Rapid molecular diagnosis of the Gilbert's syndrome-associated exon 1 mutation within the UGT1A1 gene.

Gilbert's syndrome is suspected in patients with unconjugated hyperbilirubinemia caused by decreased activity of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene in the absence of abnormal liver function and hemolysis. The major genetic variants underlying Gilbert's syndrome are TATA-box repeats of the promoter region and exon 1 G211A of the coding region, particularly in Asians. The efficacy of DNA melting curve analysis, however, has not been established for the G211A mutation. For rapid and accurate molecular diagnosis of Gilbert's syndrome, DNA melting curve analysis was evaluated for its genotyping capability not only for TATA-box repeats of the UGT1A1 promoter, but also for G211A of UGT1A1 exon 1. TA repeats within the TATA-box sequence and the exon 1 G211A mutation of the UGT1A1 gene were analyzed by DNA melting curve analysis. To evaluate the assay reliability, direct sequencing or polyacrylamide gel electrophoresis was used as a comparative method. All homozygous and heterozygous polymorphisms of A(TA)7TAA within the TATA-box allele and of exon 1 G211A mutants of the UGT1A1 gene were successfully identified with DNA melting curve analysis. DNA melting curve analysis is, therefore, an effective molecular method for the rapid diagnosis of Gilbert's syndrome, as it detects not only TATA-box polymorphisms but also the exon 1 G211A mutation located within the UGT1A1 gene.

Efficacy and tolerability of bevacizumab plus capecitabine as first-line therapy in patients with advanced hepatocellular carcinoma.

BACKGROUND: Molecularly targeted agents with anti-angiogenic activity, including bevacizumab, have demonstrated clinical activity in patients with advanced/metastatic hepatocellular carcinoma (HCC). This multicentre phase II study involving patients from several Asian countries sought to evaluate the safety and efficacy of bevacizumab plus capecitabine in this population. METHODS: Histologically proven/clinically diagnosed advanced HCC patients received bevacizumab 7.5 mg kg(-1) on day 1 and capecitabine 800 mg m(-2) twice daily on days 1-14 every 3 weeks as first-line therapy. RESULTS: A total of 45 patients were enrolled; 44 (96%) had extrahepatic metastasis and/or major vessel invasion and 30 (67%) had hepatitis B. No grade 3/4 haematological toxicity occurred. Treatment-related grade 3/4 non-haematological toxicities included diarrhoea (n=2, 4%), nausea/vomiting (n=1, 2%), gastrointestinal bleeding (n=4, 9%) and hand-foot syndrome (n=4, 9%). The overall response rate (RECIST) was 9% and the disease control rate was 52%. Overall, median progression-free survival (PFS) and overall survival (OS) were 2.7 and 5.9 months, respectively. Median PFS and OS were 3.6 and 8.2 months, respectively, for Cancer of the Liver Italian Programme (CLIP) score < or =3 patients, and 1.4 and 3.3 months, respectively, for CLIP score 4 patients. CONCLUSION: The bevacizumab-capecitabine combination shows good tolerability and modest anti-tumour activity in patients with advanced HCC.

Genetic analysis of haemophilia A in Taiwan.

Haemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene. Identification of these mutations is becoming increasingly important in a variety of clinical settings. The purpose of this report is to describe our experience of FVIII gene mutation analysis in the largest cohort of patients in Taiwan including the discovery of 21 novel mutations. We tested 115 HA patients from 91 unrelated families, including 79 severe, 15 moderate and 21 mild types starting with an assay for the intron 22 inversion by long range-PCR followed if necessary by additional genetic studies. Intron 22 inversion accounted for 27.8% of the total and 36.7% of severe HA patients respectively while intron 1 inversion comprised 7.6% of severe patients. These were clearly different from the known data in caucasian populations. Of 75 patients without intron 22 or 1 inversion, 70 from 62 unrelated families revealed 56 different mutations by denaturing high-performance liquid chromatography (DHPLC), of which 21 were novel. Also, the only female patient with severe HA was found to have heterozygous non-sense mutation (c.6683G>A) of exon 24. Seven patients, including five without amplified PCR product and two without encoded DNA defect turned out to have exon(s) deletion or insertion by reverse transcript PCR (RT-PCR). In our study, the combination of various molecular techniques including LR-PCR, multiplex PCR, DHPLC and RT-PCR analysis enabled definitive detection of the causative FVIII gene defects in 112 of 113 (99%) HA patients.

Prevalence of von Willebrand disease in women with iron deficiency anaemia and menorrhagia in Taiwan.

Iron deficiency anaemia (IDA) is a frequently encountered disease, which can be attributed to menorrhagia. Most female patients with von Willebrand disease (VWD) have menorrhagia. The aim of this study was to investigate the prevalence of VWD in women with both IDA and menorrhagia in Taiwan. From January to December 2005 and November 2006 to January 2007, 56 consecutive patients with both IDA and menorrhagia were enrolled in this study. Their median age was 41 years (range 18-53). IDA was diagnosed by anaemia plus either low ferritin or transferrin saturation. Menorrhagia was evaluated by patient's menses history. Both von Willebrand factor antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) were measured for each patient. Bleeding time (BT) and platelet function analyser (PFA)-100 assay were determined as ancillary tests. The VWD diagnosis was established if: (i) both VWF:Ag (<50%) and VWF:RCo (<50%) were low; (ii) either VWF:Ag or VWF:RCo was low plus prolonged BT or prolonged PFA closure times. VWF multimer analysis was performed for subtype confirmation of VWD. Nine of the 56 (16.1%) patients were identified to have VWD. VWD patients with menorrhagia might develop IDA at younger age (34.3 vs. 39.7, P = 0.09) and had more IDA recurrence (75% vs. 16%, P = 0.03) than those patients without VWD. Of the eight VWD patients with VWF multimer analyses, all were revealed to have type I VWD. Our study demonstrates that VWD was not uncommon in women with both IDA and menorrhagia in Taiwan.

Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz-1 domain.

Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

Influence of temperature on the ontogenetic expression of neural development-related genes from developing tilapia brain expressed sequence tags.

The developing central neural circuits in teleosts are genetically controlled and temperature-initiated. We compiled a list of transcripts expressed in the developing tilapia (Oreochromis mossambicus) brain using expressed sequence tags derived from the developing brain, and investigated genes with thermosensitive ontogenetic expression. Of 1084 clones, 893 were unique genes, 445 of which were known. Fourteen of the latter were neural development-related, and the ontogenetic expression of nine was temperature-influenced. Discs large homolog 5, myelin expression factor 2, plasticity-related protein-2, tsc2 gene product-related genes, and an inhibitor of differentiation protein 2 (Id2) were differentially temperature-influenced according to their developmental stages. Endothelial differentiation-related factor 1, midkine-related growth factor b, and mitogen-activated protein kinase 14b were specifically influenced by elevated temperature, and beta-catenin-like isoform 1 by lower temperature. Neural development-related genes, particularly those with thermosensitive ontogenetic expression, might be important for developing central neural circuits in teleosts.

Acute pneumonitis after subcutaneous injections of silicone for augmentation mammaplasty.

In a period of 3 years, seven cases of acute pneumonitis have been found after patients have been subcutaneously injected with silicone for the sole purpose of augmentation mammaplasty. Adverse symptoms following these silicone injections were fever, hypoxemia, hemoptysis, and abnormal diffuse bilateral alveolar infiltrates in both lungs. Pulmonary hemorrhaging occurred, and this was substantiated by using the bronchoscope with the bronchoalveolar lavage (BAL). The alveolar macrophage obtained from the BAL contained large quantities of pleomorphic cytoplasmic particles, which in actual fact were silicone particles. They were identified as silicone by scanning electron microscopy and energy-dispersive analysis of x-rays. This evidently showed that silicone diffusion into the circulatory system and subsequent embolization of the lung. Pulmonary function studies had shown restrictive changes with increase or normal single-breath carbon monoxide diffusing capacity (Dsb). Perfusion lung scans were interpreted as showing diffuse abnormalities consisting of decreased peripheral uptake. Acute hypoxemic respiratory failure was noted in four of these patients. Silicone injections of this nature were therefore a respiratory risk and caused the inducement of pneumonitis.

Temperature influences the ontogenetic expression of aromatase and oestrogen receptor mRNA in the developing tilapia (Oreochromis mossambicus) brain.

Water temperature has a differential influence on the development of central neurotransmitter systems according to the developmental period in tilapia (Oreochromis mossambicus). Aromatase and oestrogen receptors (ERs) represent important components of the mechanism of brain differentiation. Gene expression of aromatase and ERs is modulated by neurotransmitters in the developing brain. In the present study, the quantitative reverse transcription-polymerase chain reaction method was used to investigate the effects of temperature on the ontogenetic expression of aromatase and ERs in the developing tilapia brain. Before day 10 posthatching, exposure to a higher temperature (32 degrees C) resulted in a significant increase in the expression of brain aromatase; conversely, a lower temperature (20 degrees C) resulted in a decrease. ERalpha expression was depressed in accordance with the decrease of temperature, but ERbeta was unaffected by temperature. Between days 10 and 20, neither brain aromatase nor ERalpha expression was altered by temperature, whereas ERbeta expression was significantly enhanced by exposure to 32 degrees C. Between days 20 and 30, brain aromatase significantly increased at the higher temperature and decreased at 20 degrees C, but neither ERalpha nor ERbeta was affected by temperature. The expression of both brain aromatase and ERs, differentially regulated according to the temperature and to the developmental period, could be related to brain-sex differentiation.

Diagnosis of fungemia in bone marrow transplantation patients by examination of peripheral blood smears.

Two patients who received BMT for treatment of severe aplastic and AML-M2, developed fungemia during leukopenia. The organisms responsible for the infections were Candida parapsilosis and Rhodotorula glutinis, respectively. Early diagnosis of fungemia in these two patients was made by visualization of fungal blastospores in peripheral blood (PB) smears. These two cases illustrate that cytologic examination of PB smears is a useful method for early detection of fungus infection in BMT patients with leukopenia and unexplained fever in spite of appropriate antibiotic treatment.

The high value of high-resolution computed tomography in predicting the activity of pulmonary tuberculosis.

SETTING: A 2500-bed medical centre in southern Taiwan. OBJECTIVE: To study the clinical value of high-resolution computed tomography (HRCT) in predicting the activity of pulmonary tuberculosis (TB). DESIGN: HRCTs were performed prospectively in 148 patients whose chest radiographs (CXRs) showed highly suspicious signs of pulmonary TB, predominantly upper lung field infiltration. The HRCT findings, interpreted independently by a pulmonologist and a radiologist, were used to predict the activity of pulmonary TB. RESULTS: Pulmonologist-interpreted and radiologist-interpreted HRCTs showed high sensitivity (both 93%), specificity (83 vs. 88%), accuracy (86 vs. 90%), positive predictive values (76 vs. 83%) and negative predictive values (both 95%). Kappa statistic indicates good inter-reader agreement. CONCLUSION: HRCT has a high value in predicting the activity of pulmonary TB. It is a useful tool in this regard when a patient with suspected pulmonary TB lacks microbiologic proof, when clinical condition makes invasive diagnosis impossible or when a patient has completed anti-tuberculosis treatment with no compatible unequivocal CXR.

Cytology of fine needle aspirates of primary extragonadal germ cell tumors.

OBJECTIVE: To describe the characteristic cytologic features of fine needle aspirates (FNAs) of primary extragonadal germ cell tumors (PEGCTs). STUDY DESIGN: Thirteen patients with PEGCTs, including 2 seminomas, 2 mixed germ cell tumors, 3 immature teratomas, 1 choriocarcinoma and 5 yolk sac tumors (YSTs) were studied. The final diagnosis of PEGCT in all cases was established by histologic examination of the tumor tissues. Fine needle aspiration was done on either the primary tumor or metastatic foci. The aspirates were stained with one of the Romanovsky stains and Papanicolaou stain. RESULTS: Each type of PEGCT has its own morphologic characteristics. In seminoma, the tumor cells are large and noncohesive, with one to several distinct nucleoli; some lymphocytes are also present. YSTs show many pleomorphic cells with vacuoles in the cytoplasm and nuclei; tumor cells frequently aggregate in a microglandular or papillary pattern. Choriocarcinoma consists of syncytiotrophoblasts and cytotrophoblasts. The former are very large cells with eosinophilic cytoplasm, one to several nuclei and distinct nucleoli; the latter are medium-sized cells with vacuolated, basophilic cytoplasm and eccentric nuclei. Immature teratomas are composed of a mixture of cell types, including elongated epithelioid cells, mesenchymal cells and many large, naked, amorphous nuclei with a homogeneous chromatin pattern. Diagnosis of mixed germ cell tumor is difficult but can be made if two or more subtypes of tumor cells are observed in the FNA. CONCLUSION: Cytologic examination of FNAs of primary or metastatic lesions of PEGCTs, stained either with Romanovsky or Papanicolaou stain, is of diagnostic value for such diseases. The use of immunochemistry can help to confirm the cytologic impression.

Primary cecal lymphoma. Report of a case with preoperative diagnosis by fine needle aspiration and immunocytochemistry.

Fine needle aspiration of a cecal mass was performed on a patient with a cecal tumor and iron-deficiency anemia. Cytologic studies of the air-dried smears showed large cell lymphoma. The diagnosis of large cell lymphoma of the B-cell type was affirmed by immunocytochemical studies and at laparotomy and resection of the tumor. Even under unusual circumstances, the diagnosis of such a rare abdominal lesion as primary cecal lymphoma can be made with certainty by cytologic and immunologic studies of fine needle aspirates.

Imprint cytology of nasopharyngeal biopsies.

OBJECTIVE: To use an instant cytologic diagnostic method to examine touch imprints of nasopharyngeal (NP) biopsies. This method aimed to ensure the adequacy of biopsied specimens for histologic examination. This paper describes the morphologic findings of NP lesions examined by this method. STUDY DESIGN: Imprints were made from NP biopsies from patients suspicious for nasopharyngeal carcinoma (NPC). These imprints were air dried, stained with Diff-Quik and examined immediately. The adequacy of the specimens was assessed, and the findings of the imprints were interpreted as positive-, negative- or suspicious for NPC. Repeat biopsies and cytologic studies were done as indicated. In selected cases, immunocytochemical staining was done to identify cells on the imprints. Histologic examination of the biopsied specimens served as the control. RESULTS: With this method we could interpret the imprints within five minutes of their receipt and determine if repeat biopsy was needed. In benign lesions, the imprints often contained many cells, most mature and reactive lymphocytes. These cells and the numerous lymphoglandular bodies (fragments of lymphoid cytoplasm) intermingled with the ciliated and squamous epithelial cells. In cases of NPC, the appearance was discrete, or clusters of, carcinoma cells or naked nuclei. They were less cellular than those of benign lesions. The lymphocytes were markedly depleted. Cells of dubious lineage were identified by the additional use of immunocytochemical studies. CONCLUSION: Cytologic examination of imprints of NP biopsies helps to determine the adequacy of the specimen for histologic examination. It is a rapid, practical method with high diagnostic accuracy.

Diagnosis of primary cardiac lymphoma. Report of a case with cytologic examination of pericardial fluid and imprints of transvenously biopsied intracardiac tissue.

BACKGROUND: Primary cardiac lymphoma (PCL) is a treatable disease when appropriately diagnosed. Therefore, a prompt, safe method with high diagnostic accuracy is prerequisite to successful therapy for PCL. CASE: A 57-year-old male presented with exertional dyspnea and atrial fibrillations. A pericardial effusion (PE) and several tumor masses occupying both atria were found. Cytologic examinations of PE and of imprints of the tissues obtained by transvenous biopsy of the cardiac tumors revealed numerous small, round tumor cells and lymphoglandular bodies, suggestive of malignant lymphoma. This cytologic impression was confirmed by immunocytochemical studies on the same cytologic material. Histologic studies reaffirmed the diagnosis of B-cell lymphoma. The patient received eight courses of chemotherapy, with complete remission of the illness. CONCLUSION: Cardiac lymphoma can be quickly and safely diagnosed by cytologic examination of PE or transvenously biopsied cardiac tissue, with confirmation by immunocytochemical studies. Exploratory thoracotomy for biopsy can be avoided.

Intrathoracic meningocele in association with neurofibromatosis: report of a case.

A case of intrathoracic meningocele associated with neurofibromatosis is reported. Computed tomography (CT) demonstrated that the paravertebral mass had a characteristic low attenuation coefficient compatible with liquid content. CT-myelography further confirmed the diagnosis. We recommend that though intrathoracic meningocele is rare, it should be kept in mind when making a differential diagnosis in cases where the patient has neurofibromatosis with a posterior mediastinal mass.

Bronchial foreign bodies in adults.

Aspiration of foreign bodies into the bronchial tree is infrequently seen in adults. We reviewed 40 such cases during an 8-year period at Chang Gung Memorial Hospital, Kaohsiung. Only six patients had underlying conditions that contributed to the aspiration of foreign bodies. A positive history of aspiration was found in 18 patients (48%). Clinical manifestations were nonspecific. Chest roentgenograms were useful in 11 of the 40 patients. Bronchoscopic findings were classified into three groups: foreign bodies in the bronchial tree without granulation tissue, foreign bodies in the bronchial tree with marked granulation tissue and foreign bodies embedded in the granulation tissue. The diagnoses and removal of foreign bodies were successful in 37 patients (92%) using grasping forceps or biopsy forceps and in one patient using basket type grasping forceps. Complications of bronchoscopy were rare and not serious. The diagnosis of occult foreign bodies is often difficult and demands a high index of suspicion. Removal of endobronchial foreign bodies is usually possible with a flexible fiberoptic bronchoscope and has a high success rate.

Cyclosporine-induced encephalopathy in a patient with relapsed acute myeloid leukemia treated with unrelated allogeneic bone marrow transplantation.

Cyclosporine (CSP) is the most frequently used immunosuppressive agent for prevention of graft versus host disease (GVHD) in allogeneic bone marrow transplantation (BMT). Some adverse effects such as hepatic and renal toxicity have been frequently encountered, but central nervous system (CNS) toxicity caused by CSP is rare. We report an adult male patient with acute myeloid leukemia who developed CSP-induced encephalopathy under treatment for allogeneic BMT from an unrelated donor. Methotrexate and CSP were used for GVHD prophylaxis. Leukocyte and platelet engraftment were successfully achieved on days 21 and 24 after BMT, respectively. Abrupt onset of mental confusion and disorientation occurred on day 25, followed by a generalized tonic clonic seizure and consciousness disturbance. The whole blood CSP level was 160.65 ng/mL. Magnetic resonance (MR) imaging revealed high signal intensities in the bilateral occipital lobes with predominant involvement of the cortical areas. The patient recovered from the CNS toxicity, but with slight memory impairment, 6 days after CSP was discontinued. When patients receiving CSP treatment for allogeneic BMT develop mental confusion, consciousness disturbance, or seizure, CSP-induced CNS toxicity should be taken into consideration.

A phase II study of erlotinib in combination with bevacizumab versus chemotherapy plus bevacizumab in the first-line treatment of advanced non-squamous non-small cell lung cancer.

BACKGROUND: Molecularly targeted agents for non-small cell lung cancer (NSCLC) can provide similar efficacy to chemotherapy without chemotherapy-associated toxicities. Combining two agents with different modes of action could further increase the efficacy of these therapies. The TASK study evaluated the efficacy and safety of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in combination with the anti-angiogenic agent bevacizumab as first-line therapy in unselected, advanced non-squamous NSCLC patients. METHODS: Patients were recruited from December 2007 to September 2008. Planned sample size was 200 patients, a total of 124 patients were randomized. Patients were randomized using a minimization algorithm 1:1 to receive bevacizumab (iv 15 mg/kg day 1 of each 21-day cycle) plus chemotherapy (gemcitabine/cisplatin or carboplatin/paclitaxel standard doses, 4-6 cycles) (BC arm) or bevacizumab plus erlotinib (p.o. 150 mg/day; BE arm) until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS). If the hazard ratio (HR) of PFS for BE relative to BC was above 1.25 at the pre-planned interim analysis in favor of BC, the study would be re-evaluated. Secondary endpoints included overall survival, response rate and safety. RESULTS: All randomized patients (n = 63 BE; n = 61 BC) were evaluated for the efficacy analyses. At the updated interim analysis, median PFS was 18.4 weeks (95% confidence interval [CI] 17.0-25.1) versus 25.0 weeks (95% CI 20.6-[not reached]) for BE versus BC, respectively (HR for death or disease progression, BE relative to BC, 2.05, p = 0.0183). The incidence of death was 19% for BE treatment compared with 11.5% for BC treatment. The HR for PFS at the updated interim analysis was above 1.25, therefore patients on the BE arm were permitted to change arms or switch to another drug and the study was terminated. Adverse events reported were as expected. CONCLUSIONS: The TASK study did not show a benefit in terms of PFS for the combination of erlotinib with bevacizumab in unselected first-line advanced non-squamous NSCLC compared with chemotherapy plus bevacizumab.