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Purpose: In acute myeloid leukemia (AML), somatic mutations (commonly missense, nonsense, and frameshift indels) in RUNX1 are associated with a dismal clinical outcome. Inherited RUNX1 mutations cause familial platelet disorder. As approximately 5%–10% of germline RUNX1 mutations are large exonic deletions, we hypothesized that such exonic RUNX1 aberrations may also be acquired during the development of AML. Experimental Design: Sixty patients with well-characterized AML were analyzed with multiplex ligation-dependent probe amplification (n = 60), microarray (n = 11), and/or whole-genome sequencing (n = 8). Results: In total, 25 (42% of the cohort) RUNX1-aberrant patients (defined by the presence of classical mutations and/or exonic deletions) were identified. Sixteen patients (27%) carried only exonic deletions, 5 (8%) carried classical mutations, and 4 (7%) carried both exonic deletions and mutations. No significant difference was observed between patients with classical RUNX1 mutations and RUNX1 exonic deletions in median overall survival (OS, 53.1 vs. 38.8 months, respectively, P = 0.63). When applying the European Leukemia Net (ELN) classification including the RUNX1-aberrant group, 20% of the patients initially stratified as intermediate-risk (5% of the whole cohort) were reassigned to the high-risk group, which improved the performance of ELN classification regarding OS between intermediate- and high-risk groups (18.9 vs. 9.6 months, P = 0.09). Conclusions: Somatic RUNX1 exonic deletions constitute a novel recurrent aberration in AML. Our findings have important clinical implications regarding AML classification, risk stratification, and treatment decision. Moreover, they argue in favor of further investigating such genomic aberrations not only in RUNX1 but also in other genes implicated in cancer biology and management.

Over the last decades, rapid technological and scientific advances have led to a merge of molecular sciences and clinical medicine, resulting in a better understanding of disease mechanisms and the development of novel therapies that exploit specific molecular lesions or profiles driving disease. Precision oncology is here used as an example, illustrating the potential of precision/personalized medicine that also holds great promise in other medical fields. Real-world implementation can only be achieved by dedicated healthcare connected centers which amass and build up interdisciplinary expertise reflecting the complexity of precision medicine. Networks of such centers are ideally suited for a nation-wide outreach offering access to precision medicine to patients independent of their place of residence. Two of these multicentric initiatives, Genomic Medicine Sweden (GMS) and the Centers for Personalized Medicine (ZPM) initiative in Germany have teamed up to present and share their views on core concepts, potentials, challenges, and future developments in precision medicine. Together with other initiatives worldwide, GMS and ZPM aim at providing a robust and sustainable framework, covering all components from technology development to clinical trials, ethical and legal aspects as well as involvement of all relevant stakeholders, including patients and policymakers in the field.

Precision medicine is revolutionising patient care in cancer. As more knowledge is gained about the impact of specific genetic lesions on diagnosis, prognosis and treatment response, diagnostic precision and the possibility for optimal individual treatment choice have improved. Identification of hallmark genetic aberrations such as the BCR::ABL1 gene fusion in chronic myeloid leukaemia (CML) led to the rapid development of efficient targeted therapy and molecular follow-up, vastly improving survival for patients with CML during recent decades. The assessment of translocations, copy number changes and point mutations are crucial for the diagnosis and risk stratification of acute myeloid leukaemia and myelodysplastic syndromes. Still, the often heterogeneous and complex genetic landscape of haematological malignancies presents several challenges for the implementation of precision medicine to guide diagnosis, prognosis and treatment choice. This review provides an introduction and overview of the important molecular characteristics and methods currently applied in clinical practice to guide clinical decision making in haematological malignancies of myeloid and lymphoid origin. Further, experimental ways to guide the choice of targeted therapy for refractory patients are reviewed, such as functional precision medicine using drug profiling. An example of the use of pipeline studies where the treatment is chosen according to the molecular characteristics in rare solid malignancies is also provided. Finally, the future opportunities and remaining challenges of precision medicine in the real world are discussed.

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Despite improvement of current treatment strategies and novel targeted drugs, relapse and treatment resistance largely determine the outcome for acute myeloid leukemia (AML) patients. To identify the underlying molecular characteristics, numerous studies have been aimed to decipher the genomic- and transcriptomic landscape of AML. Nevertheless, further molecular changes allowing malignant cells to escape treatment remain to be elucidated. Mass spectrometry is a powerful tool enabling detailed insights into proteomic changes that could explain AML relapse and resistance. Here, we investigated AML samples from 47 adult and 22 pediatric patients at serial time-points during disease progression using mass spectrometry-based in-depth proteomics. We show that the proteomic profile at relapse is enriched for mitochondrial ribosomal proteins and subunits of the respiratory chain complex, indicative of reprogrammed energy metabolism from diagnosis to relapse. Further, higher levels of granzymes and lower levels of the anti-inflammatory protein CR1/CD35 suggest an inflammatory signature promoting disease progression. Finally, through a proteogenomic approach, we detected novel peptides, which present a promising repertoire in the search for biomarkers and tumor-specific druggable targets. Altogether, this study highlights the importance of proteomic studies in holistic approaches to improve treatment and survival of AML patients. Title in the list of papers of Svea Stratmann thesis: Proteogenomic analysis of relapsed acute myeloid leukemia in adults and childrenAuthors in the list of papers of Svea Stramann: S. Stratmann, M. Vesterlund, H.M. Umer, A. Skaftason, M. Krogh Herlin, C. Sundström, A. Eriksson, M. Höglund, J. Palle, J. Abrahamson, K. Jahnukainen, M. Cheng Munthe-Kaas, B. Zeller, K. Pokrovskaja Tamm, L. Cavelier, J. Lehtiö, L. Holmfeld

Germline pathogenic variants in

Germline pathogenic variants in RUNX1 are associated with familial platelet disorder with predisposition to myeloid malignancies (FPD/MM) with intragenic deletions in RUNX1 accounting for almost 7% of all reported variants. We present two new pedigrees with FPD/MM carrying two different germline RUNX1 intragenic deletions. The aforementioned deletions encompass exons 1-2 and 9-10 respectively, with the exon 9-10 deletion being previously unreported. RNA sequencing of patients carrying the exon 9-10 deletion revealed a fusion with LINC00160 resulting in a change in the 3 sequence of RUNX1. Expression analysis of the transcript isoform demonstrated altered RUNX1a/b/c ratios in carriers from both families compared to controls. Our data provide evidence on the impact of intragenic RUNX1 deletions on transcript isoform expression and highlight the importance of routinely performing copy number variant analysis in patients with suspected MM with germline predisposition. Funding Agencies|Lions Cancer Research Foundation, Uppsala

Clonal haematopoiesis of indeterminate potential (CHIP) may predispose for the development of therapy-related myeloid neoplasms (t-MN). Using target next-generation sequencing (t-NGS) panels and digital droplet polymerase chain reactions (ddPCR), we studied the myeloid gene mutation profiles of patients with chronic lymphocytic leukaemia (CLL) who developed a t-MN after treatment with chemo-(immuno)therapy. Using NGS, we detected a total of 30 pathogenic/likely pathogenic (P/LP) variants in 10 of 13 patients with a t-MN (77%, median number of variants for patient: 2, range 0–6). The prevalence of CHIP was then backtracked in paired samples taken at CLL diagnosis in eight of these patients. Six of them carried at least one CHIP-variant at the time of t-MN (median: 2, range: 1–5), and the same variants were present in the CLL sample in five cases. CHIP variants were present in 34 of 285 patients from a population-based CLL cohort, which translates into a significantly higher prevalence of CHIP in patients with a CLL who developed a t-MN, compared to the population-based cohort (5/8, 62.5% vs. 34/285, 12%, p=0.0001). Our data show that CHIP may be considered as a novel parameter affecting treatment algorithms in patients with CLL, and highlight the potential of using chemo-free therapies in CHIP-positive cases.

Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed.The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact.Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care.[https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

Myelodysplastic syndromes (MDS) are inherently resistant to chemotherapy leaving allogeneic stem cell transplantation (HCT) as the only curative treatment option. To date it remains unclear how to ...

Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy in clinical routine, and improved assay techniques are needed for broad adoption. We describe herein a highly sensitive and selective molecule amplification technology - superRCA assays - for rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples. Using a standard flow cytometer we demonstrate precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells against up to a 100,000-fold excess of DNA from normal cells in either bone marrow or peripheral blood, to follow the course of patients treated for acute myeloid leukemia (AML). We also demonstrate that sequence variants located in a high-GC region may be sensitively detected, and we illustrate the potential of the technology for early detection of disease recurrence as a basis for prompt change of therapy.

Angioimmunoblastic T-cell lymphoma (AITL) is characterized by poor prognosis and low response rate to conventional chemotherapy [1]. Genotyping has revealed a high frequency of mutations in genes i...

Precision diagnostics and therapy have been implemented rather early in clinical hematology due to the easy accessibility of blood and bone marrow, allowing not only for consecutive genetic analysis at diagnosis, remission and relapse, but also for culturing these cells and testing new drugs in vitro. One contributing factor has also been the relatively low number of »driver« mutations in hematologic malignancies and that some of them are gain of function mutations that are relatively easy to target by drugs. Examples of this development are ABL1-, JAK2-, and FLT3-inhibitors for the treatment of chronic myeloid leukemia, myeloproliferative neoplasms, and acute myeloid leukemia, respectively. More recently, gene panel sequencing has been introduced in clinical routine to identify genetic alterations with diagnostic, prognostic and predictive impact, and more sensitive techniques to monitor minimal residual disease are emerging. Whole genome and transcriptome sequencing are currently evaluated as the next diagnostic tool. Finally, a large number of targeted therapies are currently under development and/or undergoing clinical trials.

Background Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2, as well as Indoleamine 2,3-deoxygenase (IDO1) can be expressed both by tumor and microenvironmental cells and are crucial for tumor immune escape. We aimed to evaluate the role of PD-1, its ligands and IDO1 in a cohort of patients with primary diffuse large B-cell lymphoma of the CNS (PCNSL). Material and methods Tissue microarrays (TMAs) were constructed in 45 PCNSL cases. RNA extraction from whole tissue sections and RNA sequencing were successfully performed in 33 cases. Immunohistochemical stainings for PD-1, PD-L1/paired box protein 5 (PAX-5), PD-L2/PAX-5 and IDO1, and Epstein-Barr virus encoding RNA (EBER) in situ hybridization were analyzed. Results High proportions of PD-L1 and PD-L2 positive tumor cells were observed in 11% and 9% of cases, respectively. High proportions of PD-L1 and PD-L2 positive leukocytes were observed in 55% and 51% of cases, respectively. RNA sequencing revealed that gene expression of IDO1 was high in patients with high proportion of PD-L1 positive leukocytes (p = .01). Protein expression of IDO1 in leukocytes was detected in 14/45 cases, in 79% of these cases a high proportion of PD-L1 positive leukocytes was observed. Gene expression of IDO1 was high in EBER-positive cases (p = .0009) and protein expression of IDO1 was detected in five of six EBER-positive cases. Conclusion Our study shows a significant association between gene and protein expression of IDO1 and protein expression of PD-L1 in the tumor microenvironment of PCNSL, possibly of importance for prediction of response to immunotherapies. Title in Thesis: PD-L1 and IDO1 are important immunosuppressive molecules in primary diffuse large B-cell lymphoma of the CNS

Introduction One third of patients with myelodysplastic syndrome (MDS) will relapse after allogeneic stem cell transplantation (SCT), with a dismal prognosis. Early detection of relapse enables pre-emptive treatment and may potentially reduce relapse risk, but is limited by the lack of sensitive markers for minimal residual disease (MRD). We developed a pipeline where patient-specific mutations, as determined by a myeloid next generation sequencing (NGS) panel are tracked using sensitive digital droplet PCR (ddPCR). Method We designed a prospective Nordic study (NMDSG14B; NCT02872662) enrolling all patients with MDS, mixed MDS / MPN or AML with myelodysplasia related disease and < 30% marrow blasts undergoing SCT in the Nordic region. We hypothesized that personalized MRD detection by ddPCR can predict clinical relapse earlier than conventional methods. Patients were included before SCT and serial bone marrow samples were collected before, and 1 and 3 months post SCT, and thereafter every third month for 2 years or until relapse or death. Blood samples were collected monthly. The MRD results were not available for the treating physicians. MRD positivity was defined based on the background noise of the specific ddPCR-assays and varied between 0.05-0.1% VAF. Results Three-hundred and sixteen patients were screened between 2016 and 2020, of which 19 were excluded due to lack of mutation or disease progression preventing SCT. We here present data of 254 patients followed ≥ 6 months after SCT. Median age was 64 (18-78) years and 59% were male. Most WHO subgroups of MDS (n=166), MDS/MPN (n=39), AML (n=8) and therapy-related disease (n=41), were represented. Risk profile according to IPSS-R was very low (n=13), low (n=32), intermediate (n=46), high (n=60) and very high (n=32). The majority of patients received pre-SCT treatment consisting of HMA (n=159) and / or intensive chemotherapy (n=59) while 60 patients did not receive disease-modifying treatment prior to SCT. The most common mutations were ASXL1 (n=69), TET2 (n=58), SRSF2 (n=57) and TP53 (n=44). No mutation was identified in 10 pts, and NGS data is still pending for 11 patients. After a median follow-up of 436 days, estimated 2 years overall survival and relapse free survival were 72% and 63%, respectively. Cumulative incidence of NRM and relapse at 2 years was 16% and 20%, respectively. Forty-six patients relapsed after a median of 170 (53-733) days, and the estimated median survival following relapse was 197 days. The most common pre-SCT mutations in the relapsed cohort were TP53 (n=19), DNMT3A (n=11) and RUNX1 (n=9). Thirty-seven patients died due to non-relapse mortality (NRM) after a median of 83 (4-754) days. To date, MRD results are available for 64 patients. Relapse was preceded by positive MRD in 14 out of 15 patients a median of 79 (21-173) days before clinical relapse. The 15th patient had an extra-medullary relapse only. Borderline positive MRD samples < 0.2% VAF within 100 days after SCT followed by negative samples were seen in 11 non-relapse patients. Twenty-four of 37 patients in continuous complete remission (CCR) were consistently MRD neg. Six CCR patients had positive MRD after 100 days; two with transient borderline peaks ( 0.1%, which turned negative when the patients developed GVHD; one patient with slowly decreasing MRD which turned negative first after 1 y, and finally one patient with prevailing KIT mutation (>700 days post-SCT) despite negative BCOR and STAG2. Two MRD+ patients died from NRM without showing signs of clinical relapse. Discussion In summary, we show that our pipeline of personalized MRD-assessment, based on patient-specific mutations is feasible with a high sensitivity to predict relapse. An update of study progression will be presented at the meeting. Figure 1 Disclosures Illman: Sanofi-Genzyme: Other: Travel Support; Celgene: Other: Travel Support. Mielke:DNA Prime: Honoraria, Other: received via my institution , Speakers Bureau; KIADIS Pharma: Honoraria, Other: received via my institution , Speakers Bureau; Miltenyi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: received via my institution , Speakers Bureau; Kite/Gilead: Honoraria, Other: received via my institution , Speakers Bureau; Bellicum: Honoraria, Other: received via my institution, Speakers Bureau; Novartis: Honoraria, Other: received via my institution, Speakers Bureau; Celgene/BMS: Honoraria, Other: received via my institution , Speakers Bureau. Ebeling:Accord Healthcare: Other: Travel Support; Amgen: Other: Travel Support; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Otsuka Pharma Scandanavia AB: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Although copy number alterations (CNAs) and translocations constitute the backbone of the diagnosis and prognostication of acute myeloid leukemia (AML), techniques used for their assessment in routine diagnostics have not been reconsidered for decades. We used a combination of 2 next-generation sequencing–based techniques to challenge the currently recommended conventional cytogenetic analysis (CCA), comparing the approaches in a series of 281 intensively treated patients with AML. Shallow whole-genome sequencing (sWGS) outperformed CCA in detecting European Leukemia Net (ELN)–defining CNAs and showed that CCA overestimated monosomies and suboptimally reported karyotype complexity. Still, the concordance between CCA and sWGS for all ELN CNA–related criteria was 94%. Moreover, using in silico dilution, we showed that 1 million reads per patient would be enough to accurately assess ELN-defining CNAs. Total genomic loss, defined as a total loss ≥200 Mb by sWGS, was found to be a better marker for genetic complexity and poor prognosis compared with the CCA-based definition of complex karyotype. For fusion detection, the concordance between CCA and whole-transcriptome sequencing (WTS) was 99%. WTS had better sensitivity in identifying inv(16) and KMT2A rearrangements while showing limitations in detecting lowly expressed PML-RARA fusions. Ligation-dependent reverse transcription polymerase chain reaction was used for validation and was shown to be a fast and reliable method for fusion detection. We conclude that a next-generation sequencing–based approach can replace conventional CCA for karyotyping, provided that efforts are made to cover lowly expressed fusion transcripts.

Structural chromosomal rearrangements that can lead to in-frame gene-fusions are a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic leukemia (ALL). Traditional methods such as karyotyping and FISH struggle to accurately identify and phase such large-scale chromosomal aberrations in ALL genomes. We therefore evaluated linked-read WGS for detecting chromosomal rearrangements in primary samples of from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations and structural variants in the ALL genomes. We found that biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified previously unknown structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. We conclude that linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping and FISH.

BackgroundWe have evaluated an NGS-based method to detect recurrent gene fusions of diagnostic and prognostic importance in hematological malignancies. Our goal was to achieve a highly specific assay with a simple workflow, short turnaround time and low cost.MethodThe assay uses a commercially available anchored multiplex PCR panel for target enrichment and library preparation, followed by sequencing using a MiSeq instrument. The panel includes all recurrent gene fusions in AML and ALL and is designed to detect gene-specific fusions without prior knowledge of the partner sequence or specific break points. Diagnostic RNA samples from 27 cases with hematological malignancies encompassing 23 different transcript variants were analyzed. In addition, 12 cases from a validation cohort were assessed.ResultAll known fusion transcripts were identified with a high degree of confidence, with a large number of reads covering the breakpoints. Importantly, we could identify gene fusions where conventional methods had failed due to cryptic rearrangements or rare fusion partners. The newly-identified fusion partners were verified by RT-PCR and transcript-specific qPCR was designed for patient-specific follow-up. In addition, 12 cases were correctly assessed in a blind test, without prior knowledge of molecular cytogenetics or diagnosis.ConclusionIn summary, our results demonstrate that targeted RNA sequencing using anchored multiplex PCR can be implemented in a clinical laboratory for the detection of recurrent and rare gene fusions in hematological diagnostic samples.

The tumor cells in diffuse large B‐cell lymphomas (DLBCL) are considered to originate from germinal center derived B‐cells (GCB) or activated B‐cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC‐based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non‐GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression‐free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression‐free survival differed significantly. Importantly, patients assigned as non‐GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome.

Clonal haematopoiesis of indeterminate potential (CHIP) is defined by the detection of somatic mutations in genes recurrently mutated in myeloid neoplasms (MNs), in the blood of healthy individuals with normal blood values and lack of morphological evidence of MN. Recent studies have highlighted the potential association between CHIP and the development of MN, in particular therapy-related MN (t-MN) in patients with lymphoma treated with chemotherapy and/or autologous stem-cell transplantation. In the present study, we investigated whether the presence of CHIP is associated with a higher risk for the development of t-MN in patients with chronic lymphocytic leukemia (CLL) treated with chemo(immuno)therapy, including fludarabine and cyclophosphamide combinations. To this end, we studied 9 patients with CLL who developed a t-MN [acute myeloid leukemia (AML): n=6, myelodysplastic syndrome (MDS): n=3] after the administration of chemo(immuno)therapy (FCR: n=7, other, n=2), with available samples collected both before CLL treatment and at diagnosis of t-MN. The median interval between the two samples was 26 months (range: 9-38months). NGS was performed on DNA extracted from bone marrow mononuclear cells (MNCs) at t-MN diagnosis, using the Trusight Myeloid Sequencing Panel (n=4) and the Archer VariantPlex Myeloid kit (n=5). Backtracking of the variants detected at the t-MN phase was performed by NGS of DNA extracted from peripheral blood MNCs (n=8) or CD19+ selected cells (n=1) in the samples from the CLL phase. In case no variants were detected in the t-MN phase, targeted digital droplet PCR (ddPCR) was also performed in paired CLL samples to confirm the presence of the variants. Moreover, using the Trusight Myeloid Sequencing Panel, we evaluated the prevalence of CHIP in a population cohort of 285 patients with CLL at the time of diagnosis. The variant allele frequency (VAF) cut-off for the detection of the variants was set to 5%. Variants were reported if meeting the following criteria: (i) located within an exonic or splicing region; (ii) be non-synonymous; (iii) not listed in the gnomAD database, if not also recurrently reported in Cosmic v85. Overall, 16 variants were detected in 7/9 cases analyzed at the time of t-MN [NRAS (n=4), DNMT3A (n=3), TET2 (n=2), EH2 (n=2), TP53 (n=2), KRAS (n=1), U2AF1 (n=1) and SF3B1 (n=1)], while no variants were detected in 2 t-MN samples. In 6/7 cases with detectable variants at t-MN diagnosis, the same variants were present at the CLL phase with either lower (n=4) or similar (n=2) VAF. Overall, CHIP was detectable in 6/9 (66.7%) CLL patients who later developed a t-MN. Among the untreated CLL patients, 45 CHIP-related variants were detected in 35/285 cases (12%) as 7 patients harbored more than one variant. The median VAF was 12.7% (5.1-58.6%) with 27/45 (60%) having a VAF MTV and TP contributed equally. EF and PB contributed equally. Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scarfo:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees. Ghia:ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; Adaptive, Dynamo: Consultancy, Honoraria; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; MEI: Consultancy, Honoraria; Lilly: Consultancy, Honoraria; Celgene/Juno: Consultancy, Honoraria. Stamatopoulos:AstraZeneca: Honoraria; Janssen, Gilead, Abbvie: Honoraria, Research Funding.

Acute myeloid leukemia (AML) is the overall most common form of acute leukemia, characterized by a high relapse frequency and low long-term survival rate especially for adults. Even though children with AML have a better prognosis than adults, patients with recurrent AML - independent of age - show poorer overall survival, accelerated disease progression and they often do not respond to conventional treatment. Therefore, a more personalized treatment approach is required to prolong event free survival (EFS) and expand treatment options for relapse and primary resistant (R/PR) AML patients. During the last decade, AML research has largely been focused on improving diagnostic and prognostic tools in AML by investigating data derived from whole genome- and whole exome sequencing (WGS and WES, respectively), gene panels or differential expression analyses of individual genes and gene-fusions. Nevertheless, studies on relapsing AML incorporating WGS data are rare, although necessary to investigate the full repertoire of genetic aberrations underlying AML progression and therapy resistance. To this end, we applied a combination of WGS (WES for a subset of cases) and RNA-seq of longitudinal samples from 48 adult and 21 pediatric R/PR AML cases from the Nordic countries. These comprised tumor samples collected at diagnosis (n=49) and relapse (n=76), as well as PR specimens (n=6). Normal bone marrow (BM) derived stromal cells were cultivated from leukemic BM as a source of patient-matched constitutional DNA, while CD34+ BM cells from five healthy donors were used as a source of normal control RNA. Our findings reveal recurrent relapse specific mutations in CSF1R (2.9% of relapse cases) not previously reported in de novo AML, suggesting the use of receptor tyrosine kinase inhibitors as a novel therapeutic option for a subset of AML relapse cases. Further, we report specific differences in the mutational spectrum between adult and pediatric R/PR AML. In adults, we detected higher mutational frequencies of, for instance, ARID1A (6.3%), H3F3A (6.3%) and MGA (10.4%) compared with previous AML studies of only specimens from initial diagnosis, while these mutations were not seen in pediatric AML. In contrast, internal tandem duplications (ITDs) in UBTF were detected solely in pediatric relapsing AML (n=3 [14.3%]). IKZF1 was more frequently mutated in pediatric R/PR AML (14.3%) than previously reported (0.5-2.7%; Bolouri et al., Nat Med. 2018; Shiba et al., Br J Haematol, 2016). Also, differential gene expression analysis identified IKZF1 as downregulated in pediatric chemotherapy resistant samples in comparison with treatment responsive counterparts, independent of IKZF1 mutational status. By investigating differential gene expression patterns of longitudinal samples, we found lower expression of the complement inhibitor CR1/CD35 at relapse compared to their patient matched diagnostic samples in both adults and children. Additionally, IL1R1, encoding a key regulator of inflammation and immune response, was upregulated in both adult and pediatric diagnosis specimens from cases with short EFS, indicating a pronounced role of chronic inflammation during disease progression and AML cell survival. Finally, our findings reveal overexpression of GLI2 and SGMS2 among samples associated with short EFS. Overexpression of these genes may prevent excessive cell proliferation while increasing stemness and dormancy, leading to increased chemotherapy resistance and shorter EFS. Taken together, our results emphasize the advantage of applying a combination of WGS and RNA-seq, to be able to gain a more complete picture of alterations, including mutations, gene fusions and copy number alterations combined with gene expression analysis, when attempting to characterize AML at relapse. This is the first study of both adult and pediatric AML incorporating WGS and RNA-seq analyses on sequential AML samples. Knowledge gathered from this study has provided critical new insights into the biologic basis of this complex disease and will hopefully help to pave the way for improved and individualized treatment strategies. Disclosures No relevant conflicts of interest to declare.

Structural chromosomal rearrangements that may lead to in-frame gene-fusions represent a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic leukemia (ALL). However, short-read whole genome sequencing (WGS) technologies struggle to accurately identify and phase such large-scale chromosomal aberrations in cancer genomes. We therefore evaluated linked-read WGS for detection of chromosomal rearrangements in an ALL cell line (REH) and primary samples of varying DNA quality from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations (CNAs) and structural variants (SVs). Biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified additional structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. Thus, linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping or short-read WGS.

Induced pluripotent stem cells (iPSCs) have brought great promises for disease modeling and cell-based therapies. One concern related to the use of reprogrammed somatic cells is the loss of genomic integrity and chromosome stability, a hallmark for cancer and many other human disorders. We investigated 16 human iPSC lines reprogrammed by nonintegrative Sendai virus (SeV) and another 16 iPSC lines generated by integrative lentivirus for genetic changes. At early passages we detected cytogenetic rearrangements in 44% (7/16) of iPSC lines generated by lentiviral integration whereas the corresponding figure was 6% (1/16) using SeV-based delivery. The rearrangements were numerical and/or structural with chromosomes 5 and 12 as the most frequently involved chromosomes. Three iPSC lines with chromosome 5 aberrations were derived from one and the same donor. We present in this study the aberrant karyotypes including a duplication of chromosome 5q13q33 that restricts a candidate region for growth advantage. Our results suggest that the use of integrative lentivirus confers a higher risk for cytogenetic abnormalities at early passages when compared to SeV-based reprogramming. In combination, our findings expand the knowledge on acquired cytogenetic aberrations in iPSC after reprogramming and during culture.

The mouse Shb gene coding for the Src Homology 2-domain containing adapter protein B has recently been placed in context of BCRABL1-induced myeloid leukemia in mice and the current study was performed in order to relate SHB to human acute myeloid leukemia (AML). Publicly available AML databases were mined for SHB gene expression and patient survival. SHB gene expression was determined in the Uppsala cohort of AML patients by qPCR. Cell proliferation was determined after SHB gene knockdown in leukemic cell lines. Despite a low frequency of SHB gene mutations, many tumors overexpressed SHB mRNA compared with normal myeloid blood cells. AML patients with tumors expressing low SHB mRNA displayed longer survival times. A subgroup of AML exhibiting a favorable prognosis, acute promyelocytic leukemia (APL) with a PMLRARA translocation, expressed less SHB mRNA than AML tumors in general. When examining genes co-expressed with SHB in AML tumors, four other genes ( PAX5, HDAC7, BCORL1, TET1) related to leukemia were identified. A network consisting of these genes plus SHB was identified that relates to certain phenotypic characteristics, such as immune cell, vascular and apoptotic features. SHB knockdown in the APL PMLRARA cell line NB4 and the monocyte/macrophage cell line MM6 adversely affected proliferation, linking SHB gene expression to tumor cell expansion and consequently to patient survival. It is concluded that tumor SHB gene expression relates to AML survival and its subgroup APL. Moreover, this gene is included in a network of genes that plays a role for an AML phenotype exhibiting certain immune cell, vascular and apoptotic characteristics.

Between 1992 and 2008, 713 high hyperdiploid acute lymphoblastic leukemias in children aged 1–15 years were diagnosed and treated according to the Nordic Society for Pediatric Hematology and Oncology acute lymphoblastic leukemia 1992/2000 protocols. Twenty (2.8%) harbored t(1;19), t(9;22), der(11q23), or t(12;21). The median age of patients with “classic” high hyperdiploidy was lower than that of patients with translocation-positive high hyperdiploidy (P53/55 (P=0.020/0.024). In multivariate analyses, modal number and triple trisomies were significantly associated with superior event-free survival in separate analyses with age and white blood cell counts. When including both modal numbers and triple trisomies, only low white blood cell counts were significantly associated with superior event-free survival (P=0.009). We conclude that high modal chromosome numbers and triple trisomies are highly correlated prognostic factors and that these two parameters identify the same subgroup of patients characterized by a particularly favorable outcome.

PAX5-ESRRB is a recurrent fusion gene in B-cell precursor pediatric acute lymphoblastic leukemia

The translocation t(1;19)(q23;p13)/der(19) t(1;19) is a risk stratifying aberration in childhood B-cell precursor acute lymphoblastic leukaemia (BCP ALL) in the Nordic countries. We have identified ...

Background We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL. Results We used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (‘other’ subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5. Conclusions Our findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients. Electronic supplementary material The online version of this article (doi:10.1186/s13148-014-0039-z) contains supplementary material, which is available to authorized users.

The naturally occurring polyphenol resveratrol has been associated with the beneficial effects of red wine consumption on cardiovascular disease and shown to inhibit atherosclerosis in animal models. To determine if resveratrol affects the expression of genes that control lipid homeostasis in human macrophages, we measured expression changes in the LXR-alpha pathway, crucial to cholesterol efflux, and in genes that mediate lipoprotein uptake. Resveratrol treatment of THP-1 macrophages induced LXR-alpha at mRNA and protein levels. Increased recruitment of RNA polymerase II to the LXR-alpha promoter suggested that up-regulation was at least partly mediated by transcriptional mechanisms. Resveratrol also induced LXR-alpha in human monocyte-derived macrophages together with elevated ABCA1 and ABCG1 mRNA levels. Moreover, resveratrol repressed the expression of the lipid uptake genes LPL and SR-AII. The ability of resveratrol to modulate expression of the genes involved in lipid uptake and efflux suggests that polyphenols can potentially limit cholesterol accumulation in human macrophages.