Peritoneal Lavage CEA mRNA Levels Predict Conversion Gastrectomy Outcomes after Induction Chemotherapy with Intraperitoneal Paclitaxel in Gastric Cancer Patients with Peritoneal Metastasis.

BACKGROUND: The outcome of gastric cancer patients with peritoneal metastasis remains poor. We treated these patients with intraperitoneal and intravenous paclitaxel plus oral S-1 (tegafur/gimeracil/oteracil), followed by gastrectomy in responders. We evaluated the clinical significance of peritoneal lavage carcinoembryonic antigen (CEA) messenger RNA (mRNA) levels as a biomarker for indication of conversion gastrectomy. METHODS: The peritoneal lavage of 68 patients who received the above regimen as induction chemotherapy was repeatedly collected via intraperitoneal access ports. Gastrectomy was considered when improvement of peritoneal metastasis was confirmed by a second laparoscopic examination with negative peritoneal cytology. CEA and porphobilinogen deaminase mRNAs were chronologically quantified using the transcription reverse-transcription concerted reaction method. The CEA mRNA index (CmRI) was calculated as CEA mRNA/porphobilinogen deaminase mRNA × 10,000. RESULTS: Thirty-nine patients underwent gastrectomy and 29 patients did not (median survival time, 27.8 vs. 10.7 months, respectively; P < 0.001). In gastrectomy-positive patients, the outcome largely differed according to CmRI values immediately prior to surgery. Patients with a preoperative CmRI value <100 (n = 20) were associated with a significantly longer survival than those with a preoperative CmRI value >100 (n = 19) (41.8 vs. 20.1 months, respectively; P < 0.001). A preoperative CmRI value <100 was confirmed as an independent predictor of survival for gastrectomy-positive patients in the multivariate analysis. CONCLUSIONS: The CmRI reflects the response of peritoneal metastases to induction intraperitoneal chemotherapy. It may be a useful biomarker for indicating gastrectomy in gastric cancer patients with peritoneal metastasis.

[The Significance of Genetic Testing for Acute Promyelocytic Leukemia].

The majority of patients with acute promyelocytic leukemia (APL) harbor the t (15;17) (q22;q12) transloca- tion, which results in the expression of PML-RARA mRNA. All-trans retinoic acid (ATRA) is a representa- tive molecular-targeted drug and is directed against PML-RARA. Therefore, the detection of PML-RARA mRNA has become indispensable for the diagnosis of APL and the decision regarding the treatment policy. Once the diagnosis is confirmed by genetic testing, ATRA-based induction therapy can be initiated. This is also applicable in atypical cases such as the M3 variant. Furthermore, after ATRA-based induction therapy, the curative effect is evaluated by quantitative PCR analysis. Thus, genetic testing is important in the follow-up of patients with APL. [Review].

The shortest isoform of C/EBPß, liver inhibitory protein (LIP), collaborates with Evi1 to induce AML in a mouse BMT model.

Ecotropic viral integration site 1 (Evi1) is one of the master regulators in the development of acute myeloid leukemia (AML) and myelodysplastic syndrome. High expression of Evi1 is found in 10% of patients with AML and indicates a poor outcome. Several recent studies have indicated that Evi1 requires collaborative factors to induce AML. Therefore, the search for candidate factors that collaborate with Evi1 in leukemogenesis is one of the key issues in uncovering the mechanism of Evi1-related leukemia. Previously, we succeeded in making a mouse model of Evi1-related leukemia using a bone marrow transplantation (BMT) system. In the Evi1-induced leukemic cells, we identified frequent retroviral integrations near the CCAAT/enhancer-binding protein ß (C/EBPß) gene and overexpression of its protein. These findings imply that C/EBPß is a candidate gene that collaborates with Evi1 in leukemogenesis. Cotransduction of Evi1 and the shortest isoform of C/EBPß, liver inhibitory protein (LIP), induced AML with short latencies in a mouse BMT model. Overexpression of LIP alone also induced AML with longer latencies. However, excision of all 3 isoforms of C/EBPß (LAP*/LAP/LIP) did not inhibit the development of Evi1-induced leukemia. Therefore, isoform-specific intervention that targets LIP is required when we consider C/EBPß as a therapeutic target.

[Idiopathic dysplasia of undetermined/uncertain significance (IDUS) initially presenting erythroblastosis and hyperferritinemia].

A 64-year-old man was referred to our hospital because of mild anemia, erythroblasts in peripheral blood, and hyperferritinemia. In our hospital, no evidence of cytopenia was found. The peripheral blood smear showed pseudo-Pelger-Huët anomaly and giant platelets. The serum ferritin level was extremely high (1,405 ng/ml). A bone marrow examination was performed for further evaluation and revealed trilineage dysplasia. Since the patient did not fulfill the diagnostic criteria for cytopenias associated with myelodysplastic syndromes, he was diagnosed as having idiopathic dysplasia of undetermined/uncertain significance (IDUS). The SF3B1 mutation was identified in this patient, suggesting that he might be at a stage prior to myelodysplastic syndrome. Some IDUS patients reportedly progress to myeloid neoplasms, making careful observation essential. If morphological dysplasia in peripheral blood and hyperferritinemia are present, these findings suggest bone marrow failure syndromes. In such cases, further evaluation including a bone marrow examination may be required.

Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells.

BACKGROUND: The migration of T cell to atherosclerotic lesions is proposed to be involved in the pathogenesis of the atherosclerosis. Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from activated platelets, exerts a variety of responses such as cell migration and proliferation, and reportedly induces T cell migration. Accordingly, platelet-T cell interactions may exist based on T cell responses triggered by platelet-derived S1P. METHODS: S1P was measured using two-step lipid extraction followed by high-performance liquid chromatography (HPLC) separation while other phospholipids were determined by an enzymatic assay. The expression of S1P and lysophosphatidic acid receptors on Jurkat T cells was examined by RT-PCR and flow cytometry. Jurkat cell migration by S1P and the supernatant of activated platelets (SAP) was evaluated by a modified Boyden's chamber assay. RESULTS: S1P1 receptor was confirmed to be expressed on Jurkat T cell by RT-PCR and flow cytometry. S1P at 10-100 nM induced strong Jurkat cell migration, which was inhibited by the S1P1 (and S1P3) antagonist VPC23019 and the Gi inactivator pertussis toxin (PTX). We found that the supernatant (releasate) of human platelets activated by collagen stimulation, which contains S1P abundantly, induced Jurkat cell migration and that the migration was inhibited by VPC23019 and PTX. In addition, human serum, into which platelet contents (including S1P) are fully released, induced the Jurkat cell migration, which was also inhibited by VPC23019. CONCLUSIONS: Our findings suggest that platelet-derived S1P induces Jurkat T cell migration possibly via S1P1. S1P may be a key molecule involved in the responses triggered by platelet-T cell interactions, including atherosclerosis.

[The current status of genetic testing for leukemia].

The pathogenic chromosome translocations present in various hematological malignancies result in the formation of fusion genes, which are detected by a reverse transcription-polymerase chain reaction method. Furthermore, with this method, it is possible to sensitively detect minimal residual disease (MRD), which is difficult by a morphological testing. It has now been established that the detection of MRD is important for the diagnosis, treatment policy, evaluation of the prognosis, and monitoring of leukemia. In particular, quantitative analysis of MRD is important for evaluation of the curative effect and prediction of recurrence. In addition, mutation analysis is valuable to decide on the therapeutic protocol for imatinib-resistant patients, and the stratification of treatment for acute myeloid leukemia. At present, however, there is no standard laboratory procedure for genetic testing for leukemia. Here, the problems related to external precision management and analytical error are discussed.

Correlation between variant call accuracy and quality parameters in comprehensive cancer genomic profiling tests.

Background: Comprehensive genomic profiling (CGP) tests have been widely utilized in clinical practice. In this test, the variant list automatically output from the data analysis pipeline often contains false-positive variants, although the correlation between the quality parameters and prevalence of false-positive variants remains unclear. Methods: We analyzed 125 CGP tests performed in our laboratory. False-positive variants were manually detected via visual inspection. The quality parameters of both wet and dry processes were also analyzed. Results: Among the 125 tests, 52 (41.6%) required more than one correction of the called variants, and 21 (16.8%) required multiple corrections. A significant correlation was detected between somatic false-positive variants and quality parameters in the wet (ΔΔCq, pre-capture library peak size, pre-capture library DNA amount, capture library peak size, and capture library concentration) and dry processes (total reads, mapping rates, duplication rates, mean depth, and depth coverage). Capture library concentration and mean depth were strong independent predictors of somatic false-positive variants. Conclusions: We demonstrated a correlation between somatic false-positive variants and quality parameters in the CGP test. This study facilitates gaining a better understanding of CGP test quality management.

Antagonism of sphingosine 1-phosphate receptor 2 causes a selective reduction of portal vein pressure in bile duct-ligated rodents.

UNLABELLED: Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1-phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P(2) ). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P(2) antagonism on portal hypertension was examined. Intravenous infusion of the S1P(2) antagonist, JTE-013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P(2) antagonist did not alter portal vein pressure and mean arterial pressure in control sham-operated rats. Rho kinase activity in the livers was enhanced in bile duct-ligated rats compared to sham-operated rats, and this enhanced Rho kinase activity in bile duct-ligated livers was reduced after infusion of the S1P(2) antagonist. S1P(2) messenger RNA (mRNA) expression, but not S1P(1) or S1P(3) , was increased in bile duct-ligated livers of rats and mice and also in culture-activated rat hepatic stellate cells. S1P(2) expression, determined in S1P 2LacZ/+ mice, was highly increased in hepatic stellate cells of bile duct-ligated livers. Furthermore, the increase of Rho kinase activity in bile duct-ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P 2-/- mice. CONCLUSION: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P(2) . The S1P(2) antagonist merits consideration as a novel therapeutic agent for portal hypertension.

Perihepatic lymph node enlargement is a negative predictor for sustained responses to pegylated interferon-α and ribavirin therapy for Japanese patients infected with hepatitis C virus genotype 1.

AIM: Although perihepatic lymph node enlargement (PLNE) is reportedly associated with the negative outcome of interferon therapy for chronic hepatitis C, there were limitations in that the results were obtained in patients with various genotypes, viral loads and treatment regimens. We aimed to precisely clarify the significance of PLNE in interferon therapy for chronic hepatitis C. METHODS: Between December 2004 and June 2005, 112 patients with hepatitis C virus (HCV) genotype 1 and HCV RNA of more than 100 KIU/mL were enrolled, who underwent pegylated interferon-α plus ribavirin therapy thereafter. PLNE was defined as a perihepatic lymph node of more than 1 cm in the longest axis by ultrasonography. RESULTS: The sustained virological response (SVR) rate was lower in patients with PLNE (4/22, 18.2%) than in those without (37/90, 41.1%; P = 0.045) and viral load decline was smaller in patients with PLNE than in those without (P = 0.028). The proportion of PLNE positive patients was the smallest in the SVR group (P = 0.033) among the patient groups divided by the treatment outcome. PLNE was retained as a negative predictor for SVR by multivariate logistic regression analysis (P = 0.012). Furthermore, PLNE was not significantly associated with the mutations at HCV core protein and at interferon sensitivity-determining region, or interleukin-28B polymorphism in 45 patients with HCV genotype 1, enrolled between December 2011 and March 2012. CONCLUSION: PLNE is a negative predictor for SVR in patients with HCV genotype 1 and HCV RNA of more than 100 KIU/mL treated with pegylated interferon-α plus ribavirin, independent of other known predictors for SVR.

Increased activity of serum mitochondrial isoenzyme of creatine kinase in hepatocellular carcinoma patients predominantly with recurrence.

BACKGROUND & AIMS: Mitochondrial isoenzyme of creatine kinase (MtCK) is reportedly highly expressed in hepatocellular carcinoma (HCC). Clinical relevance of serum MtCK activity in patients with HCC was assessed using a novel immuno-inhibition method. METHODS: Among patients with cirrhosis caused by hepatitis B or C virus, 147 patients with HCC (12 with the first occurrence and 135 with recurrence) and 92 patients without HCC were enrolled. RESULTS: Serum MtCK activity was higher in cirrhotic patients with HCC than in those without HCC or healthy subjects. Elevated serum MtCK activity in HCC patients decreased after radiofrequency ablation. In case of prediction of HCC, MtCK had a sensitivity of 62.6% and a specificity of 70.7% at a cut-off point of 8.0 U/L, with an area under the receiver operating curve of 0.722 vs. 0.713 for alpha-fetoprotein (AFP) and 0.764 for des-gamma-carboxy prothrombin (DCP). Among the HCC patients, serum MtCK activity was elevated in 52.9% individuals with serum AFP level < 20 ng/ml and 63.2% individuals with serum DCP level < 40 mAu/ml. Even in patients with a single HCC ≤ 2 cm, the sensitivity of serum MtCK activity for the prediction of HCC was 64.4%, which was comparable to the overall sensitivity. This increased activity was due to an increase in ubiquitous MtCK, not sarcomeric MtCK, and the enhanced mRNA expression of ubiquitous MtCK was observed in cell lines originating from HCCs in contrast to healthy liver tissues. CONCLUSIONS: Serum MtCK activity merits consideration as a novel marker for HCC to be further tested as for its diagnostic and prognostic power.

Analysis for the combination expression of CK20, FABP1 and MUC2 is sensitive for the prediction of peritoneal recurrence in gastric cancer.

Prediction of peritoneal recurrence in gastric cancer patients is important for application of adjuvant chemotherapy. After surgery, occasional patients have peritoneal recurrence despite negative cytology of the peritoneal washings. Thus, molecular detection of a subliminal number of cancer cells in peritoneal washings may overcome the sensitivity limitation of conventional cytology. In this study, expressions of five specific marker genes, namely, TFF1, TFF2, CK20, FABP1 and MUC2, were evaluated for their usefulness as markers of micro-dissemination. It was found that reverse transcriptase-polymerase chain reaction for these five genes yielded results highly specific for the depth of invasion and disease stage. Furthermore, the expression of CK20, FABP1 and MUC2 was a reliable prognostic indicator of peritoneal metastasis. Our results suggest that evaluation of the expression of CK20, FABP1 and MUC2 in peritoneal washings is a useful tool for identifying patients at high risk of peritoneal recurrence who may need adjuvant chemotherapy.

[Study on the analytical error factors and evaluation of an internal control gene for leukemia gene expression analysis].

Quantitative analysis of the leukemia fusion gene by real-time PCR is a sensitive method to monitor minimal residual disease; the data obtained are very useful to evaluate the disease stage and prognosis, contributing to the clinical practice of hematology. However, there is no standard laboratory procedure for leukemia genetic testing. Therefore, this genetic testing has some problems related to precision management. To minimize analytical error factors, normalization by an internal control gene is necessary. Additionally, it is important to choose a gene suitable for leukemia gene expression analysis because the expression of an internal standard gene changes due to various factors. In this study, we examined analytical error factors (RNA extraction efficiency, reverse transcription reaction efficiency) and evaluated an internal control gene. As a result, in RNA extraction, the extraction efficiency of the acid-guanidium-phenol-chloroform (AGPC) method was high compared to the silica method. The reverse transcription reaction efficiency was significantly different with each reaction reagent. Furthermore, since three kinds of gene (18s rRNA, GUS, beta-actin) had few differences between samples, they were considered to be suitable as internal standards.

Increased production of ADAMTS13 in hepatic stellate cells contributes to enhanced plasma ADAMTS13 activity in rat models of cholestasis and steatohepatitis.

Although hepatic stellate cells, endothelial cells, glomerular podocytes and plateles were reported to be a source of ADAMTS13, it is not clarified which source is involved in the regulation of plasma ADAMTS13 activity. It was demonstrated previously that selective hepatic stellate cell damage in rats caused decreased plasma ADAMTS13 activity. To further elucidate the potential contribution of hepatic stellate cells to the regulation of plasma ADAMTS13 activity, this study examined plasma ADAMTS13 activity when hepatic stellate cells proliferate during the process of liver fibrosis by employing rat models of liver fibrosis due to cholestasis, bile duct ligation, and steatohepatitis, a choline-deficient L-amino acid-defined-diet. ADAMTS13 expression was increased with co-localisation with smooth muscle alpha-actin, a marker of hepatic stellate cells, in bile duct-ligated livers up to four weeks, in which a close correlation between ADAMTS13 and smooth muscle alpha-actin mRNA expressions was determined. Plasma ADAMTS13 activity, measured by a sandwich ELISA involving a specific substrate to ADAMTS13, was increased in bile duct-ligated rats with a significant correlation with ADAMTS13 mRNA expression levels in the liver. Furthermore, ADAMTS13 mRNA expression was increased with enhanced mRNA expression in smooth muscle alpha-actin in the livers of rats fed a choline-deficient L-amino acid-defined-diet for 16 weeks, in which increased plasma ADAMTS13 activity was determined. Thus, increased plasma ADAMTS13 activity in cholestasis and steatohepatitis in rats may be due, at least in part, to enhanced ADAMTS13 production in the liver, suggesting a significant role of hepatic stellate cells in the regulation of plasma ADAMTS13 activity.

[The current status of leukemia genetic testing and the trial of its standardization].

The pathogenic chromosome translocations present in various hematological malignancies result in the formation of fusion genes, which are detected by a reverse transcription-polymerase chain reaction (RT PCR) method. Furthermore, with this method, it is possible to detect minimal residual disease (MRD) sensitively, which is difficult with morphological testing. It has been established that the detection of MRD is important for diagnosis, evaluation of prognosis, and monitoring of leukemia. In particular, quantitative analysis of MRD load transition during the initial phase of treatment is of high prognostic value. At present, however, there is no standard laboratory procedure for leukemia genetic testing. Here, the problems related to external precision management are discussed.

[Establishment of a pharmacogenomic testing system for the realization of individual pharmacotherapy].

The metabolism of and sensitivity to drugs differ from individual to individual, and one base polymorphism of drug-metabolizing enzymes is known to play an important role in this difference between individuals. The genotyping of drug-metabolizing enzymes prior to drug administration would help to predict individual reactivity to drugs and possible adverse reactions that may occur, which is essential to realize tailor-made therapy for individual patients. In July 2005, the Pharmacogenomics Working Group, composed of members of the Clinical Genomic Medicine Unit, Pharmaceutical Department, Medical Informatics Department, Clinical Laboratory, Gastroenterology, Cardiovascular Medicine, and Department of Neurology, was established in our university hospital in an attempt to introduce such pharmacogenomic testing. The project was approved by the Institutional Research Ethics Committee of the Faculty of Medicine, the University of Tokyo, and, in August 2006, testing for CYP2C19*2 and CYP2C19*3, enzymes involved in the metabolism of proton pump inhibitors, was started. Furthermore, in August 2007, testing for CYP2C9*3, the enzyme involved in the metabolism of Warfarin, and vitamin K epoxidereductase1 (VKORC1) 6484 C>T was started. In the CYP2C19 genotyping, the high incidence of poor metabolizers has been demonstrated; it was speculated that the test could confirm the adverse effects of the drug, i.e., after administration of the drug to patients. Moreover, testing for CYP2C9*3 and VKORC1 6484 C>T was shown to be useful for the safe administration of warfarin. The pharmacogenomic testing system was successfully established in our university hospital, and the Pharmacogenomics Working Group is still active, playing an important role in this project.

Significance of carcinoembryonic antigen mRNA in peritoneal lavage determined by transcription-reverse transcription concerted method in patients with low rectal cancer.

BACKGROUND/OBJECTIVE: Positive carcinoembryonic antigen (CEA) messenger RNA (mRNA) in peritoneal lavage is associated with poor prognosis in patients with colon cancer. However, there are no reports about rectal cancer. Therefore we aimed to evaluate the frequency of positive CEA mRNA in peritoneal lavage and the significance of CEA mRNA in patients with low rectal cancer. METHODS: A total of 55 patients with low rectal cancer who received curative surgical resection were enrolled. CEA mRNA in peritoneal lavage was measured using the transcription-reverse transcription concerted method, a quantitative RNA amplification method. The correlation between CEA mRNA and overall and peritoneal recurrence-free survival was evaluated. RESULTS: Among 55 patients, 6 (10.9%) had positive CEA mRNA in peritoneal lavage. Patients with positive CEA mRNA resulted in significantly higher recurrence rate than those with negative CEA mRNA (p=0.007). Similarly, the local recurrence rate was significantly higher in the positive CEA mRNA group than in the negative CEA mRNA group (p=0.0009). Lymph node metastasis and positive CEA mRNA were independent risk factors for overall and local recurrence. CONCLUSION: Positive CEA mRNA in low rectal cancer is a factor that predisposes patients to a high risk for overall recurrence, especially for local recurrence.

Bosutinib as a fourth-line therapy for a patient with T315I-positive lymphoid blastic phase chronic myeloid leukemia: A case report.

A 35-year-old male was diagnosed with chronic myeloid leukemia in the chronic phase and was prescribed 100 mg daily dasatinib. However, dasatinib was discontinued due to thrombocytopenia, and within six months, the disease progressed to the lymphoid blastic phase. Hyper-cyclophosphamide, vincristine, adriamycin and dexamethasone chemotherapy combined with 140 mg dasatinib or 600 mg imatinib was prescribed. The two inhibitors were soon discontinued due to severe thrombocytopenia and jaundice, respectively. Myelosuppression persisted subsequent to the nadir. Bone marrow (BM) aspiration and biopsy revealed hypercellular marrow filled with blasts. Sequencing of the leukemia cells revealed overlapping peaks for the wild-type sequence and the T315I mutant sequence. The patient was treated with 500 mg bosutinib (which was later reduced to 300 mg) for pretransplant cytoreduction. After 5 months, the patient's spleen exhibited a reduction in volume and the percentage of blasts in the BM decreased from 96.1 to 17.5%. The patient successfully underwent cord blood transplantation. The patient has been disease-free for 5 months subsequent to transplantation. This case suggests that bosutinib may be effective for cytoreduction prior to stem cell transplantation, unless the leukemia cells consistently harbor the T315I mutation.