the human leukemic cells were highly consistent among littermates, and specific for each tested leukemia. Circulating leukemic cells were firstly detected in the peripheral blood of animals at a median time of 22.5 days (range 14 - 150). In vivo growth allowed expansion of infused AMLs in bone marrows and spleens of the animal, with a median fold increase of 3.5 (range 0.1 - 351.4). The gene expression profile of xenografts was reproducible amongst littermates and recapitulated the features of parental AML: genes deregulated in xenografts accounted for 9.1% of the transcript assessed, with substantial overlap in the genes and processes deregulated in each of the studied cases. GO and GSEA demonstrated the selective deregulation of genes involved in cell proliferation (CDC20, AURKA), syster chromatyde organization (CENPF CEP170) and myeloid differentiation (AZU1, MPO, MYADM, CTSG). Of note, the ability to generate xenografts was conserved when AML cells were challenged at different time-points during the clinical history of the patients, with leukemia harvested at relapse after transplantation displaying a more aggressive behavior. Similarly, upon serial transfer AML exhibited an accelerated growth kinetic. Engraftment in mice significantly correlated with poor patient prognosis: AML engrafters had dramatically lower leukemia free-survival rates compared to non-engrafters (median 5.9 vs. 21.8 months after induction chemotherapy, p=0.0022, Fig. 1A), confirmed also by multivariate analysis (p=0.002). Also the mutational profile differed greatly between engrafters and non-engrafters, as summarized in Fig. 1B. In particular, while the presence of an aberrant karyotype was not associated with PDX generation, FLT3 internal tandem duplication, DNMT3A and NPM1 mutation were all significantly associated to engraftment (p=0.0244, p=0.009 and p=0.0437 respectively). In particular the co-occurrence of mutations in these three genes, recently reported to confer very poor prognosis to AML patients (Papaemmanuil et al, NEJM 2016), markedly enhanced the ability to generate PDXs (Fig.1C). Conclusion: These data show that engraftment into immunodeficient mice mirrors the biology of primary human leukemia, providing a proxy to select cases with a higher chance to generate PDXs. Further comparisons between AML capable or not to generate PDXs might provide novel markers of leukemia aggressiveness and rationales for targeted therapies. Figure 1 Figure 1. Disclosures Bonini: TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.

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Blakemore, Stuart J., Ruth Clifford, Helen Parker, Pavlos Antoniou, Ewa Stec-Dziedzic, Marta Larrayoz, Zadie Davis et al. « Clinical significance of TP53, BIRC3, ATM and MAPK-ERK genes in chronic lymphocytic leukaemia : data from the randomised UK LRF CLL4 trial ». Leukemia 34, n^o 7 (3 février 2020) : 1760–74. http://dx.doi.org/10.1038/s41375-020-0723-2.

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Abstract Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.

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Resar, Linda, Donna Marie Williams, Zhizhuang Joe Zhao, Ophelia Rogers, Lingling Xian, Jerry L. Spivak et Alison R. Moliterno. « High Mobility Group A1/2 Chromatin Remodeling Proteins Associate with Polycythemia Vera Transformation to Acute Leukemia in Humans and a JAK2 V617F Transgenic Mouse Model ». Blood 128, n^o 22 (2 décembre 2016) : 1958. http://dx.doi.org/10.1182/blood.v128.22.1958.1958.

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Abstract Introduction: The MPN are clonal hematopoietic stem cell (HSC) disorders characterized by an overproduction of blood cells and an increased risk of transformation to an aggressive phase with myelofibrosis (MF) and/or acute myeloid leukemia (AML). Polycythemia vera (PV) is the most common clinical subtype, and while PV starts as an indolent process, nearly 25% of patients will progress to MF and/or AML. PV is caused by acquired mutations of JAK2, yet JAK2 mutations alone do not account for MF or AML transformation. Mutations in genes encoding epigenetic regulators are associated with MPN transformation, but the mechanism of action is not understood. HMGA1/2 chromatin binding proteins are potent oncogenes that drive tumor progression by activating oncogenic and stem cell transcriptional networks. Both HMGA1/2 are overexpressed in acute leukemia and have been shown to be drivers of clonal expansion in myeloid disease in humans and in murine myeloproliferative disease models. We hypothesized that HMGA proteins could be critical drivers of transformation in PV and therefore tested the association of HMGA1/2 expression to transformation in human and murine PV. Methods: We examined the HSC genomic context and clonal evolution in 49 JAK2V617F-positive PV patients using standard and SNP-array karyotyping and a targeted resequencing panel of 163 genes associated with myeloid cancers. We examined HSC clonal burden by examining JAK2V617F HSC genotypes on a single cell basis. We measured HMGA1 and HMGA2 expression in a JAK2V617F positive human cell line, in isolated CD34+ HSCs from PV patients during chronic and transformation phases, in JAK2V617F transgenic murine models of PV (tgJAK2V617F) and PV-AML (tgJAK2V617F/MPLSV; Blood 2015;126:484) using a real-time quantitative RT-PCR (qRT-PCR) assay. Results: Both HMGA1 and HMGA2 mRNA were up-regulated in all JAK2V617F-positive contexts. In primary human PV CD34+ HSCs, HMGA1 and HMGA2 were found to be increased by 7 and 100 fold, respectively, compared to controls. Moreover, there was a dramatic up-regulation in both HMGA1/2 in patients who transformed from PV to MF or AML compared to chronic phase PV, whether analyzed cross-sectionally (Figure) or prospectively in selected patients. In addition to disease phase, over-expression of HMGA1/2 correlated with clonal dominance of JAK2V617F-homozygous stem cells, and additional mutations of epigenetic regulators including EZH2 and SETBP1. Similarly, when assessed in unfractionated bone marrow or in tumor samples in the two transgenic mouse models for PV and PV-AML, Hmga1/2 were overexpressed compared to wild-type littermates, with highest levels in the PV-AML transgenic mouse model. Conclusion: HMGA1 and HMGA2 are overexpressed in PV, and higher levels associate with disease progression to MF and AML, both in human PV and in transgenic murine models of PV. These data suggest HMGA proteins are critical drivers of PV transformation and that the mechanism of HMGA1/2 overexpression is a consequence of aberrant JAK/STAT signaling and epigenetic dysregulation. Our findings indicate that HMGA1/2 overexpression may function as a necessary molecular switch for PV leukemic transformation. Therefore, HMGA proteins and their transcriptional pathways offer novel therapeutic targets aimed at the prevention of PV progression to MF and AML. Disclosures No relevant conflicts of interest to declare.

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Xue, Mengxing, Zhao Zeng, Qinrong Wang, Lijun Wen, Yi Xu, Jundan Xie, Qian Wang, Changgeng Ruan, Depei Wu et Suning Chen. « Mutational Profiles during the Progression of Chronic Myeloid Leukemia ». Blood 138, Supplement 1 (5 novembre 2021) : 3596. http://dx.doi.org/10.1182/blood-2021-154273.

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Abstract Background: Despite significant improvements in the prognosis of chronic myeloid leukemia (CML) achieved by targeted therapy with tyrosine kinase inhibitors (TKIs), a small proportion of cases may not respond to TKIs or may relapse after an initial response, and then progress from chronic phase (CP) to blastic crisis (BC), characterized by a dismal prognosis. It remained uncertain whether the genetic lesions in addition to the BCR-ABL1 fusion could predict clinical outcomes of CML in the TKI era. Aim: To study the mutational profiles at each stage of CML and the prognostic significance of somatic mutations in addition to the BCR-ABL1 fusion in the TKI era. Patients and Methods: We performed targeted sequencing in 81 CML patients chosen retrospectively. 10 patients had optimal response to TKIs by European LeukemiaNet criteria and maintained durable major molecular response more than 5 years. 71 patients had progressed to accelerated phase (AP) or BC, of whom 43 had sequencing performed at paired CP and AP/BC samples, 28 at AP or BC samples. Totally, we analyzed 53 CP, 20 AP, and 61 BC samples. The targeted resequencing gene panel, covering 386 genes which were recurrently mutated in hematologic malignancies, were performed on a HiSeq 4000 NGS platform (Illumina). Results: Among the 53 CP samples, 20 (37.7%) had mutations involving 14 genes, and the number of mutated genes in each patient was 0-3 (median 0). ASXL1 was the most commonly mutated gene, 10/53 (18.9%) patients had this mutation, followed by KMT2D (4/53, 7.5%), PC (2/53, 3.8%), ERBB4 (2/53, 3.8%). ASXL1 mutation mainly existed in 43 patients with progressed disease , while only one case carried this mutation in 10 patients responsive to TKIs (20.9% vs 10%). 17/20 (85%) AP samples (including 10 patients progressed to AP and the other 10 patients who eventually progressed to BC from AP ) carried mutations involving 18 genes, the number of mutated genes in each patient was 0-6 (median 1.5). ABL1 was the most commonly mutated gene, and 8/20 (40%) patients had this mutation. The second was the ASXL1 mutation, 7 (7/20, 35%) patients carried this mutation. The other genes mutated in more than 2 patients included BCORL1 (3/20, 15%), RUNX1 (2/20, 10%), PHF6 (2/20, 10%), KMT2D (2/20, 10%), ATM (2/20, 10%). 54/61 (88.5%) BC samples (44 with myeloid crisis, 14 with lymphoid crisis, 3 with mixed phenotypic crisis) carried mutations, involving 41 genes, and the number of mutated genes in each patient was 0-9 (median 2). Similar to the mutation status in AP, the most commonly mutated gene was also ABL1, 24/61 (39.3%) patients carried this gene mutation, followed by ASXL1 mutation (13/61, 21.3%), and the other genes were in order, RUNX1 (11/61, 18.0%), WT1 (8/61, 13.1%), GATA2 (6/61, 9.8%), MED12 (5/61, 8.2%), IDH1 (5/61, 8.2%), TP53 (4/61 , 6.6%), KMT2D (4/61, 6.6%), etc. (Figure 1A) Among all the samples, 34 nonsynonymous variants in the ASXL1 gene were identified in 31 samples of 21 patients ( 3 samples with two variants). All the variants were frameshift and nonsense mutations, localized at the last exon of the ASXL1 gene. 13/21 patients with ASXL1 mutations had multi-stage samples. The median VAF of the ASXL1 mutations in the advanced stage was 31.4% (0-47.0%), which was significantly higher than that in CP at diagnosis (7.0%, 0-27.2%, P=0.033). Most of the ASXL1 mutations detected in CP expanded at the advanced disease, and were accompanied with other additional gene abnormalities, such as ABL1, RUNX1 and WT1 mutations, with the VAF similar to or lower than that of the ASXL1 mutations. In a few cases, the ASXL1 mutant clones in the CP disappeared, suggesting that some ASXL1 mutations may be clonal hematopoiesis unrelated to disease progression.(Figure 1B) In order to evaluate the effects of ASXL1 mutations on sensitivity to TKIs in vitro. We co-expressed P210-BCR-ABL1 fusion and ASXL1 mutation (G646Wfs*12) in Ba/F3 cells. Compared to Ba/F3 cells co-expressing BCR-ABL1 fusion and ASXL1 mutation (Ba/F3-BA/As), Ba/F3-BCR-ABL1 cells without ASXL1 mutation (Ba/F3-BA/Ve) showed higher sensitivity to TKIs, including imatinib, dasatinib and nilotinib.(Figure 1C) Conclusions: These results demonstrated the genetic lesions accumulated during the progression of CML from CP to BC. ASXL1 mutations were the most common genetic lesion in CP at diagnosis and may confer a poor prognosis, as it reduced the sensitivity to TKIs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Rasi, Silvia, Sara Monti, Manuela Zanni, Carmela Ciardullo, Fary Diop, Lavinia Martuscelli, Elisa Spaccarotella et al. « Liquid Biopsy As a Tool for Monitoring the Genotype of Diffuse Large B-Cell Lymphoma ». Blood 126, n^o 23 (3 décembre 2015) : 127. http://dx.doi.org/10.1182/blood.v126.23.127.127.

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Abstract Introduction. Accessible and real-time genotyping for diagnostic, prognostic or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Since DLBCL lacks a leukemic phase, tumor genotyping has so far relied on the analysis of the diagnostic tissue biopsy. Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of cancer-gene somatic mutations. Accessing the blood has obvious sampling advantages in the monitoring of mutations in real-time. Also, cfDNA is representative of the entire tumor heterogeneity, thus allowing to identify mutations from tumor cells residing in non-biopsied sites. Here we aimed at tracking the DLBCL genetic profile using plasma cfDNA. Methods. The study was based on 26 consecutive DLBCL patients (age >65=15, male:female ratio=11:15) presenting in different Ann Arbor stages (III-IV=13) and age-adjusted IPI risk scores (2-3=13), and provided with cfDNA from plasma collected at diagnosis, during R-CHOP course, at the end of treatment and at progression. Paired normal genomic DNA from granulocytes was also collected for comparative purposes to filter out polymorphisms. A targeted resequencing panel including the coding exons and splice sites of 59 genes (207 kb) that are recurrently mutated in mature B-cell tumors was specifically designed to allow the recovery of at least one mutation in >90% of DLBCL. Ultra-deep next-generation-sequencing (NGS) of the gene panel was performed on MiSeq (Illumina) (coverage >2000x in >80% of the target) using a SeqCap library preparation strategy (NimbleGen). The somatic function of VarScan2 was used to call non-synonymous somatic mutations, and a stringent bioinformatic pipeline was developed and applied to filter out sequencing errors. The study cohort was divided in a training set of 17 patients provided with paired tumor DNA from the diagnostic tissue biopsy that was used for the set up of the ultra-deep NGS strategy, and an extension set of 9 patients lacking the tissue biopsy. Results. A total of 76 cfDNA samples (26 pretreatment, 28 during treatment, 18 at the end of treatment, and 4 at time of progression) were evaluated. Pretreatment cfDNA genotyping disclosed somatic mutations of heterogeneous abundance (median mutated molecules/ml of plasma: 3168, range 1.73-6.5x104) in known DLBCL-associated genes, including MLL2 (33%), TP53 (25%), CREBBP and TNFAIP3 (21%), EZH2, TBL1XR1, PIM1 (17%), B2M, BCL2, CARD11, CCND3, FBXW7 and STAT6 (13%) (Fig. 1A). The results of genotyping on cfDNA from plasma and of genomic DNA from tumor cells of the diagnostic biopsy (gold standard) were compared to derive the diagnostic accuracy of cfDNA genotyping (Fig. 1B). Genotyping of the paired plasma cfDNA correctly identified 79% of the tumor biopsy mutations. Most of the tumor variants not discovered in the cfDNA had a low representation in the tumor biopsy (median allelic abundance=5.7%; range 0.8-54%). Consistently, ROC analysis showed that cfDNA genotyping had the highest sensitivity (92%) if mutations were represented in >15% of the alleles of the tumor biopsy. Plasma cfDNA genotyping also disclosed a number of additional somatic mutations (~2 per case, range 1-6) that were not detectable in the tissue biopsy, including mutations of clinically relevant genes. Longitudinal analysis of plasma samples under R-CHOP chemotherapy showed a rapid clearance of the DLBCL mutations in the cfDNA already after the first cycle among responding patients (Fig. 1C). Among patients that were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations appeared in cfDNA that conceivably marked resistant clones selected during the clonal evolution process taking place under the pressure of treatment (Fig 1D). Conclusions. Overall, these results provide the proof of principle that cfDNA genotyping of DLBCL: i) is as accurate as genotyping of the diagnostic biopsy to detect somatic mutations of allelic abundance >15% in DLBCL; ii) allows the identification of mutations that are otherwise absent in the tissue biopsy conceivably because restricted to clones that are anatomically distant from the biopsy site; and iii) is a real-time and non-invasive way to track clonal evolution and emergence of treatment resistant clones. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Nazha, Aziz, David J. Seastone, Priyanka A. Pophali, Tomas Radivoyevitch, Hetty E. Carraway, Anjali S. Advani, Matt E. Kalaycio et al. « Different Genomic Patterns in Patients with Primary Acute Myeloid Leukemia (AML) Compared to Secondary AML in Patients with Normal Karyotype ». Blood 124, n^o 21 (6 décembre 2014) : 1054. http://dx.doi.org/10.1182/blood.v124.21.1054.1054.

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Abstract Background For decades, cytogenetic analysis has played an essential role in AML risk stratification. Among the 50% of AML patients (pts) with normal karyotype (NK), outcome can vary widely. More recently, whole genome sequencing (WGS) and whole exome sequencing (WES) have identified several recurrent mutations that play an important role in AML pathogenesis and impact outcome. Pts with secondary AML (sAML) have a particularly poor prognosis, are not as responsive to standard induction chemotherapy, and often are referred in first complete remission to hematopoietic stem cell transplantation. We hypothesized that different genomic patterns exist between primary AML (pAML) and sAML that can distinguish the two, and can alter treatment recommendations. To negate the impact of chromosomal abnormalities, we focused our analyses on pts with NK. Methods We performed WES and multi-amplicon targeted deep sequencing on samples from bone marrow and peripheral blood of pts diagnosed with sAML at our institution between 1/2003- 1/2013 and who had NK cytogenetics. We compared them to pts with NK primary AML (pAML) whose data were extracted from The Cancer Genome Atlas (TCGA). A panel of 62 gene mutations that has been described as recurrent mutations in myeloid malignancies was included. Mutations were considered individually and grouped based on their functional pathways: RNA splicing (SF3B1, U2AF1/2, SRSF2, ZRSR2), DNA methylation (TET2, DNMT3A, IDH1/2), chromatin modification (ASXL1, EZH2, MLL, SUZ12, KDM6A), transcription (RUNX1, CEBPA, NPM1, BCOR/BCORL1, SETBP1, ETV6), activating signaling (FLT3, JAK2), cohesion (STAG2, SMC3, RAD21), RAS superfamily (K/NRAS, NF1, PTPN11, CBL) and tumor suppressor genes (TP53, APC, WT1, PHF6). Using deep sequencing methodology for resequencing or targeted sequencing, variant allelic frequency (VAF) was measured for each mutation detected. VAF was adjusted by zygosity evaluated by SNP-array karyotyping. For confirmation of clonal architecture, serial sample sequencing and single colony PCR were applied. Differences were compared using Fisher-exact test and Mann-Whitney U test for categorical and continues variables respectively. Results: Of 143 pts included, 101 (71%) had pAML and 42 (29%) had sAML. Compared to pAML, sAML pts were older (59 vs 69 years, p <.001), and had lower white blood cell count (28 vs 3.5 X 109/L, p <.001). Median hemoglobin (10 vs 10) g/dl and platelet counts (57 vs 60) k/uL were similar between the two groups. With a median follow up of 26.4 months (mo, range, .93-95.4), median OS was shorter for sAML than for pAML (12.9 vs 16.2 mo, p= .03). Overall, the most common mutations were: NPM1 (35%), DNMT3A (27%), FLT3 (25%), RUNX1 (14%), IDH1 (12%), IDH2 (12%), STAG2 (12%), TET2 (11%), NRAS (8%), ASXL1 (8%), U2AF1 (8%), PTPN11 (7%), WT1 (6%), BCOR (5%), and PHF6 (5%). Mutations in SF3B1, U2AF1/2, BCOR/BCORL1, ETV6, ASXL1, JAK2, STAG2, and APC were more common in sAML compared to pAML, whereas mutations in DNMT3A, NPM1, CEBPA, and FLT3 were more common in pAML. Mutations in activated pathways in splicing machinery, transcription, chromatin modification, cohesion and RAS pathway were more prominent in sAML, while mutations in DNA methylation and signaling pathways occurred more frequently in pAML. Serial sample analyses at multiple time points demonstrated intra-tumor heterogeneity in most cases of sAML, which was supported by additional cross sectional analyses of VAF in multiple gene mutations in each case. These findings prompted us to evaluate secondary events in the cohort of pts whose sAML originated from an initial MDS stage, defined by ancestral mutations. Among genes frequently affected by mutations, TET2 and ASXL1 were identified as founder events, whereas STAG2, NRAS and PTPN11 were observed in subclonal sAML derived from founder MDS clones. In pAML, however, TET2 and ASXL1 mutations were found to be secondary lesions, while IDH1 and DNMT3A were identified as ancestral events. Conclusion Clear genomic variations exist between sAML and pAML that suggest differences in the pathophysiology of both diseases. Specific therapies should be directed to the activated pathways according to the unique clonal hierarchy in each AML subtype. Disclosures No relevant conflicts of interest to declare.

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