Early mobilisation in critically ill children: Does routine patient screening reduce time to commencing mobilisation?

OBJECTIVE: The objective of this study was to investigate the impact of daily screening for medical readiness to participate in early mobilisation in the paediatric intensive care unit (PICU), on reducing time to mobilisation and to explore the safety-, feasibility-, and patient-level barriers to the practice. METHODS: An interventional study with a historical control group was conducted in a PICU in a tertiary teaching hospital in Australia. The Early Mobilisation Screening Checklist was applied at 24-48 h of PICU stay with the aim to reduce time to commencing mobilisation. All patients aged term to 18 years admitted to the PICU for >48 h were included in this study. Data on time to mobilisation and patient characteristics were collected by an unblinded case note audit of children admitted to the PICU over 5 months in 2018 for the baseline group and over a corresponding period in 2019 for the intervention group. MEASUREMENTS AND MAIN RESULTS: A total of 71 children were enrolled. Survival analysis was used to compare time to mobilisation between groups, and a cox regression model found that children in the intervention group were 1.26 times more likely to participate in mobility, but this was not statistically significant (P = 0.391, log rank test for equality of survival functions). Early mobilisation was safe, with no adverse events reported in 177 participant mobilisation days. Feasibility was demonstrated by 62% of participants mobilising within 72 h of admission. Mechanical ventilation during stay (P = 0.043) and days receiving sedation infusion (% of days) (P = 0.042) were associated with a decreased likelihood of participating in mobility. CONCLUSIONS: Implementation of routine screening alone does not significantly reduce time to commencing mobility in the PICU. Early mobilisation in the PICU is safe and feasible and resulted in no adverse events during mobilisation. Patient characteristics influencing participation in mobility warrant further exploration.

[Inv(16)-type acute myeloid leukemia with repeated skin infiltration without bone marrow relapse before and after allogeneic hematopoietic stem cell transplantation].

We report a 40-year-old woman diagnosed as having acute myeloid leukemia with CBFB-MYH11. Before and after stem cell transplantation in the phase of molecular remission of the marrow, CBFB-MYH11-positive cells were detected by RT-PCR analysis in skin lesions. The former was pathologically diagnosed as leukemic infiltration, while the latter was considered to be graft-versus-host disease. We can speculate that a low level of leukemic stem cells not detectable by RT-PCR analysis remained in the bone marrow, at least prior to transplantation. This case may suggest interesting biological features of inv(16)-type acute myeloid leukemia.

[CD20-positive peripheral T-cell lymphoma, not otherwise specified].

We report a 69-year-old male with CD3-positive peripheral T-cell lymphoma, not otherwise specified (PTCL-nos). Interestingly, tumor cells slightly expressed CD20 as well. Southern analyses of the tumor cells showed rearrangement for only the T cell receptor gene but not the immunoglobulin genes. This patient achieved partial remission with a treatment regimen of THP-COP excluding prednisolone, but died of pneumonia. Although CD20-positive PTCL is rare, a review of the reported cases suggests that CD20-positive PTCL has a poor prognosis and that bone marrow infiltration of tumor cells results in a poorer prognosis in CD20-positive PTCL than in usual PTCL. By accumulating cases of this rare entity of lymphoma, we need to clarify the biological nature of the tumor cells and usefulness of rituximab combined with standard chemotherapy.

[CAG-GO therapy for patients with relapsed or primary refractory CD33-positive acute myelogenous leukemia].

We previously tested a less toxic CAG regimen consisting of low-dose cytarabine, aclarubicin and granulocyte-colony stimulating factor for the treatment of patients with relapsed or refractory myeloid malignancies or elderly patients with untreated ones, obtaining a satisfactory complete remission rate of 62%. Gemtuzumab ozogamicin, an anti-CD33 monoclonal antibody conjugated to calicheamicin, has recently been approved as a single agent in Japan for the treatment of relapsed/refractory CD33-positive acute myelogenous leukemia (9 mg/m(2) on days1 and 15). Complete remission rate was reported as 30% in a phase 2 trial in Japan. In this study, effectiveness and safety of combining dose-attenuated gemtuzumab ozogamicin (3 mg/m(2) on day5) and original CAG regimen were assessed in nine patients with relapsed/refractory CD33-positive acute myelogenous leukemia and a median age of 70 years. Rate of complete remission with or without platelet recovery was 44% (4/9). The median duration of complete remission and overall survival were 5.5 and 16 months, respectively. Reversible myelosuppression and liver toxicity were the main adverse events, but no regimen-related death was recorded. Although only a small number of cases were included in this preliminary study, this CAG-GO regimen was found to be feasible and useful even in high-risk relapsed or refractory patients.

[Extranodal NK/T-cell lymphoma, nasal type, developed in a patient with rheumatoid arthritis].

It is well known that patients with rheumatoid arthritis (RA) have a higher risk of developing malignant lymphoma (ML) than the general population. Most of these lymphomas occur in patients receiving immunosuppressive (IS) agents such as methotrexate (MTX). Spontaneous regression of tumors is often observed after the discontinuation of IS drugs, especially in patients with Epstein-Barr virus-positive lymphoma. Here we encountered an RA patient who developed extranodal NK/T-cell lymphoma, nasal type during treatment of RA with MTX and etanercept. Despite the discontinuation of MTX and etanercept, the tumor did not show any regression. Complete response was achieved after treatment with concurrent chemoradiotherapy. ML of NK-cell origin is extremely rare, while the majority of ML cases associated with RA are of B-cell origin. This report describes extranodal NK/T-cell lymphoma, nasal type case associated with RA. Such cases should be accumulated to evaluate the mechanism of onset and clinical characteristics of NK/T-cell lymphoma associated with RA.

Leukemia-related transcription factor TEL/ETV6 expands erythroid precursors and stimulates hemoglobin synthesis.

TEL/ETV6 located at chromosome 12p13 encodes a member of the E26 transformation-specific family of transcription factors. TEL is known to be rearranged in a variety of leukemias and solid tumors resulting in the formation of oncogenic chimeric protein. Tel is essential for maintaining hematopoietic stem cells in the bone marrow. To understand the role of TEL in erythropoiesis, we generated transgenic mice expressing human TEL under the control of Gata1 promoter that is activated during the course of the erythroid-lineage differentiation (GATA1-TEL transgenic mice). Although GATA1-TEL transgenic mice appeared healthy up to 18 months of age, the level of hemoglobin was higher in transgenic mice compared to non-transgenic littermates. In addition, CD71+/TER119+ and c-kit+/CD41+ populations proliferated with a higher frequency in transgenic mice when bone marrow cells were cultured in the presence of erythropoietin and thrombopoietin, respectively. In transgenic mice, enhanced expression of Alas-e and beta-major globin genes was observed in erythroid-committed cells. When embryonic stem cells expressing human TEL under the same Gata1 promoter were differentiated into hematopoietic cells, immature erythroid precursor increased better compared to controls as judged from the numbers of burst-forming unit of erythrocytes. Our findings suggest some roles of TEL in expanding erythroid precursors and accumulating hemoglobin.

Role of the RUNX1-EVI1 fusion gene in leukemogenesis.

RUNX1-EVI1 is a chimeric gene generated by t(3;21)(q26;q22) observed in patients with aggressive transformation of myelodysplastic syndrome or chronic myelogenous leukemia. RUNX1-EVI1 has oncogenic potentials through dominant-negative effect over wild-type RUNX1, inhibition of Jun kinase (JNK) pathway, stimulation of cell growth via AP-1, suppression of TGF-beta-mediated growth inhibition and repression of C/EBPalpha. Runx1-EVI1 heterozygous knock-in mice die in uteri due to central nervous system (CNS) hemorrhage and severe defects in definitive hematopoiesis as Runx1-/- mice do, indicating that RUNX1-EVI1 dominantly suppresses functions of wild-type RUNX1 in vivo. Acute myelogenous leukemia is induced in mice transplanted with bone marrow cells expressing RUNX1-EVI1, and a Runx1-EVI1 knock-in chimera mouse developed acute megakaryoblastic leukemia. These results suggest that RUNX1-EVI1 plays indispensable roles in leukemogenesis of t(3;21)-positive leukemia. Major leukemogenic effect of RUNX1-EVI1 is mainly through histone deacetyltransferase recruitment via C-terminal binding protein. Histone deacetyltransferase could be a target in molecular therapy of RUNX1-EVI1-expressing leukemia.

RUNX1/EVI1, which blocks myeloid differentiation, inhibits CCAAT-enhancer binding protein alpha function.

The RUNX1/EVI1 chimeric transcription factor produced by t(3;21) causes leukemic transformation in hematopoietic stem cell tumors, possibly through a differentiation block of malignant myeloid progenitors. A dominant negative effect over wild-type RUNX1 has been shown to constitute one of the underlying molecular mechanisms. We introduced RUNX1/EVI1 cDNA into LG-3 cells that differentiate along the myeloid lineage upon exposure to granulocyte colony stimulating factor, and confirmed that RUNX1/EVI1 suppressed the differentiation. To further investigate the molecular mechanisms of RUNX1/EVI1-mediated differentiation block, we analyzed RUNX1/EVI1's effect on the functions of CCAAT-enhancer binding protein alpha (C/EBPalpha), a key transcriptional regulator that induces granulocytic differentiation. RUNX1/EVI1 was found to associate with C/EBPalpha. By using a reporter assay with the CEBPA promoter, we observed a dominant negative effect of RUNX1/EVI1 over C/EBPalpha-mediated transcriptional activation via the carboxyl terminal-binding protein (CtBP)-binding site in the EVI1 portion. In a gel-shift assay, RUNX1/EVI1 downregulated the DNA-binding activity of C/EBPalpha. Therefore, recruitment of histone deacetylase via CtBP and disruption of DNA binding could be likely scenarios for the RUNX1/EVI1-induced dominant repression on C/EBPalpha. Importantly, coexpression of C/EBPalpha restored the differentiation ability of the RUNX1/EVI1-expressing LG-3 cells. All of these data argue that inhibition of C/EBPalpha function may be causatively related to the leukemogenic potential of RUNX1/EVI1.

Leukemia-related transcription factor TEL is negatively regulated through extracellular signal-regulated kinase-induced phosphorylation.

TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser(213) and Ser(257). TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser(213) and Ser(257) functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.

Cloning and characterization of the novel chimeric gene TEL/PTPRR in acute myelogenous leukemia with inv(12)(p13q13).

We have cloned a novel TEL/protein tyrosine phosphatase receptor-type R (PTPRR) chimeric gene generated by inv(12)(p13q13). PTPRR is the first protein tyrosine phosphatase identified as a fusion partner of TEL. The chimeric gene fused exon 4 of the TEL gene with exon 7 of the PTPRR gene, and produced 10 isoforms through alternative splicing. Two isoforms that were expressed at the highest level in the leukemic cells could have been translated into COOH-terminally truncated TEL protein possessing the helix-loop-helix domain (tTEL) and TEL/PTPRR chimeric protein linking the helix-loop-helix domain of TEL to the catalytic domain of PTPRR. These two mutant proteins exerted a dominant-negative effect over transcriptional repression mediated by wild-type TEL, although they themselves did not show any transcriptional activity. Heterodimerization with wild-type TEL might be an underlying mechanism in this effect. TEL/PTPRR did not exhibit any tyrosine phosphatase activity. Importantly, overexpression of TEL/PTPRR in granulocyte macrophage colony-stimulating factor-dependent UT7/GM cells resulted in their factor-independent proliferation, whereas overexpression of tTEL did not. After cytokine depletion, phosphorylated signal transducers and activators of transcription 3 (STAT3) significantly declined in mock cells, but remained in both tTEL- and TEL/PTPRR-overexpressing cells. Loss of tumor suppressive function of wild-type TEL and maintenance of STAT3-mediated signal could at least partly contribute to the leukemogenesis caused by inv(12)(p13q13).

[Asian variant of intravascular large B-cell lymphoma diagnosed by bone marrow biopsy].

A 63-year-old male presented with fever and general malaise in June 2004. On admission hepatosplenomegaly was apparent, but without lymphadenopathy. The laboratory examination revealed pancytopenia and increased levels of lactate dehydrogenase, direct bilirubin and soluble interleukin-2 receptor. Histological analysis of the bone marrow biopsy specimen demonstrated proliferation of atypical lymphoid cells positive for CD20 in the small capillaries, leading to the diagnosis of the Asian variant of intravascular large B-cell lymphoma (AIVL). The presence of rearrangement of the immunoglobulin gene confirmed the diagnosis. The patient responded well to CHOP therapy followed by seven courses of rituximab-combined CHOP therapy and has remained in complete remission up to the present. This case implies that bone marrow biopsy could be a useful examination for diagnosing AIVL and that rituximab-combinedchemotherapy could improve survival in patients with the disease.

The t(3;21) fusion product, AML1/Evi-1 blocks AML1-induced transactivation by recruiting CtBP.

AML1/Evi-1 is a chimeric protein that is derived from t(3;21), found in blastic transformation of chronic myelogenous leukemia. It is composed of the N-terminal AML1 portion with the DNA-binding Runt domain and the C-terminal Evi-1 portion. It has been shown to dominantly repress AML1-induced transactivation. The mechanism for it has been mainly attributed to competition with AML1 for the DNA-binding and for the interaction with PEBP2beta (CBFbeta), a partner protein which heterodimerizes with AML1. It was recently found that Evi-1 interacts with C-terminal binding protein (CtBP) to repress TGFbeta-induced transactivation. Here, we demonstrate that AML1/Evi-1 interacts with CtBP in SKH1 cells, a leukemic cell line which endogenously overexpresses AML1/Evi-1 and that AML1/Evi-1 requires the interaction with CtBP to repress AML1-induced transactivation. The association with CtBP is also required when AML1/Evi-1 blocks myeloid differentiation of 32Dcl3 cells induced by granulocyte colony-stimulating factor. Taken together, it is suggested that one of the mechanisms for AML1/Evi-1-associated leukemogenesis should be an aberrant recruitment of a corepressor complex by the chimeric protein.

Leukemia-related transcription factor TEL accelerates differentiation of Friend erythroleukemia cells.

TEL belongs to a member of the ETS family transcription factors that represses transcription of target genes such as FLI-1. Although TEL is essential for establishing hematopoiesis in neonatal bone marrow, its role in erythroid lineage is not understood. To investigate a role for TEL in erythroid differentiation, we introduced TEL into mouse erythroleukemia (MEL) cells. Overexpressing wild-type-TEL in MEL cells enhanced differentiation induced by hexamethylene bisacetamide or dimethylsulfoxide, as judged by the increased levels of erythroid-specific delta-aminolevulinate synthase and beta-globin mRNAs. TEL bound to a corepressor mSin3A through the helix-loop-helix domain. A TEL mutant lacking this domain still bound to the ETS binding site, but lost its transrepressional effect. This mutant completely blocked erythroid differentiation in MEL cells. Moreover, it showed dominant-negative effects over TEL-mediated transcriptional repression and acceleration of erythroid differentiation. Endogenous TEL mRNA was found to increase during the first 3 days in differentiating MEL cells and drastically decrease thereafter. All these data suggest that TEL might play some role in erythroid cell differentiation.

Runx1/AML1 in normal and abnormal hematopoiesis.

Runx1/AML1 (also known as CBFA2 and PEBP23B) is a Runt family transcription factor critical for normal hematopoiesis. Runx1 forms a heterodimer with CBF3 and binds to the consensus PEBP2 sequence through the Runt domain. Runx1 enhances gene transcription by interacting with transcriptional coactivators such as p300 and CREB-binding protein. However, Runx1 can also suppress gene transcription by interacting with transcriptional corepressors, including mSin3A, TLE (mammalian homolog of Groucho), and histone deacetylases. Runx1 not only is critical for definitive hematopoiesis in the fetus but also is required for normal megakaryocytic maturation and T-lymphocyte and B-lymphocyte development in adult mice. Runx1 has been identified in leukemia-associated chromosomal translocations, including t(8;21) (Runx1-ETO/MTG8), t(16;21) (Runx1-MTG16), t(3;21) (Runx1-Evi1), t(12;21) (TEL-Runx1), and t(X;21) (Runx1-Fog2). The molecular mechanism of leukemogenesis by these fusion proteins is discussed. Various mutant mice expressing these fusion proteins have been created. However, expression of the fusion protein is not sufficient by itself to cause leukemia and likely requires additional events for leukemogenesis. Point mutations in a Runx1 allele cause haploinsufficiency and a biallelic null for Runx1, which are associated with familial platelet disorder with a propensity for acute myeloid leukemia (FPD/AML) and AML-M0, respectively. Thus, the correct protein structure and the precise dosage of Runx1 are essential for the maintenance of normal hematopoiesis.

Mutational analyses of the AML1 gene in patients with myelodysplastic syndrome.

The AML1 gene is the most frequent target of translocations associated with human leukemias. We recently found somatic point mutations of the AML1 gene, V105ter and R139G, in two cases of myelodysplastic syndrome (MDS). Both mutations are present in the region encoding the Runt domain of AML1, and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with PEBP2beta/CBFbeta. On the other hand, the R139G mutant acts as a dominant negative inhibitor through competing with wild-type AML1 for interaction with PEBP2beta/CBFbeta. In this review, we summarize mutational changes of the AML1 gene in hematological malignancies, especially in MDS and discuss the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

[Philadelphia chromosome-positive acute lymphoblastic leukemia showing normal karyotype at presentation].

21-year-old male was diagnosed as having acute lymphoblastic leukemia (ALL) in March 2001. Standard G-banding analysis revealed normal karyotype. He achieved complete remission after the first course of induction therapy and underwent allogeneic PBSCT in August 2001. However, leukemic cells appeared in the peripheral blood demonstrating complex chromosomal abnormalities with Philadelphia chromosome (Ph1) and expressing minor BCR/ABL transcripts. We thus retrospectively examined the bone marrow sample at presentation with both interphase FISH and RT-PCR methods. Surprisingly, almost all leukemic blasts at presentation turned out to possess the minor type of BCR/ABL fusion gene. In spite of the second allogeneic PBSCT, the patient died of pneumonia and graft-versus-host disease in the liver in September 2002. This case is rare in that cytogenetic analysis failed to detect Ph1 due to leukemic cells dormancy. The application of both FISH and RT-PCR studies would appear to be essential to avoid overlooking Ph1-positive ALL.

[Double t (3; 21) in acute myelomonocytic leukemia transformed from chronic myelomonocytic leukemia].

A 63-year-old male was diagnosed as chronic myelomonocytic leukemia with normal karyotype in September 1998. He developed acute myelogenous leukemia (AML-M4Eo) in September 2001. The cytogenetic analysis disclosed double t(3;21) at a ratio of 1/20, and the reverse transcriptase-polymerase chain reaction showed AML1/EVI-1 mRNA. He could not achieve complete remission after two courses of induction chemotherapy, and his leukemia cells carrying double t(3;21) were relatively increased. He died of interstitial pneumonia in December 2001. This is the first leukemia case with double t(3;21) and this chromosomal abnormality might play a role in leukemia cell proliferation by generating two AML1/EVI-1 genes.

[Development of overt hemolytic anemia after splenectomy for thrombocytopenia in Evans syndrome with negative Coombs test].

A 69-year-old man was diagnosed as having idiopathic thrombocytopenic purpura (ITP) in April 2000, and treated with prednisolone (PSL) without effect. Splenectomy performed in June 2000 had only a transient and marginal influence on his platelet count. Two months later, he developed autoimmune hemolytic anemia (AIHA) without Coombs test positivity, and his diagnosis was changed to Coombs-negative Evans syndrome. Treatment with PSL led to recovery of his hemoglobin level, but not his platelet count. Although the mechanism responsible for development of AIHA after splenectomy in this patient with ITP remains unknown, close observation is required for any association with other autoimmune diseases such as SLE.