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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.

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.

Early recognition of adult-onset immunodeficiency associated with neutralizing anti-interferon gamma autoantibodies (anti-IFNγ Abs) remains difficult, and misdiagnoses have been reported. Although febrile lymphadenopathy is among the most common initial manifestations of this disorder, no comprehensive clinicopathologic analysis of lymphadenopathy in patients with anti-IFNγ Abs has been reported. Here, we describe 26 lymph node biopsy specimens from 16 patients. All patients exhibited concurrent disseminated nontuberculous mycobacterial infections, and 31% received a tentative diagnosis of lymphoma at initial presentation. We found 3 distinct histomorphologic patterns: well-formed granuloma (46%), suppurative inflammation or loose histiocytic aggregates (31%), and lymphoproliferative disorder (LPD, 23%). The latter shared some of the features of malignant T-cell lymphoma, IgG4-related disease, and multicentric Castleman disease. Half of the specimens with LPD had monoclonal T cells, and 33.3% were indistinguishable from angioimmunoblastic T-cell lymphoma as per current diagnostic criteria. All lymphadenopathy with LPD features regressed with antibiotics without administration of cytotoxic chemotherapy or immunotherapy. The median follow-up time was 4.3 years. Our study highlights the substantial challenge of distinguishing between lymphoma and other benign lymphadenopathy in the setting of neutralizing anti-IFNγ Abs. Increased vigilance and multidisciplinary discussion among clinicians and pathologists are required to achieve the most appropriate diagnosis and management.

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.

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.

Aberrant alternative splicing (AS) is a hallmark of cancer development. However, there are limited data regarding its clinical implications in myelodysplastic syndrome (MDS). In this study, we performed an in-depth analysis of global AS in 176 primary MDS patients with 20 normal marrow transplant donors as reference. We found that 26.9% of the expressed genes genome-wide were aberrantly spliced in MDS patients compared with normal donors. These aberrant AS genes were related to pathways involved in cell proliferation, cell adhesion and protein degradation. A higher degree of global aberrant AS was associated with male gender and U2AF1 mutation, and predicted shorter overall survival and time to leukemic change. Moreover, it was an independent unfavorable prognostic factor irrespective of age, revised international prognostic scoring system (IPSS-R) risk, and mutations in SRSF2, ZRSR2, ASXL1, TP53, and EZH2. With LASSO-Cox regression method, we constructed a simple prognosis prediction model composed of 13 aberrant AS genes, and demonstrated that it could well stratify MDS patients into distinct risk groups. To our knowledge, this is the first report demonstrating significant prognostic impacts of aberrant splicing on MDS patients. Further prospective studies in larger cohorts are needed to confirm our observations.

Homeodomain-only protein homeobox (HOPX) is the smallest homeodomain protein. It was regarded as a stem cell marker in several non-hematopoietic systems. While the prototypic homeobox genes such as the HOX family have been well characterized in acute myeloid leukemia (AML), the clinical and biological implications of HOPX in the disease remain unknown. Thus we analyzed HOPX and global gene expression patterns in 347 newly diagnosed de novo AML patients in our institute. We found that higher HOPX expression was closely associated with older age, higher platelet counts, lower white blood cell counts, lower lactate dehydrogenase levels, and mutations in RUNX1, IDH2, ASXL1, and DNMT3A, but negatively associated with acute promyelocytic leukemia, favorable karyotypes, CEBPA double mutations and NPM1 mutation. Patients with higher HOPX expression had a lower complete remission rate and shorter survival. The finding was validated in two independent cohorts. Multivariate analysis revealed that higher HOPX expression was an independent unfavorable prognostic factor irrespective of other known prognostic parameters and gene signatures derived from multiple cohorts. Gene set enrichment analysis showed higher HOPX expression was associated with both hematopoietic and leukemia stem cell signatures. While HOPX and HOX family genes showed concordant expression patterns in normal hematopoietic stem/progenitor cells, their expression patterns and associated clinical and biological features were distinctive in AML settings, demonstrating HOPX to be a unique homeobox gene. Therefore, HOPX is a distinctive homeobox gene with characteristic clinical and biological implications and its expression is a powerful predictor of prognosis in AML patients.

Plant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

Background Additional sex combs-like 1 (ASXL1) is frequently mutated in myeloid malignancies. Recent studies showed that hematopoietic-specific deletion of Asxl1 or overexpression of mutant ASXL1 resulted in myelodysplasia-like disease in mice. However, actual effects of a “physiological” dose of mutant ASXL1 remain unexplored. Methods We established a knock-in mouse model bearing the most frequent Asxl1 mutation and studied its pathophysiological effects on mouse hematopoietic system. Results Heterozygotes (Asxl1 tm/+) marrow cells had higher in vitro proliferation capacities as shown by more colonies in cobblestone-area forming assays and by serial re-plating assays. On the other hand, donor hematopoietic cells from Asxl1 tm/+ mice declined faster in recipients during transplantation assays, suggesting compromised long-term in vivo repopulation abilities. There were no obvious blood diseases in mutant mice throughout their life-span, indicating Asxl1 mutation alone was not sufficient for leukemogenesis. However, this mutation facilitated engraftment of bone marrow cell overexpressing MN1. Analyses of global gene expression profiles of ASXL1-mutated versus wild-type human leukemia cells as well as heterozygote versus wild-type mouse marrow precursor cells, with or without MN1 overexpression, highlighted the association of in vivo Asxl1 mutation to the expression of hypoxia, multipotent progenitors, hematopoietic stem cells, KRAS, and MEK gene sets. ChIP-Seq analysis revealed global patterns of Asxl1 mutation-modulated H3K27 tri-methylation in hematopoietic precursors. Conclusions We proposed the first Asxl1 mutation knock-in mouse model and showed mutated Asxl1 lowered the threshold of MN1-driven engraftment and exhibited distinct biological functions on physiological and malignant hematopoiesis, although it was insufficient to lead to blood malignancies. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0508-x) contains supplementary material, which is available to authorized users.

Supplementary methods, figures and table. (PDF 913 kb)

Background: Myelodysplastic syndromes (MDS) are highly heterogeneous regarding pathogenesis and clinical outcome. Traditionally, the revised international prognostic scoring system (IPSS-R)incorporating clinical features and cytogenetic abnormalities has been the standard for prognostication, while in recent years, recurrent somatic mutations are becoming increasingly important for this purpose. Additionally, gene expression profiling of coding genes and microRNAs has also emerged as a powerful tool to separate patients into distinct prognostic categories. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides without protein-coding capacity. lncRNAs not only participate in normal hematopoiesis, but perturbation of their expressions may contribute to development of acute leukemia. However, the impact of lncRNA expression profiling on the prognosis of MDS patients has not yet been explored to date. Aims: This study was aimed to evaluate lncRNAs expression profiling in MDS and to find out lncRNAs whose expression levels were associated with clinical outcomes. A scoring system was constructed to better risk-stratify MDS patients. We also tried to seek clues to the functionality of these lncRNAs. Materials & Methods: By using the Affymetrix Human Transcriptome Array 2.0 platform, we obtained the global expression profiles of 24120 lncRNAs in 176 adult patients with de novo MDS diagnosed according to the 2008 WHO classification. Through mathematical modeling, we identified six lncRNAs whose expression levels were significantly associated with overall survival (OS). We then constructed a risk scoring system with the weighted sum of each of these six lncRNAs, and evaluated the correlation of the scores with clinical features,cytogenetic abnormalities, gene mutations, andtreatment outcomesof the MDS patients.The reliability of our lncRNA scoring system was assured based on the coefficient of variance obtained from a permutation test, and further validated by using a five-fold cross-validation, in which 80% of the patients were used as the training samples to develop the scoring model, whose prediction performances were evaluated by using the rest of the 20% of the patients. Analysis of mutations in 21 genes was performed by conventional Sanger sequencing technique. Results: Higher lncRNA scores were positively associated with refractory anemia with excess of blasts (RAEB)-1 or RAEB-2 subtypes, complex cytogenetic changes, and IPSS-R high and very high risks, but inversely associated with RCUD, RARS, RCMD subtypes, a normal karyotype, and IPSS-R low risk. Patients with higher lncRNA scores had more frequent RUNX1, ASXL1, TP53, EZH2, SRSF2 and ZRSR2 mutations, shorter OS (median 14.0 months vs. 77.3 months, P Conclusion: Our integrated 6-lncRNA risk scoring system provides distinct insights into the clinical and biological implications of lncRNAs expression in de novo MDS patients. The scoring system represents an independent prognostic factor for survival and leukemic transformation. It may assist improving risk stratification of newly-diagnosed MDS patients. Further prospective trials are necessary to confirm the findings of our study. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.