5-Fluorouracil metabolic pathway genes predict recurrence risk following adjuvant S-1 therapy: Results of an ancillary analysis from a phase III trial of resected biliary tract cancer (JCOG1202A1).

BACKGROUND: S-1, an oral fluoropyrimidine derivative, is standard adjuvant therapy for resected biliary tract cancer (BTC), based on the results of the JCOG1202, a phase III trial evaluating the survival benefit with adjuvant S-1 following curative resection for BTC compared to surgery alone. This multicenter ancillary study of the JCOG1202 aimed to evaluate the prognostic impact of the 5-fluorouracil (5-FU) metabolic pathway genes including thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). METHODS: The 5-FU metabolic pathway genes were measured in tumor cells from formalin-fixed paraffin-embedded resected specimens from 183 patients (surgery alone: n = 94; adjuvant S-1: n = 89). We randomly divided them into training (n = 96) and validation sets (n = 87) for evaluating the interaction between gene levels and RFS benefits in the treatment arm. RESULTS: RFS benefits of adjuvant S-1 were observed in the low DPD (HR = 0.440 and 0.748, respectively in the training and validation sets) and the low TP groups (HR = 0.709 and 0.602, respectively). Clinicopathological characteristics were well balanced between low and high DPD populations. More advanced stage tumors were observed in high TP populations as compared to those in low TP populations (p = .0332). CONCLUSION: The results suggest the RFS benefit of adjuvant S-1 in resected BTC patients with low DPD and low TP gene expressions.

A classification model for resectability in hepatocellular carcinoma patients.

AIM: Some patients undergoing liver resection for hepatocellular carcinoma (HCC) have poor outcomes. Therefore, we aimed to propose a new resectability classification for patients with HCC. METHODS: We classified patients into three categories: resectable (R), borderline resectable (BR), and unresectable (UR). Patients (n = 409) who underwent hepatectomy for HCC were assigned to the non-UR (R and BR classes combined; n = 285) and UR-HCC classes (n = 68; training cohort). Patient characteristics in the BR-HCC and R-HCC groups were compared. The new criteria were tested in a validation cohort (n = 295). RESULTS: Of the 285 patients, 229 and 56 were classified into the R- and BR-HCC classes, respectively, using macrovascular invasion, tumor size, and future liver remnant/modified albumin-bilirubin scores. Patients with BR-HCC demonstrated significantly worse progression-free and overall survival (p < 0.0001 and p < 0.0001, respectively) than patients with R-HCC in the training cohort. Similar results were observed in the validation cohort. Multivariate analysis of the non-UR-HCC group in the training cohort revealed that the tumor number and BR-HCC were independent predictive factors for poor overall survival. CONCLUSIONS: This classification can help select patients with BR-HCC for preoperative treatment before considering surgery.

A patient who underwent conversion surgery after atezolizumab plus bevacizumab for hepatocellular carcinoma with portal vein thrombosis and perihepatic lymph node metastases achieved a pathological complete response.

Here, we present a patient with hepatocellular carcinoma complicated by tumor thrombosis into the main portal trunk and perihepatic lymph node metastases who was treated with atezolizumab plus bevacizumab. Shrinkage of the main tumor, portal vein thrombosis, and lymph node metastases were achieved; therefore, hepatectomy with lymphadenectomy could be performed. Final pathology indicated a complete pathological response in the main tumor, portal vein thrombosis, and perihepatic lymph nodes. The patient is currently alive with no evidence of recurrence on radiological assessment at 3 months after surgery.

Surgical Intervention After Lenvatinib Treatment in Patients With Advanced Hepatocellular Carcinoma.

BACKGROUND/AIM: The survival and prognostic factors in patients with advanced hepatocellular carcinoma (HCC) who underwent surgical intervention after lenvatinib treatment is not well-understood. PATIENTS AND METHODS: Seventy-six patients with advanced HCC who had lenvatinib treatment were retrospectively analyzed. RESULTS: Of 70 patients who were treated with lenvatinib, 14 patients underwent surgical intervention after lenvatinib treatment for 4-28 weeks. Progression-free survival (PFS) was significantly longer in patients who underwent surgical intervention than in patients with non-surgical treatment (median, 8.6 vs. 5.1 months, p=0.019). Non-significantly longer overall survival (OS) was also observed in patients with surgical intervention compared to patients with non-surgical treatment (median, unreached vs. 21.0 months, p=0.206). In patients who underwent surgical intervention, two patients had a partial response, and 12 had stable disease according to RECIST ver. 1.1 criteria. The serum alpha-fetoprotein (AFP) level was significantly lower after lenvatinib treatment than before lenvatinib treatment (median, 19.2 vs. 196.5 ng/ml, p=0.0081). Eleven patients underwent curative surgery with a 14% major postoperative complication (Clavien-Dindo ≥IIIa) rate. Patients who exhibited decreases in AFP levels or maintained AFP levels within the normal range during lenvatinib treatment had significantly longer PFS (median, 8.6 vs. 3.0 months, p=0.0009) and OS (median, unreached vs. 12.4 months, p=0.012) compared to those with persistently elevated AFP levels beyond the normal range. CONCLUSION: Surgical intervention after lenvatinib treatment for advanced HCC was associated with longer PFS. Patients exhibiting decreased AFP levels or maintaining AFP levels within the normal limit may be suitable candidates for surgical intervention after lenvatinib treatment for advanced HCC.

Impact of conversion surgery after chemotherapy in patients with initially unresectable and recurrent biliary tract cancer.

Purpose: Gemcitabine, cisplatin, and S-1 chemotherapy was superior to gemcitabine and cisplatin chemotherapy for progression-free survival and overall survival for unresectable and recurrent biliary tract cancer in a randomized phase III trial (KHBO1401). This study aimed to evaluate the outcome of conversion surgery after chemotherapy in biliary tract cancer patients (ancillary study, KHBO1401-3C). Methods: A total of 246 patients were enrolled in KHBO1401. We compared progression-free and overall survivals between the conversion surgery and non-conversion surgery groups. Results: Eight patients (3.3%) underwent conversion surgery with chemotherapy, seven of whom were diagnosed with unresectable disease and one with recurrence. Six and two patients received gemcitabine, cisplatin, and S-1 chemotherapy as well as gemcitabine and cisplatin chemotherapy, respectively. Three patients in the conversion surgery group who received gemcitabine, cisplatin, and S-1 chemotherapy showed no disease progression and survived without postoperative chemotherapy. Preoperative carbohydrate antigen 19-9 (CA19-9) level was a prognostic factor for conversion surgery. After correcting for immortal time bias, 1-year progression-free survival rates in the conversion surgery and non-conversion surgery groups were 50.0% and 19.0%, respectively (hazard ratio 0.343, 95% confidence interval 0.286-0.843, p = 0.0092). One-year overall survival rates in the conversion surgery and non-conversion surgery groups were 87.5% and 56.0%, respectively (hazard ratio 0.222, 95% confidence interval 0.226-0.877, p = 0.0197). Conclusions: Conversion surgery might be an option for the treatment of unresectable and recurrent biliary tract cancer in patients with normal preoperative CA19-9 level.

Is 6 Months the Optimal Duration of Adjuvant Chemotherapy for Pancreatic Cancer?

Aim: This study evaluated the relationship between the relative dose intensity (RDI) and the prognosis to assess the optimal duration of adjuvant chemotherapy for pancreatic cancer. Patients and Methods: From 2013 to 2018, 119 patients with pancreatic cancer underwent radical surgery. After excluding five patients who underwent R2 resection, three with stage IV disease, and two with adjuvant chemotherapy other than S-1, 109 cases were evaluated. They were classified into four groups based on the RDI for the total dosage of S-1: group 1: <50%, group 2: 50% to <80%, group 3: 80% to ≤125%, and group 4: >125%. Results: The number of patients in each group were 48, 20, 30 and 11, with median ages of 74, 73, 66 and 74, respectively. Median estimated glomerular filtration rate was 75, 72, 89 and 77 ml/min/1.73 m2, respectively, demonstrating statistically significant differences. The corresponding median and 5-year overall survival rates were: 378 days and 17.9%; 1,011 days and 35.1%; 1,246 days and 41.6%; 1,389 days and 10.6%. Using group 1 as a reference, the adjusted hazard ratio was 0.39 for group 2, 0.36 for group 3, and 0.30 for group 4; all were statistically significant. Conclusion: The higher the RDI of S-1 in adjuvant chemotherapy, the better the overall survival. Therefore, 1 year of adjuvant chemotherapy with S-1 in pancreatic cancer may be preferable to 6 months.

A large deletion within intron 20 sequence of single-copy PolA1 gene as a useful marker for the speciation in Oryza AA-genome species.

Oryza AA-genome complex comprises five wild species, O. rufipogon, O. barthii, O. longistaminata, O. glumaepatula, and O. meridionalis. Evolutionary relationships among these five wild species have remained contentious and inconclusive. We found that intron 20 of PolA1, a single-copy nuclear gene, was short (S-type: 141-142 bp) in O. rufipogon, O. barthii, and O. glumaepatula, while long (L-type: ca. 1.5 kb) introns were apparent in O. longistaminata and O. meridionalis. Because Oryza species containing BB, CC, EE, FF, and GG genome showed L-type introns, the S-type intron was probably derived from the L-type intron by the deletion of a 1.4 kb fragment through intramolecular homologous recombination between two tandem TTTTGC repeats. Excluding the large deletion sequence, intron 20 sequence of O. barthii was identical to that of O. longistaminata. As more than 3,470 accessions of O. rufipogon and O. sativa also contained the same intron 20 sequence with O. longistaminata except for single T-nucleotide deletion, which was shared with O. glumaepatuala, the deletion of the T-nucleotide probably occurred in the L-type intron 20 of O. logistaminata. Deletions of a large 1.4 kb fragment and single T-nucleotide within the intron 20 of PolA1 gene were considered as useful DNA markers to study the evolutionary relationships among Oryza AA-genome species.

Impact of anatomical liver resection on patient survival in KRAS-wild-type colorectal liver metastasis: A multicenter retrospective study.

BACKGROUND: Liver resection is a standard therapy for colorectal liver metastasis. However, the impact of anatomical resection and nonanatomical resection on the survival in patients with Kirsten rat sarcoma-wild-type and Kirsten rat sarcoma-mutated colorectal liver metastasis remain unclear. We investigated whether anatomical resection versus nonanatomical resection improves survival in colorectal liver metastasis stratified by Kirsten rat sarcoma mutational status. METHODS: Among 639 consecutive patients with colorectal liver metastasis who underwent primary liver resection between January 2008 and December 2017, 349 patients were excluded due to their unknown Kirsten rat sarcoma mutational status, or due to receiving anatomical resection with concomitant non-anatomical resection, radiofrequency, or R2 resection. Accordingly, 290 patients with colorectal liver metastasis were retrospectively assessed. The relationships between resection types and survival were investigated in Kirsten rat sarcoma-wild-type and -mutated groups. RESULTS: Anatomical resection was performed in 77/186 (41%) and 44/104 (42%) patients with Kirsten rat sarcoma-wild-type and Kirsten rat sarcoma-mutated genetic statuses, respectively. For both, the clinical-pathologic factors were comparable, except a larger maximum tumor size and surgical margin were observed in anatomical resection cases. Anatomical resection patients had significantly longer recurrence-free survival and overall survival than nonanatomical resection cases in the Kirsten rat sarcoma-wild-type group (recurrence-free survival, P < .001; overall survival, P = .005). No significant recurrence-free survival or overall survival differences were observed between Kirsten rat sarcoma-mutated anatomical resection and non-anatomical resection (recurrence-free survival, P = .132; overall survival, P = .563). Although, intrahepatic recurrence in Kirsten rat sarcoma-wild-type and -mutated colorectal liver metastasis was comparable (P = .973), extrahepatic recurrence was increased in Kirsten rat sarcoma-mutated versus -wild-type colorectal liver metastasis (P < .001). CONCLUSION: In contrast to Kirsten rat sarcoma-mutated colorectal liver metastasis with higher extrahepatic recurrence after liver resection, local liver control via anatomical resection improved the postoperative survival in patients with Kirsten rat sarcoma-wild-type colorectal liver metastasis.

Impact of the aberrant right hepatic artery on local recurrence of pancreatic ductal adenocarcinoma after pancreaticoduodenectomy.

BACKGROUND: The influence and risk associated with an aberrant right hepatic artery, a common anatomical variation, during pancreatoduodenectomy for pancreatic ductal adenocarcinoma has not been fully investigated. The present study analyzed the impact of an aberrant right hepatic artery on local recurrence after pancreatoduodenectomy for pancreatic ductal adenocarcinoma. METHODS: A total of 169 patients with pancreatic ductal adenocarcinoma who underwent pancreatoduodenectomy at 2 separate Japanese medical institutions were retrospectively analyzed. RESULTS: Thirty of 169 patients (17.7%) presented with an aberrant right hepatic artery. The incidence of local recurrence was higher in the aberrant right hepatic artery group than in the normal right hepatic artery group (43.3 vs 21.5%, P = .017). The local recurrence-free survival was significantly poorer in the aberrant right hepatic artery group than in the normal right hepatic artery group (P = .011). A multivariate analysis found that the aberrant right hepatic artery was an independent risk factor for local recurrence (hazard ratio: 3.74, P = .017). In the aberrant right hepatic artery group, more frequent local recurrence was observed in patients with tumors situated ≤10 mm from the aberrant right hepatic artery root. However, local recurrence was not observed in 2 out of 3 patients with tumors ≤10 mm from the aberrant right hepatic artery root who underwent pancreatoduodenectomy with combined resection of the aberrant right hepatic artery. CONCLUSION: The presence of an aberrant right hepatic artery in patients undergoing pancreatoduodenectomy for pancreatic ductal adenocarcinoma may be associated with an increased risk of postoperative local recurrence. Combined resection of the aberrant right hepatic artery may reduce local recurrence, especially for tumors near the root of the aberrant right hepatic artery.

Liver stiffness measured by virtual touch quantification predicts the occurrence of posthepatectomy refractory ascites in patients with hepatocellular carcinoma.

PURPOSE: This study assessed the significance of measuring liver stiffness using virtual touch quantification before hepatectomy to predict posthepatectomy refractory ascites. METHODS: A total of 267 patients with hepatocellular carcinoma who underwent hepatectomy were prospectively analyzed. Liver stiffness was defined as the median value of the virtual touch quantification (Vs; m/s) by acoustic radio-force-impulse-based virtual touch. RESULTS: A multivariate analysis showed that Vs and the aspartate aminotransferase-to-platelet ratio index were independent risk factors for postoperative refractory ascites (odds ratio = 3.27 and 3.08, respectively). The cutoff value for Vs was 1.52 m/s (sensitivity: 59.5%, specificity: 88.6%) as determined by the analysis of the receiver-operating characteristic curve, and the area under the receiver-operating characteristic curve was 0.79. The cutoff value for the aspartate aminotransferase-to-platelet ratio was 0.952 (sensitivity: 65.5%, specificity: 82.9%), and the area under the receiver-operating characteristic curve was 0.75. CONCLUSIONS: Vs is an independent risk factor for refractory ascites after hepatectomy. The measurement of liver stiffness by virtual touch quantification before hepatectomy can help estimate the risk of postoperative refractory ascites. Nonsurgical treatments should be considered for the management of patients who are at high risk for refractory ascites.

Sulfatase-2 Regulates Liver Fibrosis through the TGF-ß Signaling Pathway.

Transforming growth factor-ß (TGF-ß) activates hepatic stellate cells (HSCs), which drive liver fibrosis via the production and deposition of extracellular matrix (ECM). We aimed to elucidate the mechanistic role of sulfatase-2 (SULF2) in liver fibrosis. To this end, we induced liver fibrosis in wild-type (WT) and SULF2 knockout (Sulf2-KO) mice (6-8 weeks-old) via bile duct ligation (BDL), intraperitoneal injection of carbon tetrachloride (CCl4) or thioacetamide (TAA). The levels of fibrosis in the liver sections were assessed via Sirius red and Masson's trichrome staining, immunohistochemistry and immunoblotting for α-smooth muscle actin (α-SMA) and hydroxyproline. To evaluate the interaction between TGF-ß and SULF2, we transfected human HSCs with scrambled control shRNA and shRNA constructs targeting SULF2 and measured α-SMA expression following treatment with TGF-ß1 ligand. We show here that knockout of SULF2 significantly decreases collagen content, as well as bands of bridging fibrosis, as demonstrated by Sirius red, Masson's trichrome and α-SMA staining after BDL, CCl4 and TAA injection in Sulf2-KO versus WT mice. In all three models of liver fibrosis, we observed significantly lower levels of hydroxyproline in the Sulf2-KO mice compared to the WT mice. HSCs with reduced levels of SULF2 failed to significantly express α-SMA and collagen type I following treatment with TGF-ß1. Furthermore, SULF2 co-localizes with TGFBR3 and the in vitro knockdown of SULF2 in HSCs decreases the release of TGF-ß1 from TGFBR3. Together, these data suggest that SULF2 regulates liver fibrosis via the TGF-ß signaling pathway. Pharmacologic inhibition of SULF2 may represent a novel therapeutic approach to improve liver fibrosis.

Risk stratification and prognostic factors in patients with unresectable undifferentiated carcinoma of the pancreas.

BACKGROUND: Undifferentiated carcinoma (UC) of the pancreas has been considered a highly aggressive malignancy. However, only a few studies have systematically described the clinical course of UC patients. The aim of this study was to clarify the prognosis and construct a prognostic model for patients with unresectable UC. METHODS: This study was conducted at 17 institutions in Japan, and a total of 55 patients were analyzed. RESULTS: The median overall survival (OS) of patients with unresectable UC was 3.95 months. In the multivariate Cox proportional hazards (CPH) model, age ≥65 years, Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2, and C-reactive protein (CRP) >10 mg/L were independent prognostic factors for OS (age ≥65 years: hazard ratio [HR], 2.732; 95% confidence interval [CI], 1.353-5.515; ECOG PS ≥ 2: HR, 7.866; 95% CI, 1.981-31.241; CRP >10 mg/L: HR, 1.956; 95% CI, 1.013-3.775). Based on the ß coefficients from the CPH model, the prognostic scores were defined as follows: age ≥65 years (3 points), ECOG PS ≥ 2 (6 points), and CRP >10 ml/L (2 points). The final prognostic model was the sum of the points. The derived prognostic model stratified patients into high-risk (score ≥4) and low-risk (score 0-3) groups, with significant differences in OS (1.45 vs. 8.19 months, respectively; p < 0.001). CONCLUSIONS: The prognostic model stratified patients into high-risk and low-risk groups. These findings suggest that this model can serve as a tool for patient information and decision-making with regard to the therapeutic strategy for UC.

Enhanced patterns on intraoperative contrast-enhanced ultrasonography predict outcomes after curative liver resection in patients with hepatocellular carcinoma.

PURPOSE: This study aimed to clarify what hepatocellular carcinoma (HCC) phenotype, as categorized by intraoperative contrast-enhanced ultrasonography (CEUS), showed a high risk of recurrence after hepatic resection. METHODS: Patients who underwent initial curative hepatectomy with intraoperative CEUS for a single HCC nodule were retrospectively assigned to three patterns of fine (FI), vascular (VA), and irregular (IR) according to the maximum intensity projection pattern based on intraoperative CEUS. Staining was performed for Ki-67, pyruvate kinase type M2 (PKM2), and vascular endothelial growth factor (VEGF) to assess the tumor proliferative activity, tumor glucose metabolism, and angiogenesis, respectively. RESULTS: Of 116 patients, 18, 50, and 48 were assigned to the FI, VA and IR patterns, respectively. IR patients demonstrated a significantly worse prognosis for both the recurrence-free survival (RFS) and overall survival (OS) (P = 0.0002, 0.0262, respectively) than did patients with other patterns. A multivariate analysis revealed an IR pattern in intraoperative CEUS to be an independent predictive factor for a poor RFS, and major hepatectomy and an IR pattern were independent predictive factors for a poor OS. An IR pattern was closely related to the tumor size (≥ 3.3 cm) and poor histological differentiation and showed a high Ki-67 index, low VEGF expression, and high PKM2 expression. CONCLUSION: IR-pattern HCCs as classified by intraoperative CEUS may be associated with a higher risk of recurrence and worse outcomes in HCC patients after hepatic resection than other patterns.

Over-Expression of a Melon Y3SK2-Type LEA Gene Confers Drought and Salt Tolerance in Transgenic Tobacco Plants.

Climate change, with its attendant negative effects, is expected to hamper agricultural production in the coming years. To counteract these negative effects, breeding of environmentally resilient plants via conventional means and genetic engineering is necessary. Stress defense genes are valuable tools by which this can be achieved. Here we report the successful cloning and functional characterization of a melon Y3SK2-type dehydrin gene, designated as CmLEA-S. We generated CmLEA-S overexpressing transgenic tobacco lines and performed in vitro and in vivo drought and salt stress analyses. Seeds of transgenic tobacco plants grown on 10% polyethylene glycol (PEG) showed significantly higher germination rates relative to wild-type seeds. In the same way, transgenic seeds grown on 150 mM sodium chloride (NaCl) recorded significantly higher germination percentages compared with wild-type plants. The fresh weights and root lengths of young transgenic plants subjected to drought stress were significantly higher than that of wild-type plants. Similarly, the fresh weights and root lengths of transgenic seedlings subjected to salt stress treatments were also significantly higher than wild-type plants. Moreover, transgenic plants subjected to drought and salt stresses in vivo showed fewer signs of wilting and chlorosis, respectively. Biochemical assays revealed that transgenic plants accumulated more proline and less malondialdehyde (MDA) compared with wild-type plants under both drought and salt stress conditions. Finally, the enzymatic activities of ascorbate peroxidase (APX) and catalase (CAT) were enhanced in drought- and salt-stressed transgenic lines. These results suggest that the CmLEA-S gene could be used as a potential candidate gene for crop improvement.

Cytoglobin-expressing cells in the splenic cords contribute to splenic fibrosis in cirrhotic patients.

BACKGROUND AND AIM: Among several noninvasive evaluation methods of portal hypertension (PH), the measurement of spleen stiffness is a reliable method for predicting esophageal variceal bleeding; however, the underlying mechanisms for increased stiffness remain unclear. We attempted to elucidate the pathological changes to the spleen and the underlying mechanisms in patients with PH. METHODS: Histological examination was performed using splenic tissues from 42 patients with PH who underwent laparoscopic splenectomy, and the results were compared with those from patients without PH. RESULTS: In addition to splenic sinus congestion, diffuse fibrosis was detected in the splenic cords in the red pulp of patients with PH. The degree of the fibrosis was well correlated with severity in thrombocytopenia and splenomegaly. Cells expressing α-smooth muscle actin dramatically increased in the splenic cord. Cytoglobin (Cygb) expression was detected in human splenic cords as reported in animal reticular cells, and fluorescent double immunostaining revealed that these cells expressed α-smooth muscle actin in patients with PH, suggesting transformation of Cygb-expressing cells to myofibroblastic cells. Expression levels of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 2, nitrotyrosine, and transforming growth factor-ß were markedly upregulated in the red pulp of patients with PH, implying a significant role of oxidative stress in the mechanism for splenic fibrosis. CONCLUSION: Splenic fibrosis progresses along with advancement of PH. Cygb-expressing cells in the splenic cord possibly participate in this process through mechanisms including oxidative stress.

Efficient elimination of Yam mosaic virus (YMV) from white yam (Dioscorea rotundata Poir.) by cryotherapy of axillary buds.

Dioscorea rotundata is an economically important food crop in many tropical countries as many people in this region depend on it for food and livelihood. Viral diseases, especially Yam mosaic virus (YMV), constitute a major constraint in the cultivation of this crop as they perpetuate through generations in the vegetatively propagated planting materials. Getting resistant or at least virus-free planting materials for farmers thus becomes crucial. This study was aimed at eliminating YMV in Dioscorea rotundata by cryotherapy of axillary buds. Enlarged axillary buds of YMV-infected TDr 2269 were frozen in liquid nitrogen for 1 h, re-warmed at 40 °C and cultured to regenerate plantlets. Approximately 76.33% plantlet regeneration and 100% YMV eradication were obtained for cryo-treated buds, against 95% and 0% obtained respectively for non-treated buds. RT-PCR and RT-qPCR analyses did not reveal detectable quantity of YMV in treated plants but did in control plants. Plants from cryo-treated buds showed no mosaic symptoms and produced slightly more tubers, and heavier mini-tubers (20.48±3.11 g) under greenhouse conditions contrary to non-treated plants that showed severe mosaic symptoms with significantly smaller tubers (1.91±0.39 g) (P < 0.05). This is the first report showing the elimination of YMV from infected white yam stock plant by cryotherapy and would be useful for producing clean planting materials.

Transgene Stacking as Effective Tool for Enhanced Disease Resistance in Plants.

Introduction of more than one gene into crop plants simultaneously or sequentially, called transgene stacking, has been a more effective strategy for conferring higher and durable insect and disease resistance in transgenic plants than single-gene technology. Transgenes can be stacked against one or more pathogens or for traits such as herbicide tolerance or anthocyanin pigmentation. Polygenic agronomic traits can be improved by multiple gene transformation. The most widely engineered stacked traits are insect resistance and herbicide tolerance as these traits may lead to lesser use of pesticides, higher yield, and efficient control of weeds. In this review, we summarize transgene stacking of two or more transgenes into crops for different agronomic traits, potential applications of gene stacking, its limitations and future prospects.

Predictive factors for hepatocellular carcinoma occurrence or recurrence after direct-acting antiviral agents in patients with chronic hepatitis C.

BACKGROUND AND AIMS: Direct-acting antiviral agents (DAAs) and the risk of hepatocellular carcinoma (HCC) is controversially reported in the literature. The primary endpoints of this study were to clarify the cumulative incidence and recurrence rate of HCC after DAA treatment. The secondary endpoints were to identify the factors associated with the occurrence or recurrence of HCC after DAAs treatment. METHODS: Of 234 HCV patients, 211 with no history of HCC (no-HCC-history group) and 23 with previous treated HCC history (HCC-history group) were treated with DAAs and followed for more than 24 weeks to determine the incidence of HCC. Platelet count, albumin, α-fetoprotein (AFP) level, L3%, the FIB-4 index and APRI scores were analyzed as possible factors associated with HCC occurrence and recurrence. An intergroup comparison was made of the cumulative incidence of HCC. Cox proportional hazards regression was used to determine associations between blood test values and risk of HCC. RESULTS: The median observation period was 21 months. Cumulative incidence of HCC was higher in the HCC-history group than in the no-HCC-history group (p < 0.0001, 19.0 and 0.52 per 100 patient-years, respectively). Univariate analysis revealed platelet count, albumin, α-fetoprotein (AFP) level, AFP-L3%, and FIB-4 index and APRI scores at the end of DAA treatment as being significantly associated with occurrence/recurrence of HCC. Multivariate analysis revealed that AFP levels before and after the administration of DAAs and AFP-L3% after DAA were independently associated with the occurrence/recurrence of HCC (p = 0.045, 0.043, 0.005, respectively). CONCLUSION: The HCC occurrence rate after DAA treatment was very low, and the recurrence rate lower than that in previous interferon reports. The AFP level and AFP-L3% were identified as important factors in predicting occurrence/recurrence of HCC. Careful observation is needed when increased levels of AFP or AFP-L3% after DAAs treatment are observed.

Genetic diversity among perennial wild rice Oryza rufipogon Griff., in the Mekong Delta.

Oryza rufipogon Griff. is a perennial species of wild rice widely distributed along the channels and rivers of the Mekong Delta, Vietnam. This study attempted to find centers of diversity among wild rice populations in this area and their inter-relationships. The highest genetic diversity was found in the Dong Thap population and the lowest in the Can Tho population. Maternal diversity evaluated using chloroplast INDELs detected ten plastid types, five of which were novel relative to other Asian countries. The mitochondrial genome suggested two unique deletions. One 699-bp deletion via short tandem repeats was accompanied by another deletion including orf153. All accessions carrying the mitochondrial type were found in a particular plastid type. This unique maternal lineage was confined to specific channels where it showed vigorous vegetative growth in comparison to upstream areas where various maternal lineages and maximum genetic diversity occurred. This area along the Mekong Delta is a center of not only nuclear but also maternal diversity.

Immunohistochemical characterization of cancer-associated fibroblasts at the primary sites and in the metastatic lymph nodes of human intrahepatic cholangiocarcinoma.

Cancer-associated fibroblasts (CAFs) are an important constituent of the cancer stroma. In intrahepatic cholangiocarcinoma (ICC), the features of CAFs at the primary site and in the metastatic lymph nodes (Met-LNs) and their origin have been unclear. In the present study, we characterized CAFs at the primary site (n = 42) and in the Met-LNs (n = 10) of human ICC by immunohistochemistry using potential molecular markers of CAFs, portal fibroblasts (PFs), hepatic stellate cells (HSCs), and bone marrow-derived fibrocytes (BMDFs). At the primary site, the stroma was strongly positive for α-smooth muscle actin (α-SMA; marker for CAFs), platelet-derived growth factor receptor-ß (PDGFR-ß) (common marker for HSCs and PFs), fibulin-2, and thymus cell antigen-1 (Thy-1; PF marker), whereas immunoreactivity for fascin (HSC marker) was scarce. Most of the α-SMA-positive cells were found to express PDGFR-ß, Thy-1, and fibulin-2 by double immunostaining. A small population of BMDF marker-positive (α-SMA+CD45+CD34+) cells was found by triple immunostaining. In the micro-Met-LNs, α-SMA-positive cells were absent in cancer aggregates of the LN sinus, whereas they were present in the invasion area of cancer cells from the LN sinus to the LN parenchyma. In the macro-Met-LNs, there were abundant α-SMA-positive cells that were also positive for PDGFR-ß and Thy-1 but negative for fibulin-2 and fascin. Thus, regarding the expression of molecular markers, CAFs at the primary site of ICC are similar to PFs and different from those of HSCs or CAFs in the Met-LNs. CAFs at the primary sites and in the Met-LN are thought to be derived from PFs/BMDFs and resident cells of LNs, respectively.