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Invasive pulmonary aspergillosis (IPA) is an emerging and life-threatening infectious disease in patients admitted to the intensive care unit (ICU). Most diagnostic studies are conducted in hematological patients and results cannot readily be transferred to ICU patients lacking classical host factors.

The contribution of molecular alterations in bone marrow mesenchymal stromal cells (BM-MSC) to the pathogenesis of acute myeloid leukemia (AML) is poorly understood. Thus we assessed genome-wide genetic, transcriptional and epigenetic alterations in BM-MSC derived from AML patients (AML BM-MSC). Whole-exome sequencing (WES) of AML BM-MSC samples from 21 patients revealed a non-specific pattern of genetic alterations in the stromal compartment. The only mutation present in AML BM-MSC at serial time points of diagnosis, complete remission and relapse was a mutation in the PLECgene encoding for cytoskeleton key player Plectin in one AML patient. Healthy donor controls did not carry genetic alterations as determined by WES. Transcriptional profiling using RNA sequencing revealed deregulation of proteoglycans and adhesion molecules as well as cytokines in AML BM-MSC. Moreover, KEGG pathway enrichment analysis unravelled deregulated metabolic pathways and endocytosis in both transcriptional and DNA methylation signatures in AML BM-MSC. Taken together, we report molecular alterations in AML BM-MSC suggesting global changes in the AML BM microenvironment. Extended investigations of these altered niche components may contribute to the design of niche-directed therapies in AML.

We evaluated the impact of clinical and molecular characteristics on overall survival (OS) in 108 patients with indolent (n=41) and advanced systemic mastocytosis (SM) (advSM, n=67). Organomegaly was measured by magnetic resonance imaging-based volumetry of the liver and spleen. In multivariate analysis of all patients, an increased spleen volume ?450?ml (hazard ratio (HR), 5.2; 95% confidence interval (CI), (2.1–13.0); P=0.003) and an elevated alkaline phosphatase (AP; HR 5.0 (1.1–22.2); P=0.02) were associated with adverse OS. The 3-year OS was 100, 77, and 39%, respectively (P<0.0001), for patients with 0 (low risk, n=37), 1 (intermediate risk, n=32) or 2 (high risk, n=39) parameters. For advSM patients with fully available clinical and molecular data (n=60), univariate analysis identified splenomegaly ?1200?ml, elevated AP and mutations in the SRSF2/ASXL1/RUNX1(S/A/R) gene panel as significant prognostic markers. In multivariate analysis, mutations in S/A/R (HR 3.2 (1.1–9.6); P=0.01) and elevated AP (HR 2.6 (1.0–7.1); P=0.03) remained predictive adverse prognostic markers for OS. The 3-year OS was 76 and 38%, respectively (P=0.0003), for patients with 0–1 (intermediate risk, n=28) or 2 (high risk, n=32) parameters. We conclude that splenomegaly, elevated AP and mutations in the S/A/R gene panel are independent of the World Health Organization classification and provide the most relevant prognostic information in SM patients.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Treatment of chronic myeloid leukemia (CML) has been profoundly improved by the introduction of tyrosine kinase inhibitors (TKIs). Long-term survival with imatinib is excellent with a 8-year survival rate of ∼88%. Long-term toxicity of TKI treatment, especially carcinogenicity, has become a concern. We analyzed data of the CML study IV for the development of secondary malignancies. In total, 67 secondary malignancies were found in 64 of 1525 CML patients in chronic phase treated with TKI (n=61) and interferon-α only (n=3). The most common malignancies (n⩾4) were prostate, colorectal and lung cancer, non-Hodgkin’s lymphoma (NHL), malignant melanoma, non-melanoma skin tumors and breast cancer. The standardized incidence ratio (SIR) for all malignancies excluding non-melanoma skin tumors was 0.88 (95% confidence interval (0.63–1.20)) for men and 1.06 (95% CI 0.69–1.55) for women. SIRs were between 0.49 (95% CI 0.13–1.34) for colorectal cancer in men and 4.29 (95% CI 1.09–11.66) for NHL in women. The SIR for NHL was significantly increased for men and women. An increase in the incidence of secondary malignancies could not be ascertained. The increased SIR for NHL has to be considered and long-term follow-up of CML patients is warranted, as the rate of secondary malignancies may increase over time.

Most patients with KITD816V+advanced systemic mastocytosis (SM) are characterized by somatic mutations in additional genes. We sought to clarify the prognostic impact of such mutations. Genotype and clinical characteristics of 70 multi-mutated KITD816V+advanced SM patients were included in univariate and multivariate analyses. The most frequently identified mutated genes were TET2(n=33 of 70 patients), SRSF2(n=30), ASXL1(n=20), RUNX1(n=16) and JAK2(n=11). In univariate analysis, overall survival (OS) was adversely influenced by mutations in SRSF2(P<0.0001), ASXL1(P=0.002) and RUNX1(P=0.03), but was not influenced by mutations in TET2or JAK2. In multivariate analysis, SRSF2and ASXL1remained the most predictive adverse indicators concerning OS. Furthermore, we found that inferior OS and adverse clinical characteristics were significantly influenced by the number of mutated genes in the SRSF2/ASXL1/RUNX1(S/A/R) panel (P<0.0001). In conclusion, the presence and number of mutated genes within the S/A/R panel are adversely associated with advanced disease and poor survival in KITD816V+SM. On the basis of these findings, inclusion of molecular markers should be considered in upcoming prognostic scoring systems for patients with SM.

ABSTRACTAlthough it is a severe complication in immunocompromised patients, diagnosing invasive fungal disease (IFD), especially invasive aspergillosis (IA), remains difficult. In certain clinical scenarios, examining tissue samples for identification of the infectious organism becomes important. As culture-based methods rarely yield results, the performance of an Aspergillus-specific nested PCR in fresh tissue or pleural effusion samples was evaluated. Fresh tissue (n= 59) and effusion (n= 47) specimens from 79 immunocompromised patients were subjected to an Aspergillus-specific PCR assay. Twenty-six patients had proven (n= 20) or probable (n= 6) IFD, according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, while the remaining patients were classified as having either possible IFD (n= 30) or no IFD (n= 23). IA was identified as the underlying IFD in 21/26 proven/probable cases. PCR positivity was observed for 18/21 proven/probable and 6 possible IA cases; cases classified as no IA did not show positive signals. Patients with proven IFD (n= 5) with cultures positive for non-Aspergillusmolds also had negative AspergillusPCR results. AspergillusPCR performance analysis yielded sensitivity and specificity values of 86% (95% confidence interval [CI], 65% to 95%) and 100% (95% CI, 86% to 100%), respectively, thus leading to a diagnostic odds ratio of >200. In this analysis, good diagnostic performance of the PCR assay for detection of IA was observed for tissue samples, while effusion samples showed lower sensitivity rates. PCR testing represents a complementary tool; a positive PCR result strengthens the likelihood of IA, whereas IA seems unlikely in cases with negative results but findings could indicate non-AspergillusIFD. Thus, PCR testing of these specimens enhances the diagnostic capabilities.

Abstract:  Used as single agents, ATRA, G-CSF, and IFN-α have shown a moderate benefit in patients with low-risk MDS, with a response rate of 10%. The aim of the present study was to evaluate the efficacy of a combination of these agents. The effect on hemoglobin (Hb), platelets, and absolute neutrophil count (ANC), as well as on transfusion frequency, was examined in 25 patients with MDS (11 RA, four RARS, eight RAEB, two CMML). The median age was 61 years (range 44–81), and the male/female ratio was 14/11. Treatment consisted of ATRA at 25 mg/m2/day p.o. for months 1, 3, 5, 7, 9, and 11, IFN-α at 1.5 MIU twice a week s.c. for 52 weeks, and, in patients with initial ANC <500/μl, G-CSF at 100–480 μg daily s.c. according to the degree of ANC. The duration of therapy was scheduled for 12 months. Two patients achieved ongoing CR (+19 months; +16 months), one patient with RA after 3 months and one with CMML after 7 months of treatment. In all patients, the mean ANC increased significantly from 1400±200/μl before the start of therapy to 3500±600/μl at the end of treatment (p=0.025). In two patients an increase of Hb was observed, and one patient ceased to require transfusions. In an additional patient with RA and 5q-syndrome, the platelet count normalized following administration of ATRA/IFN-α, increasing from 89,000/μl to 293,000/μl. The eight RAEB patients were nonresponders. We conclude that therapy with ATRA, IFNα, and G-CSF is effective in approximately 35% of low-risk MDS patients (in this study: six of 17) and may induce complete remission in individual cases.

Abstract:  The toxicity and outcome after high-dose ara-C/daunorubicin (HDara-C/DNR) consolidation therapy in de novo AML was compared in 11 patients who received an idarubicin-containing induction therapy (IDA; from June 1995 to March 1997) and 16 patients pretreated with daunorubicin (DNR; from July 1990 to May 1995) for induction. The DNR group consisted of two cohorts, one (n=6) of patients who had received, as had the IDA group, two induction and one intermediate-dose ara-C consolidation courses, and another (n=10) of patients who had been pretreated with one induction and one consolidation course prior to HDara-C/DNR. There was no difference in the relative dose between the three cohorts. Following HDara-C/DNR, the IDA-pretreated patients experienced a more prolonged myelosuppression during consolidation therapy compared with the DNR group. Duration of neutropenia (<500 neutrophils/μl) following HDara-C/DNR was 31.2 ± 16 days (mean ±SEM) in the IDA group compared with 18.7 ± 5 days in the DNR group (p<.001 Mann-Whitney U-test). The duration of thrombocytopenia (platelets <25 000/μl) was 34.8 ± 20 days in the IDA group vs. 18.5 ± 6 days in the DNR group (p<.005). The more prolonged myelosupression was associated with a longer duration of fever (18.9 ± 24 vs. 6.9 ± 5.2 days). A greater incidence, length (11 ± 8 vs. 1.2 ± 2 days), and severity of diarrhea were observed in the IDA-pretreated group. Three of 11 IDA patients experienced WHO grade III-IV diarrhea. In the IDA group two patients developed severe enterocolitis with Candida septicemia, and one of these patients died. One patient in the IDA group died during prolonged aplasia. In the DNR group 6/16 patients experienced grade I-II diarrhea. Two patients in each group died during consolidation therapy. The CR rate was 87% in the IDA group and 79% in the DNR group. Relapse-free survival after HDara-C is 50% at a median follow-up of 60 months in the DNR group and 45% after a median follow-up of 17 months in the IDA group. Whether the advantage of the superior response rate in the IDA-treated patients may be lost during HDara-C consolidation treatment due to increased toxicity remains to be proven in larger trials.

Correction to: Leukemia (2016) 30, 1672–1681; doi:10.1038/leu.2016.69 Following the publication of this article, the authors have noted that three genes: FLT3, NRAS and ARID1A (each with mutational frequency of 5%) have been incorrectly labelled in Figure 4a showing the mutational landscape of APL relapse.

CpG islands in the 5′ regulatory regions of genes are generally protected from cytosine methylation as methylation in a promoter can result in transcriptional silencing of the associated gene. Failure of a cell to prevent methylation in the promoter regions of tumor suppressor genes contributes to the onset and progression of cancers. The demethylating agent 5-aza-2′deoxycytidine (DAC) and the histone deacetylase inhibitor suberoyl anilide bishydroxamide (SAHA) possess potent antitumorigenic properties against myeloid disorders. Understanding the alterations of the transcriptome mediated by these drugs should prove vital in uncovering potential tumor suppressor genes epigenetically silenced in myeloid disorders. To this end, we used DAC and SAHA to induce expression of methylated genes in the CD34+ AML cell line KG-1. Expression levels of over 22,000 genes were compared between normal CD34+ cells and treated and untreated KG-1 cells using Affymetrix HG-U133A GeneChip® micoarrays. Statistical analyses revealed 76 genes constitutively expressed in normal CD34+ stem cells, absent in KG-1 cells but whose expression was induced in KG-1 after drug treatment. 39 (51%) of these genes harbored a CpG island in their 5′ regulatory regions, representing potentially methylated tumor suppressor genes in AML. To fit the tumor suppressor paradigm, we hypothesized that any gene possessing antitumorigenic properties would not be expressed in a number of AML patient samples. We examined the expression level of our 39 genes in 120 AML patient samples using microarray analyses. 20 patients belonging to each of the following AML karyotypic groups were analyzed: t(8;21), t(15:17), inv(16), 11q23/MLL, complex and normal karyotpye. Of special note were 8 genes, whose expression was markedly diminished in a subset of patients across all AML karyotypes examined: DAR22, TFIIS, EH-3, ENO2, MXA, DRAL, ASTML and MG50. These represent strong candidates for tumor suppressor genes in AML. Unsupervised clustering analyses using our original 39 genes were performed upon microarray data obtained from patients with myeloproliferative disease (MPD). A subset of 10 genes discriminated between granulocyte samples obtained from healthy donors and those obtained from a subset of agnogenic myeloid metaplasia, essential thronbocythemia and polycythemia vera patients. One of these genes, α-catenin, is located at q31 of chromosome 5, a hot spot for deletion in MDS and AML. α-catenin was expressed in all 120 primary AML samples, including those harboring deletions in chromosome 5. However, Real Time PCR analysis of 32 MDS patients harboring a 5q deletion in the region of α-catenin showed a marked decrease in expression of this gene compared to 20 non 5q- MDS patients. Neighboring genes in the deleted region of 5q did not show as marked a decrease in expression, suggesting loss of expression of both α-catenin alleles in these patients. These findings imply a double hit mechanism in 5q- MDS, where loss of one allele of α-catenin through deletion is supplemented by epigenetic silencing (directly or indirectly) of the second allele. In summary, we have uncovered groups of genes that may be involved in the pathogenesis of AML and various MPDs by virtue of their transcriptional repression through epigenetic events. Importantly, we have identified α-catenin as a key gene on chromosome 5, whose expression is lost in 5q- MDS.

are prepared by annealing mixtures of the elements at 750‐800 °C for about one week.

New ternary arsenides, LnNi2As2 (Ln: La, Ce, Pr, Nd, Sm, and U), are prepared by annealing stoichiometric mixtures of the elements in evacuated silica tubes at 660‐1000 °C for 1‐2 weeks.