Multicolor detection of rare tumor cells in blood using a novel flow cytometry-based system.

The presence and number of circulating tumor cells (CTCs) in the blood of patients with solid tumors are predictive of their clinical outcomes. To date, the CellSearch system is the only US Food and Drug Administration-approved CTC enumeration system for advanced breast, prostate, and colon cancers. However, sensitivity issues due to epithelial cellular adhesion molecule (EpCAM)-based enrichment and limited capability for subsequent molecular analysis must be addressed before CTCs can be used as predictive markers in the clinical setting. We have developed a multicolor CTC detection system using cross-contamination-free flow cytometry, which permits the enumeration and characterization of CTCs for multiple molecular analyses. Tumor cell lines with different expression levels of EpCAM were spiked into peripheral blood obtained from healthy donors. Spike-in samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, and this was followed by fixation and labeling with CD45-Alexa Fluor 700, EpCAM-phycoerythrin, cytokeratin (CK)-fluorescein isothiocyanate antibodies, and/or 7-aminoactinomycin D for nuclei staining. Excellent detection (slope = 0.760-0.888) and a linear performance (R(2) = 0.994-0.998) were noted between the observed and expected numbers of tumor cells, independent of EpCAM expression. The detection rate was markedly higher than that obtained using the CellSearch system, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Additionally, the incorporation of an epithelial-mesenchymal transition (EMT) marker allowed us to detect EpCAM-/CK- cells and EMT-induced tumor cells. Taken together, our multicolor CTC detection system may be highly efficient in detecting previously unrecognized populations of CTCs.

Activation of NOTCH signaling impedes cell proliferation and survival in acute megakaryoblastic leukemia.

The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.

Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis.

The RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1(-/-) mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1(-/-) HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1(-/-) HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications.

A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells.

Runx1 is essential for the generation of hematopoietic stem cells (HSCs) and is frequently mutated in human leukemias. However, the cis-regulatory mechanisms modulating the Runx1 gene expression remain to be elucidated. Herewith, we report the identification of an intronic Runx1 enhancer, Runx1 +24 mouse conserved noncoding element (mCNE), using a combinatorial in silico approach involving comparative genomics and retroviral integration sites mapping. The Runx1 +24 mCNE was found to possess hematopoietic-specific enhancer activity in both zebrafish and mouse models. Significantly, this enhancer is active specifically in hemogenic endothelial cells (ECs) at sites where the de novo generation of HSCs occurs. The activity of this enhancer is also strictly restricted to HSCs within the hematopoietic compartment of the adult bone marrow. We anticipate that Runx1 +24 mCNE HSC enhancer will serve as a molecular handle for tracing and/or manipulating hemogenic ECs/HSCs behavior in vivo, and consequently become an invaluable tool for research on stem cell and cancer biology.

A case of perforated multiple gastric duplication cysts with five-layered appearance on ultrasonography.

A case of perforated multiple gastric duplication cysts is presented. One of the gastric duplication cysts without perforation was seen on ultrasonography (US) with the following specific findings of gastric duplication cysts: a five-layered appearance, fold-like structures in the wall, and blood supply from the gastroepiploic artery. Other cysts with perforation presented with irregular and thin walls with a lack of the "double-wall sign" on US. These characteristic findings may be specific to perforated multiple gastric duplication cysts.

Secondary aortoduodenal fistula identified by ultrasonography.

A 73-year-old man underwent coronary artery bypass grafting, abdominal aortic aneurysm resection, and prosthetic implantation as a single procedure in 2002. His progress was favorable until April 2008, when he was admitted to our hospital with melena. B-mode ultrasonography revealed a 5-mm defect in the abdominal aorta at the graft anastomosis, and an umbilicated lesion was seen projecting between the posterior wall of the third part of the duodenum and the abdominal aorta. A color signal was noticed at this site on color Doppler ultrasonography, leading to the diagnosis of a secondary aortoduodenal fistula (ADF). We resected the inflammatory mass comprising the graft and the third part of the duodenum, and performed prosthetic re-implantation, omentopexy, and duodenojejunostomy. We could not find any previous reports of successful identification of secondary ADF using ultrasonography. When a patient with gastrointestinal hemorrhage following reconstructive aortic surgery is encountered in the emergency department, ultrasonography may be considered to be a useful modality in the diagnosis of secondary ADF.

Runx1 protects hematopoietic stem/progenitor cells from oncogenic insult.

The RUNX1/AML1 gene encodes a transcription factor essential for the generation of hematopoietic stem cells and is frequently targeted in human leukemia. In human RUNX1-related leukemias, the RAS pathway is often concurrently mutated, but the mechanism of the synergism remains elusive. Here, we found that inactivation of Runx1 in mouse bone marrow cells results in an increase in the stem/progenitor cell fraction due to suppression of apoptosis and elevated expression of the polycomb gene Bmi-1, which is important for stem cell self-renewal. Introduction of oncogenic N-RAS into wild-type cells, in contrast, reduced the stem/progenitor cell fraction because of senescence, apoptosis, and differentiation. Such detrimental events presumably occurred because of the cellular fail-safe program, although hyperproliferation was initially induced by an oncogenic stimulus. Runx1 insufficiency appears to impair such a fail-safe mechanism, particularly in the stem/progenitor cells, thereby supporting the clonal maintenance of leukemia-initiating cells expressing an activated oncogene. Disclosure of potential conflicts of interest is found at the end of this article.

Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons specifically project axons to central and peripheral targets according to their sensory modality. The Runt-related genes Runx1 and Runx3 are expressed in DRG neuronal subpopulations, suggesting that they may regulate the trajectories of specific axons. Here we report that Runx3-deficient (Runx3(-/-)) mice displayed severe motor uncoordination and that few DRG neurons synthesized the proprioceptive neuronal marker parvalbumin. Proprioceptive afferent axons failed to project to their targets in the spinal cord as well as those in the muscle. NT-3-responsive Runx3(-/-) DRG neurons showed less neurite outgrowth in vitro. However, we found no changes in the fate specification of Runx3(-/-) DRG neurons or in the number of DRG neurons that expressed trkC. Our data demonstrate that Runx3 is critical in regulating the axonal projections of a specific subpopulation of DRG neurons.

Contrast-enhanced ultrasonography with Sonazoid for evaluation of renal microcirculation.

PURPOSE: The renal medullary microcirculation plays an important role in regulating sodium and water excretion, and its impairment is closely associated with various renal diseases. Contrast-enhanced ultrasonography (CEUS) using Sonazoid has not yet been reported as a method for evaluating the renal microcirculation; consequently, this study was carried out to reveal the characteristics of renal microcirculation using CEUS with Sonazoid. METHODS: CEUS was performed on three healthy volunteers after they had fasted for at least 6 h. A GE LOGIQ7 ultrasound unit was used with a 2.0- to 5.5-MHz convex probe. Within approximately 1 min of intravenous injection of 0.0050 ml/kg Sonazoid, contrast images of the right kidney were acquired using a coded phase-inversion mode. Time-intensity curves were calculated for the cortex and medulla. RESULTS: Peak contrast intensity was significantly higher in the cortex (-56.4 ± 1.9 dB) than in the medulla (-66.7 ± 1.7 dB; P < 0.005). Peak times were significantly earlier in the cortex (17.4 ± 3.7 ms) than in the medulla (28.8 ± 6.3 ms; P < 0.05). CONCLUSION: CEUS using Sonazoid enables differentiation between the cortical and medullary microcirculation and is useful in clarifying renal pathophysiology and pharmacology.

Cystic echinococcosis in immigrant from Peru: first case treated with percutaneous treatment in Japan.

We report a case of cystic echinococcosis (CE) caused by Echinococcusgranulosus, for which a modified percutaneous evacuation (PEVAC) treatment was applied. The patient had immigrated from Peru to Japan and had 2 hydatid cystic masses, 1 located in segment (S)5 of the liver and the other in S3 (5.3 and 3.5 cm in diameter, respectively), both of which were visualized as pseudotumors by ultrasound (US) examinations. Albendazole treatment showed no effects and surgical treatment was refused. After punctuation of the S5 cyst under US guidance and S3 with CT guidance, 10- and 12-French gauge catheters, respectively, with multiple side holes were inserted. About 60 ml of the cyst contents was drawn out from the S5 lesion and 2 ml from the S3 lesion. Using repetitive manual injections and aspiration of small amounts of hypertonic saline, the remaining cyst content was removed as much as possible, after which 20 and 10 ml of 98% ethanol was injected into the S5 and S3 lesions, respectively. A short-term evaluation during the 4 month-period following the procedure using US revealed nearly complete evacuation of the S5 lesion, whereas that at S3 remained as a pseudo-solid mass. We consider that percutaneous treatment is a safe therapeutic modality for hydatid cysts. This is the first case report of CE treated percutaneously in Japan.

Increased dosage of Runx1/AML1 acts as a positive modulator of myeloid leukemogenesis in BXH2 mice.

The RUNX1/AML1 gene on chromosome 21 is most frequently inactivated in human leukemias. In addition, an increased dose of RUNX1 is suggested as a basis for several kinds of leukemias. Amplifications of chromosome 21 or the RUNX1 gene are shown to be associated with leukemias with lymphoid lineage, whereas its involvement in myeloid lineage remains unclear. In this study, we generated GATA-1 promoter-driven Runx1 transgenic (Tg) mice, which showed a transient mild increase of megakaryocyte marker-positive myeloid cells but no spontaneous leukemia. These mice were then crossed with BXH2 mice, which have a replication-competent retrovirus in the mouse and develop myeloid leukemia due to insertional mutagenesis by random integration of the virus. Overexpressed Runx1 transgene in BXH2 mice resulted in shortening of the latency of leukemia with increased frequency of megakaryoblastic leukemia, suggesting that increased Runx1 dosage is leukemogenic in myeloid lineage. Identifications of retroviral integration sites revealed the genetic alterations that may cooperate with Runx1 overdose in myeloid leukemogenesis. This mouse model may be useful for analysing the pathogenesis of myeloid leukemias with RUNX1 overdose, especially to examine whether an extra-copy of RUNX1 by trisomy 21 is causally related to Down's syndrome-related acute megakaryoblastic leukemia (DS-AMKL).

Disruption of Runx1 and Runx3 leads to bone marrow failure and leukemia predisposition due to transcriptional and DNA repair defects.

The RUNX genes encode transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here, we show that Runx1;Runx3 double-knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes such as Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independently of CBFß, suggesting a nontranscriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes the development of leukemias and other cancers.

Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation.

RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (-0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.

Haploinsufficiency of Runx1/AML1 promotes myeloid features and leukaemogenesis in BXH2 mice.

Haploinsufficiency of RUNX1/AML1 is associated with familial platelet disorder with a predisposition to acute myeloid leukaemia (FPD/AML), but the causal relationship remains to be addressed experimentally. Mice heterozygous for the Runx1 null mutation, Runx1+/-, are considered to be genetically comparable with human FPD/AML patients but do not develop spontaneous leukaemia. To induce additional genetic alterations, retroviral insertional mutagenesis was employed with the use of BXH2 mice, which develop myeloid leukaemia because of the random integration of retrovirus present in the mouse. Heterozygous disruption of Runx1 in BXH2 mice resulted in a shortening of the latency period of leukaemia. In addition, BXH2-Runx1+/- mice exhibited more marked myeloid features than control mice. Moreover, the c-Kit gene, mutated in human RUNX leukaemias, was recurrently activated in BXH2-Runx1+/- mice, and a colony-forming assay revealed synergism between the Runx1+/- status and c-KIT overexpression. In conclusion, the BXH2-Runx1+/- system is a promising mouse model to investigate the mechanism of leukaemogenesis in FPD/AML.

Runx3 is essential for the target-specific axon pathfinding of trkc-expressing dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons project their axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality (Neuron 30 (2001), 707; Cell 101 (2000), 485; Neuron 31 (2001), 59; J. Comp. Neurol. 380 (1997), 215; Sensory Neurons, Oxford Univ. Press, New York, 1992, p. 131). Expression of runt-related Runx/AML genes (Mech. Dev. 109 (2001), 413) on subtypes of DRG neurons suggests their involvement in lamina-specific afferent differentiation and maturation. Here we show that Runx3-/- mice display severe limb ataxia and abnormal posture and that most of them die shortly after birth. They show that proprioceptive afferent axons fail to reach the ventral horn and have a smaller dorsal funiculus in their spinal cords. Despite the strong resemblance of this phenotype to that of knockout mice deficient in neurotrophin-3 (NT-3) (Cell 77 (1994), 503; Nature 369 (1994), 658) and its receptor, trkC, (Nature 368 (1994), 249), which show proprioceptive afferent loss through selective neuronal cell death, Runx3-/- mice maintain normal number of TrkC/trkC positive DRG neurons throughout development. Our results suggest that Runx3 controls the target-specific axon pathfinding of trkC-expressing DRG neurons in the spinal cord.

Causal relationship between the loss of RUNX3 expression and gastric cancer.

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.