Course of colonization by multidrug-resistant organisms after allogeneic hematopoietic cell transplantation.

Multidrug-resistant organisms (MDRO) have been developing as an emerging problem in allogeneic hematopoietic cell transplantation (HCT). Since no data are available on the course of MDRO colonization after HCT, we investigated in this retrospective, single-center study, persistence and clearance of MDRO after HCT. From June 2010 to December 2015, 121 consecutive HCT patients were included. Patients received a MDRO screening before conditioning as well as surveillance cultures after HCT. In MDRO-colonized patients, surveillance specimens were taken until MDRO were no longer detectable. Thirty-three patients (27%) were found to be colonized by at least one MDRO at any time point until day 100 post HCT. Day 100 (2-year) non-relapse mortality (NRM) and overall survival (OS) of MDRO-colonized (MDRO+) versus non-colonized (MDRO-) patients were essentially the same. NRM is 15% (21%) versus 15% (24%). Two-year OS is 60 versus 55% for MDRO+ versus MDRO- patients. Out of the 33 MDRO+ patients, 21 cleared the MDRO. Median time to non-detectability of MDRO was 6 months. In 12 patients, the MDRO persisted. There was a significant (p < 0.0001) survival difference between patients who cleared the MDRO versus those with MDRO persistence (2-year OS 80 vs 40%). Except for the length of antibiotic therapy as a potential risk factor for MDRO persistence after HCT, no other conventional factors could be identified. (a) colonization by MDRO per se had no negative impact on the outcome, (b) MDRO can be cleared by the majority of patients after allogeneic HCT, and (c) to increase the probability to clear MDRO, the use of antibiotics in MDRO+ patients should be reviewed critically.

Molecular alterations in bone marrow mesenchymal stromal cells derived from acute myeloid leukemia patients.

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 PLEC gene 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.

Splenomegaly, elevated alkaline phosphatase and mutations in the SRSF2/ASXL1/RUNX1 gene panel are strong adverse prognostic markers in patients with systemic mastocytosis.

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.

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

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.

Secondary malignancies in chronic myeloid leukemia patients after imatinib-based treatment: long-term observation in CML Study IV.

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.

Allogeneic hematopoietic cell transplantation without fluconazole and fluoroquinolone prophylaxis.

Fluoroquinolone (FQ) and fluconazole prophylaxis is recommended for patients undergoing allogeneic hematopoietic cell transplantation (alloHCT). However, due to an uncertain scientific basis and the increasing emergence of resistant germs, this policy should be questioned. Therefore, FQ and fluconazole prophylaxis was omitted in alloHCT at our center. In this retrospective analysis, all consecutive patients (n = 63) who underwent first alloHCT at our institution from September 2010 to September 2013 were included. Patients neither received FQ nor fluconazole prophylaxis. Day 100 mortality, incidence of febrile neutropenia, bacterial infections, and invasive fungal diseases (IFD) were assessed. Sixteen patients who started conditioning under antimicrobial treatment/prophylaxis due to pre-existing neutropenia (3/16), IFD (12/16), or aortic valve replacement (1/16) were excluded from the analysis. Finally, 47 patients were transplanted without prophylaxis as intended. Day 100 mortality was 9 %. Febrile neutropenia occurred in 62 % (29/47); 17/47 patients (36 %) experienced a blood stream infection (BSI) with detection of Gram-positive bacteria in 14 patients, Gram-negative bacteria in five patients, and candida in one patient, respectively. Coagulase-negative staphylococci were the most frequently isolated Gram-positive bacteria; 12/21 isolated Gram-positive and 3/6 Gram-negative bacteria were FQ resistant. In 21 % (10/47) of the patients, IFD (1x proven, 1x probable, and 8x possible) were diagnosed. To conclude, all three criteria, day 100 mortality, the incidence of IFD, and BSI, are in the range of published data for patients transplanted with FQ and fluconazole prophylaxis. These data demonstrate that alloHCT is feasible without FQ and fluconazole prophylaxis.

Additional mutations in SRSF2, ASXL1 and/or RUNX1 identify a high-risk group of patients with KIT D816V(+) advanced systemic mastocytosis.

Most patients with KIT D816V(+) 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 KIT D816V(+) 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 TET2 or JAK2. In multivariate analysis, SRSF2 and ASXL1 remained 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 KIT D816V(+) SM. On the basis of these findings, inclusion of molecular markers should be considered in upcoming prognostic scoring systems for patients with SM.

Identification of invasive fungal diseases in immunocompromised patients by combining an Aspergillus specific PCR with a multifungal DNA-microarray from primary clinical samples.

The increasing incidence of invasive fungal diseases (IFD), most of all invasive aspergillosis (IA) in immunocompromised patients emphasises the need to improve the diagnostic tools for detection of fungal pathogens. We investigated the diagnostic performance of a multifungal DNA-microarray detecting 15 different fungi [Aspergillus, Candida, Fusarium, Mucor, Rhizopus, Scedosporium and Trichosporon species (spp.)] in addition to an Aspergillus specific polymerase chain reaction (PCR) assay. Biopsies, bronchoalveolar lavage and peripheral blood samples of 133 immunocompromised patients (pts) were investigated by a multifungal DNA-microarray as well as a nested Aspergillus specific PCR assay. Patients had proven (n = 18), probable (n = 29), possible (n = 48) and no IFD (n = 38) and were mostly under antifungal therapy at the time of sampling. The results were compared to culture, histopathology, imaging and serology, respectively. For the non-Aspergillus IFD the microarray analysis yielded in all samples a sensitivity of 64% and a specificity of 80%. Best results for the detection of all IFD were achieved by combining DNA-microarray and Aspergillus specific PCR in biopsy samples (sensitivity 79%; specificity 71%). The molecular assays in combination identify genomic DNA of fungal pathogens and may improve identification of causative pathogens of IFD and help overcoming the diagnostic uncertainty of culture and/or histopathology findings, even during antifungal therapy.

Arsenic trioxide-based therapy of relapsed acute promyelocytic leukemia: registry results from the European LeukemiaNet.

In 2008, a European registry of relapsed acute promyelocytic leukemia was established by the European LeukemiaNet. Outcome data were available for 155 patients treated with arsenic trioxide in first relapse. In hematological relapse (n=104), 91% of the patients entered complete hematological remission (CR), 7% had induction death and 2% resistance, 27% developed differentiation syndrome and 39% leukocytosis, whereas no death or side effects occurred in patients treated in molecular relapse (n=40). The rate of molecular (m)CR was 74% in hematological and 62% in molecular relapse (P=0.3). All patients with extramedullary relapse (n=11) entered clinical and mCR. After 3.2 years median follow-up, the 3-year overall survival (OS) and cumulative incidence of second relapse were 68% and 41% in hematological relapse, 66% and 48% in molecular relapse and 90 and 11% in extramedullary relapse, respectively. After allogeneic or autologous transplantation in second CR (n=93), the 3-year OS was 80% compared with 59% without transplantation (n=55) (P=0.03). Multivariable analysis demonstrated the favorable prognostic impact of first remission duration ⩾1.5 years, achievement of mCR and allogeneic or autologous transplantation on OS of patients alive after induction (P=0.03, P=0.01, P=0.01) and on leukemia-free survival (P=0.006, P<0.0001, P=0.003), respectively.

Molecular profiling of myeloid progenitor cells in multi-mutated advanced systemic mastocytosis identifies KIT D816V as a distinct and late event.

To explore the molecular profile and its prognostic implication in systemic mastocytosis (SM), we analyzed the mutation status of granulocyte-macrophage colony-forming progenitor cells (CFU-GM) in patients with KIT D816V(+) indolent SM (ISM, n=4), smoldering SM (SSM, n=2), aggressive SM (ASM, n=1), SM with associated clonal hematologic non-mast cell lineage disorder (SM-AHNMD, n=5) and ASM-AHNMD (n=7). All patients with (A)SM-AHNMD (n=12) carried 1-4 (median 3) additional mutations in 11 genes tested, most frequently TET2, SRSF2, ASXL1, CBL and EZH2. In multi-mutated (A)SM-AHNMD, KIT D816V(+) single-cell-derived CFU-GM colonies were identified in 8/12 patients (median 60%, range 0-95). Additional mutations were identified in CFU-GM colonies in all patients, and logical hierarchy analysis indicated that mutations in TET2, SRSF2 and ASXL1 preceded KIT D816V. In ISM/SSM, no additional mutations were detected and CFU-GM colonies were exclusively KIT D816V(-). These data indicate that (a) (A)SM-AHNMD is a multi-mutated neoplasm, (b) mutations in TET2, SRSF2 or ASXL1 precede KIT D816V in ASM-AHNMD,

Velocity of early BCR-ABL transcript elimination as an optimized predictor of outcome in chronic myeloid leukemia (CML) patients in chronic phase on treatment with imatinib.

UNLABELLED: Early assessment of response at 3 months of tyrosine kinase inhibitor treatment has become an important tool to predict favorable outcome. We sought to investigate the impact of relative changes of BCR-ABL transcript levels within the initial 3 months of therapy. In order to achieve accurate data for high BCR-ABL levels at diagnosis, beta glucuronidase (GUS) was used as a reference gene. Within the German CML-Study IV, samples of 408 imatinib-treated patients were available in a single laboratory for both times, diagnosis and 3 months on treatment. In total, 301 of these were treatment-naïve at sample collection. RESULTS: (i) with regard to absolute transcript levels at diagnosis, no predictive cutoff could be identified; (ii) at 3 months, an individual reduction of BCR-ABL transcripts to the 0.35-fold of baseline level (0.46-log reduction, that is, roughly half-log) separated best (high risk: 16% of patients, 5-year overall survival (OS) 83% vs 98%, hazard ratio (HR) 6.3, P=0.001); (iii) at 3 months, a 6% BCR-ABL(IS) cutoff derived from BCR-ABL/GUS yielded a good and sensitive discrimination (high risk: 22% of patients, 5-year OS 85% vs 98%, HR 6.1, P=0.002). Patients at risk of disease progression can be identified precisely by the lack of a half-log reduction of BCR-ABL transcripts at 3 months.

Assessment of Aspergillus-specific PCR as a screening method for invasive aspergillosis in paediatric cancer patients and allogeneic haematopoietic stem cell recipients with suspected infections.

Invasive aspergillosis (IA) remains difficult to diagnose in immunocompromised patients, because diagnostic EORTC/MSG criteria are often not met. As biomarkers might elucidate the pathogen, we analysed the performance of an Aspergillus PCR assay in blood for diagnosis of IA in immunocompromised paediatric patients with suspected infections. Ninety-five haemato-oncological paediatric patients were included over a period of 3 years, the underlying diseases consisting of acute leukaemia, solid tumours, non-malignant immunocompromising disorders and haematopoietic stem cell transplantation recipients. We retrospectively analysed 253 consecutive episodes of suspected infections. Thirty-eight patients had possible IA, none of the patients fulfilled EORTC/MSG criteria of probable/proven IA. PCR positivity was observed in 97/967 analyses. Sensitivity, specificity, positive and negative predictive value of the PCR per episode were 34%, 78%, 31% and 81% using possible IA as endpoint. Taken together, an undirected blood screening by Aspergillus-specific PCR is of little diagnostic value in a heterogenous paediatric patient cohort. Harnessing PCR for diagnosis of IA should thus be focused on blood analyses of more homogenous high-risk patients and/or analyses of bronchoalveolar lavage, tissue or cerebrospinal fluid specimens.

Expression of transketolase-like gene 1 (TKTL1) depends on disease phase in patients with chronic myeloid leukaemia (CML).

PURPOSE: Overexpression of transketolase-like gene 1 (TKTL1) on RNA and protein level has been linked to tumour progression, metastasis and unfavourable patient outcome in many solid tumours. Chronic myeloid leukaemia (CML) cells show metabolic characteristics resembling deviations observed in TKTL1 overexpressing solid tumour cells. We therefore sought to evaluate TKTL1 gene expression in different phases of CML. METHODS: A total of 120 peripheral blood samples from 69 patients in various phases of CML and 21 healthy individuals were investigated. TKTL1 expression levels were determined by real-time quantitative polymerase chain reaction using LightCycler technology and normalised against beta-glucuronidase expression. RESULTS: A significantly lower TKTL1 expression was found in chronic phase (CP) CML patients compared to healthy controls. Lowest expression levels were observed in patients during blast crisis (BC). Baseline TKTL1 expression in CP patients did not have value in prognostication of subsequent favourable or dismal outcome. Further, more mature granulocytes showed significantly higher TKTL1 expression compared to immature CD34+ and CD34-/CD33+ cells both in healthy controls and in CML patients. CONCLUSION: TKTL1 expression levels appear to decline in the course of CML with lowest levels during BC. A potential reason is a shift of TKTL1-high-expressing mature granulocytes towards TKTL1-low-expressing immature cells and blasts.

MDR1 expression predicts outcome of Ph+ chronic phase CML patients on second-line nilotinib therapy after imatinib failure.

In the face of competing tyrosine kinase inhibitors (TKIs), identification of chronic myeloid leukemia (CML) patients expecting favorable response to second-line treatment is warranted. At the time of imatinib resistance, the investigation of multidrug-resistance protein 1 (MDR1) and BCR-ABL yielded the following results: (i) Patients with high MDR1 transcript levels showed superior response at 48 months as compared with low-level MDR1 patients: major molecular response (MMR) in 41% vs 16% (P=0.014), complete cytogenetic response (CCyR) in 58% vs 39% (P=0.044), and progression-free survival (PFS) in 67% vs 46% (P=0.032). (ii) Patients with BCR-ABL(IS) <28% achieved higher MMR rates (48% vs 21%, P=0.009). (iii) PFS at 48 months was associated with in vitro resistance of BCR-ABL kinase domain mutations: 63% (no mutation) vs 61% (sensitive, intermediately sensitive or unknown IC50 (median inhibitory concentration)) vs 23% (resistant, P=0.01). (iv) Single-nucleotide polymorphisms (SNPs) at positions 1236 and 2677 were associated with higher MDR1 expression in comparison to wild type. (v) Nilotinib was able to impede proliferation of MDR1-overexpressing imatinib-resistant cells. High MDR1 gene expression might identify patients whose mode of imatinib resistance is essentially determined by increased efflux activity of MDR1 and therefore can be overcome by second-line nilotinib treatment.

Whole-body CT for lymphoma staging: feasibility of halving radiation dose and risk by iterative image reconstruction.

OBJECTIVES: Patients with lymphoma are at higher-risk of secondary malignancies mainly due to effects of cancer therapy as well as frequent radiological surveillance. We thus aimed to investigate the objective and subjective image quality as well as radiation exposure and risk of full-dose standard (FDS), full-dose iterative (FDI), and half-dose iterative (HDI) image reconstruction in patients with lymphoma. MATERIAL AND METHODS: In 100 lymphoma patients, contrast-enhanced whole-body staging was performed on a dual-source CT. To acquire full-dose and half-dose CT data simultaneously, the total current-time product was equally distributed on both tubes operating at 120 kV. HDI reconstructions were calculated by using only data from one tube. Quantitative image quality was assessed by measuring image noise in different tissues of the neck, thorax, and abdomen. Overall diagnostic image quality was assessed using a 5-point Likert scale. Radiation doses and risks were estimated for a male and female reference person. RESULTS: For all anatomical regions apart from the lungs image noise was significantly lower and the overall subjective image quality significantly better when using FDI and HDI instead of FDS reconstruction (p<0.05). For the half-dose protocol, the risk to develop a radiation-induced cancer was estimated to be less than 0.11/0.19% for an adult male/female. CONCLUSIONS: Image quality of FDI and more importantly of HDI is superior to FDS reconstruction, thus enabling to halve radiation dose and risk to lymphoma patients.

Patients with malignant hematological disorders treated on a palliative care unit: prognostic impact of clinical factors.

A reliable estimation of prognosis in patients receiving palliative care is desirable in order to facilitate clinical decision finding. For patients with advanced hematological malignancies, only few data are available to estimate prognosis of the individual's remaining life span. Here, we sought to investigate potential clinical prognostic parameters in patients with hematological malignancies admitted to a palliative care unit. Using a prospectively collected database, we analyzed clinical and laboratory parameters regarding their prognostic impact in 290 patients with malignant hematological diseases. The parameters included patient-related factors such as Eastern Cooperative Oncology Group (ECOG) performance status, need for transfusions, parenteral nutrition or analgetics, and laboratory values (hemoglobin, platelet count, lactic dehydrogenase (LDH), albumin, total protein, calcium, and C-reactive protein (CRP)) as well as referral symptoms (including anemia, infection, fever, fatigue, and dyspnea). In univariate analyses, LDH (>248 U/l), albumin corrected calcium (>2.55 mmol/l), CRP (>50 mg/l), albumin (<30 g/l), platelet count (<90 × 10(9)/l), total protein (≤60 g/l), hemoglobin (<10 g/dl), opioid treatment, performance status (ECOG >2), and need for parenteral nutrition or blood transfusion significantly correlated with impaired survival. Multivariate analysis showed that low performance status, low platelet count, opioid based pain therapy, high LDH, and low albumin were associated with poor prognosis. Using these five parameters, patients were divided into three "risk groups": low risk (presence of zero to one factor), intermediate risk (two to three factors), and high risk. Median survival for the poor risk patients was 10 days, and the intermediate and low risk patients survived a median of 63 and 440 days, respectively (p < 0.0001). Several clinical and laboratory parameters were associated with a poor prognosis of patients with hematological malignancies treated on a palliative care unit. These parameters might help clinicians to estimate prognosis of remaining life span and individualize treatment and/or end-of-life care options for patients.