BCR-ABL induces gene expression levels of SOCS proteins.

<p><b>A.</b> Ba/F3 cells were transduced with BCR-ABL or an empty vector control. Prior to RNA extraction, cells were starved over night from IL-3. RT-PCR was performed for SOCS family members. Relative gene expression normalized to <i>RNA pol II</i> is depicted. The experiment was performed three times, the error bars are indicating the standard deviation. <b>B.</b> Sca-1+ cells were enriched from murine BM and transduced with BCR-ABL or an empty vector control. GFP-sorted cells were starved over night from cytokines prior to RNA extraction. Relative gene expression of SOCS family members is shown. Relative gene expression normalized to <i>RNA pol II</i> is depicted. The experiment was performed three times, the error bars are indicating the standard deviation. *P ≤ 0.05, **P ≤ 0.009, ***P ≤ 0.001.</p

Mouse transplantation model.

<p>Transduction efficiency of Sca-1⁺ cells was normalized according to GFP expression and 2.5 x 10⁴ GFP-positive cells were transplanted into the tail vein of sub-lethally irradiated recipient mice. Moribund mice were sacrificed and analyzed. All data shown are from time of death. <b>A.</b> Disease free survival of transplanted animals is plotted in Kaplan-Meier curves. <b>B.</b> White blood counts and hemoglobin content in the peripheral blood. <b>C.</b> Spleen and liver weights of analyzed mice. <b>D.</b> Spleen samples were stained with H&E. Representative images are shown.</p

Gene expression levels of SOCS proteins after BCR-ABL kinase inhibition.

<p><b>A-B.</b> Philadelphia chromosome positive (Ph+) or negative (Ph-) long term cultures of primary human acute lymphoblastic leukemia cells were treated with 1 μM imatinib for 16h. RNA was extracted and RT-PCR was performed for SOCS family members. Relative gene expression normalized to B2M is shown. The experiment was performed three times, the error bars are indicating the standard deviation. **P ≤ 0.009, ***P ≤ 0.001 <b>C.</b> K562 cells were treated with 2 μM imatinib for 16h. RNA was extracted and RT-PCR performed for SOCS family members. Gene expression relative to B2M is depicted. The experiment was performed three times, the error bars are indicating the standard deviation. *P ≤ 0.02, **P ≤ 0.009, ***P ≤ 0.001 <b>D.</b> K562 cells were washed with PBS and resuspended in RPMI medium containing 1% FCS. BCR-ABL was inhibited for 16h with 2 μM imatinib or 20 nM dasatinib. Total cell lysates were immunoblotted and probed with indicated antibodies. Tubulin is shown as loading control. Inhibition of BCR-ABL was assessed by its phosphorylation status. The experiment was performed three times.</p

Multivariate analysis for overall survival.

<p>95% CI, 95% confidence interval; allo-HSCT, allo-HSCT, allogeneic hematopoietic stem cell transplantation; ELN, European Leukemia Net. Of all <i>S</i>.<i>maltophilia</i> colonized patients (n = 20), 15 (75%) were also colonized by an MDRO as described previously[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201169#pone.0201169.ref019" target="_blank">19</a>].</p

Baseline patient characteristics.

<p>P-values reveals differences between colonized and non-colonized patients. Allo-HSCT, allogeneic hematopoietic stem cell transplantation; AML, acute myeloid leukemia; ELN, European Leukemia Net; CR, complete remission; MAC, myeloablative conditioning; PBSC, peripheral blood stem cells; MRD, matched related donor; MUD, matched unrelated donor; MMUD, mismatched unrelated donor; CMV, cytomegalovirus; GvHD, Graft versus Host Disease, HCT-CI, hemtaopoetic stem cell transplantation comorbidity index.</p

<i>Stenotrophomonas maltophilia</i> colonization during allogeneic hematopoietic stem cell transplantation is associated with impaired survival - Fig 2

<p><b>Cumulative incidence of non-relapse related mortality (A) and cumulative incidence of relapse (B)</b> after allogeneic hematopoietic stem cell transplantation, stratified by colonized (dotted line) and non-colonized (solid line) patients.</p

Transplant-related characteristics and outcomes.

<p>P-values reveal differences between colonized and non-colonized patients. ANC, absolute neutrophil count; PLT, platelets; aGvHD, acute Graft versus Host Disease; cGvHD, chronic Graft versus Host Disease MDRO, multidrug resistant organisms; BIS, bloodstream infection; allo-HSCT, allogeneic hematopoietic stem cell transplantation. OS, Overall survival, 95% CI, 95% confidence interval; NRM, non-relapse mortality; GvHD, Graft versus Host Disease.</p

<i>Stenotrophomonas maltophilia</i> colonization during allogeneic hematopoietic stem cell transplantation is associated with impaired survival

<div><p>Allogeneic hematopoietic stem cell transplantation (allo-HSCT) offers potential cure to acute myeloid leukemia (AML) patients. However, infections with commensal bacteria are an important cause for non-relapse mortality (NRM). We have previously described the impact of multidrug-resistant organism (MDRO) colonization on the survival of allo-HSCT patients. In the aforementioned publication, according to consensus, we there did not consider the opportunistic gram-negative bacterium <i>Stenotrophomonas maltophilia</i> (<i>S</i>. <i>maltophilia</i>) to be an MDRO. Since rate of <i>S</i>. <i>maltophilia</i> colonization is increasing, and it is not known whether this poses a risk for allo-HSCT patients, we here analyzed here its effect on the previously described and now extended patient cohort. We report on 291 AML patients undergoing allo-HSCT. Twenty of 291 patients (6.9%) were colonized with <i>S</i>. <i>maltophilia</i>. Colonized patients did not differ from non-colonized patients with respect to their age, remission status before allo-HSCT, donor type and HSCT-comorbidity index. <i>S</i>. <i>maltophilia</i> colonized patients had a worse overall survival (OS) from 6 months up to 60 months (85% vs. 88.1% and 24.7% vs. 59.7%; p = 0.007) due to a higher NRM after allo-HSCT (6 months: 15% vs. 4.8% and 60 months: 40.1% vs. 16.2% p = 0.003). The main cause of mortality in colonized patients was infection (46.2% of all deaths) and in non-colonized patients relapse (58.8% of all deaths). 5/20 colonized patients developed an invasive infection with <i>S</i>. <i>maltophilia</i>. The worse OS after allo-HSCT due to higher infection related mortality might implicate the screening of allo-HSCT patients for <i>S</i>. <i>maltophilia</i> and a closer observation of colonized patients as outpatients.</p></div

CONSORT statement.

<p>The patient flow through this dose finding pilot trial is shown.</p

Hematotoxicity of induction and consolidation therapy.

<p>The time from start of chemotherapy until regeneration of leukocytes >1,000/µl, neutrophils >500/µl and plateletes >20,000/µl (transfusion independent) is shown. A: induction therapy, B: consolidation therapy.</p