A genetically defined signature of responsiveness to erlotinib in early-stage pancreatic cancer patients: Results from the CONKO-005 trial.

BACKGROUND: high recurrence rates of up to 75% within 2 years in pancreatic ductal adenocarcinoma (PDAC) patients resected for cure indicate a high medical need for clinical prediction tools and patient specific treatment approaches. Addition of the EGFR inhibitor erlotinib to adjuvant chemotherapy failed to improve outcome but its efficacy in some patients warrants predictors of responsiveness. PATIENTS AND METHODS: we analysed tumour samples from 293 R0-resected patients from the randomized, multicentre phase III CONKO-005 trial (gemcitabine ± erlotinib) with targeted sequencing, copy number, and RNA expression analyses. FINDINGS: a total of 1086 mutations and 4157 copy-number aberrations (CNAs) with a mean of 17.9 /tumour were identified. Main pathways affected by genetic aberrations were the MAPK-pathway (99%), cell cycle control (92%), TGFß signalling (77%), chromatin remodelling (71%), and the PI3K/AKT pathway (65%). Based on genetic signatures extracted with non-negative matrix factorization we could define five patient clusters, which differed in mutation patterns, gene expression profiles, and survival. In multivariable Cox regression analysis, SMAD4 aberrations were identified as a negative prognostic marker in the gemcitabine arm, an effect that was counteracted when treated with erlotinib (DFS: HR=1.59, p = 0.016, and OS: HR = 1.67, p = 0.014). Integration of differential gene expression analysis established SMAD4 alterations with low MAPK9 expression (n = 91) as a predictive biomarker for longer DFS (HR=0.49; test for interaction, p = 0.02) and OS (HR = 0.32; test for interaction, p = 0.001). INTERPRETATION: this study identified five biologically distinct patient clusters with different actionable lesions and unravelled a previously unappreciated association of SMAD4 alteration status with erlotinib effectiveness. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that SMAD4 status might guide addition of erlotinib treatment in early-stage PDAC patients.

Long-term outcome of 6-month maintenance chemotherapy for acute lymphoblastic leukemia in children.

In the treatment of childhood acute lymphoblastic leukemia (ALL), excess shortening of maintenance therapy resulted in high relapse rate, as shown by our previous trial, TCCSG L92-13, in which maintenance therapy was terminated at 1 year from initiation of treatment. In this study, we aimed to confirm the long-term outcome of L92-13, and to identify who can or cannot be cured by shorter duration of maintenance therapy. To obtain sentinel cytogenetics information that had been missed before, we performed genetic analysis with genomic microarray and target intron-capture sequencing from diagnostic bone marrow smear. Disease-free survival (DFS) at 10 years from the end of therapy was 66.0±2.8%. Females (n=138) had better DFS (74.6±3.7%) than males (n=142, 57.5±4.2%, P=0.002). Patients with TCF3-PBX1 (n=11) and ETV6-RUNX1 (n=16) had excellent DFS (90.9±8.7% and 93.8±6.1%, respectively), whereas high hyperdiploidy (n=23) was the most unfavorable subgroup, with 56.6±10.3% of DFS. Short duration of therapy can cure more than half of pediatric ALL, especially females, TCF3-PBX1 and ETV6-RUNX1. Our retrospective observations suggest a gender/karyotype inhomogeneity on the impact of brief therapy.

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.

Protein synthesis using poly(2'-halogeno-2'-deoxyadenylic acids) as messenger.

Poly(2'-fluoro-2'-deoxyadenylic acid), poly(2'-chloro-2'-deoxyadenylic acid) and poly(2'-bromo-2'-deoxyadenylic acid) are used as messenger RNAs in protein synthesizing systems in vitro. All polynucleotides were active as messengers and [14 C]lysine was incorporated into polypeptides. The initial velocity of polylysine formation was greater using poly(2'-fluoro-2'-deoxyadenylic acid) as messenger than in the case of poly(rA), and all synthetic messengers lived longer in the protein synthetic system.

2'-Fluoro-2'-deoxypolynucleotides as templates and inhibitors for RNA- and DNA-dependent DNA polymerases.

Poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) and poly(2'-deoxycytidylic acid) (poly(dCfl)) were tested as templates in DNA synthesis reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha, mouse cell DNA polymerase gamma and avian myeloblastis virus (AMV) reverse transcriptase. Poly(dAfl).(dT)12 can fully substitute for poly(rA).(dT)12 as template with DNA polymerase gamma, to 50% with reverse transcriptase, but was poorly recognized by DNA polymerase alpha. DNA synthesis by reverse transcriptase with poly(dCfl).(dG)12 as template was 50% of that with poly(rC).(dG).(dG)12. The use of 2'-fluoropolymers as templates was more efficient at 37 degrees C than at 25 degrees C. No appreciable differences on the fidelity of DNA synthesis by reverse transcriptase were observed when dCMP misincorporation was measured with poly(dAfl).(dT)12 or poly(rA).(dT)12 as template primers. Poly(C) and poly-2'-O-methylcytidylic acid had no significant effect on the reaction catalyzed by DNA polymerase gamma and reverse transcriptase, independent of the synthetic polynucleotide complex utilized as template. On the other hand, poly(dCfl) was an inhibitor when poly(rA).(dT)12 or poly(dA).(dT)12 were used as templates, but not when poly(dAfl).(dT)12 was employed. Analogous results have been obtained with activated DNA and AMV 70 S RNA as templates in the reverse transcriptase reaction. The inhibition by poly(dCfl) was noncompetitive with regard to TTP, poly(dA) and poly(rA). Xenopus laevis oocytes DNA polymerase alpha was not inhibited by poly(dCfl).

Non-peptide inhibitors of HCV serine proteinase.

We screened a chemical library of 2000 compounds for inhibitors of hepatitis C virus (HCV) serine proteinase using an in vitro screening method measuring the hydrolysis of the peptide substrate. Three compounds were found to be the most potent inhibitors (IC50 < 10(-5) M). Two of them had a similar structure, that of halogenated benzanilide, and were not inhibitory for common serine proteinases. They inhibited the enzyme non-competitively with the substrate. Together with the result of the analogous compounds in the chemical library, the presumed structural requirements of the inhibition are pointed out.

Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli.

The serine protease domain of HCV comprising amino acids 1027-1218 (deltaNS3) was expressed in E. coli with a His tag at its N-terminal end. The protease was purified to apparent homogeneity by a single step affinity chromatography resulting in high yields (approximately 3 mg/l of cultured cells). The deltaNS3 efficiently cleaves a 17-mer peptide corresponding to the NS5A-NS5B junction with kcat/Km = 160 x 10(-3) min(-1) microM(-1) in the presence of NS4A peptide. Our deltaNS3 represents the minimal domain possessing highly active protease of NS3 constructed so far. The deltaNS3 protein also efficiently processed a longer substrate corresponding to NS5A/5B junction (2203-2506 amino acids) that was synthesized by in vitro transcription and translation system.

Template activity of poly(2'-fluoro-2'-deoxyinosinic acid) for murine leukemia virus reverse transcriptase.

The 2'-substituted analog of poly(inosinic acid) ((I)n), poly(2'-fluoro-2'-deoxyinosinic acid) ((dIfl)n) served as an effective template for the RNA-directed DNA polymerase (reverse transcriptase) from Moloney murine leukemia virus. When assayed under the same conditions, the parent compound (I)n showed little, if any, template activity. In the presence of other templates, i.e. poly (2-O-methylcytidylic acid), (dIfl)n could also assume the role of primer for the reverse transcriptase reaction, whereas, again, (I)n failed to do so.

Influence of various 2- and 2'-substituted polyadenylic acids on murine leukemia virus reverse transcriptase.

Several newly synthesized polyadenylic acid [(A)n] analogues, including poly(2-methyladenylic acid) [(m2A)n], poly(2-ethyladenylic acid) [(e2A)n], poly(2-isopropyladenylic acid) [(i-pro2A)n], poly(2-methylthioadenylic acid) [(ms2A)n], poly(2-ethylthioadenylic acid) [(e2A)n], poly(2'-fluoro-2'-deoxyadenylic acid) [(dAfl)n] and poly(2'-azido-2'-deoxyadenylic acid) [(dAz)n] have been evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukemia virus; (m2A)n and (e2A)n did not markedly affect reverse transcriptase activity, (dAfl)n served as an efficient template for the reverse transcriptase reaction, and (i-pro2A)n, (ms2A)n, (es2A)n and (dAz)n strongly inhibited reverse transcriptase activity. (dAfl)n also served as an efficient template (Km : 0.025 micron) for the reverse transcriptase of avian myeloblastosis virus.

Cleavage activity of hepatitis C virus serine proteinase.

To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.

Inhibition of hepatitis C virus serine protease in living cells by RNA aptamers detected using fluorescent protein substrates.

Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.

A high throughput assay of the hepatitis C virus nonstructural protein 3 serine proteinase.

A simple assay was developed based on intramolecular fluorescence resonance energy transfer for detection of the activity of hepatitis C virus (HCV) serine proteinase. Two quenched-fluorogenic substrates, (7-methoxycoumarin-4-yl)acetyl (Mca) Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-(2,4-dinitrophenyl, Dnp) Lys (Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Lys[Dnp], QF-1) and Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Lys(Dnp)-Arg-Arg (QF-2), which derived from the NS5A/5B junction of the HCV polyprotein, were designed. Kinetic studies revealed that QF-1 and QF-2 had high affinity for a recombinant enzyme which is a fusion protein of maltose binding protein and almost entire nonstructural protein (MBP-NS3), with Km values comparable to that of longer substrate based on the same cleavage site. QF-1 and QF-2 were cleaved by MBP-NS3 efficiently with kcat values of 7.5 and 4.2 min(-1), respectively. QF-2 was also found to be a good substrate of deltaNS3 which contained serine proteinase part of NS3 with kcat value of 4.3 min(-1). The cleavage reaction is detected continuously by the elevation of the fluorescence due to release from quenching. The fluorescence of the substrates increases in proportion to progress of the cleavage reaction under the standard conditions. This method was applied for screening of HCV serine protease inhibitors using a fluorescence multiwell plate reader. A group of natural occurring products, flavonoids, was subjected to be screened. Two flavonoids out of 25 were found to inhibit the enzyme moderately at a concentration of 100 microM. The data agreed with those obtained by high-performance liquid chromatography (HPLC). This method is suited to sensitive quantitation of the enzyme reaction as well as the high throughput analysis of the inhibitors.

Anti-HIV-1 and anti-HIV-1-protease substances from Ganoderma lucidum.

A new highly oxygenated triterpene named ganoderic acid alpha has been isolated from a methanol extract of the fruiting bodies of Ganoderma lucidum together with twelve known compounds. The structures of the isolated compounds were determined by spectroscopic means including 2D-NMR. Ganoderiol F and ganodermanontriol were found to be active as anti-HIV-1 agents with an inhibitory concentration of 7.8 micrograms ml-1 for both, and ganoderic acid B, ganoderiol B, ganoderic acid C1, 3 beta-5 alpha-dihydroxy-6 beta-methoxyergosta-7,22-diene, ganoderic acid alpha, ganoderic acid H and ganoderiol A were moderately active inhibitors against HIV-1 PR with a 50% inhibitory concentration of 0.17-0.23 mM.

Cross-sectional study on respiratory effect of toner-exposed work in manufacturing plants, Japan: pulmonary function, blood cells, and biochemical markers.

The aim of the study is to examine the relationship between toner-exposed work and health indices related to respiratory disorders and to confirm the baseline of a cohort study to clarify the effect of toner exposure in manufacturing plants. Subjects were 1614 male workers (809 toner-exposed workers and 805 referents) who were engaged in toner manufacturing plants in Japan (Fuji Xerox Co., Ltd). The age of subjects was from 19 to 59 years, and the average age was 40.2 years(median 40 years, SD 7.67). We conducted a pulmonary function test (PEFR, VC, FVC, FEV(1.0)%, V25/Ht) and a blood cell test (RBC, Hb, Hct, Plt, WBC, cell contents of WBC) and measured biochemical indices in blood (ALT, AST, gamma-GTP, CRP, IgE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine. Student t-test and logistic regression analysis were applied to compare between the toner-exposed workers and the referents and to analyze the relationship among indices of effects and independent factors. There was no significant difference between the two groups in blood cell count and biochemical indices. Inflammation- and allergy-related markers such as 8OHdG and IgE also showed no significant difference between toner-exposed workers and the referents. The influence of smoking on pulmonary function indices was observed, but there was no relationship between the pulmonary function and toner-exposed work. In this article, we report a preliminary cross-sectional analysis in the subjects of a cohort study. No difference in pulmonary function indices was observed between the toner-exposed workers and the referents, and there was no consistent relationship between the exposure status and examined indices; however, the prevalence of subjective respiratory symptoms was higher in the exposed workers as presented in another report. Further analysis is important in the ongoing cohort study to clarify the effect of toner exposure on respiratory systems.

Polynucleotides. XLV Synthesis and properties of poly(2'-azido-2'-deoxyinosinic acid).

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality. In solutions of high Na+ ion concentration, poly (Iz) forms a multi-stranded complex and its Tm at 1.0M Na+ ion concentration was 43 degrees. Upon mixing with poly (C), poly (Iz) forms a 1:1 complex having a Tm lower than that of poly (I)-poly (C) complex in the same conditions. The effect of substitution at the 2'-position of the poly (I) strand was discussed in relation to the interferon-inducing activity.

Polynucleotides. LII. Synthesis and properties of poly(2'-deoxy-2'-fluoroadenylic acid).

2'-Deoxy-2'-fluoroadenosine was chemically transformed to its 5'-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2'-deoxy-2'-fluoroadenylic acid) [poly(Af)]. Polymerization proceeded smoothly as in the case of poly(A) and the yield of the polymerization was 55%. The UV absorption spectra of poly(Af) closely resembled those of poly(A) and the hypochromicity was 32% at pH 7.0. The CD profile at 25 degrees and neutrality showed similar pattern to that of other poly(2'-deoxy-2'-halogenoadenylic acids) with somewhat larger [theta] values both in the positive and negative maxima. Acid titration of poly(Af) showed a transition point at pH 5.2 and the Tm of the acid form was 37 degrees which was significantly lower than that of poly(A), but similar to that of poly(2'-azido-2'-deoxyadenylic acid). Poly(Af) formed 1:1 and 1:2 complexes with poly-(U) having Tm of 49 degrees and 62 degrees at 0.04M and 0.15M Na(+) concentration, respectively. Poly(Af) also formed a 1:2 complex with poly(I) and its Tm was 36 degrees at 0.05M Na(+) concentration. These data showed that poly(Af) has rather similar properties to those of poly(A), but not to poly(dA).

Polynucleotides. LVI. Synthesis and properties of poly(2-deoxy-2'-fluoroinosinic acid).

Poly (2'-deoxy-2'-fluoroinosinic acid) [ poly(If)] was synthesized by polymerization of 2'-deoxy-2'-fluoroinosine 5'-diphosphate catalyzed by Escherichia coli polynucleotide phosphorylase. Although the UV absorption properties of poly(If) closely resembled those of poly(I), thermal melting curves at Na+ concentrations of 0.15M and 0.75M suggested two ordered structures for poly(If) neutral form. CD psectra taken at 0.15M Na+ concentration showed rather larger amplitudes in both a peak at 273 nm and a trough at 246 nm, suggesting rather strong vertical stacking of bases. When complexed with poly(C), poly(If) forms a double-stranded complex, poly(If).poly(C) which has Tm's higher by 10-20 degrees than those of poly(If).poly(C) measured under the same conditions. The CD spectrum of this complex resembled that of poly(I).poly(C). The effect of the fluorine atom at the 2'-position on thermal stability of polynucleotides is discussed.

Differential stabilization by netropsin of inducible B-like conformations in deoxyribo-, ribo- and 2'-deoxy-2'-fluororibo-adenosine containing duplexes of (dA)n . (dT)n and (dA)n . (dU)na.

Six polynucleotide duplexes containing polydeoxyadenylic acid, polyadenylic acid or poly-2'-deoxy-2'-fluoro-adenylic acid in one strand, and polydeoxyuridylic acid or polydeoxythymidylic acid in the other strand have been studied by circular dichroism, ionic strength dependence of melting temperatures and binding of the DNA specific antibiotic netropsin. Circular dichroism spectra of (dA)n . (dT)n and (dA)n . (dU)n indicated the presence of the B-form of DNA, while those of (dAfl)n . (dT)n and (rA)n . (dT)n (and the corresponding (dU)n hybrids) indicated the presence of the A-form. (dAfl)n . (dT)n and (dAfl)n . (dU)n bound netropsin only slightly less than the (dA)n containing duplexes, while replacement by (rA)n decreased netropsin binding to a large degree. Since netropsin requires B-DNA for binding, it is concluded that the A to B transition is facilitated in the case of fluorine substitution in the sugar moiety, while the 2'-OH group greatly limits this conformational change.

Polynucleotides. LVII. Synthesis and properties of poly (2'-chloro-2'-deoxyinosinic acid).

Poly (2'-chloro-2'-deoxyinosinic acid) [poly(Icl)] was synthesized from Icl 5'-DP by polymerization with polynucleotide phosphorylase. UV absorption properties of poly(Icl) are very similar to those of poly(I). Poly(Icl) adopted a multi-stranded ordered form in the presence of 0.95M Na ion. The Tm value of this form was 36 degrees, which resembles that of poly(I) quadruple-stranded form at high salt. CD spectra also suggested presence of these two forms. Upon mixing with poly(C), poly-(Icl) forms a double-stranded 1 : 1 complex, which had very similar Tm-log[Na+] relationship to that of poly(I) . poly(C). Thus it was concluded that the chlorine substitution at 2'-position of the polynucleotide had the similar effect to OH on physical properties of polynucleotides.