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Funding Information: This study was supported by the Cancer Foundation Finland, the Helsinki Institute of Life Science (HiLIFE) Fellow grants, the Academy of Finland (grant 1320185 ), the Sigrid Jusélius Foundation , the Finnish Cancer Institute , the Finnish Medical Foundation , University of Helsinki , and the Finnish special governmental subsidy for health sciences, research, and training . The FIMM High Throughput Biomedicine Unit is financially supported by the University of Helsinki (HiLIFE) and Biocenter Finland. O.D. was supported by the K. Albin Johansson Foundation , the Emil Aaltonen Foundation , and the Juhani Aho foundation . Publisher Copyright: © 2023 The American Society of Hematology Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL–selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

In adult patients, the treatment outcome of acute lymphoblastic leukemia (ALL) remains suboptimal. Here, we used an ex vivo drug testing platform and comprehensive molecular profiling to discover new drug candidates for B-ALL. We analyzed sensitivity of 18 primary B-ALL adult patient samples to 64 drugs in a physiological concentration range. Whole-transcriptome sequencing and publicly available expression data were used to examine gene expression biomarkers for observed drug responses. Apoptotic modulators targeting BCL2 and MDM2 were highly effective. Philadelphia chromosome-negative (Ph-) samples were sensitive to both BCL2/BCL-W/BCL-XL-targeting agent navitoclax and BCL2-selective venetoclax, whereas Ph-positive (Ph+) samples were more sensitive to navitoclax. Expression of BCL2 was downregulated and BCL-W and BCL-XL upregulated in Ph+ ALL compared with Ph- samples, providing elucidation for the observed difference in drug responses. A majority of the samples were sensitive to MDM2 inhibitor idasanutlin. The regulatory protein MDM2 suppresses the function of tumor suppressor p53, leading to impaired apoptosis. In B-ALL, the expression of MDM2 was increased compared with other hematological malignancies. In B-ALL cell lines, a combination of BCL2 and MDM2 inhibitor was synergistic. In summary, antiapoptotic proteins including BCL2 and MDM2 comprise promising targets for future drug studies in B-ALL.

To integrate mRNA and miRNA expression profiles of mucoepidermoid carcinomas (MECs) and normal salivary gland (NSGs) tissue samples and identify potential drivers.Gene and miRNA expression arrays were performed in 35 MECs and six NSGs.We found 46 differentially expressed (DE) miRNAs and 3,162 DE mRNAs. Supervised hierarchical clustering analysis of the DE transcripts revealed two clusters in both miRNA and mRNA profiles, which distinguished MEC from NSG samples. The integrative miRNA-mRNA analysis revealed a network comprising 696 negatively correlated interactions (44 miRNAs and 444 mRNAs) involving cell signaling, cell cycle, and cancer-related pathways. Increased expression levels of miR-205-5p and miR-224-5p and decreased expression levels of miR-139-3p, miR-145-3p, miR-148a-3p, miR-186-5p, miR-338-3p, miR-363-3p, and miR-4324 were significantly related to worse overall survival in MEC patients. Two overexpressed miRNAs in MEC (miR-22 and miR-205) were selected for inhibition by the CRISPR-Cas9 method. Cell viability, migration, and invasion assays were performed using an intermediate grade MEC cell line. Knockout of miR-205 reduced cell viability and enhancedThe

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm accounting for ∼15% of all leukemia. Progress of the disease from an indolent chronic phase to the more aggressive accelerated phase or blast phase (BP) occurs in a minority of cases and is associated with an accumulation of somatic mutations. We performed genetic profiling of 85 samples and transcriptome profiling of 12 samples from 59 CML patients. We identified recurrent somatic mutations in ABL1 (37%), ASXL1 (26%), RUNX1 (16%), and BCOR (16%) in the BP and observed that mutation signatures in the BP resembled those of acute myeloid leukemia (AML). We found that mutation load differed between the indolent and aggressive phases and that nonoptimal responders had more nonsilent mutations than did optimal responders at the time of diagnosis, as well as in follow-up. Using RNA sequencing, we identified other than BCR-ABL1 cancer-associated hybrid genes in 6 of the 7 BP samples. Uncovered expression alterations were in turn associated with mechanisms and pathways that could be targeted in CML management and by which somatic alterations may emerge in CML. Last, we showed the value of genetic data in CML management in a personalized medicine setting.

The oncogenic protein Bcr-Abl has two major isoforms, p190Bcr-Abl and p210Bcr-Abl. While p210Bcr-Abl is the hallmark of chronic myeloid leukemia (CML), p190Bcr-Abl occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) patients. In CML, p190Bcr-Abl occurs in a minority of patients associating with distinct hematological features and inferior outcomes, yet the pathogenic role of p190Bcr-Abl and potential targeting therapies are largely uncharacterized. We employed next generation sequencing, phospho-proteomic profiling, and drug sensitivity testing to characterize p190Bcr-Abl in CML and hematopoietic progenitor cell line models (Ba/f3 and HPC-LSK). p190Bcr-Abl CML patients demonstrated poor response to imatinib and frequent mutations in epigenetic modifiers genes. In contrast with p210Bcr-Abl, p190Bcr-Abl exhibited specific transcriptional upregulation of interferon, interleukin-1 receptor, and P53 signaling pathways, associated with hyperphosphorylation of relevant signaling molecules including JAK1/STAT1 and PAK1 in addition to Src hyperphosphorylation. Comparable to p190Bcr-Abl CML patients, p190Bcr-Abl cell lines demonstrated similar transcriptional and phospho-signaling signatures. With the drug sensitivity screening we identified targeted drugs with specific activity in p190Bcr-Abl cell lines including IAP-, PAK1-, and Src inhibitors and glucocorticoids. Our results provide novel insights into the mechanisms underlying the distinct features of p190Bcr-Abl CML and promising therapeutic targets for this high-risk patient group.

The

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies., Article, NATURE COMMUNICATIONS.9:1567(2018)

Only half of chronic myeloid leukemia (CML) patients in deep molecular remission are able to maintain treatment free remission (TFR) after tyrosine kinase inhibitor (TKI) discontinuation. Identifying predictive markers for TFR remains a key issue. The immunological control or eradication of TKI insensitive leukemic stem cells may contribute to successful cessation, as recent work has demonstrated that differences in immune cell compositions can be associated with better probability of TFR. In order to understand the precise immunological changes in CML, we profiled over 170,000 cells from 25 CML samples from different clinical phases (untreated, during TKI therapy and after TKI cessation) using single-cell RNA and T cell receptor (TCR) αβ sequencing (scRNA+TCRab-seq). We profiled both CD45+ sorted peripheral blood samples (n=20) and CD34+ sorted bone marrow samples (n=5). To understand antigen-specific responses, we profiled TCRs specific to tumor-antigen PR1 (n=12) and compared these to unsorted TCRβ-sequenced samples from CML (n=137) and healthy donors (n=786). To understand the distinctive immunological features in CML, we compared the immune system in CML (n=20) to those in other hematological and solid malignancies profiled with scRNA+TCRab-seq (n=9). We discovered NK cells to be the most unique feature in CML, as NK CD56dim cells were more abundant in CML patients (Padj We sought to understand whether the immunological activity differs before and after TKI discontinuation in different clinical outcomes following discontinuation. Therefore, we reclustered the discontinuation samples from three different outcomes: TFR, early relapse (6 months). In all clinical outcomes, TKI cessation invigorated NK cell exhaustion and the most upregulated pathways included IFNg and TNFa via NFkB pathways. Patients with successful TFR had less differences in their immune system after cessation in comparison to relapsing patients, which could hint that their immune subsets were better prepared for the cessation. Unlike the early relapse patients' quiescent immune cells, the late relapse patients' immune subsets were active, but under pronounced inhibitory Treg signals, providing a clinically interesting approach to alter the outcome of these patients. Next, to understand how malignant CML cells evade or interact with the immune system, we analyzed CD34+ cells and calculated a BCR-ABL1 activity score. The most primitive CD34+ cells had lower BCR-ABL1 score and not as many immunological ligand-receptor pairs as their less primitive, highly BCR-ABL1 pathway expressing, counterparts (P Finally, we found that tumor-antigen PR1 is preferentially expressed on CD34+ cells with high BCR-ABL1 score. Most TCRs recognizing PR1 are restricted to individuals, but they share short amino-acid motifs that are shared across patients. Thus, we were able to generate an in silico tool to recognize PR1-specific TCRs from unsorted TCR-seq samples with high accuracy (AUC 0.91 from 10-fold cross validation). With our tool, we were able to show that anti-PR1 responses were more frequent in CML than in healthy donors (P In conclusion, our results provide high-resolution insights into anti-leukemic immune responses in CML and how we could harness and monitor them to enable successful TKI cessations. Figure Disclosures Olsson-Strömberg: Pfizer: Research Funding. Hjorth-Hansen:Pfizer: Honoraria, Research Funding; Austrian Orphan Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Mustjoki:Pfizer: Research Funding; Novartis: Research Funding; BMS: Honoraria, Research Funding.

BACKGROUND Wilms Tumor 1 (WT1) and Survivin genes are important leukemia-associated antigens (LAAs) in AML with potential prognostic impact. METHODS We investigated WT1 and Survivin expression levels by RT-PCR in 61 AML patients in correlation with clinical characteristics and outcomes. RESULTS WT1 was overexpressed in 45 patients (73.8%), associated with higher BM blasts (p = 0.017), lower incidence of favorable-prognosis cytogenetics (p = 0.035), and higher incidence of Flt3-ITD mutations (p = 0.026). Survivin was overexpressed in 17 patients (27.9%) with higher mean WBC count (p = 0.049). Patients with overexpression of either gene showed inferior complete remission (CR) rates and survival rates, patients with overexpression of both genes showed higher mean WBCs (p = 0.035) and higher BM blasts (p = 0.029) while the double negative group showed higher incidence of favorable cytogenetic events (p = 0.021), better CR rates and survival rates. CONCLUSIONS Our findings support the introduced prognostic impact of WT1 and Survivin genes in AML patients and its potential use in MRD monitoring and immunotherapy.

Telomere length (TL) in peripheral blood (PB) cellsofpatientswith chronic myeloid leukemia(CML) has been shown to correlate with disease stage, prognostic scores, response to therapy, and disease progression. However, due to considerable genetic interindividual variability, TL varies substantially between individuals, limiting its use as a robust prognostic marker in individual patients. Here, we compared TL of BCR-ABL(-), nonleukemic CD34(+)CD38(-) hematopoietic stem cells (HSC) in the bone marrow of CML patients at diagnosis to their individual BCR-ABL(+) leukemic stem cell (LSC) counterparts. We observed significantly accelerated telomere shortening in LSC compared with nonleukemic HSC. Interestingly, the degree of LSC telomere shortening was found to correlate significantly with the leukemic clone size. To validate the diagnostic value of nonleukemic cells as internal controls and to rule out effects of tyrosine kinase inhibitor (TKI) treatment on these nontarget cells, we prospectively assessed TL in 134 PB samples collected in deep molecular remission after TKI treatment within the EURO-SKI study (NCT01596114). Here, no significant telomere shortening was observed in granulocytes compared with an age-adjusted control cohort. In conclusion, this study provides proof of principle for accelerated telomere shortening in LSC as opposed to HSC in CML patients at diagnosis. The fact that the degree of telomere shortening correlates with leukemic clone's size supports the use of TL in leukemic cells as a prognostic parameter pending prospective validation. TL in nonleukemic myeloid cells seems unaffected even by long-term TKI treatment arguing against a reduction of telomere-mediated replicative reserve in normal hematopoiesis under TKI treatment.

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is

BACKGROUND FLT3-ITD mutations are common in AML subgroups, particularly in Acute Promyelocytic Leukemia (APL). It infers poor prognosis in AML patients; however, its prognostic value in APL is still controversial. We aimed to assess the distribution and prognostic value of FLT3-ITD mutation within AML subgroups, focusing on APL. METHODS In NCI, Cairo University, 346 newly diagnosed AML patients were included. Morphological, immunophenotypic and cytogenetic analysis were done at presentation and fixed follow-up points with monitoring in follow up visits of patients. FLT3-ITD mutations were detected using RT-PCR. RESULTS FLT3-ITD mutations were detected in 18.5% of AML patients with significant higher incidence in M3 patients (35.5%, p = 0.027) in comparison to other types. There was significant negative association between FLT3ITD mutations and CD34 expression (p = 0.026). FLT3 wild patients had a significantly better response on Day 35 than the mutant group (p = 0.046). Overall Survival (OS) of the wild group was significantly better than that of the mutant group (p = 0.003). In APL patients, OS of wild-FLT3 patients was significantly better than of those with mutant FLT3 (p = 0.018). In non-APL patients, favorable cytogenetic changes - t(8;21) and inv(16) - are more common in FLT3 wild group (12.8%) than in FLT3 mutant group (6.3%). In non-APL patients, FLT3-ITD mutation retained its adverse prognostic effect (p = 0.016). CD34 expression affected survival in both FLT3 mutant and wild groups. CONCLUSIONS We conclude that FLT3-ITD mutations infer poor prognostic effects both in AML patients generally and in the APL group specifically. CD34 may have a prognostic impact in FLT3-ITD mutant patients, which is to be further investigated.

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

Somatic mutations have a central role in cancer but their role in other diseases such as autoimmune disorders is poorly understood. Earlier work has provided indirect evidence of rare somatic mutations in autoreactive T-lymphocytes in multiple sclerosis (MS) patients but such mutations have not been identified thus far. We analysed somatic mutations in blood in 16 patients with relapsing MS and 4 with other neurological autoimmune disease. To facilitate the detection of somatic mutations CD4 +, CD8 +, CD19 + and CD4 −/CD8 −/CD19 − cell subpopulations were separated. We performed next-generation DNA sequencing targeting 986 immune-related genes. Somatic mutations were called by comparing the sequence data of each cell subpopulation to other subpopulations of the same patient and validated by amplicon sequencing. We found non-synonymous somatic mutations in 12 (60%) patients (10 MS, 1 myasthenia gravis, 1 narcolepsy). There were 27 mutations, all different and mostly novel (67%). They were discovered at subpopulation-wise allelic fractions of 0.2%–4.6% (median 0.95%). Multiple mutations were found in 8 patients. The mutations were enriched in CD8 + cells (85% of mutations). In follow-up after a median time of 2.3 years, 96% of the mutations were still detectable. These results unravel a novel class of persistent somatic mutations, many of which were in genes that may play a role in autoimmunity (ATM, BTK, CD46, CD180, CLIP2, HMMR, IKFZF3, ITGB3, KIR3DL2, MAPK10, CD56/NCAM1, RBM6, RORA, RPA1 and STAT3). Whether some of this class of mutations plays a role in disease is currently unclear, but these results define an interesting hitherto unknown research target for future studies., Highlights • Nonsynonymous somatic mutations were detectable in the blood in more than half of the patients in this study. • The mutant clones were rare, allelic fractions mostly

// Ahmed Al-Samadi 1 , Shady Adnan Awad 2, 3, * , Katja Tuomainen 1, 4, * , Yue Zhao 1 , Abdelhakim Salem 5 , Mataleena Parikka 6, 7 and Tuula Salo 1, 8, 9 1 Department of Oral and Maxillofacial Diseases, Clinicum, University of Helsinki, Helsinki, Finland 2 Hematology Research Unit, Department of Hematology, University of Helsinki and Helsinki University Central Hospital Comprehensive Cancer Center, Helsinki, Finland 3 Clinical Pathology Department, National Cancer Institute, Cairo University, Cairo, Egypt 4 Department of Otorhinolaryngology, Helsinki University Hospital, Helsinki, Finland 5 Department of Internal Medicine, Clinicum, University of Helsinki, Helsinki, Finland 6 Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland 7 Oral and Maxillofacial Unit, Tampere University Hospital, Tampere, Finland 8 Cancer and Translational Medicine Research Unit, University of Oulu, Oulu, Finland 9 Medical Research Center, Oulu University Hospital, Oulu, Finland * These authors have contributed equally in this work Correspondence to: Ahmed Al-Samadi, email: ahmed.al-samadi@helsinki.fi Keywords: tongue cancer, immune cells, extracellular vesicles, in vitro model, cytotoxicity Received: December 14, 2016 Accepted: April 19, 2017 Published: May 10, 2017 ABSTRACT The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8 + T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8 + T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.

The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8+ T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8+ T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.

Despite the advances in the treatment of acute lymphoblastic leukemia (ALL), a major fraction of adult patients still succumb to leukemia- or treatment-related events. In particular, the outcome of elderly ALL patients remains dismal. Our aim was to discover new or repurposed drugs for B-cell ALL in a clinically relevant ex vivo drug sensitivity testing platform. We analyzed 19 primary B-ALL samples using a well-established drug sensitivity and resistance testing platform and a drug panel including 65 drugs in five different concentrations. The main drug classes were glucocorticoids, MDM2 antagonists, and inhibitors of BCR-ABL1, VEGFR, BCL-2, BCL-XL, BET, MEK, JAK, Aurora kinase, PI3K, MTOR, IGF1R, ERK, STAT3, STAT5, HSP90 and NAMPT proteins. The samples were viably frozen bone marrow (BM) mononuclear cells collected at diagnosis. The cohort included both Philadelphia-positive (Ph+) (n=10) and Ph-negative (Ph-) (n=9) patients with a median age of 43 years (range 22-68). Cell viability (CellTiter-Glo) was measured after plating and after a three-day incubation with the drugs. A drug sensitivity score (DSS) was calculated from the viability readouts, which takes into account the area under the dose response curve, measuring both drug efficacy and potency. DSS values >10 are considered effective and >20 highly effective. As an overall view of drug sensitivity, a heatmap and dendrograms from DSS values are shown in Figure 1A. As expected, most patients were sensitive to glucocorticoids and tyrosine kinase inhibitors (TKIs) showed efficacy in Ph+ ALL. In addition, two Ph-negative patients were sensitive to TKIs, suggesting a Philadelphia-like disease. Drugs that showed pan-ALL efficacy included BCL-2 family inhibitors, idasanutlin (MDM2 inhibitor), luminespib (HSP90 inhibitor), daporinad (NMPRT inhibitor) and plicamycin (antineoplastic antibiotic). For the other drugs, only individual patients showed sensitivity, in line with the diverse molecular background of ALL. Strikingly, 17/19 (89%) of patients in our cohort were highly sensitive (DSS>20) to navitoclax (a BCL-2, BCL-XL and BCL-W inhibitor), whereas the BCL-2-specific inhibitor venetoclax was effective only in a distinct subset of patients (Figure 1B). 6/19 (32%) of patients were highly sensitive (DSS>20) to venetoclax and represented all risk classes based on age, white blood cell counts and karyotype, but interestingly, all were Ph-negative. Overall, response to venetoclax correlated with response to navitoclax (Spearman, r=0.85; P To examine differential gene expression of anti-apoptotic proteins between Ph+ and Ph- patients, we analyzed microarray gene expression data from ArrayExpress public database (www.ebi.ac.uk/arrayexpress, E-MTAB-5035). The analyzed cohort included 96 Ph- and 41 Ph+ adult B-ALL patients. Ph-negative samples were characterized with higher BCL-2 expression, whereas Ph-positive samples showed higher BCL-W expression and a trend to higher BCL-XL expression (Figure 1C). Thus, lack of venetoclax efficacy ex vivo in Ph-positive ALL indicated dependence on BCL-W and BCL-XL, as also reflected in the gene expression analyses. Inhibitors of BCL-2, such as navitoclax and venetoclax, potently induce apoptosis in a variety of cancer cells. Both inhibitors showed promising efficacy in our B-ALL samples. Dose-limiting thrombocytopenia has limited the use of navitoclax in solid tumors. However, in our assay navitoclax showed more uniform potency, particularly in Ph+ samples suggesting a rational combination with tyrosine kinase inhibitors. Similar to conventional cytotoxic agents used in ALL, a therapeutic window may exist for safe use of navitoclax in acute leukemia. In conclusion, targeting the multidomain anti-apoptotic proteins (BCL-2, BCL-XL, BCL-W, MCL-1) and TP53 with MDM2, possibly in combination, is a promising strategy for improving outcome of adult B-ALL. Figure 1. Figure 1. Disclosures Hohtari: Incyte: Research Funding. Heckman:Novartis: Research Funding; Celgene: Research Funding; Orion Pharma: Research Funding. Wennerberg:Novartis: Research Funding. Mustjoki:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Porkka:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Background Identification of prognostic factors in acute lymphoblastic leukemia (ALL) patients is important for stratifying patients into risk groups and tailoring treatment accordingly. Molecular and cytogenetic abnormalities are the most important prognostic factors. Minimal residual disease (MRD) is also an important predictor of relapse in ALL. However, the correlation of both prognostic variables has not been thoroughly studied. Methods We investigated the correlation between defined cytogenetic abnormalities and selected new MRD markers (CD79b, CD123, and CD200) in 56 newly diagnosed Egyptian pediatric B-cell ALL patients. Results CD123 found to be expressed in 45% of patients, CD200 in 80.3%, and CD79b in 67.9%. MRD analysis during treatment showed stable expression patterns of CD200. There was significant association of CD123 expression with the hyperdiploid ALL group ( P = .017). Another association ( P = .029) was found between CD79b negativity and the t(12;21) group. CD200 was widely expressed in all groups. Conclusion There is a significant correlation between some markers, and certain ALL recurrent cytogenetic subgroups (CD123 and hyperdiploidy, CD79b negativity, and ETV-RUNX1 group) have good prognostic value. CD200 can be used as MRD markers in ALL patients and can also can serve as therapy targets.

Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukaemia have been reported to induce immunomodulatory effects. We aimed to assess peripheral blood (PB) and bone marrow (BM) lymphocyte status at the diagnosis and during different TKI therapies and correlate it with treatment responses. BM and PB samples were acquired from 105 first-line TKI-treated patients. Relative number of BM lymphocytes was evaluated from MGG-stained BM aspirates, and immunophenotypic analyses were performed with multicolour flow cytometry. Early 3-month expansion of BM lymphocytes was found during all different TKIs (imatinib n = 71, 20 %; dasatinib n = 25, 21 %; nilotinib n = 9, 22 %; healthy controls n = 14, 12 %, p 10 % (23 vs. 17 %, p

Introduction: Chronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the bcr-abl translocation. Recent data provides evidence that CML chronic phase (CP) can be classified into early and late CP depending on the degree of expansion of the leukemic stem cell (LSC) clone. Patients in late CP have a higher LSC burden going along with an inferior response to TKI therapy. Telomeres shorten with each cell division and telomere length (TL) reflects the replicative history of a cell. We postulate that the LSC burden correlates with accelerated telomere shortening due to clonal replicative expansion of bcr-abl positive cells. Previous studies evaluating TL in peripheral blood cells of CML patients already revealed a correlation of age-adapted TL with disease stage, response to treatment and duration of CP. However, the high intra-individual, mostly genetic inter-individual variability in TL limits the predictive value of TL measurements when no patient specific bcr-abl negative cells were available for comparison. The aim of our study was to analyze TL in the LSC and non-clonal HSC compartment of patients with CP using a modified Q-FISH technique allowing the TL analysis in bcr-abl positive and negative cells. Methods and Patients: 11 patients diagnosed with CML in CP of the NCT00852566 study (Nordic CML Group) were included into our retrospective analysis. Mean age of the patients was 58.9 years. Bone marrow samples from initial diagnosis were sorted for CD34+/CD38- cells reflecting the leukemic stem cell compartment. Samples were analyzed with the FISH method using dual fusion dual color BCR-ABL1 probe following standard procedures. After capturing the bcr-abl staining using confocal microscopy, samples were re-processed for TL analysis by Q-FISH using established protocols. Optimized microscopy techniques allowed for TL to be assessed in all previously captured cells allowing the identification of bcr-abl positive and negative cells within the same sample. Analysis and quantification of bcr-abl FISH staining and TL measurement by Q-FISH were performed in single-blinded fashion. Results: Bcr-abl negative cells represent the non-clonal hematopoiesis and were used to correct TL. We observed significant shortened TL in the bcr-abl positive cells compared to bcr-abl negative cells (-2.18 ± 2.08 kilobases (kb), p=0.01) in line with previously published results. Next, we correlated the clone size (i.e. the proportion of bcr-abl positive cells) with the degree of telomere shortening in the leukemic stem cell compartment. Mean clone size of the patients was 67.4 ± 21.7 % S.D. Despite of the relatively small sample size studied, we found a significant negative correlation (R²=0.45, p=0.04) between TL and clone size strongly supporting the notion that increased expansion of the bcr-abl positive LSC pool leads to accelerated telomere shortening. Conclusions: In this study, we provide evidence for accelerated telomere shortening in bcr-abl positive LSC as compared to their normal CD34+/CD38- counterpart in CP CML samples at diagnosis. Furthermore, the degree of TL shortening correlates with the clone size in the HSC compartment, i.e. a parameter that reflects duration of CP. Thus, this allows the discrimination of early vs. late CP and might as such be used as a prognostic (and potentially predictive) biomarker in CML. Disclosures Richter: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Fioretos:Cantargia: Equity Ownership. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Brümmendorf:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties.

Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

INTRODUCTION Natural killer (NK) cell malignancies are rare aggressive neoplasms that are classified by the WHO as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK-cell leukemia (ANKL). Recently, genome and exome level studies in NKTCL have shed light on the mutational spectrum of the disease. However, somatic mutations in ANKL have not been characterized. Here, we identified somatic mutations in 14 cases of ANKL to further clarify the genetic landscape underlying malignant NK cell proliferation. We compared the discovered variants to those detected in NKTCL to understand whether the two diseases harbor common molecular alterations. Moreover, we used high-throughput drug screening and RNA sequencing on NK cell lines derived from ANKL, NKTCL and other malignant NK cell proliferations to identify therapeutically actionable drivers of malignant NK cell growth. METHODS We performed whole-exome sequencing on genomic DNA extracted from peripheral blood or bone marrow samples of 14 ANKL patients. To compare the mutational profiles in ANKL and NTKCL, we re-analyzed the published whole-exome NKTCL data from Jiang et al. (Nat Genet 2015) using our somatic variant calling pipeline. For profiling of drug responses, we used a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs to screen the ANKL cell lines IMC-1, KHYG-1 and NK-92, NKTCL cell lines NK-YS and SNK-6 as well as DERL-7, KAI3, NKL, YT and IL-2-stimulated NK cells from healthy donors. All drugs were tested in 384-well format in 5 concentrations over a 10,000-fold concentration range for 72 h, cell viability was measured and normalized dose response curve values were used to calculate a drug sensitivity score (DSS) for each drug. Finally, we performed amplicon sequencing of known cancer driver genes and RNA sequencing on the cell lines and healthy NK cells to identify driver mutations and integrate gene expression profiles with drug sensitivity patterns. RESULTS We identified recurrent somatic activating mutations in STAT3 in 21% (3 of 14) of ANKL patients. Other mutated genes included RAS-MAPK pathway molecules (BRAF, NRAS, KRAS), protein phosphatases regulating JAK-STAT and PI3K-AKT-mTOR pathways (PTPRT, PTPRK, INPP5D) as well as several epigenetic modifiers (TET2, ARID2, KDM2B, SETD7, SETD2) and the tumor suppressor TP53. Interestingly, we detected mutations in genes recurrently mutated in NKTCL, such as the RNA helicase DDX3X and the cell surface receptor FAS. Re-analysis of the published NKTCL data revealed a high frequency of missense mutations in receptor type and non-receptor type protein phosphatases (e.g. PTPRC, PTPRR, PTPRT, PTPN1, PTPN2, PTPN3), many of which have established roles as negative regulators of JAK-STAT signaling. These findings potentially expand the subset of NK cell tumors where the JAK-STAT pathway is somatically activated and implicate deregulated JAK-STAT signaling as a major driver in these diseases. The malignant NK cell lines used in drug sensitivity profiling frequently harbored mutations in same genes and pathways, including STAT3 (N=3), STAT5B (N=1), DDX3X (N=2), KRAS (N=1), FAS (N=2) and several epigenetic modifiers, thus validating these cell lines as relevant disease models. The drug sensitivities in NK cell lines showed a high correlation and the cell lines formed a distinct group from other lymphoid and myeloid leukemia cell lines in unsupervised hierarchical clustering, suggesting an NK-cell specific drug response pattern. The most effective targeted drugs across all NK cell lines included HDAC inhibitors, inhibitors of Aurora and Polo-like kinases, JAK inhibitors, HSP inhibitors and CDK inhibitors as well as the Bcl-2 inhibitor navitoclax. Compared to other leukemia and lymphoma cell lines, JAK inhibitors, navitoclax and methotrexate emerged as the most NK-cell specific compounds. CONCLUSIONS Our genetic data demonstrate extensive heterogeneity in the mutational spectrum of ANKL and implicate JAK-STAT and RAS-MAPK signaling as well as disruption of epigenetic modifiers in the disease pathogenesis. Integrated drug sensitivity and gene expression profiling corroborates the JAK-STAT pathway as a major therapeutically actionable driver of malignant NK cell proliferation and identifies other potential novel targeted therapy options such as Bcl-2 inhibition in NK cell malignancies. Disclosures Suzumiya: Chugai: Honoraria, Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko kirin: Research Funding; Astellas: Research Funding; Eisai: Honoraria, Research Funding; Takeda: Honoraria. Ohshima:Chugai: Research Funding, Speakers Bureau; Kyowa Kirin: Research Funding, Speakers Bureau. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

Introduction: Progression of chronic myeloid leukemia (CML) to blast crisis (BC) results from the acquisition of additional driver mutations, which still are poorly understood. In the tyrosine kinase inhibitor (TKI) era, BC-CML remains a challenging clinical entity with very poor prognosis and short survival. In addition, there is an unmet need for identification of progression-related genetic mutations and potentially targetable pathways. Patients and methods: Bone marrow samples from 16 patients with BC-CML (myeloid n=13, lymphoid n=2, unknown phenotype n=1) and 2 patients with accelerated phase (AP) were collected from the Helsinki University Hospital, Finland and National Cancer Institute, Cairo University, Egypt. In addition, skin biopsy samples were collected for germline variant controls. Whole exome sequencing (WES) (n=8) was done with Agilent or NimbleGen exome capture kits and deep-targeted sequencing with the NimbleGen (SeqCap EZ Design) comprehensive cancer gene panel (n=10) using an Illumina HiSeq instrument. The panel was comprised of 578 driver genes with documented association to common and rare cancers (gathered from Sanger and NCBI tests databases). All mutated genes identified by WES were included in the panel, as well as genes previously reported to be mutated in BC-CML. Results: We identified 55 mutations in 33 driver genes (average: 3 per patient, range 0-7). Of the identified 33 mutated genes, 27 were ranked ≥1 in the Gene Ranker Cancer scoring system (http://cbio.mskcc.org/tcga-generanker), where genes with a score of 1 have a documented association with cancer in a cancer gene database and higher scores indicate more frequent incidence of gene mutations in different cancers. Core-binding factor (CBF) aberrations (RUNX1 mutations and inv [16]) were the most recurrent variants (n=6 in 5 patients, 27.7% of the patients) followed by ABL1 mutations (n=4 in 4 patients, 22%) and BCOR mutations (n=4 in 3 patients, 16.7%). Other recurrent mutations included FLT3, IKZF1, and NOTCH1 mutations which all were found in 2 cases. Some of the discovered mutations have not been reported in BC-CML patients before, such as mutations to MTOR, PTPRJ, CD274 (PD-L1), IL21R, SETD2 and ZRSR2 genes. In silico analysis of the targeted genes showed that many of the affected genes interact with each other in different pathways and also with ABL1. The top pathways affected were associated with key biological functions: regulation of hematopoiesis (11 genes affected), leucocyte differentiation (9 genes) and transcriptional regulation (11 genes). Conclusion: The genomic landscape of advanced phases of CML (BC and AP) shows complex heterogeneity with a broad range of genes affected leading to dysregulation of multiple molecular pathways that have an impact on treatment responses and disease biology. Such complexity suggests that a personalized approach maybe the best treatment option for these patients. Disclosures Heckman: Celgene: Research Funding; Pfizer: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.