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A complete coupled simulation of the convection chamber and tubes with dual stage steam feed mixing of an industrial ethylene cracking furnace has been carried out with the computational fluid dynamics (CFD) method for the first time. In the convection chamber, the standard k – e model and discrete ordinates (DO) radiation model were respectively used in the descriptions of turbulence characteristics and radiative heat transfer. In the tubes, renormalization group (RNG) k – e model and volume of fluid (VOF) model were respectively applied to the turbulence flow and the liquid–vapor two phases flow. Simulation results agree well with the industrial data. Based on the coupled result, a dynamic simulation was calculated in the feedstock preheater (FPH). Simulation results show that the velocity and temperature fields are inhomogeneous distributions along the width direction due to the asymmetrical structure of convection chamber. Two recirculation zones occur at the corner both near and away from the entrance to the convection chamber, which will cause a longer residence time of flue gas and local overheating in furnace wall of convection chamber. The process gas temperature, tube skin temperature and heat flux profiles are respectively different along the axial and radial direction of the high temperature coil (HTC-I). The changes of flow pattern from bubble flow to spray flow are effected by gravity and centrifugal force during evaporation. The results will be helpful for the design and operation in cracking furnace.

We present the case of a 25-year-old woman at 16 weeks of gestation who presented with non-comatose autoimmune acute liver failure and was at high risk of developing fulminant hepatitis. Predictive formulas indicated a high probability of developing fulminant hepatitis. Unenhanced computed tomography showed marked hepatic atrophy and broadly heterogeneous hypoattenuating areas. The course of her illness was subacute, and the etiology of liver injury was unclear. Considering all of the above, we predicted a poor prognosis. Plasma exchange (PE) and continuous hemodiafiltration (CHDF) therapy were initiated just after admission. A few days after admission, a high titer (×80) of antinuclear antibody was noted. Because autoimmune hepatitis (AIH) was considered a cause of liver failure, treatment with moderate prednisolone (30 mg/day) doses was administrated, with careful consideration of her pregnancy. Thereafter, her laboratory findings and clinical course gradually improved without the need for liver transplantation. A liver biopsy at 18 days after admission indicated a diagnosis of AIH. She continued the pregnancy and delivered a healthy baby without any complications. Eventually, prednisolone doses were decreased to 10 mg, after which her liver function worsened. The second liver biopsy also indicated a diagnosis of AIH. Accordingly, low-dose prednisolone and azathioprine doses (50 mg/day) were administrated to recover her liver function, after which her liver function regained normalcy. This case illustrates that a pregnant woman with non-comatose autoimmune acute liver failure in the first or second trimester of pregnancy and her fetus can be rescued by PE/CHDF therapy and safe moderate doses of prednisolone.

A novel membrane distillation (MD) process enhanced by an asymmetric electric field was proposed. By applying an asymmetric electric field across a membrane with an increased field intensity in the direction of permeation, the permeation of water molecules through the membrane will be facilitated by the electric potential gradient, resulting in a higher flux than that achieved with the conventional process. The effectiveness of using asymmetric electric field to enhance mass transport was confirmed experimentally, and the effects of electric field parameters (including voltage, electrode distance, and electrode geometry) on water permeation were investigated. The novel process was tested for water desalination by MD and the results showed that both permeation flux and salt rejection were increased by the electric field. © 2014 American Institute of Chemical Engineers AIChE J, 60: 2307–2313, 2014

Graft fibrosis is a common finding during protocol biopsy examinations after pediatric liver transplantation. We evaluated the clinical utility of liver stiffness measurements by acoustic radiation force impulse (ARFI) imaging, a novel ultrasound-based elastography method, for assessing graft fibrosis after pediatric living donor liver transplantation (LDLT). We performed 73 liver stiffness measurements by ARFI imaging in 65 pediatric LDLT recipients through the upper midline of the abdomen (midline value) and the right intercostal space (intercostal value) around the time of protocol biopsy examinations. Fifty-nine of these liver stiffness measurements could be compared with histopathological findings. Graft fibrosis was assessed according to the degrees of portal and pericellular fibrosis. Significant fibrosis, which was defined as F2 or worse portal fibrosis and/or moderate or worse pericellular fibrosis, was observed in 14 examinations, which had significantly higher midline (P = 0.005) and intercostal values (P < 0.001) than the others. Liver stiffness measurements by ARFI imaging significantly increased with increases in the portal and pericellular fibrosis grades. For the diagnosis of significant fibrosis, the areas under the receiver operating characteristic curve (AUROCs) were 0.760 (P = 0.005) and 0.849 (P < 0.001) for the midline and intercostal values, respectively. The optimal cutoff values were 1.30 and 1.39 m/second for midline and intercostal values, respectively. Slight but significant elevations were noted in the results of biochemical liver tests: serum levels of γ-glutamyltransferase showed the highest AUROC (0.809, P = 0.001) with an optimal cutoff value of 20 IU/L. In conclusion, liver stiffness measurements by ARFI imaging had good accuracy for diagnosing graft fibrosis after pediatric LDLT. The pericellular pattern of fibrosis was frequently observed after pediatric LDLT, and moderate pericellular fibrosis was detectable by ARFI imaging.

Background Sivelestat sodium hydrate is a specific neutrophil elastase inhibitor effective in acute lung injury (ALI) associated with systemic inflammatory response syndrome. Bowel ischemia reperfusion injury (IRI) induced by supravisceral aortic clamping is associated with an excessive systemic inflammatory response, resulting in remote organ damage, including ALI. In this study, we investigated whether sivelestat can attenuate neutrophil sequestration in the lung, alleviate ALI, and improve survival in a rat bowel IRI model. Methods Adult male Sprague-Dawley rats underwent bowel IRI induced by supravisceral aortic clamping and were randomly assigned to receive sivelestat or saline (control) and monitored for survival. We randomly assigned other rats to undergo laparotomy alone (sham operation), IRI alone, or IRI and sivelestat treatment. We evaluated blood samples for organ function, cytokine levels, and neutrophil elastase activity after reperfusion. Organs were analyzed histologically. We also determined lung injury in another set of rats. Results Bowel IRI induced a significant increase in serum variables indicative of organ function, cytokine concentrations, neutrophil elastase activity, and lung permeability and edema, which reflected the presence of both systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome. Treatment with sivelestat significantly improved survival rate, lung permeability and edema, and significantly decreased levels of creatinine, interleukin 6, interleukin 10, and neutrophil elastase activity. Histological studies showed that sivelestat-treated rats had less bowel IRI-induced damage to lung and liver tissue than controls. Conclusion In a rat model, administration of sivelestat attenuated the effects of bowel IRI induced by supravisceral aortic clamping, and improved the survival rate.

With the highest cancer incidence rate, lung cancer, especially non-small cell lung cancer (NSCLC), is the leading cause of cancer death in the world. Tea (leaves of Camellia sinensis) has been widely used as a traditional beverage beneficial to human health, including anti-NSCLC activity. Theabrownin (TB) is one major kind of tea pigment responsible for the beneficial effects of tea liquor. However, its effect on NSCLC is unknown. The aim of the present study was to evaluate anti-proliferative and apoptosis-inducing effect of TB on NSCLC (A549) cells, using MTT assay, morphological observation (DAPI staining), in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and annexin-V/PI flow cytometry. Subsequently, the expression of several genes associated with cell proliferation and apoptosis were detected by real time PCR assay to explore its potential underlying mechanism. TB was revealed to inhibit cell proliferation of A549 cells in a concentration-dependent and time-dependent manner. Morphological observation, TUNEL assay and flow cytometric analysis evidenced an apoptosis-inducing effect of TB on A549 cells in a concentration-dependent manner. The real time PCR assay demonstrated that TB down-regulated the expression of TOPO I, TOPO II, and BCL-2, and up-regulated the expression of E2F1, P53, GADD45, BAX, BIM, and CASP 3,7,8,9, which suggests an activation of P53-mediated apoptotic (caspase-dependent) pathway in response to TB treatment. The western blot analysis showed a similar trend for the corresponding protein expression (P53, Bax, Bcl-2, caspase 3,9, and PARP) and further revealed DNA damage as a trigger of the apoptosis (phosphorylation of histone H2A.X). Accordingly, TB can be speculated as a DNA damage inducer and topoisomerase (Topo I and Topo II) inhibitor that can up-regulate P53 expression and subsequently modulate the expression of the downstream genes to induce cell proliferation inhibition and apoptosis of A549 cells. Our results indicate that TB exhibits its anti-NSCLC activity via a P53-dependent mechanism, which may be a promising candidate of natural product for anti-cancer drug development in the treatment of NSCLC.

Green tea, the fresh leaves of Camellia sinensis, is not only a health-promoting beverage but also a traditional Chinese medicine used for prevention or treatment of cancer, such as lung cancer. Theabrownin (TB) is the main fraction responsible for the medicinal effects of green tea, but whether it possesses anti-cancer effect is unknown yet. This study aimed to determine the in vitro and in vivo anti-lung cancer effect of TB and explore the underlying molecular mechanism, by using A549 cell line and Lewis lung carcinoma-bearing mice. In cellular experiment, MTT assay was performed to evaluate the inhibitory effect and IC50 values of TB, and flow cytometry was conducted to analyze the cell cycle progression affected by TB. In animal experiment, mice body mass, tumor incidence, tumor size and tumor weight were measured, and histopathological analysis on tumor was performed with Transferase dUTP nick-end labeling staining. Real time PCR and western blot assays were adopted to detect the expression of C-MYC associated genes and proteins for mechanism clarification. TB was found to inhibit A549 cell viability in a dose- and time-dependent manner and block A549 cell cycle at G0/G1 phase. Down-regulation of c-myc, cyclin A, cyclin D, cdk2, cdk4, proliferation of cell nuclear antigen and up-regulation of p21, p27, and phosphate and tension homolog in both gene and protein levels were observed with TB treatment. A c-myc-related mechanism was thereby proposed, since c-myc could transcriptionally regulate all other genes in its downstream region for G1/S transitions of cell cycle and proliferation of cancer cells. This is the first report regarding the anti-NSCLC effect and the underlying mechanism of TB on cell cycle progression and proliferation of A549 cells. The in vivo data verified the in vitro result that TB could significantly inhibit the lung cancer growth in mice and induce apoptosis on tumors in a dose-dependent manner. It provides a promising candidate of natural products for lung cancer therapy and new development of anti-cancer agent.

Hepatocellular carcinoma (HCC) occurs mainly in the liver associated with chronic hepatitis and hepatic cirrhosis as a result of prolonged viral infection. Transforming growth factor-beta (TGF-beta) induces the fibrosis in hepatic cirrhosis, although it is also an inhibitor of hepatocyte proliferation. To understand the role of TGF-beta signaling in HCC progression, we analyzed gene expression in HCC cells in relation to TGF-beta signaling using a two-way clustering algorithm. By the analysis, five HCC cell lines were classified into two groups according to their metastatic capacity. TGF-beta receptor II (TGFBR2) was downregulated in metastatic cells, which did not show a response to TGF-beta. Immunohistochemistry demonstrated clear membrane distribution of TGFBR2 in noncancerous hepatocytes, whereas reduced TGFBR2 expression was observed in 34 of 136 HCCs. In clinical cases, reduced TGFBR2 expression correlated with larger tumor size (P

肝細胞癌に対するラジオ波焼灼療法（以下RFA）後に，肝内に多発性に再発する症例が報告されている．我々は同区域多発再発した肝細胞癌に対し体幹部定位放射線治療（以下SBRT）を施行し，著効した症例を経験したので報告する．症例は77歳男性．2002年1月に肝S6の腫瘤を認めた．同年2月に腫瘍生検を施行しNASH（非アルコール性脂肪肝炎）に合併した肝細胞癌と診断，同年3月にRFAを施行した．2004年9月に肝S6に同区域多発再発病変が認められたため，線量35 Gy/5回/1週間にてSBRTを施行した．SBRT後，CTにて再発病変は消失し，PIVKA-IIも262 mAU/mlから29 mAU/mlと正常に復した．多発性の再発病変も病巣範囲がある程度限局していれば，SBRTによりコントロール可能となることもあり，本症例を通じRFA後の同区域多発再発病変に対してSBRTは検討すべき治療法の一つであることが示唆された．

Hepatocyte growth factor (HGF) promotes cell growth and motility and also increases neovascularization. Multiple myeloma (MM) cells produce HGF, and the plasma concentration of HGF is significantly elevated in patients with clinically active MM, suggesting that HGF might play a role in the pathogenesis of MM. NK4, an antagonist of HGF, is structurally homologous to angiostatin, and our previous report showed that NK4 inhibited the proliferation of vascular endothelial cells induced by HGF stimulation. The purposes of this study were to elucidate the contribution of HGF to the growth of MM cells as well as to investigate the possibility of the therapeutic use of NK4. In vitro study showed that NK4 protein stabilized the growth of MM cell lines and regulated the activation of c-MET, ERK1/2, STAT3, and AKT-1. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was injected intramuscularly into lcr/scid mice bearing tumors derived from HGF-producing MM cells. AdCMV.NK4 significantly inhibited the growth of these tumors in vivo. Histologic examination revealed that AdCMV.NK4 induced apoptosis of MM cells, accompanied by a reduction in neovascularization in the tumors. Thus, NK4 inhibited the growth of MM cells via antiangiogenic as well as direct antitumor mechanisms. The molecular targeting of HGF by NK4 could be applied as a novel therapeutic approach to MM.

Purpose: Hepatocellular carcinoma (HCC) associated with chronic liver disease is known to show an obvious multistage process of tumor progression. We previously identified heat shock protein 70 as a molecular marker of early HCC during investigation of expression profiling in multistage hepatocarcinogenesis. In this report, we examined cyclase-associated protein 2 (CAP2), which is also listed as an up-regulated gene in early HCC. Experimental Design: We measured the level of CAP2 mRNA by real-time quantitative PCR. We raised a polyclonal antibody against CAP2 and we confirmed the expression of CAP2 by immunoblotting and immunohistochemistry in HCC cell lines and HCC tissues. Results: According to real-time quantitative PCR, the level of CAP2 mRNA was up-regulated in early HCC when compared with noncancerous liver tissue, and it was further up-regulated in progressed HCC. We raised a polyclonal antibody against CAP2, which showed a single 53-kDa band of strong intensity in the human HCC cell lines and HCC tissues but only a weak band in the noncancerous liver tissues in Western blot analysis. Immunohistochemical examination of CAP2 revealed its significant overexpression in early HCC when compared with noncancerous and precancerous lesions and in progressed HCC when compared with early HCC. Conclusion: Our findings show that CAP2 is up-regulated in HCC when compared with noncancerous and precancerous lesions. This is the first report that proves that CAP2 is up-regulated in human cancers and that this is possibly related to multistage hepatocarcinogenesis.

背景.1999年World Health Organization (WHO) により胸腺腫の新分類が提唱された. このなかでtype AおよびABとされるものは予後が良好で, 従来胸腺腫の進行度分類に用いられてきた正岡分類では殆どの症例がII期までに相当する. また, 正岡IVb期のものでも胸腔外への転移はまれである. 今回, WHO新分類でtyp e ABと考えられた脳転移を伴う胸腺腫の1症例を経験した. 症例. 71歳, 男性. 胸部単純写真で偶然発見された前縦隔腫瘍を9年にわたり放置していた. 後に左片麻痺によって発見された脳腫瘍を切除され, 胸腺腫の転移巣と診断された. この時点で前縦隔腫瘍は増大し, 両側肺にも多数の転移巣を伴っていた. 原発巣の切除により, 前縦隔腫瘍は正岡rVb期に相当するWHO新分類でtyp e ABの胸腺腫と診断された. 結論. 本症例は長期間にわたり原発巣を放置していたことにより, 遠隔転移を生じたtype ABの胸腺腫の極めてまれな1例であると考えられた.

We present the case of a 25-year-old woman at 16 weeks of gestation who presented with non-comatose autoimmune acute liver failure and was at high risk of developing fulminant hepatitis. Predictive formulas indicated a high probability of developing fulminant hepatitis. Unenhanced computed tomography showed marked hepatic atrophy and broadly heterogeneous hypoattenuating areas. The course of her illness was subacute, and the etiology of liver injury was unclear. Considering all of the above, we predicted a poor prognosis. Plasma exchange (PE) and continuous hemodiafiltration (CHDF) therapy were initiated just after admission. A few days after admission, a high titer (×80) of antinuclear antibody was noted. Because autoimmune hepatitis (AIH) was considered a cause of liver failure, treatment with moderate prednisolone (30 mg/day) doses was administrated, with careful consideration of her pregnancy. Thereafter, her laboratory findings and clinical course gradually improved without the need for liver transplantation. A liver biopsy at 18 days after admission indicated a diagnosis of AIH. She continued the pregnancy and delivered a healthy baby without any complications. Eventually, prednisolone doses were decreased to 10 mg, after which her liver function worsened. The second liver biopsy also indicated a diagnosis of AIH. Accordingly, low-dose prednisolone and azathioprine doses (50 mg/day) were administrated to recover her liver function, after which her liver function regained normalcy. This case illustrates that a pregnant woman with non-comatose autoimmune acute liver failure in the first or second trimester of pregnancy and her fetus can be rescued by PE/CHDF therapy and safe moderate doses of prednisolone.

Graft fibrosis is a common finding during protocol biopsy examinations after pediatric liver transplantation. We evaluated the clinical utility of liver stiffness measurements by acoustic radiation force impulse (ARFI) imaging, a novel ultrasound-based elastography method, for assessing graft fibrosis after pediatric living donor liver transplantation (LDLT). We performed 73 liver stiffness measurements by ARFI imaging in 65 pediatric LDLT recipients through the upper midline of the abdomen (midline value) and the right intercostal space (intercostal value) around the time of protocol biopsy examinations. Fifty-nine of these liver stiffness measurements could be compared with histopathological findings. Graft fibrosis was assessed according to the degrees of portal and pericellular fibrosis. Significant fibrosis, which was defined as F2 or worse portal fibrosis and/or moderate or worse pericellular fibrosis, was observed in 14 examinations, which had significantly higher midline (P = 0.005) and intercostal values (P0.001) than the others. Liver stiffness measurements by ARFI imaging significantly increased with increases in the portal and pericellular fibrosis grades. For the diagnosis of significant fibrosis, the areas under the receiver operating characteristic curve (AUROCs) were 0.760 (P = 0.005) and 0.849 (P0.001) for the midline and intercostal values, respectively. The optimal cutoff values were 1.30 and 1.39 m/second for midline and intercostal values, respectively. Slight but significant elevations were noted in the results of biochemical liver tests: serum levels of γ-glutamyltransferase showed the highest AUROC (0.809, P = 0.001) with an optimal cutoff value of 20 IU/L. In conclusion, liver stiffness measurements by ARFI imaging had good accuracy for diagnosing graft fibrosis after pediatric LDLT. The pericellular pattern of fibrosis was frequently observed after pediatric LDLT, and moderate pericellular fibrosis was detectable by ARFI imaging.

CD26 is a type II glycoprotein known as dipeptidyl peptidase IV and has been identified as one of the cell surface markers associated with various types of cancers and a subset of cancer stem cells. Recent studies have suggested that CD26 expression is involved in tumor growth, tumor invasion, and metastasis. The CD26 is shown in an extensive intracellular distribution, ranging from the cell surface to the nucleus. We have previously showed that the humanized anti-CD26 monoclonal antibody (mAb), YS110, exhibits inhibitory effects on various cancers. However, functions of CD26 on cancer cells and molecular mechanisms of impaired tumor growth by YS110 treatment are not well understood. In this study, we demonstrated that the treatment with YS110 induced nuclear translocation of both cell-surface CD26 and YS110 in cancer cells and xenografted tumor. It was shown that the CD26 and YS110 were co-localized in nucleus by immunoelectron microscopic analysis. In response to YS110 treatment, CD26 was translocated into the nucleus via caveolin-dependent endocytosis. It was revealed that the nuclear CD26 interacted with a genomic flanking region of the gene for POLR2A, a subunit of RNA polymerase II, using a chromatin immunoprecipitation assay. This interaction with nuclear CD26 and POLR2A gene consequently led to transcriptional repression of the POLR2A gene, resulting in retarded cell proliferation of cancer cells. Furthermore, the impaired nuclear transport of CD26 by treatment with an endocytosis inhibitor or expressions of deletion mutants of CD26 reversed the POLR2A repression induced by YS110 treatment. These findings reveal that the nuclear CD26 functions in the regulation of gene expression and tumor growth, and provide a novel mechanism of mAb-therapy related to inducible translocation of cell-surface target molecule into the nucleus.

Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma.

Background Interleukin-18 (IL-18) is a potent proinflammatory cytokine that augments both innate and acquired immune responses. It is also a crucial regulator of lymphocyte production of interferon-γ (IFN-γ), which can promote acute cellular rejection of transplanted solid organs. Methods To evaluate the role of IL-18 in liver transplantation, we constructed an adenoviral vector encoding IL-18 binding protein (Adex-IL18bp), which specifically suppressed the biologic activity of IL-18, and examined the effect of this suppression on liver allografts by using a high-responder rat model (ACI to Lewis) of orthotopic liver transplantation (OLTx). Donor rats were given one intravenous injection of Adex-IL18bp or Adex-LacZ (control vector) 2 d before OLTx. Results Seven days after OLTx, overexpression of IL-18bp resulting from the adenovirus gene transfer was associated with significantly decreased serum alanine aminotransferase levels and less histologic hepatic injury in recipient rats with Adex-IL18bp-pretreated donors compared with Adex-LacZ controls. Adex-IL18bp pretreatment also significantly prolonged rat/allograft survival, inhibited expression of IFN-γ, and reduced levels (versus control values) of both CXCL10 and CX3CL1, which can be induced by IFN-γ. Conclusion These results suggest that IL-18 has an important role in liver allograft rejection through IFN-γ and chemokines and that specific suppression of IL-18 may improve liver function early after transplantation.

Objective: Hepatocellular carcinoma (HCC) is characterized by a multistage process of tumor progression. This study addressed its molecular features to identify novel protein candidates involved in HCC progression. Methods: Using liquid chromatography-tandem mass spectrometry, proteomes of 4 early HCCs and 4 non-HCC tissues derived from 2 cases of liver transplant surgery were compared with respect to the separation profiles of their tryptic peptides. Immunohistochemistry was performed on 106 HCC nodules to confirm the results of the proteomic analysis. Results: Statistical analysis of the profiles selected the peptide peaks differentiating HCC from non-HCC. A database search of the tandem mass spectrometry data from those peptide peaks identified 61 proteins, including a cytoskeletal protein, talin-1, as upregulated in HCC. Talin-1 expression levels in HCC nodules were significantly associated with the dedifferentiation of HCC (p = 0.001). A follow-up survey of the examined clinical cases revealed a correlation between talin-1 upregulation and a shorter time to recurrence after resection (p = 0.039), which may be related to the higher rate of portal vein invasion in HCCs with talin-1 up-regulation (p = 0.029). Conclusions: Proteomic analysis led to identification of talin-1 as a promising HCC marker. Talin-1 upregulation is associated with HCC progression and may serve as a prognostic marker.

(Cancer Sci 2010; 101: 666–672) Overexpression of “stemness gene”Bmi-1 has been identified in some solid tumors. We investigated Bmi-1 expression in hepatocellular carcinoma (HCC) and ATP-binding cassette transporter B1 (ABCB1) as a new potential target for Bmi-1. Bmi-1 was highly expressed in HCC cell lines and the most well differentiated cell line, KIM-1, showed the highest expression. Immunohistochemical, immunocytochemical, and immunoelectron microscopic analysis showed the Bmi-1 protein as having a high intensity of small dots within the nucleus which reflected concentrated sites of Bmi-1 repressive activity. Clear “dot-pattern” staining was observed in 24 of 37 (65%) well differentiated HCC (including 13 of 21 early nodules [62%]), in 32 of 71 (45%) moderately differentiated HCC, and 7 of 14 (50%) poorly differentiated HCC. A similar expression was not observed in non-cancerous background regions. High Bmi-1 expression was observed in the early and well differentiated HCC. Furthermore, overexpression and suppression of Bmi-1 was followed by a respective increase and decrease in ABCB1 expression. As with Bmi-1, high ABCB1 expression was also observed in the early and well differentiated HCC. A strong correlation between ABCB1 and Bmi-1 mRNA expression was seen in HCC cell lines and clinical samples (Pearson’s correlation coefficient 0.95 and 0.90, respectively). The Bmi-1 gene is upregulated in HCC, and in particular is highly expressed in early and well differentiated HCC. The fact that this expression correlated with that of ABCB1 suggests a new regulation target for Bmi-1, and gives new insight into early hepatocarcinogenesis mechanisms and potential targets for future HCC treatment.

Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

The Schwannian stroma in neuroblastomas is related to patient prognosis. There is debate surrounding the origin of Schwannian stroma in neuroblastomas: one theory is that the Schwann cells are derived from neoplastic cells, and the other is that they arise from normal cells surrounding the neuroblastoma. We examined whether human bone marrow stromal cells (hBMSCs) or human mesenchymal stem cells (hMSCs) could differentiate into Schwann cells in neuroblastomas. hBMSCs or hMSCs along with enhanced green fluorescent protein (EGFP) were injected into xenotransplanted neuroblastomas in nonobese diabetic mice with severe combined immunodeficiency and the resulting tumors were analyzed using immunohistochemistry. HBMSCs and hMSCs were co-cultured with neuroblastoma cells, and the induction of Schwann cell-specific molecules, S100beta and Egr-2, was monitored. S100beta-positive Schwannian stroma was observed only in neuroblastomas containing either hBMSCs or hMSCs, but not in neuroblastomas lacking these cells. Double staining with anti-S100 and anti-EGFP antibodies showed that S100-positive cells in neuroblastomas were also EGFP-positive. By contrast, hBMSCs did not develop into Schwann cells in Ewing's sarcoma, demonstrating that differentiation of transplanted hBMSCs or hMSCs into Schwann cells occurs specifically in neuroblastomas. Both S100beta and Egr-2 were expressed in hBMSCs or hMSCs co-cultured with neuroblastoma cells. HBMSCs or hMSCs may contribute to the formation of human tumor stroma. The Schwannian stroma of neuroblastomas appears to be derived from nonneoplastic stromal cells rather than neuroblastoma cells, further clarifying its developmental origins.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. HCC occurs mainly in chronically diseased livers, e.g. following hepatitis B and C infection. These high-risk patients are closely followed up, and increasing numbers of small equivocal lesions are detected by imaging diagnosis. They are now widely recognized as precursor or early-stage HCCs and are classified as dysplastic nodule or early HCC. These lesions lack typical imaging and histology of ordinary HCC and do not show elevated serum markers of α-fetoprotein and PIVKA-II, for example. Molecular analysis of these lesions would help to develop molecular markers for objective histological diagnosis of early HCC and possibly new serum markers for early detection of HCC. It has been reported that HSP70, CAP2, glypican 3 and glutamine synthetase could serve as molecular markers for early HCC. Further analysis is expected to evaluate their usefulness in routine pathological diagnosis including biopsy diagnosis and also as serum markers for early detection of HCC.

Objective Rho GTPase may be involved in human cancer invasion via the augmentation of cell motility and adhesion. We report on two point mutations of the D4-guanine diphosphate (GDP)–dissociation inhibitor (GDI) gene, one of the Rho-GDIs, which were found in a human leukemic cell line, Reh, and the mutated D4-GDI functions as an accelerator of leukemic cell invasion. Material and Methods We investigated the altered activity of GDP dissociation by mutated (mt) D4-GDI and the functions of this mt and wild-type (wt) D4-GDI in invasion. The mice inoculated with wt or mt D4-GDI vector–transfected Raji cells were observed and examined pathologically. Adhesiveness and cell motility of wt or mt D4-GDI vector–transfected Raji cells were examined. Finally, it was examined whether Rho activation was changed by mutation of D4-GDI under the condition of Rho-GDI knockdown. Results Two point mutations of the D4-GDI gene were found in Reh cells. The region of mutations is conserved among members of the Rho-GDI family at the amino acid level. D4-GDI with two mutations (V68L and V69A) functioned in a dominant negative manner in the inhibition of GDP dissociation from Rho. Severe combined immune-deficient mice inoculated with Raji cells developed hemiparalysis. The Raji cells were present in bone marrow and peripheral blood, and hepatic invasion was observed in 20% of the mice. Mice inoculated with wt D4-GDI vector–transfected Raji cells (wt D4) showed later paralysis and none developed hepatic invasion. Mice inoculated with mt D4-GDI–transfected Raji cells (mt D4) showed a 5-day reduction in the time to paraplegia and death. In addition, hepatic invasion was evident in 80% of mice transplanted with mt D4 cells. There were no differences in growth rates and amounts of guanine triphosphate (GTP)–bound Rho, cdc42, or Rac among all clones, however, GTP-bound Rho in mt D4 clone with short hairpin RNA (shRNA) vector for Rho-GDI knockdown was increased compared with wt D4 clone with shRNA vector for Rho-GDI knockdown. The mt D4 cells showed an augmentation of adhesiveness and cell motility. On the other hand, wt D4 cells showed a decreased ability of cell motility. Conclusion These results suggest the mutated D4-GDI functions as a dominant negative molecule against the wt D4-GDI and accelerates invasion via regulation of cytoskeletal machinery.

Angiogenesis in solid tumors is important to tumor growth, invasion and metastasis. Recently, it has been suggested that angiogenesis plays a certain role in the development of hematopoietic malignancies, including leukemia and multiple myeloma. We evaluated tumor angiogenesis in the bone marrow (BM) of multiple myeloma (MM) patients by calculating microvessel density (MVD) in needle-biopsy specimens obtained from 51 cases of untreated MM or monoclonal gammopathy of undetermined significance (MGUS). The MVD in the BM of donors for transplantation and patients with non-hematological diseases was calculated as a control. There was an obvious increase in MVD in the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.

We describe a patient with impairment of mitochondrial fatty acid P-oxidation. A Japanese baby boy was delivered in the 38th week of gestation by emergency cesarean section due to fetal asphyxia. His birth weight was 1,985 g (10th percentile), length 44.8 cm (10th percentile), and head circumference 31.0 cm (10th percentile). His Apgar scores were 3 and 5 at 1 min and 5 min, respectively. Blood glucose was 12 mg/dl at 1 hour after birth, requiring glucose administration. On day 1 his serum CK was 20,780 IU/l, which was thought to be due to asphyxia. His serum CK levels gradually began to decrease. At 3 months of age, he sucked poorly, had poor body weight gain, and muscle hypotonia was observed. On day 117 his general condition was impaired, and marked hepatomegaly was observed. The blood glucose level was 43 mg/dl. The patient's urine was negative for ketone bodies. His serum triglyceride level was 3,670 mg/dl. Abdominal CT scan revealed a fatty liver. Serum levels of acyl carnitine from very-long chain fatty acid increased. On day 118 he died due to ventricular fibrillation. On necropsy, massive lipid deposition was observed in the liver, cardiac muscle, kidney, skeletal muscle, and intestinal mucosa. The ratio of very-long chain acyl-CoA dehydrogenase (VLCAD) activity for C16/C8 fatty acid was 0.50 (normal control 1.29), suggesting abnormal VLCAD. He was diagnosed as having impairment of mitochondrial fatty acid beta-oxidation, presumably due to the VLCAD deficiency.

The 90-kDa heat shock protein, HSP90, is an abundant molecular chaperone which functions in cellular homeostasis in prokaryotes and eukaryotes. It is well known that HSP90 plays a critical and indispensable role in regulating cell growth through modulations of various signal transduction pathways, but its roles in cell cycle control are not so well known. We transferred human HSP90 (wild-type or mutated types) expression vectors into NIH-3T3 cells in order to study certain functions of HSP90 in the cell cycle and cell growth under physiological conditions. We found that the exogenous expression of HSP90 (wild-type) induced a decrease in cell growth via retardation of the G1/S transition. The inhibition of cell growth was caused by reduced expressions of cyclin D3 and cyclin A mRNA and protein. On the other hand, no stable transfectants with the three types of mutated HSP90 were obtained. Unexpectedly, exogenous HSP90 expression impaired the heat shock response by inhibiting both heat shock transcription factor 1(HSF1) activation and transportation of HSF1 into the nucleus. The HSF1 function was disrupted by the direct association between HSF1 and exogenous HSP90, which was present as a monomer. These results reveal important roles of HSP90 in cell cycle control and in the stress response of nontransformed cells.

Recent advances in the treatment of multiple myeloma (MM) using newly developed drugs, such as thalidomide, lenalidomide and bortezomib, have improved the survival of the patients with refractory disease. However, it remains difficult to treat certain populations of MM patients; these patients are considered high-risk cases. Tumor cells obtained from these patients conferred characteristic chromosomal abnormalities, including deletion of chromosome 13 (del 13), t(4;14) translocation and deletion of chromosome 17, on which tumor suppressor gene TP53 is localized.1, 2, 3, 4 Here we report the establishment of a novel myeloma cell line, MUM24, which was obtained from the bone marrow of a 64-year-old female patient with immunoglobulin G (IgG) (k)-type MM. She was refractory to melphalan plus prednisolone, vincristine plus doxorubicin plus dexamethasone and high-dose dexamethasone as well as thalidomide. A bone marrow sample was obtained in September 2002, and was used for establishing the MUM24 cell line. The patient passed away in December 2002 due to systemic progression of the MM, including intracranial invasion. Microscopic pictures of MUM24 cells are provided in Figure 1a. Most of the cells were mononuclear but occasionally bi- or multinuclear cells were observed. The cells had irregularly shaped cytoplasm and vacuoles were observed in some cells. The karyotype analysis of MUM24 cells by Giemsa staining revealed complex chromosomal changes together with del 13 and other abnormalities (Figure 1b). The representative abnormalities were 45, X, add(X)(p22), add(1)(p11), del(1)(q32q42), add(2)(q11), del(2)(q31q33), add(4)(q21), i(5)(p10), der(6;11)(p10;p10),+8,−13,−14, add(16)(q22), add(17)(q25), del(17)(p11), der(21)t(1;21)(q11;p12), add(22)(q12),+mar. The fluorescence in situ hybridization (FISH) analysis showed that the p53 gene is deleted in the MUM24 cell line (Figure 1c). The t(4;14) fusion signals were also detected using fibroblast growth factor receptor 3 (FGFR3) and immunoglobulin heavy chain probes. Figure 1 Morphological and biological characterization of MUM24 cells. May-Gruenwald-Giemsa-stained cytospin specimens are shown (a). Typical chromosomal aberration (b). Multicolor FISH analyses were performed using RB1 (13q14) red, LEU (13q14) red and PIXB (13q34) ... We also examined the genomic DNA sequences of residual TP53 gene of the MUM24 cells and found a nonsense mutation from ‘tgg' to ‘tga' at codon 273 in exon 4 (Figure 1d). Thus, no functional TP53 gene exists in MUM24 cells. The cell surface antigen characteristics for myeloma cells, such as CD38, CD56, Ia (DR) and CD138 were all positive in nearly 100% of the MUM24 cells. B-cell antigens, CD19, CD20 and cell-surface IgG were negative. Cereblon (CRBN), a component of the E3 ubiquitin ligase complex, was identified as a thalidomide-binding protein, which is considered to be a responsible molecule for thalidomide-induced teratogenicity.5 CRBN is also known as a critical target for the antimyeloma effect of lenalidomide and pomalidomide. In MUM24 cells, CRBN transcripts were detected (data not shown). The involvement of CRBN expression in thalidomide resistance is still unclear. It was reported that bone marrow angiogenesis have a role in the progression of myeloma cells.6 We previously demonstrated that MM cells produce and secrete angiogenic growth factors such as fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF).7 The plasma concentrations of these growth factors in MM patients are associated with disease activity and prognosis.7 Thus, we examined the concentrations of these growth factors in the culture medium of MUM24 cells. An enzyme-linked immunosorbent assay showed that MUM24 cells (5 × 105/ml) secreted large amounts of VEGF (1.82 μg/ml). FGF-2 (8.21 ng/ml) and HGF (2.67 μg/ml) were also detected in the culture medium. We also conducted reverse transcription-PCR to detect the expression of FGFs and their receptor family. The MUM24 cells expressed FGF-1, -2 and -4 as well as FGFR1, 2, 3 and 4 (data not shown). In addition, as reflected by the FISH analysis results, t(4;14) translocation resulted in the expression of FGFR3. We therefore speculate that angiogenic growth factors also mediate the interaction of MM cells with bone marrow interstitial cells. The microenvironment in the bone marrow niche may protect MM cells from exogenous noxious stimuli such as antimyeloma drugs including thalidomide. In order to examine the response of MUM24 cells to the newly developed drugs, we incubated MUM24 cells with various concentrations of thalidomide, lenalidomide or bortezomib, and then evaluated the growth suppression (Figure 1e). As a reference, KMS-21 cells (which do not have high-risk chromosomal changes such as t(4;14) or del 13 and 17) were also used. MUM24 cells did not respond to thalidomide even at concentrations that were much higher than therapeutic plasma concentrations in vitro (1–10 μℳ)(data not shown). Lenalidomide and bortezomib inhibited the growth of MUM24 cells in slightly higher concentrations compared with KMS-21 cells. Namely, the IC50 values of lenalidomide and bortezomib for the MUM24 cells were over 25μm and 3.5 nℳ, whereas those for the KMS-21 cells were ∼3 μℳ and 2.4 nℳ, respectively. To examine tumorigenicity, 1 × 107 MUM24 cells were subcutaneously injected in 5-week-old male NOD/Shi-SCID, IL-2Rg KO Jic (NOG) mice, and skin plasmacytomas were established in 2 weeks. Macroscopic and microscopic pictures of a typical skin plasmacytoma are shown in Figure 2a and b. Plasma cells with eccentric oval nuclei with prominent nucleoli were significantly proliferated. Immunohistochemical staining showed that the tumors were positive for CD138 staining (Figure 2c). To evaluate tumor angiogenesis, we also stained endothelial cells, and we found that CD34-positive endothelial cells proliferated in the tumors (Figure 2d). Figure 2 Xenograft model in mice: 1 × 107 MUM24 cells were inoculated subcutaneously in NOD/Shi-scid, IL-2R SCID (NOG) mice. Macroscopic (a) and microscopic (b) pictures of subcutaneous plasmacytomas. Immunohistological staining of CD138 antigen (c). Microvessels ... Taken together, the results of the present study demonstrate that the MUM24 cell line, established from a thalidomide-resistant MM patient, harbored high-risk cytogenetic changes and produced VEGF. Further studies using MUM24 and other MM cells on the expression of multiple myeloma SET and CRBN proteins, intracellular signaling via angiogenic growth factors and antitumor activities of immunomodulatory drugs in vivo are necessary to elucidate molecular mechanisms for thalidomide resistance. As mentioned earlier, overcoming high-risk myeloma is a present unmet clinical need. MUM24 cells are expected to be useful as a tool for studying the mechanisms of thalidomide resistance, as well as for the screening of novel drugs to overcome high-risk MM.

Abstract 1841 PURPOSE: Despite recent advances in the treatment of multiple myeloma (MM), patients with high-risk chromosomal changes such as del13q, t(4;14) or del17p revealed significantly shorter survival. In addition, bone disease markedly reduces quality of life of the patients with MM. To overcome these problems, we have designed and screened synthetic phthalimides which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purposes of this study are to explore novel drugs which possess anti-tumor activity against high-risk MM cells and to examine the anti-osteoclastogenic activity and to isolate directly binding molecules. METHODS AND RESULTS: Thirty synthetic phthalimides were screened for anti-proliferative effect on KMS34 cells with t(4;14) and deletion of chromosome 17. A phthalimide derivative, 2-(2,6-diisopropylphenyl)- 5-amino- 1H-isoindole- 1,3- dione (TC11) significantly inhibited growth of KMS34 cells as well as other MM cells lines with high-risk chromosomal abnormalities (IC50 to KMS34 cells= 4μM). TC11 increased annexin V fraction and induced apoptosis in a caspase-dependent manner. In vivo anti-myeloma activity was evaluated using KMS34-bearing lcr/SCID mice by intraperitoneal injection of TC11. Twenty mg/kg of TC11 significantly inhibited growth of TC11-derived tumor cells, and apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not suppressed, suggesting low hematopoietic toxicity. In pharmacokinetic study using lcr/SCID mice, the plasma concentrations of TC11 was examined; Cmax=18.1μM, Tmax=1.5hr, T1/2 =4.5hr when 100mg/kg of TC11 was injected, and Cmax=2.1μM Tmax=1.0hr, T1/2 =4.5hr when 20mg/kg was injected. In order to examine efficacy to bone disease, anti-osteoclastogenic activity was examined by adding TC11 to M-CSF/RANK ligand-induced osteoclastogenic culture of mouse bone marrow mononuclear cells. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of 1μM of TC11. It was also found that TC11 inhibited bone resorption by pit assay. We also tried to isolate directly binding proteins to TC11 by our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. We identified nucleophosmin 1 (NPM1) as a TC11-binding molecule. Knockdown assay introducing siRNA for NPM1 into HeLa cells induced emergence of the cells with multipolar spindles, suggesting centrosomal disruption during cell division. Since NPM1 gene localizes at chromosome 5q, anti-MDS effect of TC11 was also examined. TC11 also inhibited growth of MDS-L cells (IC50=7μM). CONCLUSION: A novel phthalimide derivative, TC11, has anti-MM activity in vivo and is a potentially effective drug for high-risk MM with bone lesions. TC11 directly binds to NPM1 and induces apoptosis of MM and MDS cells with low toxicity to normal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

We screened 46 novel anilinoquinazoline derivatives for activity to inhibit proliferation of a panel of human cancer cell lines. Among them, Q15 showed potent in vitro growth-inhibitory activity towards cancer cell lines derived from colorectal cancer, lung cancer and multiple myeloma. It also showed antitumor activity towards multiple myeloma KMS34 tumor xenografts in lcr/scid mice in vivo. Unlike the known anilinoquinazoline derivative gefitinib, Q15 did not inhibit cytokine-mediated intracellular tyrosine phosphorylation. Using our mRNA display technology, we identified hCAP-G2, a subunit of condensin II complex, which is regarded as a key player in mitotic chromosome condensation, as a Q15 binding partner. Immunofluorescence study indicated that Q15 compromises normal segregation of chromosomes, and therefore might induce apoptosis. Thus, our results indicate that hCAP-G2 is a novel therapeutic target for development of drugs active against currently intractable neoplasms.

Abstract 4076 Purpose: Despite recent advances in the use of newly developed drugs, high-risk multiple myeloma (MM) patients harboring del13q, t(4;14) or del17p revealed significantly shorter survival. To overcome the limitation, we have screened forty synthetic anilinoquinazoline (AQ) derivatives, and found a novel compound, Q15, which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purpose of this study is to examine anti-tumor and anti-osteoclastogenic activities of Q15 and to clarify the possibility of development of new drug effective for high-risk MM and bone diseases. Methods and Results: Forty AQ derivatives were synthesized and screened for anti-proliferative effect on KMS34 cells. Q15 strongly inhibited growth of t(4;14)-positive KMS34 cells and induced apoptosis in much lower concentration (IC50=78nM) compared with gefitinib (IC50=2500nM), a representative AQ. Q15 also inhibited growth of other MM cell lines harboring high-risk chromosomal abnormalities. It was also found that Q15 did not inhibit intracellular tyrosine phosphorylation induced by EGF, FGF-2, HGF and IL-6, suggesting that Q15 showed anti-tumor activity in a different mechanism from that of gefitinib. In vivo anti-myeloma activity was evaluated by intraperitoneal injection of Q15 into KMS34-bearing lcr/SCID mice. Twenty mg/kg Q15 significantly delayed the tumor growth in these mice. Histopathological examinations revealed apoptosis of MM cells in Q15-treated mice. Growth of colony-forming cells was not suppressed by much higher concentrations (25μ M) of Q15 than IC50, suggesting low hematopoietic toxicity of Q15. In pharmacokinetic study using high-performance liquid chromatography (HPLC), the plasma concentration of Q15 in mice reached a maximum (Cmax=4.5μ M) at 1.5hr after injection, and its half life (T1/2) was 4.5hr. In addition, anti-osteoclastogenic activity was also examined by adding Q15 to M-CSF/RANK ligand-induced osteoclastogenic culture of bone marrow mononuclear cells from C57BL/6JJcl mice. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of Q15. Conclusion: Q15, a novel AQ derivative, has anti-MM activity in vivo and is a potentially safe and effective drug for high-risk MM with bone lesions. Disclosures: No relevant conflicts of interest to declare.

Background and Objective: Disease control of refractory or relapsed multiple myeloma (MM) remains a therapeutic challenge. Classification of the patients in the molecular basis and development of targeted therapy are crucial to overcome the situation. We previously reported that 20–30% of MM cells produce hepatocyte growth factor (HGF), and its serum concentration was specifically elevated in symptomatic MM patients. The purpose of this study is to elucidate the clinical characteristics of HGF-producing MM (HGF myeloma) and to explore a novel therapy. Patients & Methods: Serum concentrations of HGF obtained from 76 patients with MM were measured by ELIZA. Less than 0.3μg/L was considered as a normal range. Association of serum HGF levels with various clinical parameters for disease activity was examined, including age, ISS stage, type of M protein, hemoglobin level, serum creatinine, Ca, β2M, albumin, LDH, CRP, bone marrow MM cell %, chromosomal abnormality, presence of lytic bone lesion and extramedullar plasmacytoma. Phase 2 thalidomide monotherapy was also conducted in 56 patients with refractory MM. HGF-specific competitive inhibitor, NK4 protein, was used for in vitro experiments such as growth inhibition by MTT assay, induction of apoptosis by flow cytometer and activation of intracellular signaling molecules by Western blot analysis. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was intramuscularly injected into lcr/scid-mice bearing tumors derived from HGFproducing KMS11 and 34 cells. Results: Elevated serum HGF level was significantly associated with the presence of anemia (p=0.03) and lytic bone lesion (p=0.009). Seven out of eight patients with high CRP levels without infection and five out of nine patients with extramedullar plasmacytoma also demonstrated elevated serum HGF level. Furthermore, in thalidomide monotherapy for refractory MM, increase in pre- and posttreatment serum HGF level was significant poor prognostic factors for overall survival (OS) in univariate analyses, p=0.025 and 0.01, respectively. Elevation of post-treatment HGF level was also independent poor prognostic factor for OS in multivariate analysis. We next examined the possibility of molecular targeted therapy of HGF using NK4. NK4 protein stabilized the growth of HGF-producing MM cell lines and primary bone marrow MM cells in vitro. NK4 also increased in annexin V+ apoptotic cell fraction and regulated the activation of c-Met, ERK1/2, STAT3, and AKT-1. AdCMV.NK4 significantly delayed growth of KMS 11 and 34-derived tumors in vivo. Histological examination revealed that AdCMV.NK4 induced apoptosis of the tumor cells, accompanied by a reduction in neovascularization in the tumors. AdCMV.NK4 injection prolonged survival of KMS 11 tumor-bearing mice. Conclusion: HGF myeloma is characterized by increased disease activity such as anemia, bone lesion, high CRP level and extramedullar plasmacytoma and also by shorter survival by conventional therapy including thalidomide, and it should be recognized as a distinct clinical entity. Since NK4 showed direct anti-myeloma effect as well as anti-angiogenic activity against HGF-producing MM cells, molecular targeted therapy, for example using NK4, is to be established to improve the therapeutic outcome of HGF myloma.