Effects of Molecular Weight of Functionalized Liquid Butadiene Rubber as a Processing Aid on the Properties of SSBR/Silica Compounds.

Liquid butadiene rubber (LqBR) which used as a processing aid play a vital role in the manufacturing of high-performance tire tread compounds. However, the studies on the effect of molecular weight, microstructure, and functionalization of LqBR on the properties of compounds are still insufficient. In this study, non-functionalized and center-functionalized liquid butadiene rubbers (N-LqBR and C-LqBR modified with ethoxysilyl group, respectively) were synthesized with low vinyl content and different molecular weights using anionic polymerization. In addition, LqBR was added to the silica-filled SSBR compounds as an alternative to treated distillate aromatic extract (TDAE) oil, and the effect of molecular weight and functionalization on the properties of the silica-filled SSBR compound was examined. C-LqBR showed a low Payne effect and Mooney viscosity because of improved silica dispersion due to the ethoxysilyl functional group. Furthermore, C-LqBR showed an increased crosslink density, improved mechanical properties, and reduced organic matter extraction compared to the N-LqBR compound. LqBR reduced the glass transition temperature (Tg) of the compound significantly, thereby improving snow traction and abrasion resistance compared to TDAE oil. Furthermore, the energy loss characteristics revealed that the hysteresis loss attributable to the free chain ends of LqBR was dominant.

Targeting KRAS4A splicing through the RBM39/DCAF15 pathway inhibits cancer stem cells.

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.

Establishment and characterization of 13 human colorectal carcinoma cell lines: mutations of genes and expressions of drug-sensitivity genes and cancer stem cell markers.

Thirteen human colorectal cancer (CRC) cell lines were established from 10 primary tumors and 3 metastatic tumors obtained from 13 Korean patients. Characteristics of the cell lines including morphology in vivo and in vitro; mutations of the K-ras, p53, APC and MMR genes and microsatellite instability (MSI) status in vitro were determined. Expression of drug-sensitivity genes including MDR1, MXR, MRP1 and COX2 was also analyzed. The cell lines were unique as judged by DNA fingerprinting using 16 short tandem repeats. Eleven of the cell lines grew as adherent populations and the remaining two as floating aggregates. None of the cell lines were contaminated with Mycoplasma or bacteria. All cell lines showed high viability with relatively long doubling times. Six cell lines contained mutations at K-ras. Seven cell lines displayed p53 gene missense, nonsense and frameshift mutations. MSI was found in three cell lines and two cell lines with an MSI-high phenotype-possessed hMLH1 mutations. Nine cell lines had an APC mutation. MRP1 was highly expressed in all cell lines, and high expression of MDR1, MXR and COX2 evident in eight, six and six cell lines, respectively. Embryonal stem cell markers (MELK, SOX4 and OCT4) were expressed in most of cell lines. The cancer stem cell biomarkers CD133, CD44 and Lgr5 were expressed in 12, 13 and 13 cell lines, respectively. The presently well-characterized CRC cell lines should be useful in investigations of the biological characteristics of CRC, particularly for investigations related to gene alterations associated with CRC and biology of cancer stem cells.

Vulcanizate Structures of SBR Compounds with Silica and Carbon Black Binary Filler Systems at Different Curing Temperatures.

The tire industry has shown an increasing demand for the reduction in rolling resistance. Efforts have been made to improve the viscoelastic properties of tire compounds and reduce the weight of tires through optimization of the vulcanizate structure, which has become extremely complex. In this study, vulcanizates using carbon black and silica as binary fillers were prepared at various curing temperatures. Vulcanizate structures with respect to curing temperature were classified according to the chemical crosslink density by sulfur, carbon black bound rubber (i.e., physical crosslink due to carbon black), and silica-silane-rubber network. All properties exhibited a decreasing trend under the application of high curing temperatures, and the decrease in the crosslink density per unit content of filler with an increase in curing temperature was shown to be greater in carbon black than in silica. Mechanical and viscoelastic properties were also measured to evaluate the impact that the compound variates have on tire tread performance. These results serve as a guideline for determining the content and filler type and for setting the cure condition during the design of actual compound formulations to increase the crosslink density of rubber while retaining the necessary mechanical and viscoelastic properties for practical application.

Development of High Dielectric Electrostrictive PVDF Terpolymer Blends for Enhanced Electromechanical Properties.

Electroactive polymers with high dielectric constants and low moduli can offer fast responses and large electromechanical strain under a relatively low electric field with regard to theoretical driving forces of electrostriction and electrostatic force. However, the conventional electroactive polymers, including silicone rubbers and acrylic polymers, have shown low dielectric constants (ca. < 4) because of their intrinsic limitation, although they have lower moduli (ca. < 1 MPa) than inorganics. To this end, we proposed the high dielectric PVDF terpolymer blends (PVTC-PTM) including poly(vinylidene fluoride-trifluoroethylene-chlorofluoro-ethylene) (P(VDF-TrFE-CFE), PVTC) as a matrix and micelle structured poly(3-hexylthiophene)-b-poly(methyl methacrylate) (P3HT-b-PMMA, PTM) as a conducting filler. The dielectric constant of PVTC-PTM dramatically increased up to 116.8 at 100 Hz despite adding only 2 wt% of the polymer-type filler (PTM). The compatibility and crystalline properties of the PVTC-PTM blends were examined by microscopic, thermal, and X-ray studies. The PVTC-PTM showed more compatible blends than those of the P3HT homopolymer filler (PT) and led to higher crystallinity and smaller crystal grain size relative to those of neat PVTC and PVTC with the PT filler (PVTC-PT). Those by the PVTC-PTM blends can beneficially affect the high-performance electromechanical properties compared to those by the neat PVTC and the PVTC-PT blend. The electromechanical strain of the PVTC-PTM with 2 wt% PTM (PVTC-PTM2) showed ca. 2-fold enhancement (0.44% transverse strain at 30 Vpp µm-1) relative to that of PVTC. We found that the more significant electromechanical performance of the PVTC-PTM blend than the PVTC was predominantly due to the electrostrictive force rather than electrostatic force. We believe that the acquired PVTC-PTM blends are great candidates to achieve the high-performance electromechanical strain and take all benefits derived from the all-organic system, including high electrical breakdown strength, processibility, dielectrics, and large strain, which are largely different from the organic-inorganic hybrid nanocomposite systems.

Thermal and Flame Retardant Properties of Phosphate-Functionalized Silica/Epoxy Nanocomposites.

We report a flame retardant epoxy nanocomposite reinforced with 9,10-dihydro-9-oxa-10-phosphaphenantrene-10-oxide (DOPO)-tethered SiO2 (DOPO-t-SiO2) hybrid nanoparticles (NPs). The DOPO-t-SiO2 NPs were successfully synthesized through surface treatment of SiO2 NPs with (3-glycidyloxypropyl)trimethoxysilane (GPTMS), followed by a click reaction between GPTMS on SiO2 and DOPO. The epoxy nanocomposites with DOPO-t-SiO2 NPs as multifunctional additive exhibited not only high flexural strength and fracture toughness but also excellent flame retardant properties and thermal stability, compared to those of pristine epoxy and epoxy nanocomposites with a single additive of SiO2 or DOPO, respectively. Our approach allows a facile, yet effective strategy to synthesize a functional hybrid additive for developing flame retardant nanocomposites.

Alkaline Hydrolysis and Dyeing Characteristics of Sea-Island-Type Ultramicrofibers of PET Tricot Fabrics with Black Disperse Dye.

In this study, we investigated conditions for the alkaline hydrolysis and black-disperse dyeing of sea-island-type polyethylene terephthalate (PET) ultramicrofiber tricot fabric. We examined the weight loss ratios and tensile strengths according to the NaOH content (10%-30% on mass of fabric (omf)) during treatment; the optimal conditions used 25% omf NaOH for 30 min at 100 °C for an average weight loss ratio of 23.47%. By scanning electron microscope (SEM) analysis, the 'sea' components are extracted with increasing NaOH concentration until 25% omf NaOH, and damage of the 'island' components above 25% omf NaOH leads to a reduction in tensile strength. The dyeing conditions, including temperature (95-135 °C), time (20-60 min), pH buffer solution concentration (1-9 g/L), and contents of dispersant (1-9 g/L) and UV-absorbent (5%-25% omf) were also explored. The optimal dyeing conditions were established as a dye concentration of 8% omf with 1 g/L dispersant, 1 g/L pH buffer solution concentration, and 10% omf UV-absorbent at 135 °C for 40 min at a 1:10 goods-to-liquor ratio. The rubbing colorfastness values for the fabrics dyed with the black disperse dye spanned four grades under dry and wet conditions. The light colorfastness values of the dyed fabrics were good to excellent in the range of 4-5 grades.

Identification of genes with differential expression in chemoresistant epithelial ovarian cancer using high-density oligonucleotide microarrays.

A major obstacle in treatment of epithelial ovarian cancer is chemoresistance. The aim of this study was to determine whether distinct gene expression profiles are associated with chemoresistance in epithelial ovarian carcinoma. We performed global gene expression analysis in 13 primary epithelial ovarian cancer tissues including 5 primary chemosensitive tumors and 8 primary chemoresistant tumors using Affymetrix HGU133A microarray. The gene expression patterns of chemosensitive tumors were compared with those of chemoresistant tumors using fold change. Validity of microarray results was examined by semiquantitative RT-PCR. We identified over 320 genes differentially expressed in chemoresistant epithelial ovarian cancer (> or = twofold). Upregulated genes in chemoresistant tumors included cell cycle regulating genes (TOP2A, BCAT1, CDCA8, CCNA2, CENPE), and genes with previously known mechanisms in tumorigenesis (S100A9, APOA1, RNF125, IFI16). Downregulated genes in chemoresistant tumors included genes related to cell adhesion (MUC5B, CITED2), transcription regulating genes (FOXD1, MAD1L1, PAX2), genes involving signal transduction (SOSTDC1, SNX1, SFRP1, FOXA2, PLK2), and stress protein gene (TP53AP1). These data show that gene expression profiling can discriminate primary chemoresistant from primary chemosensitive ovarian cancers. This type of molecular profiling could provide a basis for additional functional studies.

Incorporation of a Molecular Prognostic Classifier Improves Conventional Non-Small Cell Lung Cancer Staging.

INTRODUCTION: Despite adoption of molecular biomarkers in the management of NSCLC, the recently adopted eighth edition of the TNM staging system utilized only clinicopathologic characteristics and validated improvement in risk stratification of early-stage disease has remained elusive. We therefore evaluated the integration of a clinically validated molecular prognostic classifier into conventional staging. METHODS: A novel staging system, the TNMB (with the B denoting biology) system, which integrates a 14-gene molecular prognostic classifier into the eighth edition of the TNM staging system, was developed by using data from 321 patients with NSCLC at the University of California, San Francisco. The TNMB staging system was subsequently validated in an independent, multicenter cohort of 1373 patients, and its implementation was compared with adoption of the seventh and eighth edition staging systems utilizing metrics of reclassification. RESULTS: Compared with staging according to the eighth edition of the TNM system, the TNMB staging system enhanced the identification of high-risk patients, with a net reclassification improvement of 0.33 (95% confidence interval [CI]: 0.24-0.41). It better predicted differences in survival, with a relative integrated discrimination improvement of 22.1% (95% CI: 8.8%-35.3%), and it improved agreement between observed and predicted survival, with a decrease in the reclassification calibration statistic of from 39 to 21. The seventh and eighth editions failed to change the net reclassification improvement (0.01 [95% CI: -0.04 to 0.03] and 0.03 [95% CI: 0.00 to 0.06], respectively) or relative integrated discrimination improvement (2.1% [95% CI: -5.8 to 9.9] and -2.5% [95% CI: -17.6 to 12.4], respectively); in addition, the eighth edition worsened calibration, with an increase in the reclassification calibration statistic from 23 to 25. CONCLUSIONS: Incorporation of a molecular prognostic classifier significantly improved identification of high-risk patients and survival predictions compared with when conventional staging is used. The TNMB staging system may lead to improved survival of early-stage disease through more effective application of adjuvant therapy.

Coding region polymorphisms in the CHFR mitotic stress checkpoint gene are associated with colorectal cancer risk.

CHFR was recently identified as an early mitotic checkpoint that delays transition to metaphase in response to mitotic stress. Although studies have shown that CHFR is relevant to tumorigenesis, no previous report has investigated whether polymorphisms in the CHFR gene are associated with the risk of cancer development. Here, we genotyped polymorphisms in the CHFR gene and analyzed the possible associations of single polymorphisms and haplotypes with the risk and clinicopathological characteristics of colorectal cancer. Six coding SNPs in the CHFR gene were genotyped in 462 colorectal cancer patients and 245 healthy normal controls, using either the TaqMan assay or direct sequencing. Our results revealed that the V539M polymorphism was significantly associated with a lower risk of colorectal cancer (P=0.03; OR, 0.533; 95% CI, 0.302-0.94), and significantly correlated with no distant metastasis (M0 stage), different TNM stage, and microsatellite instability (MSI) among the colorectal cancer patients. Among the five tested haplotypes, hap 10 (TGACTA) was significantly associated with a lower risk of colorectal cancer (P=0.017; OR, 0.496; 95% CI, 0.279-0.883), and colorectal cancer patients carrying this haplotype showed no distant metastasis, different TNM stage, and microsatellite instability at a significantly higher frequency. These results reveal for the first time that polymorphisms in the CHFR gene are associated with colorectal cancer susceptibility.

DHPLC analysis of adenomatous polyposis coli (APC) mutations using ready-to-use APC plates: simple detection of multiple base pair deletion mutations.

The adenomatous polyposis coli (APC), which is the susceptible gene for familial adenomatous polyposis (FAP) and sporadic colorectal cancer, spans 15 exons. The open reading frame of APC is 8529 bp, which encodes 2843 amino acids. Conventional genetic screening involves extensive time as well as high cost and labor. Thus, we developed a novel APC ready-to-use plate for high-throughput mutational analysis by denaturing high performance liquid chromatography (DHPLC). To prepare the ready-to-use APC plate, all 38 primer pairs and PCR mixtures were aliquoted into individual wells of a 96-well plate, and frozen at -20 degrees C until use. All 38 PCR primers were designed to be amplified at the same temperature (52 degrees C). We examined a total of 27 FAP patient samples with APC germline mutations (17 for multiple bp deletions, 1 for 1 bp deletion, 9 for nonsense mutations) and 50 APC-negative noncarriers. All 17 multiple bp deletion mutations were detected during the initial 50 degrees C running analysis and thus ruled out for further analyses. All other mutations were clearly detected under specific optimized conditions. More than 50% of the APC germline mutations were multiple base pair deletions and efficiently selected by omitting time-consuming partial denaturing conditions.

Development and applications of a BRAF oligonucleotide microarray.

We herein describe the development of a sensitive microarray hybridization method called competitive DNA hybridization (CDH) and its use for analysis of BRAF somatic mutations. These mutations have been identified in many human cancers, and fast, reliable BRAF mutation detection may one day facilitate directed therapy of BRAF-mutated tumors. Our fast, reliable mutation detection by CDH is based on the principle that competition among multiple fluorescent-labeled samples for binding to shared wild-type sequences should reduce nonspecific results and increase the positive signals of unshared mutated sequences. The positive signals can then be discriminated based on the labeling of each sample (ie, with Cy3, Cy5, or Alexa-594). For testing of this method, we developed a BRAF oligonucleotide microarray containing 65 mutation types (more than 95% of the known BRAF mutations) and validated this microarray with 20 colorectal cancer tissues/cancer cell lines with BRAF mutations and 60 BRAF-negative samples. In sum, we were able to screen up to nine cancer samples on a single BRAF microarray (three per CDH on three regions per slide), indicating that this method may dramatically decrease the experimental time, cost, and effort of mutation detection in BRAF and other genes amenable to microarray analysis.

Regulation of MDK expression in human cancer cells modulates sensitivities to various anticancer drugs: MDK overexpression confers to a multi-drug resistance.

MDK is a heparin-binding growth factor associated with cancer development. Here, we sought to examine the association of MDK expression with resistance and sensitivity to different chemotherapeutic agents. We established stable HeLa cell transfectants (HeLa-MDK) and tested for decreased sensitivity to chemotherapeutic agents (5-FU, doxorubicin, and cisplatin). In addition, we used siRNA to block MDK expression in SNU-638 human gastric cancer cells and examined the chemosensitizing effect. HeLa-MDK cells treated with 5-FU, doxorubicin, and cisplatin showed a fold increase in the average IC(50) and an increased cell survival. siRNA-based knockdown of MDK expression in SNU-638 cells decreased the average IC(50) by 18-44% in cells treated with three drugs. Further investigations on the molecular mechanism should be clarified, but these results indicate that MDK up- and down-regulation appears to be capable of changing the chemosensitivities of cancer cells and MDK may have possible importance as a candidate therapeutic molecule.

Genetic alterations of the TGF-beta signaling pathway in colorectal cancer cell lines: a novel mutation in Smad3 associated with the inactivation of TGF-beta-induced transcriptional activation.

To investigate genetic alterations involved in the TGF-beta signaling pathway in colorectal cancer, we assayed DNA synthesis rates after treating TGF-beta and checked for genetic alterations in TGF-betaRII, TGF-betaRI, Smad2, Smad3, and Smad4 in 12 colorectal cancer cell lines. Eleven lines, except SNU-61, show no significant change in DNA synthesis rate after TGF-beta treatment. In these 11 lines, several mutations were found in genes involved in the TGF-beta signaling pathway: (i) frameshift deletions in the poly(A)(10) tract of the TGF-betaRII gene in SNU-407, SNU-769A, SNU-769B, and SNU-1047 cell lines, (ii) a missense mutation of Smad2 (R321Q) in SNU-81, (iii) two missense mutations in TGF-betaRI (R487W in SNU-175 and A202V in SNU-1040), and (iv) a monoallelic loss at the Smad4 locus in three cell lines. Interestingly, a missense mutation (R373H) in Smad3 gene was found in SNU-769A. To our knowledge, this is the first report of Smad3 mutation in human malignancy. This mutation was found to result in the inhibition of translocation of Smad3 protein to the nucleus and a reduction in the activity of Smad3 during TGF-beta-induced transcriptional activation. These results indicate that the majority of cell lines, which are insensitive to TGF-beta, have alterations in genes involved in the TGF-beta signaling pathway in colorectal cancer cell lines.

Establishment and characterization of cell lines from three human thyroid carcinomas: responses to all-trans-retinoic acid and mutations in the BRAF gene.

We report the characteristics of three cell lines (designated, SNU-80, SNU-373 and SNU-790), which were established from two papillary carcinomas and one anaplastic carcinoma obtained from three Korean thyroid carcinoma patients. All cell lines grow as adherent cells. Electron microscopy characteristically showed cytoplasmic invaginations of nuclei and intranuclear cytoplasmic inclusions. SNU-80 and SNU-790 cells showed a positive reaction to anti-cytokeratin antibody, and SNU-790 cells positivity for CK-19. All lines were free of mycoplasma or bacteria and were proven unique by DNA fingerprinting analysis. The p15 and p16 genes are deleted in the SNU-790 line. Mutations of the p53 gene were found in two lines (SNU-80 and SNU-373), but no mutations in the RET or MEN1 genes were observed. Mutations of the BRAF gene were found in the SNU-80 (G468R) and the SNU-790 (V599E) cell lines, but no mutations in the K-ras gene were present. SNU-80 and SNU-790 cells showed a positive reaction to anti-cytokeratin antibody, and no evidence of the production of thyroglobulin or calcitonin was observed. The cell lines were unable to trap radioactive iodine but did not contain TSH receptor. In addition, we investigated the mRNA expression levels of Tg, TSHR, TTF-1, PAX-8, NIS, IL-6, and LIF, and of the alpha, beta and gamma retinoic acid receptors in these cell lines. IL-6 was down-regulated in all three cell lines by all-trans-retinoic acid treatment. RAR-alpha was expressed but RAR-beta was not expressed in the three cell lines, and RAR-gamma was not expressed in SNU-790. Interestingly, RAR-beta (SNU-80 and SNU-373) and RAR-gamma (SNU-790) was up-regulated by all-trans-retinoic acid treatment. We believe that these well-characterized thyroid carcinoma cell lines may be useful tools for investigations on the biological characteristics of thyroid carcinoma, particularly for investigations related to gene alterations, especially of the BRAF gene. These cell lines may also be useful for redifferentiation therapy studies on thyroid carcinoma using all-trans-retinoic acid.

Differential promoter methylation may be a key molecular mechanism in regulating BubR1 expression in cancer cells.

The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.

GSTT2 promoter polymorphisms and colorectal cancer risk.

BACKGROUND: Glutathione S-transferases are a group of enzymes that participate in detoxification and defense mechanisms against toxic carcinogens and other compounds. These enzymes play an important role in human carcinogenesis. In the present study, we sought to determine whether GSTT2 promoter single nucleotide polymorphisms (SNPs) are associated with colorectal cancer risk. METHODS: A total of 436 colorectal cancer patients and 568 healthy controls were genotyped for three GSTT2 promoter SNPs (-537G>A, -277T>C and -158G>A), using real-time TaqMan assay and direct sequencing. An electrophoretic mobility shift assay (EMSA) was performed to determine the effects of polymorphisms on protein binding to the GSTT2 promoter. RESULTS: The -537A allele (-537G/A or A/A) was significantly associated with colorectal cancer risk (OR = 1.373, p = 0.025), while the -158A allele (-158G/A or A/A) was involved in protection against colorectal cancer (OR = 0.539, p = 0.032). Haplotype 2 (-537A, -277T, -158G) was significantly associated with colorectal cancer risk (OR = 1.386, p = 0.021), while haplotype 4 (-537G, -277C, -158A) protected against colorectal cancer (OR = 0.539, p = 0.032). EMSA data revealed lower promoter binding activity in the -537A allele than its -537G counterpart. CONCLUSION: Our results collectively suggest that SNPs and haplotypes of the GSTT2 promoter region are associated with colorectal cancer risk in the Korean population.

Germ line mutations of mismatch repair genes in hereditary nonpolyposis colorectal cancer patients with small bowel cancer: International Society for Gastrointestinal Hereditary Tumours Collaborative Study.

PURPOSE: The aim of study was to determine the clinical characteristics and mutational profiles of the mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients with small bowel cancer (SBC). EXPERIMENTAL DESIGN: A questionnaire was mailed to 55 members of the International Society for Gastrointestinal Hereditary Tumours, requesting information regarding patients with HNPCC-associated SBC and germ line mismatch repair gene mutations. RESULTS: The study population consisted of 85 HNPCC patients with identified mismatch repair gene mutations and SBCs. SBC was the first HNPCC-associated malignancy in 14 of 41 (34.1%) patients for whom a personal history of HNPCC-associated cancers was available. The study population harbored 69 different germ line mismatch repair gene mutations, including 31 mutations in MLH1, 34 in MSH2, 3 in MSH6, and 1 in PMS2. We compared the distribution of the mismatch repair mutations in our study population with that in a control group, including all pathogenic mismatch repair mutations of the International Society for Gastrointestinal Hereditary Tumours database (excluding those in our study population). In patients with MSH2 mutations, patients with HNPCC-associated SBCs had fewer mutations in the MutL homologue interaction domain (2.9% versus 19.9%, P = 0.019) but an increased frequency of mutations in codons 626 to 733, a domain that has not previously been associated with a known function, versus the control group (26.5% versus 2.8%, P < 0.001). CONCLUSIONS: In HNPCC patients, SBC can be the first and only cancer and may develop as soon as the early teens. The distribution of MSH2 mutations found in patients with HNPCC-associated SBCs significantly differed from that found in the control group (P < 0.001).

Systematic comparison of two whole-genome amplification methods for targeted next-generation sequencing using frozen and FFPE normal and cancer tissues.

Sequencing key cancer-driver genes using formalin-fixed, paraffin-embedded (FFPE) cancer tissues is becoming the standard for identifying the best treatment regimen. However, about 25% of all samples are rejected for genetic analyses for reasons that include too little tissue to extract enough high quality DNA. One way to overcome this is to do whole-genome amplification (WGA) in clinical samples, but only limited studies have tested different WGA methods in FFPE cancer specimens using targeted next-generation sequencing (NGS). We therefore tested the two most commonly used WGA methods, multiple displacement amplification (MDA-Qiagen REPLI-g kit) and the hybrid or modified PCR-based method (Sigma/Rubicon Genomics Inc. GenomePlex kit) in FFPE normal and tumor tissue specimens. For the normalized copy number analysis, the FFPE process caused none or very minimal bias. Variations in copy number were minimal in samples amplified using the GenomePlex kit, but they were statistically significantly higher in samples amplified using the REPLI-g kit. The pattern was similar for variant allele frequencies across the samples, which was minimal for the GenomePlex kit but highly variable for the REPLI-g kit. These findings suggest that each WGA method should be tested thoroughly before using it for clinical cancer samples.

Mobile Genome Express (MGE): A comprehensive automatic genetic analyses pipeline with a mobile device.

The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or genome. However, data analysis is still the biggest bottleneck for its wide implementation. Most laboratories still depend on manual procedures for data handling and analyses, which translates into a delay and decreased efficiency in the delivery of NGS results to doctors and patients. Thus, there is high demand for developing an automatic and an easy-to-use NGS data analyses system. We developed comprehensive, automatic genetic analyses controller named Mobile Genome Express (MGE) that works in smartphones or other mobile devices. MGE can handle all the steps for genetic analyses, such as: sample information submission, sequencing run quality check from the sequencer, secured data transfer and results review. We sequenced an Actrometrix control DNA containing multiple proven human mutations using a targeted sequencing panel, and the whole analysis was managed by MGE, and its data reviewing program called ELECTRO. All steps were processed automatically except for the final sequencing review procedure with ELECTRO to confirm mutations. The data analysis process was completed within several hours. We confirmed the mutations that we have identified were consistent with our previous results obtained by using multi-step, manual pipelines.