Integrated Evaluation of Inflammatory, Nutritional, and Sarcopenia Markers to Predict Survival in Metastatic Breast Cancer Patients.

BACKGROUND/AIM: The prognosis of a cancer patient is influenced by the tumor-related factors, as well as by various patient-related factors. We evaluated the association between inflammatory and nutritional factors and their outcomes, including the prognosis and therapeutic course, in patients with metastatic breast cancer. PATIENTS AND METHODS: In this observational retrospective study, we evaluated 35 patients. The inflammatory and nutritional markers before systemic therapy included the lymphocyte count, neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), systemic immune-inflammatory index (SII), systemic inflammatory response index (SIRI), pan-immuno-inflammatory values (PIV), prognostic nutritional index (PNI), Glasgow prognostic score (GPS), and psoas muscle index (PMI). RESULTS: Triple-negative, low PNI, and GPS 2 were correlated with worse overall survival in the univariable analysis. The GPS was the only independent predictor of overall survival [hazard ratio=5.85, 95% confidence interval=1.15-29.68, p<0.01]. The time to treatment failure of first-line therapy in patients with GPS 2 was significantly shorter than that in patients with GPS 0/1 (p<0.01). CONCLUSION: The GPS was an independent predictive marker for overall survival in patients with metastatic breast cancer.

S-1 and CPT-11 Plus Ramucirumab (IRIS+Rmab) as Second-Line Chemotherapy for Patients with Oxaliplatin-Refractory Metastatic Colorectal Cancer (mCRC): A Multicenter Phase II Study in Japan (N-DOCC-F-C-1701).

This multicenter phase II N-DOCC-F-C-1701 trial is being planned in order to investigate the efficacy and safety of CPT-11+S-1 +Ramucirumab (IRIS+Rmab), which is anticipated to have a stronger anti-tumor effect than IRIS+Bmab in patients with metastatic colorectal cancer (mCRC) previously treated with oxaliplatin (L-OHP) containing regimen, in consideration of the result of RAISE, FIRIS and some phase II trials of IRIS+Bevacicizumab (Bmab). The number of patients is set at 38 for the statistical analysis, assuming an expected median PFS of 5.0 months (threshold: 3.0 months). The primary endpoint of the study is the progression free survival (PFS), and the secondary endpoints are the overall response rate (ORR), overall survival (OS), adverse events (AE), quality of life (QOL) and review of nausea and vomiting. This trial is registered in the UMIN Clinical Trials Registry as UMIN000028170. We intend to start conducting the trial in September 1, 2017. If this trial meets the endpoint, IRIS+Rmab might be supported as a new optional standard regimen for mCRC.

Long-term outcome of adipose-derived regenerative cell-enriched autologous fat transplantation for reconstruction after breast-conserving surgery for Japanese women with breast cancer.

PURPOSE: More effective methods are needed for breast reconstruction after breast-conserving surgery for breast cancer. The aim of this clinical study was to assess the perioperative and long-term outcomes of adipose-derived regenerative cell (ADRC)-enriched autologous fat grafting. METHODS: Ten female patients who had undergone breast-conserving surgery and adjuvant radiotherapy for breast cancer were enrolled. An ADRC-enriched fat graft prepared from the patient's adipose tissue was implanted at the time of adipose tissue harvest. The perioperative and long-term outcomes of the grafts, which included safety, efficacy, and questionnaire-based patient satisfaction, were investigated. RESULTS: The mean operation time was 188 ± 30 min, and the mean duration of postoperative hospitalization was 1.2 ± 0.4 days. No serious postoperative complications were associated with the procedure. Neither recurrence nor metastatic disease was observed during the follow-up period (7.8 ± 1.5 years) after transplantation. Of 9 available patients, "more than or equal to average" satisfaction with breast appearance and overall satisfaction were reported by 6 (66.7%) and 5 (55.6%) patients, respectively. CONCLUSIONS: ADRC-enriched autologous fat transplantation is thus considered to be safe perioperatively, with no long-term recurrence, for patients with breast cancer treated by breast-conserving surgery, and it may be an option for breast reconstruction, even after adjuvant radiotherapy.

Hepatic inflammatory pseudotumor associated with xanthogranulomatous cholangitis mimicking cholangiocarcinoma.

Inflammatory pseudotumor (IPT) is a rare benign condition often misdiagnosed as malignancy. An 80-year-old man was referred to our clinic for an asymptomatic hepatic mass detected on plain abdominal CT. Abdominal ultrasonography identified the lesion as a poorly defined hypoechoic mass. Although a liver biopsy did not provide any evidence of malignancy, imaging modalities suggested a diagnosis of cholangiocarcinoma. The patient underwent left lobectomy, and the pathological findings were consistent with the features of xanthogranulomatous cholangitis. This case is the first report of hepatic IPT originating from xanthogranulomatous cholangitis without symptoms and illustrates the importance of obtaining a preoperative diagnosis in order to avoid a misdiagnosis of malignant tumor.

Intraductal papillary mucinous neoplasm in an annular pancreas: a case report.

Annular pancreas is a rare anomaly in which a ring of pancreatic tissue encircles the second portion of the duodenum. We herein report a case involving a 79-year-old Japanese man with an intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Imaging studies showed that the pancreatic tissue encircled the descending part of the duodenum and that a 30-mm-diameter cystic tumor was present in the annular segment, leading to the diagnosis of pancreatic IPMN. Limited pancreatic resection was successfully performed by careful division of the annular segment from the second portion of the duodenum. The postoperative course was uneventful, and the patient's pancreatic function was retained without the need for supplementation. To the best of our knowledge, this is the first report of IPMN occurring in the annular segment of the pancreas. Limited resection of the pancreatic annular segment is a feasible surgical treatment for noninvasive IPMN of the annular pancreas.

The impact of hepatic denervation on the accumulation of hepatic progenitor cells during liver regeneration in rats.

BACKGROUND/AIMS: All autonomic hepatic nerves are transected following liver transplantation. Recent studies have shown the relationship between an inhibition of autonomic nerves and the accumulation of hepatic progenitor cells (HPC). This study aims to elucidate the influence of hepatic denervation on the accumulation of HPC in the process of liver regeneration. METHODOLOGY: Male Sprague-Dawley rats underwent hepatic denervation. Immediately after either denervation (DN group, n=30) or a sham operation (control group, n=30), a two-thirds hepatectomy was performed, and these were sacrificed chronologically. An immunohistochemical analysis of HPC was performed with a mouse monoclonal OV6 type antibody. RESULTS: The liver per body weight ratio gradually increased in both groups. On postoperative day (POD) 7, the DN group showed a significantly higher ratio. The HPC expression gradually increased in both groups. The maximal HPC number was observed on POD 7 in the DN group and on POD 3 in the control group. Although there was no significant difference in the HPC numbers between the DN and control group until POD 3, the number of HPC were significantly higher in livers of the denervated rats than in those of the sham operated rats between POD 5 and 14. CONCLUSIONS: The hepatic autonomic nerves were thus suggested to play an important role in the accumulation of HPC during liver regeneration in rats.

Management of cytomegalovirus infection after living donor liver transplantation.

BACKGROUND/AIMS: Few studies on Cytomegalovirus (CMV) infection in adult-to-adult living donor liver transplantation (LDLT) have been reported. The aim of this study was to analyze the incidence, risk factors and management of CMV infection after LDLT. METHODOLOGY: Retrospective analysis was performed with 72 consecutive adult cases. RESULTS: CMV antigenemia was demonstrated in 31 (43.1%) patients and 9 patients (12.5%) manifested fever. Twelve patients were treated with intravenous ganciclovir (GCV) injection. There was improvement in 10 patients; foscanet concomitant with CMV-IG was administered in one patient who had an adverse effect resulting in improvement and another one resulted in death from sepsis. Twelve patients were given oral valganciclovir (VGCV) and all showed improvement. ABO incompatible transplantation was associated with CMV infection after LDLT in both the univariate (p=0.005) and multivariate analyses (p=0.04). After discharge 12 out of 63 patients (19%) suffered from CMV infection and all of them were taking steroid. CONCLUSIONS: ABO incompatible transplantation was demonstrated as a risk factor for CMV infection during hospitalization. After discharge immunosuppressive status seemed to be more essential as a predictor for CMV infection. Routine examination to detect CMV antigenemia is needed especially in patients with potentially over-immunosuppressive conditions in out-patient clinics.

99mTc-Galactosyl sialyl albumin (GSA) scintigram adjusts hepatic resection range in ICG based estimation.

BACKGROUND/AIMS: Indocyanin Green (ICG)-based diagnosis is widely accepted in determination of hepatic resection range, however, we frequently encounter scattered results of preoperative assessment. The aim of this study was to clarify the role of 99mTc Galactosyl sialyl albumin (GSA) scintigram in the decision of the resection range. METHODOLOGY: One hundred and eighty patients who underwent liver resection were included in this study. The patients were divided according to ICGR15 value as follows; Group 1 (n=100): ICG R15 <15%, Group 2 (n=32): ICGR15 15-20%, Group 3 (n=32): ICGR15 20-25% and Group 4 (n=8): ICGR15 >25%. Each group was divided into two subgroups by their GSA-LHL 0.9 levels and compared with regards to liver function, portal pressure and HAI score of background liver. The frequency of complications was also compared to previous cases without GSA-LHL estimation (n=64). RESULTS: In Groups 2 and 3, the GSA-LHL ≥ 0.9 subgroup showed better platelet counts, portal pressure and lower HAI score. In groups of GSA-LHL ≥ 0.9, platelet counts showed higher value between below 20% of ICGR15 and above that, while GSA-LHL<0.9 showed no difference in groups exceeding 15% of ICGR15. Overall complications occurred less in GSA-LHL ≥ 0.9 compared to previous cases without GSA-LHL estimation. CONCLUSIONS: Levels of GSA-LHL reflects severity of portal hypertension in moderately damaged liver preoperatively and could contribute to the decision of the range of resection with low morbidity.

Identification of HLA-A*0201/-A*2402-restricted CTL epitope-peptides derived from a novel cancer/testis antigen, MCAK, and induction of a specific antitumor immune response.

Cancer immunotherapy is a potential therapeutic strategy, in addition to surgical treatment, radiotherapy, and chemotherapy. Cancer-specific immunotherapy, such as the MAGE peptide vaccine, has been utilized clinically. How-ever, there are inherent limits to the effectiveness of vaccinotherapy using a single antigen because of the expression frequency of cancer-specific antigens on tumor cells. Thus, identification of a new cancer-specific antigen is needed. In this study, we examined the possibility of using cancer-specific immunotherapy based upon mitotic centromere-associated kinesin (MCAK) which was previously identified as a novel cancer/testis antigen. To evaluate the feasibility of developing cancer immunotherapy using MCAK peptides, we studied HLA-A*0201 and *2402 as targets for CTLs in the context of HLA class I molecules. By using a peptide with a sequence of AINPELLQL (amino acid positions 63-71 in MCAK, HLA-A*0201) and FFEIYNGKL (amino acid positions 401-409 in MCAK, HLA-A*2402), CTL responses could be induced from unseparated PBMCs by stimulation of freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and also by using interleukin-7 and keyhole limpet hemocyanin in primary culture. The induced CTLs could lyse HLA-A-*0201/*2402 colon and gastric cancer cells expressing MCAK, as well as the peptide-pulsed target cells, in an HLA class l, and CD8 restricted manner. The identification of the MCAK/HLA-A*0201 and *2402 peptides suggests the possibility of designing peptide-based immunotherapeutic approaches that might prove effective in treating patients with MCAK-positive cancer.

The impact of the intra-abdominal space on liver regeneration after a partial hepatectomy in rats.

BACKGROUND: Little is known about the relationship between intra-abdominal space and liver regeneration. The present study was experimentally designed to investigate the influence of the "occupied space" or the "loss of occupied space" on a regenerating liver. METHODS: Experiment 1: Rats were randomly assigned to two groups: SO (space occupied) rats (n = 40); occupancy of intra-abdominal space followed by a two-thirds partial hepatectomy (PH) and control rats (n = 40); A PH alone. The rats in both groups were euthanized at 24, 48, 96, and 168 h after the operation. Computed tomography (CT) images were analyzed to evaluate the regenerating-direction and the shape of the regenerated remnant liver. The liver to body weight ratio and the proliferating cell nuclear antigen (PCNA) labeling index were measured at each time point. Experiment 2: A second laparotomy was performed at 168 h after the PH in both groups; occupier-removal for the SO rats and a sham operation for the control rats. The rats in both groups were euthanized at 24 and 168 h after the second operation. The liver to body weight ratio and PCNA labeling index were measured at each time point. RESULTS: Experiment 1: The remnant liver of the SO rats enlarged toward the dorsal and caudal side because liver regeneration toward the ventral side in the SO rats was inhibited with the occupier in the abdominal space at 96 h, and later, after the PH. CT images showed a statistically significant difference in the shape of the regenerated remnant liver between the control group and the SO group. The liver/body weight ratio was significantly decreased in the SO rats at 96 and 168 h after PH (P < 0.05). There was no significant difference between the groups in the PCNA labeling index. The SO rats showed a significant increase of the PCNA labeling of the inferior right lobe (10.6%) in comparison with the index of the superior right lobe (7.8%), which came in direct contact with the occupier, at 96 h after the operation (P < 0.05). The cell density of superior right lobe of the SO rats group was significantly higher than that of the control group at 168 h after operation (P < 0.05). Experiment 2: There was no statistically significant difference in the liver/body weight ratio at 168 hrs after the second operation between the groups. However, there was a statistically significant increase of the PCNA labeling index 24 h after the second operation in the occupier-removal rats in comparison with the control rats (P < 0.05). CONCLUSION: The occupied intra-abdominal space was therefore found to suppress liver regeneration after a partial hepatectomy, while the removal of such an occupied space stimulated the regeneration of the liver.

Diverse effects of FK506 on the apoptosis of hepatocytes and infiltrating lymphocytes in an allografted rat liver.

BACKGROUND: The current study investigated whether FK506 (FK) regulates the apoptotic systems in allografted rat liver and the contribution of Fas/Fas-ligand system and Bcl-2 family during acute rejection. MATERIALS AND METHODS: The recipients were divided into three groups, the allo, the allo-FK, and the syn group. Rats were euthanized 1, 3, 5, and 7 d after OLT. Apoptotic activity was explored using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The expression of Fas/Fas-ligand and Bcl-2/Bax in the grafted livers was investigated by Western blotting and immunohistochemistry. RESULTS: The apoptotic index (AI) of hepatocytes in the allo-FK group was less than that in the allo group. Fas in the allo group was more intense than that in the allo-FK group in the periportal areas on day 1 and 3, while Bcl-2 in the allo group was less intense than that in the allo-FK group in the pericaval areas at all time-points after OLT. CONCLUSION: FK provides beneficial antiapoptotic effects on hepatocytes in the grafted rat livers through both the down-regulation of Fas expression in the periportal areas and the up-regulation of Bcl-2 expression in the pericaval areas.

Management of fungal colonization and infection after living donor liver transplantation.

BACKGROUND/AIMS: Control of infection is important in liver transplant patients under immunosuppressive conditions. In particular, invasive fungal infection is often fatal if diagnosis and therapy are delayed. The aim of this study was to analyze the incidence of fungal colonization and infection after living donor liver transplantation (LDLT). METHODOLOGY: Retrospective analysis was performed with 60 consecutive adult recipients of LDLT. RESULTS: Fungi were isolated from specimens of 16 (26.7%) patients after LDLT. All the fungi were Candida species. One patient for whom Candida species were isolated in ascites and blood was complicated with systemic methicillin-resistant Staphylococcus aureus and cytomegalovirus infection. In the univariate analysis, fungal carriage before surgery (p = 0.01) was associated with fungal isolation after LDLT. In the multivariate analysis, fungal carriage was found to be an independent predictor of fungal isolation (odds ratio: 15.7, p = 0.03). Of the 60 recipients, 16 (26.7%) showed serum levels of beta-D glucan above 60 pg/ml after surgery. Among these, 4 died and were all complicated with severe bacterial infection. CONCLUSION: Preoperative fungal carriage was associated with fungal isolation after LDLT. If fungal infection was suspected after LDLT, along with treatment against fungi, control of complicated infections with other pathogens to be simultaneously considered.

Clinical significance of stanniocalcin 2 as a prognostic marker in gastric cancer.

BACKGROUND: Stanniocalcins are glycoprotein hormones that were originally found in the endocrine gland of bony fish. Microarray expression data from 32 paired samples of gastric cancer and normal mucosa in a public microarray database showed that the expression level of Stanniocalcin 2 was higher in gastric cancer than in normal gastric mucosa. The clinical significance of Stanniocalcin 2 expression has been observed for several cancers. However, the relationship between Stanniocalcin 2 and clinicopathological factors in gastric cancer has not yet been investigated. MATERIALS AND METHODS: We examined the clinical significance of Stanniocalcin 2 in gastric cancer in 108 gastric cancer samples using real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical studies were conducted with paraffin sections. The suppression analysis of Stanniocalcin 2 using siRNA was done to determine Stanniocalcin 2's biological roles. RESULTS: The level of Stanniocalcin 2 in cancer tissues was higher than in normal tissues (P = .0002). The high Stanniocalcin 2 expression group (n = 54) had more progressive lymph node metastasis (P = .07) and venous invasion (P = .028) than the low-expression group (n = 54). High Stanniocalcin 2 expression was an independent prognostic factor in gastric cancer patients (P = .02). Moreover, siRNA suppression of Stanniocalcin 2 in a gastric cancer cell line inhibited cellular proliferation (P < .05). CONCLUSIONS: The high expression level of Stanniocalcin 2 in gastric cancer tissues could be a very powerful marker of poor prognosis. Therefore, Stanniocalcin 2 is a promising candidate for a molecular target for the treatment of gastric cancer.

Regeneration of graft livers and limited contribution of extrahepatic cells after partial liver transplantation in humans.

BACKGROUND: Liver regeneration is still not fully understood. Partial liver transplantation (LT) can provide the opportunity to investigate the mechanisms of liver regeneration, including the contribution of extrahepatic cells to liver regeneration. METHODS: Of 61 patients transplanted with partial liver graft between August 1997 and October 2006, 56 patients were studied, including 49 adults and 7 children. Sequential computed tomography volumetric analysis was performed for volume measurement, while proliferating cell nuclear antigen (PCNA) labeling index was investigated for liver cell proliferation in nonprotocol liver biopsy specimens. In addition, 15 male recipients who had female liver grafts were investigated in order to detect Y chromosomes as extrahepatic cells in nonprotocol liver biopsy specimens. RESULTS: Graft volume per standard liver volume was markedly increased after adult-to-adult living-donor (LD) LT. In pediatric transplants, there was no volume increase over time. PCNA labeling index was vigorous in adult-to-adult LDLT in the early period after LDLT. No Y chromosome was evident in hepatocytes from female-donor male-recipient grafts during or after liver regeneration. However, in the cases of failing grafts of this type, many Y-chromosome-positive cells were observed in the graft liver. The character of those cells was CD34(-), CK9(-), hepatocyte-specific antigen(-), and CD68(+/-). CONCLUSION: In adult-to-adult LDLT, vigorous liver regeneration occurs in the graft liver, demonstrated by not only volumetric but cell kinetic analysis. Involvement of extrahepatic cells in normal liver regeneration seems limited.

Clinical significance of ApoE expression in human gastric cancer.

ApoE plays a key role in various biological events. The aim of this study is to clarify its clinical significance in gastric cancer. We obtained paired clinical bulk samples of tumor tissue and corresponding normal tissue from 124 gastric cancer patients. To address ApoE mRNA expression clearly, we selected four samples, and differentially dissected gastric cancer and normal epithelium using laser microdissection (LMD) system. ApoE mRNA expression was examined by real-time reverse transcription (RT)-polymerase chain reaction (PCR). ApoE protein expression was assessed by immnunohistochemistry. The relationship between ApoE mRNA expression and clinicopathologic factors was statistically analyzed. RT-PCR assay for 124 bulk samples showed that ApoE mRNA expression was more highly expressed in gastric cancer tissue than in corresponding normal mucosa (p < 0.0001). By RT-PCR assay of four LMD samples, ApoE mRNA was overexpressed in gastric cancer. Immunohistochemistry showed that ApoE was predominantly expressed in gastric cancer. Tumors with high ApoE mRNA expression showed deeper tumor invasion into the muscle layer (p < 0.0001), the serosal layer (p < 0.01), or more positive lymph node metastasis (p < 0.05). When assessed by Kaplan-Meier analysis, patients with high ApoE expression tumor had a shorter survival than those with low ApoE expression tumor (p < 0.05). Moreover, multivariate analysis indicated that high ApoE mRNA expression was an independent indicator for muscular invasion (p < 0.01). ApoE is highly expressed in gastric cancer, contributing to shorter survival. In particular, ApoE was closely correlated with muscular invasion, and may be a possible biomarker predicting muscular invasion of gastric cancer.

Metabolism for cyclosporin A during liver regeneration after partial hepatectomy in rats.

AIM: To elucidate the metabolism and the effect of the cyclosporin A (CyA) as a representative immunosuppressive drug used in transplantation in a partially hepatectomized rat model. METHODS: CyA was administered to rats that underwent a 70% hepatectomy. These rats were randomly assigned into three groups according to the dose of CyA administration as follows; (group 1) water, (group 2) 5 mg/kg CyA, (group 3) 10 mg/kg CyA. On postoperative days-1, 3, 7 and 14, the rats were killed to analyze the serum concentration of CyA, the liver regeneration ratio, biochemical or histological markers, and mRNA expression using reverse transcriptase-polymerase chain reaction method to determine albumin and cytochrome p450 expression. RESULTS: The serum concentration of CyA in group 3 was significantly higher than group 2 during liver regeneration. CyA enhanced the liver regeneration in a dose dependent manner. The mRNA expression associated with CyA metabolism was significantly decreased on day 14, while preserving the albumin producing activity. CONCLUSION: These data indicate that the p-450 activity required to metabolize the CyA may be reduced during regeneration of the remnant liver after a hepatectomy, which may, therefore, be linked to difficulty in controlling the optimal dose of CyA during early period of LDLT.

Activin A enhances MMP-7 activity via the transcription factor AP-1 in an esophageal squamous cell carcinoma cell line.

Activin A, a member of the transforming growth factor beta (TGF-beta) superfamily, is often overexpressed in solid carcinomas. We have previously reported that the expression of activin A is associated with lymph node metastasis in esophageal cancer. In the current study, our goal was to clarify the molecular mechanisms underlying the aggressive behavior of tumors expressing high levels of activin A. Using cDNA microarrays, the gene expression profile of a human esophageal carcinoma cell line (KYSE170) stably transfected with activin betaA (Act-betaA, a subunit of activin A) was compared with those of control human esophageal carcinoma cell lines. We found that expression of MMP-7 was higher in the Act-betaA transfectants than in the control cells. To reveal the mechanism of expression of MMP-7 mediated by activin A, we evaluated mRNA expression of MMP-7 and Act-betaA with or without activin A neutralizing antibody, using real-time PCR and Northern blot analysis. We also performed promoter analysis of the MMP-7 promoter and assessed c-Jun and Smad2/3 expression. MMP-7 expression in the transfectants was correlated with the level of Act-betaA expression and was reduced by activin A neutralizing antibody. The Act-betaA transfectants had higher MMP-7 promoter activity than control cells. MMP-7 promoter activity was not affected by mutation in the Smad binding site, while mutation of the AP-1 binding site did reduce activity. Moreover, the expression of c-Jun was increased in Act-betaA transfectants. These results indicate that activin A may modulate the expression of MMP-7 via AP-1 and not through Smad2/3.

CD133+CD44+ population efficiently enriches colon cancer initiating cells.

BACKGROUND: Previous reports have demonstrated that CD133(+) cells or CD44(+) cells might be cancer initiating cells (CIC) of colon cancer. However, the association between the two cell types is unclear. In this study, we evaluated the tumorigenicity of each population of human colon cancer divided by CD133 and CD44 using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. METHODS: Using the colon cancer cell lines HT29 and Caco2 we evaluated the change of expression status of CD133 or CD44 by a treatment with sodium butyrate (NaBT) that can induce cellular differentiation. Next, we prepared ten clinical samples of colon cancer and analyzed the expression and tumorigenicity of CD133 and CD44. RESULTS: With NaBT treatment, CD44 expression was greatly downregulated in both HT29 and Caco2 (HT29: nontreatment versus treatment; 77.8% versus 0.6%, Caco2: 14.0% versus 0.4%, respectively), more than CD133 expression (HT29: nontreatment versus treatment; 90.1% versus 67.7%, Caco2: 98.9% versus 76.3%, respectively). In clinical samples, the percentages of CD133(+) cells and CD44(+) cells varied from 0.3% to 82.0% (mean 35.5%), and from 11.5% to 58.4% (mean 30.0%), respectively. Subcutaneous injection of CD133(+) or CD44(+) cells made a tumor in all mice (3/3 and 4/4, respectively). The combined analysis of CD133 and CD44 revealed that only the CD133(+)CD44(+) population had the ability to produce a tumor (3/3). CONCLUSION: The findings demonstrate that, at present, the CD133(+)CD44(+ ) population may be the best to identify tumor initiating cells of human colon cancer.

The search for cancer stem cells in hepatocellular carcinoma.

BACKGROUND: Recent evidence suggests that some solid cancers originate from cancer stem cells. We have identified a subset of candidate stem cells, which are termed side population (SP) cells, in the hepatocellular carcinoma cell line Huh7. Because most stem cells reside in the G0 phase of the cell cycle, G0 cells were isolated, and the relationship between SP cells and G0 cells was investigated to clarify the biological characteristics of G0 cells. METHODS: Huh7 cells were sorted using Hoechst 33342 and Pyronin Y. The cells were then divided into G0, G1, and G2/M fractions and cultured under low-attachment conditions to obtain cellular spheres. Tumorigenetic ability was investigated using subcutaneous transplantation to NOD/SCID mice. G0 and G1 cells were analyzed for markers indicative of hepatocytic (albumin expression) and cholangiocytic (keratin 19 expression) differentiation and DNA synthesis (Ki67). RESULTS: The cell-cycle distribution of cultured Huh7 cells was 0.7% (G0), 63.8% (G1), and 34.5% (G2/M, S). The G0 cells were located within the neck of the SP fraction. The G0 cells showed spheroid formation and 3-dimensional growth. Those cells showed marked tumorigenesis in NOD/SCID transplantation. G0 cells, which did not express Ki67, were weakly positive for expression of albumin and were clearly positive for the expression of keratin 19. In contrast, G1 cells were positive for Ki67 and albumin expression but negative for keratin 19. CONCLUSION: G0 cells are present in the SP fraction of Huh7. They show self-renewal, tumorigenesis, and bidirectional lineage. These findings suggest that the G0 cells within the Huh7 cell line are promising candidates as cancer stem cells for future studies of hepatocellular carcinoma.

Prognostic relevance of Tensin4 expression in human gastric cancer.

BACKGROUND: The Tensin4 gene is involved in various biological events by mediating signal transduction. This study was designed to clarify its clinical significance in gastric cancer. METHODS: A total of 114 gastric cancer patients were enrolled in this study, and we obtained paired samples from tumor tissue and matched normal mucosa. Total RNA was extracted, and TNS4 mRNA expression was quantified using real-time reverse-transcription polymerase chain reaction. TNS4 protein expression was investigated by immunohistochemistry. Additionally, the relationship between TNS4 expression and clinicopathologic factors was statistically analyzed. RESULTS: TNS4 mRNA expression was significantly higher in tumor tissue than in normal mucosa (p < 0.0001). Additional immunohistochemical analysis demonstrated that TNS4 protein was predominantly expressed in gastric cancer. Tumors with higher TNS4 mRNA expression showed histologically poorer grade (p < 0.02), deeper invasion into the serosa (p < 0.01), more positive lymph node metastasis (p < 0.02) or peritoneal dissemination (p < 0.05). Patients with high TNS4 mRNA expression tumor were more likely to have cancer-related death (p < 0.05). A Kaplan-Meier curve also demonstrated that patients with high TNS4 expression tumor had significantly poorer prognosis (p < 0.01) than those with low TNS4 expression tumor. In particular, the 2-year overall survival rates were 48.2% and 83.1%, respectively, a very significant difference. Multivariate analysis revealed that high TNS4 mRNA expression was an independent prognostic predictor (relative risk 1.41, 95% confidence interval 1.03-1.96, p = 0.03). CONCLUSION: TNS4 was frequently overexpressed in gastric cancer, and tumors with high TNS4 mRNA expression showed biologically aggressive behavior. High TNS4 mRNA expression may be a novel prognostic predictor for those patients.