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Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2–M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages.Implications:This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

Supplementary Data from Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2–M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages. Implications: This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

The pathogenesis of acute leukemia involves interaction among genetic alterations. Mutations of IDH1/2 and PHF6 are common and co-exist in some patients of hematopoietic malignancies, but their cooperative effects remain unexplored. In this study, we addressed the question by characterizing the hematopoietic phenotypes of mice harboring neither, Phf6 knockout, Idh2 R172K, or combined mutations. We found that the combined Phf6

S100A8/A9 is a proinflammatory protein and plays an essential role in the pathogenesis of myelodysplastic syndromes (MDS) via the S100A8/A9-Toll-like receptors axis. While S100A8/A9 levels have been used as biomarkers in many inflammatory diseases, their clinical relevance has not been conclusively resolved in MDS. To address this, we used an enzyme-linked immunosorbent assay to quantify S100A8/A9 heterodimers in bone marrow (BM) plasma from 215 MDS patients and compared S100A8/A9 levels across patients with various disease risks and genotypes. S100A8/A9 levels correlated with ASXL1 variant allele frequencies significantly. Moreover, mutant ASXL1 with concurrent RUNX1, STAG2, ZRSR2, or EZH2 mutations was associated with higher S100A8/A9 levels. We further showed that higher S100A8/A9 independently predicted inferior leukemia-free survival and overall survival in MDS patients, irrespective of age, Revised International Prognostic Scoring System subgroups, and known detrimental mutations. Lastly, through deep-sequenced transcriptomic analysis, we demonstrated that higher S100A8/A9 in the BM intimated a perturbed microenvironment with enhanced myeloid-derived suppressor cell-mediated tumor immune escape signal, altered metabolism, and impairment in the functions and quantities of CD8+ T cells and NK cells. S100A8/A9 in the BM microenvironment may be a potential biomarker in the prognostication of MDS and target for novel therapy.

We present the case of a 70-year-old man diagnosed with acute myelomonocytic leukemia, albeit that his leukemic blasts at initial presentation had scant cytoplasm, inconspicuous cytoplasmic granules, and morphologically mimicked lymphoblasts. We would like to raise the recognition that acute myelomonocytic leukemia can actually present with atypical blast morphology.

Myelodysplastic syndromes (MDS) are a group of clinically and genetically diverse diseases that impose patients with an increased risk of leukemic transformation. While MDS is a disease of the elderly, the interplay between aging and molecular profiles is not fully understood, especially in the Asian population. Thus, we compared the genetic landscape between younger and older patients in a cohort of 698 patients with primary MDS to advance our understanding of the distinct pathogenesis and different survival impacts of gene mutations in MDS according to age. We found that the average mutation number was higher in the older patients than younger ones. The younger patients had more WT1 and CBL mutations, but less mutated ASXL1, DNMT3A, TET2, SF3B1, SRSF2, STAG2, and TP53 than the older patients. In multivariable survival analysis, RUNX1 mutations with higher variant allele frequency (VAF) and U2AF1 and TP53 mutations were independent poor prognostic indicators in the younger patients, whereas DNMT3A and IDH2 mutations with higher VAF and TP53 mutations conferred inferior outcomes in the older patients. In conclusion, we demonstrated the distinct genetic landscape between younger and older patients with MDS and suggested that mutations impact survival in an age-depended manner.

The increased expression of programmed death-ligands 1 and 2 (PD-L1 and PD-L2, respectively) on tumour cells contributes to immune evasion, suggesting that these proteins are attractive therapeutic targets. This study aimed to evaluate the validity of cerebrospinal fluid (CSF) soluble PD-L1 (sPD-L1) and soluble PD-L2 (sPD-L2) as biomarkers for primary central nervous system lymphoma (PCNSL). We determined the CSF concentrations of sPD-L1 and sPD-L2 in 46 patients with PCNSL using enzyme-linked immunosorbent assays (ELISAs). A control group comprised 153 patients with other brain tumours, inflammatory/infectious status, or neurodegenerative diseases. Only CSF sPD-L1 levels were significantly higher in patients with PCNSL relative to the controls. CSF sPD-L1 also exhibited superior overall discrimination performance compared to CSF sPD-L2 in diagnosing PCNSL. Compared with patients with PCNSL with low CSF sPD-L1 levels, more patients with high levels had high serum lactate dehydrogenase levels, leptomeningeal involvement, and deep-brain involvement. Furthermore, CSF sPD-L1 could predict poor survival in PCNSL but CSF sPD-L2 could not. Intriguingly, CSF sPD-L1 levels were correlated with disease status and their dynamic changes post treatment could predict time to relapse. In conclusion, this study identified CSF sPD-L1 as a promising prognostic biomarker, indicating a therapeutic potential of PD-L1 blockade in PCNSL.

The S100 family proteins are involved in a variety of important biological processes, most notably immune and inflammatory responses. Their dysregulation also plays a role in the pathogenesis of human cancers. S100A4, also known as metastasin, has long been regarded as a biological marker in tumor progression and metastasis in multiple solid cancers, but its clinical significance in acute myeloid leukemia (AML) has not been extensively studied.We retrospectively studied the association between S100A4 gene expression and the clinical characteristics, mutational and transcriptomic profiles of 227 AML patients treated with standard intensive chemotherapy. Genetic mutations of myeloid disease associated genes were analyzed by Sanger sequencing. Microarray-based transcriptomic gene expression profiling was performed on archived bone marrow mononuclear cells. Bioinformatic analyses, including differential gene expression and gene set enrichment analysis, were conducted to delineate the underlying pathogenic mechanisms.Higher S100A4 expression was associated with older age, monocytic differentiation of leukemic cells, and adverse clinical outcome. S100A4 high-expressors had inferior overall survival and disease-free survival; this finding could be validated in the TCGA AML cohort (both the microarray and RNA-seq platforms). Multivariate Cox regression analysis supported S100A4 as an independent prognostic factor. Bioinformatic analysis showed that AML with higher S100A4 expression was enriched for the interferon, NLRP3 inflammasome, and epithelial-mesenchymal transition pathways.This study provides evidence that S100A4 overexpression serves as a poor prognostic biomarker in AML, holds potential to guide treatment planning in the clinic, and indicates novel therapeutic directions.

Increasing evidence supports the role of the immune microenvironment and associated signalling in the pathogenesis of myelodysplastic syndromes (MDS). Nevertheless, the clinical relevancy of immune signals in patients with MDS remains elusive. To address this, we used single-sample gene-set enrichment analysis to score immune signatures of bone marrow (BM) samples from 176 patients with primary MDS. Enhanced signatures of 'immature dendritic cells' and 'natural killer cells with cluster of differentiation (CD)56bright' were correlated with better overall survival (OS), whilst higher 'CD103+ signature' was associated with reduced survival. An MDS-Immune-Risk (MIR) scoring system was constructed based on the weighted sums derived from Cox regression analysis. High MIR scores were correlated with higher revised International Prognostic Scoring System (IPSS-R) scores and mutations in ASXL transcriptional regulator 1 (ASXL1), Runt-related transcription factor 1 (RUNX1), and tumour protein p53 (TP53). High-score patients had significantly inferior leukaemia-free survival (LFS) and OS than low-score patients. The prognostic significance of MIR scores for survival remained valid across IPSS-R subgroups and was validated in two independent cohorts. Multivariable analysis revealed that a higher MIR score was an independent adverse risk factor for LFS and OS. We further proposed a model with the combination of MIR score and gene mutations to be complementary to IPSS-R for the prognostication of LFS and OS of patients with MDS.

RUNX1 is mutated in ∼10% of adult acute myeloid leukemia (AML). Although most RUNX1 mutations in this disease are believed to be acquired, they can also be germline. Indeed, germline RUNX1 mutations result in the well-described autosomal-dominant familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD, FPD/AML, FPDMM); ∼44% of affected individuals progress to AML or myelodysplastic syndromes. Using the Leucegene RUNX1 AML patient group, we sought to investigate the proportion of germline vs acquired RUNX1 mutations in this cohort. Our results showed that 30% of RUNX1 mutations in our AML cohort are germline. Molecular profiling revealed higher frequencies of NRAS mutations and other mutations known to activate various signaling pathways in these patients with RUNX1 germline–mutated AML. Moreover, 2 patients (mother and son) had co-occurrence of RUNX1 and CEBPA germline mutations, with variable AML disease onset at 59 and 27 years, respectively. Together, these data suggest a higher than anticipated frequency of germline RUNX1 mutations in the Leucegene cohort and further highlight the importance of testing for RUNX1 mutations in instances in which allogeneic stem cell transplantation using a related donor is envisioned.

Myelodysplastic syndrome (MDS) comprised a heterogeneous group of diseases. The prognosis of patients varies even in the same risk groups. Searching for novel prognostic markers is warranted. Leukemic stem cells (LSCs) are responsible for chemoresistance and relapse in leukemia. Recently, expressions of 17 genes related to stemness of LSCs were found to be associated with prognosis in acute myeloid leukemia patients. However, the clinical impact of LSC genes expressions in MDS, a disorder arising from hematopoietic stem cells, remains unclear. We analyzed expression profile of the 17 stemness-related genes in primary MDS patients and identified expression of 4 genes (LAPTM4B, NGFRAP1, EMP1, and CPXM1) were significantly correlated with overall survival (OS). We constructed an LSC4 scoring system based on the weighted sums of the expression of 4 genes and explored its clinical implications in MDS patients. Higher LSC4 scores were associated with higher revised International Prognostic Scoring System (IPSS-R) scores, complex cytogenetics, and mutations in RUNX1, ASXL1, and TP53. High-score patients had significantly shorter OS and leukemia-free survival (LFS), which was also confirmed in 2 independent validation cohorts. Subgroup analysis revealed the prognostic significance of LSC4 scores for OS remained valid across IPSS-R lower- and higher-risk groups. Furthermore, higher LSC4 score was an independent adverse risk factor for OS and LFS in multivariate analysis. In summary, LSC4 score can independently predict prognosis in MDS patients irrespective of IPSS-R risks and may be used to guide the treatment of MDS patients, especially lower-risk group in whom usually only supportive treatment is given.

The coincident downregulation of NR4A1 and NR4A3 has been implicated in myeloid leukemogenesis, but it remains unknown how these two genes function in myeloid cells and how their combined downregulation promotes myeloid leukemogenesis. Since NR4A1 abrogation is thought to confer a survival and proliferation advantage to myeloid cells, we hypothesized that downregulation of NR4A3 may have a complementary effect on myeloid cell differentiation. First, we tested the association between differentiation status of leukemic cells and NR4A3 expression using two large clinical datasets from patients with different acute myeloid leukemia (AML) subtypes. The analysis revealed a close association between differentiation status and different subtypes of AML Then, we probed the effects of differentiation-inducing treatments on NR4A3 expression and NR4A3 knockdown on cell differentiation using two myeloid leukemia cell lines. Differentiation-inducing treatments caused upregulation of NR4A3, while NR4A3 knockdown prevented differentiation in both cell lines. The cell culture findings were validated using samples from chronic myeloid leukemia (CML) patients at chronic, accelerated and blastic phases, and in acute promyelocytic leukemia (APL) patients before and after all trans-retinoic acid (ATRA)-based differentiation therapy. Progressive NR4A3 downregulation was coincident with impairments in differentiation in patients during progression to blastic phase of CML, and NR4A3 expression was increased in APL patients treated with ATRA-based differentiating therapy. Together, our findings demonstrate a tight association between impaired differentiation status and NR4A3 downregulation in myeloid leukemias, providing a plausible mechanistic explanation of how myeloid leukemogenesis might occur upon concurrent downregulation of NR4A1 and NR4A3.

Key Points CIBERSORTx analytical approach reveals M0, M2, and eosinophil fractions in the BM to be the most relevant prognostic factors in patients with MDS.High-risk immune cell scores correlate with NF-κB signaling, oxidative stress, and leukemic stem cell signature pathways., Visual Abstract, Aside from cell intrinsic factors such as genetic alterations, immune dysregulation in the bone marrow (BM) microenvironment plays a role in the development and progression of myelodysplastic syndromes (MDS). However, the prognostic implications of various immune cells in patients with MDS remain unclear. We adopted CIBERSORTx to estimate the relative fractions of 22 subtypes of immune cells in the BM of 316 patients with MDS and correlated the results with clinical outcomes. A lower fraction of unpolarized M0 macrophages and higher fractions of M2 macrophages and eosinophils were significantly associated with inferior survival. An immune cell scoring system (ICSS) was constructed based on the proportion of these 3 immune cells in the BM. The ICSS high-risk patients had higher BM blast counts, higher frequencies of poor-risk cytogenetics, and more NPM1, TP53, and WT1 mutations than intermediate- and low-risk patients. The ICSS could stratify patients with MDS into 3 risk groups with distinct leukemia-free survival and overall survival among the total cohort and in the subgroups of patients with lower and higher disease risk based on the revised International Prognostic Scoring System (IPSS-R). The prognostic significance of ICSS was also validated in another independent cohort. Multivariable analysis revealed that ICSS independently predicted prognosis, regardless of age, IPSS-R, and mutation status. Bioinformatic analysis demonstrated a significant correlation between high-risk ICSS and nuclear factor κB signaling, oxidative stress, and leukemic stem cell signature pathways. Further studies investigating the mechanistic insight into the crosstalk between stem cells and immune cells are warranted.

Expression of long non-coding RNA KIAA0125 has been incorporated in various gene expression signatures for prognostic prediction in acute myeloid leukemia (AML) patients, yet its functions and clinical significance remain unclear. This study aimed to investigate the clinical and biological characteristics of AML bearing different levels of KIAA0125. We profiled KIAA0125 expression levels in bone marrow cells from 347 de novo AML patients and found higher KIAA0125 expression was closely associated with RUNX1 mutation, but inversely correlated with t(8;21) and t(15;17) karyotypes. Among the 227 patients who received standard chemotherapy, those with higher KIAA0125 expression had a lower complete remission rate, shorter overall survival (OS) and disease-free survival (DFS) than those with lower expression. The prognostic significance was validated in both TCGA and GSE12417 cohorts. Subgroup analyses showed that higher KIAA0125 expression also predicted shorter DFS and OS in patients with normal karyotype or non-M3 AML. In multivariable analysis, higher KIAA0125 expression remained an adverse risk factor independent of age, WBC counts, karyotypes, and mutation patterns. Bioinformatics analyses revealed that higher KIAA0125 expression was associated with hematopoietic and leukemic stem cell signatures and ATP-binding cassette transporters, two predisposing factors for chemoresistance.

Long non-coding RNAs (lncRNAs) have important functions in cancer biology. Among them, lncRNA KIAA0125 is one of the genes proposed to play a critical role in leukaemia stem cell (LSC). In this study, we aimed to investigate the clinical relevance of the expression levels of lncRNA KIAA0125 in myelodysplastic syndromes (MDS), a disease with highly heterogeneous clinical and biological features. Using RNA arrays, we measured the expression of KIAA0125 in 176 primary MDS patients. We found that higher KIAA0125 expression was associated with higher risk MDS, based on the revised International Prognostic Scoring System (IPSS-R), mutations in ASXL1 and NRAS, and predicted poorer overall survival (OS) and leukaemia-free survival (LFS). Multivariate analysis revealed that higher KIAA0125 expression was an independent, unfavourable prognostic factor for OS and LFS, irrespective of IPSS-R and mutation status. Further global gene expression profile analysis suggested a close association of higher KIAA0125 expressions with LSC signatures. The expression of KIAA0125 may be a potential biomarker to guide the treatment choice in MDS patients, especially those with lower risk subtypes, in whom palliative treatment is usually used.

Expression of long non-coding RNA KIAA0125 has been incorporated in various gene expression signatures for prognostic prediction in acute myeloid leukemia (AML) patients, yet its functions and clinical significance remain unclear. This study was aimed to investigate the clinical and biological characteristics of AML bearing different levels of KIAA0125. We profiled KIAA0125 expression levels in bone marrow cells from 347 de novo AML patients and found higher KIAA0125 expression was closely associated with FLT3-ITD, RUNX1, and DNMT3A mutations, and M1 subtype by the French-American-British classification, but inversely correlated with t(8;21) and t(15;17). Among the 227 patients who received standard chemotherapy, those with higher KIAA0125 expression had a lower complete remission rate, shorter overall survival (OS) and disease-free survival (DFS) than those with lower expression. The prognostic significance was validated in both TCGA and GSE12417 cohorts. Subgroup analyses showed that higher KIAA0125 expression also predicted shorter DFS and OS in patients with normal karyotype or non-M3 AML. In multivariable analysis, higher KIAA0125 expression remained an adverse risk factor independent of age, WBC counts, karyotypes, and mutation patterns. Bioinformatics analyses revealed that higher KIAA0125 expression was associated with hematopoietic and leukemic stem cell signatures and ATP-binding cassette transporters, two predisposing factors for chemoresistance.

HOPX is a stem cell marker in hair follicles and intestines. It was shown critical for primitive hematopoiesis. We previously showed an association between higher HOPX expression and clinical characteristics related to stemness and quiescence of leukemic cells in acute myeloid leukemia (AML) patients. To further explore its physiologic functions in hematopoietic system, we generated a mouse model with hematopoietic cell-specific knockout of Hopx (Hopx-/-). In young Hopx-/- mice, the hematopoietic stem cells (HSC) showed decreased reconstitution ability after serial transplantation. Further transcriptomic study revealed decreased HSC signatures in long-term HSCs from the Hopx-/- mice. At 18 months of age, half of the Hopx-/- mice developed cytopenia and splenomegaly. Bone marrow (BM) from the sick mice showed myeloid hyperplasia with predominant mature neutrophils, and decreased progenitor cells and lymphocytes. These phenotypes suggested critical functions of Hopx in maintaining HSC quiescence. Transcriptomic study of the Hopx-/- marrow cells showed significant downregulation of the Cxcl12-Cxcr4 axis, which is critical for maintenance of HSC quiescence. We next examined the role of Hopx in AML by using the MN1 overexpression murine leukemia model. Mice transplanted with MN1-overexpressed Hopx-/- BM cells developed AML with more aggressive phenotypes compared with those transplanted with MN1-overexpressed Hopx-wild cells. Hopx-/- MN1-overexpressed leukemia cells showed higher proliferation rate and downregulation of Cxcl12 and Cxcr4. Furthermore, in human AML, BM plasma CXCL12 levels were lower in patients with lower HOPX expression. In conclusion, our study highlights the roles of Hopx in maintenance of quiescence of the hematopoietic stem cells through CXCL12 pathway in vivo and provides implication of this protein in normal and malignant hematopoiesis.

Euonymus is a highly diverse and cosmopolitan genus within Celastraceae. Floral anatomy of twenty-eight Euonymus taxa were investigated to examine whether floral features provide useful information for infrageneric classification using light microscopy and scanning electron microscopy. The floral characters were evaluated by cluster and principal coordinate analysis. Flower merosity, petal color, and ovule number per locule were of potential systematic value as supported by ordination analysis. In addition, morphological data on the ovary of Euonymus were first reported here, and three subtypes of axile placentation were detected: (1) apical-axile, (2) basal-axile, and (3) dispersed-axile. This study highlights the importance of the floral features and provides better understanding of the relationships among sections in Euonymus.

A conspicuous nectary disk is common but has a distinguishing morphology in the cosmopolitan genus Euonymus. Our study focuses on the morphology of floral nectaries in 21 representatives of Euonymus and Glyptopetalum. Two main types of nectaries were documented: a mix of inter- and extrastaminal nectaries existed between the corolla and the stigma, while the intrastaminal nectaries were distributed between the stigma and the stamen bases. The main route of nectar release in Euonymus is via modified stomata, and different nectarostomata locations were observed: in depressions, slightly raised above the epidermal surface or at the same level as the epidermis. Floral nectaries in E. sect. Echinococcus species developed into the protrusions on the fruit surface at the later stage. The development of nectaries not only explained the mystery of the origin of the echinate fruit surface, but also showed that differences in fruit surface might be inappropriate for use in infrageneric classification. These discoveries inform morphological observations of floral nectaries in Euonymus.

RUNX1 is a member of the core-binding factor family of transcription factors and is imperative for establishing definitive haematopoiesis. Mutated RUNX1 is an adverse risk factor for myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Meanwhile, high expression of RUNX1 is correlated with dismal prognosis in cytogenetically normal AML patients and critical for maintaining leukemic stem cells across AML genetic subgroups. However, the clinical relevancy of RUNX1 expression in MDS patients remains elusive. This study aimed to investigate the prognostic and biologic impacts of RUNX1 expression in MDS patients. We recruited 341 primary MDS patients who had enough bone marrow (BM) samples for RNA and next-generation sequencing. We first examined the difference in RUNX1 expression among patients with wild RUNX1 and N-terminal or C-terminal RUNX1 mutation. Among the 341 patients, 54 (15.8%) harboured RUNX1 mutation, 15 (27.8%) in N-terminal and 39 (72.2%), C-terminal. Patients with C-terminal RUNX1 mutant had higher RUNX1 expression than those with N-terminal RUNX1 mutant or wild RUNX1 (p Regarding survival, we first examined the impact of RUNX1 mutation on survival in this cohort. As expected, patients with mutated RUNX1 had significantly inferior leukaemia-free survival (LFS) and overall survival (OS) than those with unmutated RUNX1 (p=0.007, and p=0.008, respectively). We then explored the effects of RUNX1 expression on patients' survival. Patients with higher RUNX1 expression had significantly inferior LFS and OS than those with lower expression (both p Figure 1 Figure 1. Disclosures Tien: AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria. Chou: Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; IQVIA: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Bristol Myers Squibb: Honoraria, Research Funding; Kirin: Honoraria, Research Funding.

Background: Myelodysplastic syndrome (MDS) comprised a heterogeneous group of diseases. The prognosis of patients varies even in the same risk groups. Searching for novel prognostic markers is warranted. Leukemic stem cells (LSC) are responsible for chemoresistance and relapse in leukemia. Recently, expressions of 17 genes related to stemness of LSC were found to be associated with prognosis in acute myeloid leukemia patients. However, the clinical impact of LSC genes expressions on patients with MDS, a disorder arising from hematopoietic stem cells, remains unclear. Methods: We analysed expression profile of the 17 stemness-related genes in primary MDS patients and identified expressions of 4 genes were significantly correlated with overall survival (OS). We constructed an LSC4 scoring system based on the weighted sums of the four genes expressions and explored its clinical implications in MDS patients. Findings: Higher LSC4 scores were associated with higher revised international prognostic scoring system (IPSS-R) scores, complex cytogenetics, and mutations in RUNX1, ASXL1, ZRSR2, U2AF1, SRSF2, and TP53. High-score patients had significantly shorter OS and leukemia-free survival (LFS) which was also confirmed in two independent validation cohorts. Subgroups analysis revealed the prognostic significance of LSC4 scores for OS remained valid across IPSS-R lower- and higher-risk groups. Furthermore, higher LSC4 score was an independent adverse risk factor for OS and LFS. Interpretation: LSC4 score can independently predict prognosis in MDS patients irrespective of IPSS-R risks and may be used to guide the treatment of MDS patients, especially lower-risk group in whom usually only supportive treatment is given. Funding: The study was supported by grants from Ministry of Health and Welfare, and Science and Technology, Taiwan. Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by the Institutional Review Board of the NTUH.

Background: Expression of long non-coding RNAs (lncRNAs) has recently been recognized as a potential prognostic marker in acute myeloid leukemia (AML). However, it remains unclear whether incorporation of the lncRNAs expression in the 2017 European LeukemiaNet (ELN) risk classification can further improve the prognostic prediction. Methods: We enrolled 275 newly diagnosed non-M3 AML patients and randomly assigned them into the training (n=183) and validation cohorts (n=92). In the training cohort, we formulated a prognostic lncRNA scoring system composed of five lncRNAs with significant prognostic impact from the lncRNA expression profiling. Findings: Higher lncRNA scores were significantly associated with older age and adverse gene mutations. Further, the higher-score patients had shorter overall and disease-free survival than lower-score patients, which were also confirmed in both internal and external validation cohorts (TCGA database). The multivariate analyses revealed the lncRNA score was an independent prognosticator in AML, irrespective of the risk based on the 2017 ELN classification. Moreover, in the 2017 ELN intermediate-risk subgroup, lncRNA scoring system could well dichotomize the patients into two groups with distinct prognosis. Within the ELN intermediate-risk subgroup, we found that allogeneic hematopoietic stem cell transplantation could provide better outcome on patients with higher lncRNA scores. Through bioinformatics approach, we identified high lncRNA scores were correlated with leukemia/hematopoietic stem cell signatures. Interpretation: Incorporation of lncRNA scoring system in 2017 ELN classification can improve risk-stratification of AML patients and help clinical decision-making. Funding Statement: This work was supported Ministry of Science and Technology, and Ministry of Health and Welfare of Taiwan. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This study was approved by the Research Ethics Committee of the NTUH in accordance with the Declaration of Helsinki.

The ground technique is often proposed for suppressing and controlling the noise of EMI emissions. The signal grounds are defined as a low-impedance path for current flowing into the source which can be divided into different modes of ground plane. Nowadays, all the electronic products have already intend to the smaller and thinner size, therefore, the area of ground on PCB will be limited. The R&D and EMI engineers have to face the less and less ground plane which can be utilized. In this paper, the EMI reduction effectiveness for the area of ground plane and location on the PCB is studied by radiated measurement results. The coupling behaviors of the electric or magnetic fields between the traces of harmonic signal and ground plane are investigated to suppress the EMI interferences. The main contributions of this work are the experimental verification and analysis that can be used for the EMI reduction effectiveness. The area of ground plane and location will determine the effectiveness of capacitive coupling and return currents path. The designer can use this concept to achieve the best EMI performance with limited PCB ground plane.

Purpose Differential counting of blood cells is the basis of diagnostic hematology. In many circumstances, identification of cells in bone marrow smears is the golden standard for diagnosis. Presently, methods for automatic differential counting of peripheral blood are readily available commercially. However, morphological assessment and differential counting of bone marrow smears are still performed manually. This procedure is tedious, time-consuming and laden with high inter-operator variation. In recent years, deep neural networks have proven useful in many medical image recognition tasks, such as diagnosis of diabetic retinopathy, and detection of cancer metastasis in lymph nodes. However, there has been no published work on using deep neural networks for complete differential counting of entire bone marrow smear. In this work, we present the results of using deep convolutional neural network for automatic differential counting of bone marrow nucleated cells. Materials & Methods The bone marrow smears from patients with either benign or malignant disorders in National Taiwan University Hospital were recruited in this study. The bone marrow smears are stained with Liu's stain, a modified Romanowsky stain. Digital images of the bone marrow smears were taken using 1000x oil immersion lens and 20MP color CCD camera on a single microscope with standard illumination and white-balance settings. The contour of each nucleated cell was artificially defined. These cells were then divided into a training/validation set and a test set. Each cell was then classified into 1 of the 11 categories (blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophils, eosinophils and precursors, basophil, monocyte and precursors, lymphocyte, erythroid lineage cells, and invalid cell). In training/validation set, the classification of each cell was annotated once by experienced medical technician or hematologist. The annotated dataset was used to train a Path-Aggregation Network for instance segmentation task. In test set, cell classification was annotated by three medical technicians or hematologists; only over 2/3 consensus was regarded as valid. After the neural network model was fully trained, the ability of the model to classify and detect bone marrow nucleated cells was evaluated in terms of precision, recall and accuracy. During the model training, we used group normalization and stochastic gradient descent optimizer for training. Random noise, Gaussian blur, rotation, contrast and color shift were also used as means for data augmentation. Results The digital images of 150 bone marrow aspirate smears were taken for this study. They included 61 for acute leukemia, 39 for lymphoma, 2 for myelodysplastic syndrome (MDS), 2 for myeloproliferative neoplasm (MPN), 10 for MDS/MPN, 12 for multiple myeloma, 4 for hemolytic anemia, 9 for aplastic anemia, 8 for infectious etiology and 3 for solid cancers. The final data contained 5927 images and 187730 nucleated bone marrow cells, which were divided into 2 sets: 5630 images containing 170966 cells as the training/validation set, and 297 images containing 16764 cells as the test set. Among the 16764 cells annotated in test set, 15676 cells (93.6 %) reached over 2/3 consensus. The trained neural network achieved 0.832 recall and 0.736 precision for cell detection task, 0.79 mean intersection over union (IOU) for cell segmentation task, mean average precision of 0.659 and accuracy of 0.801 for cell classification. For individual cell categories, the model performs the best with "erythroid-lineage-cells" (0.971 recall, 0.935 precision) and the worst with "monocyte-and-precursors" (0.825 recall, 0.337 precision). Conclusions We have created the largest and the most comprehensive annotated bone marrow smear image dataset for deep neural network training. Compared with previous works, our approach is more practical for clinical application because it is able to take in an entire field of smear and generate differential counts without any other preprocessing steps. Current results are highly encouraging. With continued expansion of dataset, our model would be more precise and clinically useful. Figure Disclosures Yeh: aether AI: Other: CEO and co-founder. Yang:aether AI: Employment. Tien:Novartis: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Research Funding; Roche: Honoraria; Johnson &Johnson: Honoraria; Alexion: Honoraria; BMS: Honoraria; Roche: Research Funding; Celgene: Honoraria; Pfizer: Honoraria; Abbvie: Honoraria. Hsu:aether AI: Employment.