Diagnostic and Therapeutic Endoscopic Retrograde Cholangiography Using a Short-Type Double-Balloon Endoscope in Patients With Altered Gastrointestinal Anatomy: A Multicenter Prospective Study in Japan.

OBJECTIVES: To evaluate the utility and safety of a short-type double-balloon endoscope (DBE) in the treatment of biliary disease in patients with surgically altered gastrointestinal (GI) anatomy. METHODS: This study was conducted as a multicenter, single-arm, prospective trial at five tertiary academic care centers and three community-based hospitals in Japan. Consecutive patients with biliary disease with altered GI anatomy were prospectively included in this study. RESULTS: A total of 311 patients underwent double-balloon endoscopic retrograde cholangiography (ERC). The success rate of reaching the target site, the primary end point, was 97.7% (95% confidence interval (CI): 95.4-99.1). The success rate of biliary cannulation and contrast injection of the targeted duct, the secondary end point, was 96.4% (95% CI: 93.6-98.2), and the therapeutic success rate was 97.9% (95% CI: 95.4-99.2). Adverse events occurred in 33 patients (10.6%, 95% CI: 7.1-14.0) and were managed conservatively in all patients with the exception of 1 in whom a perforation developed, requiring emergency surgery. CONCLUSIONS: ERC using a short-type DBE resulted in an excellent therapeutic success rate and a low rate of adverse events. This treatment can be a first-line treatment for biliary disease in patients with surgically altered GI anatomy.

Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4-ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

Identification of the transforming activity of Indian hedgehog by retroviral expression screening.

To identify novel cancer-promoting genes in biliary tract cancer (BTC), we constructed a retroviral cDNA expression library from a clinical specimen of BTC with anomalous pancreaticobiliary duct junction (APBDJ), and used the library for a focus formation assay with 3T3 fibroblasts. One of the cDNAs rescued from transformed foci was found to encode Indian hedgehog homolog (IHH). The oncogenic potential of IHH was confirmed both in vitro with the focus formation assay and in vivo with a tumorigenicity assay in nude mice. The isolated IHH cDNA had no sequence alterations, suggesting that upregulation of IHH expression may contribute to malignant transformation. Quantitation of IHH mRNA among clinical specimens has revealed that the expression level of IHH in BTC with APBDJ is higher than that in BTC without APBDJ and than in non-cancerous biliary tissues. Our data thus implicate a direct role of IHH in the carcinogenesis of BTC with APBDJ.

Identification of transforming activity of free fatty acid receptor 2 by retroviral expression screening.

Gallbladder cancer (GBC) is a highly fatal malignancy in humans. Genetic alterations in KRAS or TP53 as well as overexpression of ERBB2 have been shown to contribute to the development of certain types of GBC. However, many cases of GBC do not harbor such genetic changes, with other transforming events awaiting discovery. We here tried to identify novel cancer-promoting genes in GBC, with the use of a retroviral cDNA expression library. A retroviral cDNA expression library was constructed from a surgically resected clinical specimen of GBC, and was used to infect 3T3 fibroblasts in a focus formation assay. cDNA incorporated into the transformed foci was rescued by PCR. One such cDNA was found to encode free fatty acid receptor 2 (FFAR2), a G protein-coupled receptor for short-chain fatty acids. The oncogenic potential of FFAR2 was confirmed both in vitro with the focus formation assay and by evaluation of cell growth in soft agar as well as in vivo with a tumorigenicity assay in nude mice. The isolated FFAR2 cDNA had no sequence alterations, suggesting that upregulation of FFAR2 expression may contribute to malignant transformation. Indeed, all of quantitative RT-PCR, in situ hybridization, and immunohistochemical analyses showed that the amount of FFAR2 mRNA and its protein product was increased in digestive tract cancer specimens. Furthermore, short-chain fatty acids potentiated the mitogenic action of FFAR2 in 3T3 cells. Our data thus, for the first time, implicate FFAR2 in carcinogenesis of the digestive tract.

Risk factors for recurrent bile duct stones after endoscopic papillary balloon dilation: long-term follow-up study.

BACKGROUND: Little is known about the long-term results of endoscopic papillary balloon dilation (EPBD) for bile duct stones. METHODS: Between 1995 and 2000, 204 patients with bile duct stones successfully underwent EPBD and stone removal. Complete stone clearance was confirmed using balloon cholangiography and intraductal ultrasonography (IDUS). Long-term outcomes of EPBD were investigated retrospectively in the year 2007, and risk factors for stone recurrence were multivariately analyzed. RESULTS: Long-term information was available in 182 cases (89.2%), with a mean overall follow-up duration of 9.3 years. Late biliary complications occurred in 22 patients (12.1%), stone recurrence in 13 (7.1%), cholangitis in 10 (5.5%), cholecystitis in four, and gallstone pancreatitis in one. In 11 of 13 patients (84.6%), stone recurrence developed within 3 years after EPBD. All recurrent stones were bilirubinate. Multivariate analysis identified three risk factors for stone recurrence: dilated bile duct (>15 mm), previous cholecystectomy, and no confirmation of clean duct using IDUS. CONCLUSION: Approximately 7% of patients develop stone recurrence after EPBD; however, retreatment with endoscopic retrograde cholangiopancreatography is effective. Careful follow up is necessary in patients with dilated bile duct or previous cholecystectomy. IDUS is useful for reducing stone recurrence after EPBD.

Chromosome copy number analysis in screening for prognosis-related genomic regions in colorectal carcinoma.

Colorectal carcinoma (CRC) remains the major cause of cancer death in humans. Although chromosomal structural anomaly is presumed to play an important role in the carcinogenesis of CRC, chromosomal copy number alterations (CNA) and loss of heterozygosity (LOH) have not yet been analyzed extensively at high resolution in CRC. Here we aim to identify recurrent CNA and LOH in human CRC with the use of single nucleotide polymorphism-typing microarrays, and to reveal their relevance to clinical outcome. Surgically resected CRC specimens and paired normal mucosa were obtained from a consecutive series of 94 patients with CRC, and both of them were subjected to genotyping with Affymetrix Mapping 50K arrays. CNA and LOH were inferred computationally on every single nucleotide polymorphism site by integrating the array data for paired specimens. Our large dataset reveals recurrent CNA in CRC at chromosomes 7, 8, 13, 18, and 20, and recurrent LOH at chromosomes 1p, 4q, 5q, 8p, 11q, 14q, 15q, 17p, 18, and 22. Frequent uniparental disomy was also identified in chromosomes 8p, 17p, and 18q. Very common CNA and LOH were present at narrow loci of <1 Mbp containing only a few genes. In addition, we revealed a number of novel CNA and LOH that were linked statistically to the prognosis of the patients. The precise and large-scale measurement of CNA and LOH in the CRC genome is efficient for pinpointing prognosis-related genome regions as well as providing a list of unknown genes that are likely to be involved in CRC development.

Mouse microRNA profiles determined with a new and sensitive cloning method.

MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling method, termed miRNA amplification profiling (mRAP), as well as its application both to mouse embryos at various developmental stages and to adult mouse organs. A total of 77,436 Small-RNA species was sequenced, with 11,776 of these sequences found to match previously described miRNAs. With the use of a newly developed computational prediction algorithm, we further identified 229 independent candidates for previously unknown miRNAs. The expression of some of these candidate miRNAs was confirmed by northern blot analysis and whole-mount in situ hybridization. Our data thus indicate that the total number of miRNAs in vertebrates is larger than previously appreciated and that the expression of these molecules is tightly controlled in a tissue- and developmental stage-specific manner.

Identification of a constitutively active mutant of JAK3 by retroviral expression screening.

To identify transforming genes in acute myeloid leukemia (AML) we here constructed a retroviral cDNA expression library from an AML patient, and then used this library to infect a mouse cell line 32Dcl3-mCAT. cDNA inserts of the cell clones which proliferated in the presence of granulocyte colony-stimulating factor were derived from JAK3 encoding a JAK3 mutant with a valine-to-alanine substitution at codon 674 and two additional amino acid substitutions. The transforming activity of JAK3(V674A) was confirmed by its introduction into 32Dcl3-mCAT. Sequencing of the original JAK3 cDNA derived from the patient, however, failed to detect the V674A mutation.

Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.

Biphenotypic acute leukemia (BAL) is a relatively rare subtype of acute leukemia characterized by the presence of both myeloid and lymphoid cell surface antigens. We have now screened for transforming genes in BAL blasts with the use of the focus formation assay with a retroviral cDNA expression library constructed from malignant blasts isolated from a BAL patient. Some of the retroviral inserts recovered from transformed foci were found to encode wild-type purinergic receptor P2Y, G protein coupled, 8 (P2RY8). The oncogenic potential of P2RY8 was confirmed with the in vitro focus formation assay as well as with an in vivo tumorigenicity assay in nude mice. A variety of luciferase-based reporter assays revealed that P2RY8 increased both the trans-activation activities of CREB and Elk-1 as well as the transcriptional activities of the serum response element and enhancer-promoter fragments of the c-Fos and c-Myc genes. Quantitation of P2RY8 mRNA in CD34(+) cells of bone marrow showed that P2RY8 expression is frequently increased in leukemia patients, especially in those with refractory disease. Our data thus reveal an abundant expression of P2RY8 in leukemic cells and its unexpected role in the pathogenesis of acute leukemia.

Multiple orifices and cholangiography with a "fire-like" appearance after Kasai hepatoportoenterostomy for biliary atresia.

A 21-year-old female underwent a Kasai hepatoportoenterostomy with Roux-en-Y reconstruction for typeIII biliary atresia at age 63 days. At the age of 19 years, she developed cholangitis and CT scan revealed hepatolithiasis. She presented for treatment of the intrahepatic stone and the hepatportoenterostomy was directly visualized with double-balloon endoscopy (DBE). Endoscopic findings showed multiple intrahepatic bile ducts open to the jejunum through multiple orifices. Cholangiography showed narrowing of intrahepatic bile duct branches with a "fire-like" appearance. These findings have not been previously reported, since endoscopic approaches to patients with a hepaticojejunostomy were limited. DBE was useful to directly visualize the anastomosis in a patient status-post the Kasai operation for biliary atresia with a Rouxen-Y reconstruction.

Tips and tricks of double-balloon endoscopic retrograde cholangiopancreatography (with video).

Although endoscopic retrograde cholangiopancreatography (ERCP) is technically difficult in patients with altered gastrointestinal tract, double-balloon endoscopy (DBE) allows endoscopic access to pancreato-biliary system in such patients. Balloon dilation of biliary stricture and extraction of bile duct stones, placement of biliary stent in patients with Roux-en-Y or Billroth-II reconstruction, using DBE have been reported. However, two major technical parts are required for double-balloon ERCP (DB-ERCP). One is insertion of DBE and the other is an ERCP-related procedure. The important point of DBE insertion is a sure approach to the afferent limb with Roux-en-Y reconstruction or Braun anastomosis. Short type DBE with working length 152 cm is beneficial for DB-ERCP because it is short enough for most biliary accessory devices. In this paper, we introduce our tips and tricks for successful DB-ERCP.

A case of intraductal papillary mucinous neoplasms after recurrent acute pancreatitis.

We report a case of main pancreatic duct (MPD)-type intraductal papillary mucinous neoplasms of the pancreas (IPMNs), in whom diagnostic imaging modalities showed abnormal findings after 4 episodes of acute pancreatitis. The patient was 51 years old at his first admission for acute pancreatitis. He experienced two more episodes of acute pancreatitis, though repeated computed tomography (CT) and endoscopic retrograde cholangiopancreatography (ERCP) showed no abnormality to explain the cause of the pancreatitis. After 3½ years from his first episode of pancreatitis, CT and endoscopic ultrasonography revealed pancreatic duct dilation of the pancreas head. Seven years after the first admission, a second ERCP and intraductal ultrasonography revealed a partially dilated MPD with papillary tumors. He underwent pancreaticoduodenectomy, and the pathological diagnosis was intraductal papillary mucinous adenoma with moderate atypia. This case suggests that acute pancreatitis can precede visualized IPMNs. Therefore, acute recurrent pancreatitis with unknown etiology should be followed up for the possibility of IPMNs, in order to detect neoplastic changes in the early stage to provide a better prognosis for the patient.

Autoimmune pancreatitis associated with a large pancreatic pseudocyst that disappeared after corticosteroid therapy: a case report and literature review.

A 51-year-old woman was admitted to our department because of upper abdominal pain. The serum IgG4 concentration was elevated, and abdominal computed tomography revealed diffuse enlargement of the pancreas associated with a large cyst, measuring 8 cm in diameter. Endoscopic retrograde cholangiopancreatography revealed narrowing of the main pancreatic duct (from the body to the tail), narrowing of the intrapancreatic bile duct, and dilatation of the bile ducts. The patient was given a diagnosis of autoimmune pancreatitis (AIP) associated with a pancreatic pseudocyst and intrapancreatic bile duct stenosis. Oral steroid therapy resulted in reduced pancreatic swelling, complete disappearance of the pancreatic cyst, and an improvement in biliary stenosis. AIP is rarely associated with pancreatic cyst, and only 13 cases, including ours, have been reported to date. In our patient, intense inflammation apparently led to cyst formation in association with AIP, which responded remarkably to corticosteroid therapy. Correct diagnosis of AIP associated with a pancreatic pseudocyst might save patients from undergoing unnecessary endoscopic and surgical procedures.

Screening for genetic abnormalities involved in ovarian carcinogenesis using retroviral expression libraries.

The purpose of this study was to screen for genes involved in ovarian carcinogenesis in an attempt to develop an effective molecular-targeted therapy for ovarian cancer. We constructed retroviral expression libraries for the human ovarian cancer cell lines SHIN-3 and TYK-CPr, and performed a focus formation assay with 3T3 cells. As a result, proteasome subunit beta-type 2 (PSMB2), ubiquitin-specific protease 14 (USP14), and keratin 8 (KRT8) were identified from SHIN-3, and polymerase II RNA subunit (POLR2E), chaperonin containing T-complex polypeptide 1 subunit 4 (CCT4), glia maturation factor beta (GMFB), and neuroblastoma ras viral oncogene homolog (NRAS) from TYK-CPr. NRAS gene analysis revealed a CAA --> AAA substitution at codon 61, resulting in a Glu --> Lys change at position 61. When the mutant NRAS was introduced into fibroblasts for its expression, many transformed foci were generated, confirming the transforming ability of the mutant NRAS.

Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer.

The genome of a subset of non-small-cell lung cancers (NSCLC) harbors a small inversion within chromosome 2 that gives rise to a transforming fusion gene, EML4-ALK, which encodes an activated protein tyrosine kinase. Although breakpoints within EML4 have been identified in introns 13 and 20, giving rise to variants 1 and 2, respectively, of EML4-ALK, it has remained unclear whether other isoforms of the fusion gene are present in NSCLC cells. We have now screened NSCLC specimens for other in-frame fusion cDNAs that contain both EML4 and ALK sequences. Two slightly different fusion cDNAs in which exon 6 of EML4 was joined to exon 20 of ALK were each identified in two individuals of the cohort. Whereas one cDNA contained only exons 1 to 6 of EML4 (variant 3a), the other also contained an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). The protein encoded by the latter cDNA thus contained an insertion of 11 amino acids between the EML4 and ALK sequences of that encoded by the former. Both variants 3a and 3b of EML4-ALK exhibited marked transforming activity in vitro as well as oncogenic activity in vivo. A lung cancer cell line expressing endogenous variant 3 of EML4-ALK underwent cell death on exposure to a specific inhibitor of ALK catalytic activity. These data increase the frequency of EML4-ALK-positive NSCLC tumors and bolster the clinical relevance of this oncogenic kinase.

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening.

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.