Implementation and Quality Control of Lung Cancer EGFR Genetic Testing by MALDI-TOF Mass Spectrometry in Taiwan Clinical Practice.

Molecular diagnostics in cancer pharmacogenomics is indispensable for making targeted therapy decisions especially in lung cancer. For routine clinical practice, the flexible testing platform and implemented quality system are important for failure rate and turnaround time (TAT) reduction. We established and validated the multiplex EGFR testing by MALDI-TOF MS according to ISO15189 regulation and CLIA recommendation in Taiwan. Totally 8,147 cases from Aug-2011 to Jul-2015 were assayed and statistical characteristics were reported. The intra-run precision of EGFR mutation frequency was CV 2.15% (L858R) and 2.77% (T790M); the inter-run precision was CV 3.50% (L858R) and 2.84% (T790M). Accuracy tests by consensus reference biomaterials showed 100% consistence with datasheet (public database). Both analytical sensitivity and specificity were 100% while taking Sanger sequencing as the gold-standard method for comparison. EGFR mutation frequency of peripheral blood mononuclear cell for reference range determination was 0.002 ± 0.016% (95% CI: 0.000-0.036) (L858R) and 0.292 ± 0.289% (95% CI: 0.000-0.871) (T790M). The average TAT was 4.5 working days and the failure rate was less than 0.1%. In conclusion, this study provides a comprehensive report of lung cancer EGFR mutation detection from platform establishment, method validation to clinical routine practice. It may be a reference model for molecular diagnostics in cancer pharmacogenomics.

FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis.

Engineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976-1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

Association between the apolipoprotein E genotypes and breast cancer patients in Taiwanese.

To display the association between the apolipoprotein E (APOE) genotypes and breast cancer patients, a cross sectional study including 291 patients and 148 controls was performed. The APOE genotypes were measured in all participants, and the pathological diagnosis, estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2) among breast cancer patients were collected. The results showed the APOE allele frequency in breast cancer patients was 11.7% epsilon2 carriers, 74.6% epsilon3 carriers and 13.7% epsilon4 carriers, and there was no significant difference when they were compared with those of the control group (15.5% epsilon2 carriers, 74.3% epsilon3 carriers and 10.1% epsilon4 carriers; p=0.342). Among the patients in pre-menopause, showed a higher frequency of epsilon2 carriers had the cancer site on the left than that of the epsilon3 carriers (78.6% versus 40.3%; p=0.019). Among breast cancer patients, there was no significant association between the APOE genotypes and menopausal status, pathological diagnosis, estrogen receptor, progesterone receptor, and HER-2. Our findings demonstrated that the APOE genotypes were not associated with breast cancer patients, and epsilon2 allele tended to induce breast cancer on the left site among those patients in pre-menopause.

Method for testing for human papillomavirus infection in patients with cervical intraepithelial disease.

To determine whether DNA detection of high-risk human papillomavirus could represent a reliable screening technique and a useful follow-up method, we investigated the performance of the Hybrid Capture II assay in detecting high-grade cervical lesions. The test positivity at 1.86 pg/ml had a high sensitivity (94.7%) and improved specificity. It was also a suitable follow-up method for detecting the recurrence of cervical intraepithelial disease in patients.

Lipoprotein glomerulopathy associated with psoriasis vulgaris: report of 2 cases with apolipoprotein E3/3.

Lipoprotein glomerulopathy (LPG) is a rare disease, characterized by a special histology, including dilated glomerular capillaries filled with pale-stained and meshlike lipoprotein thrombi. It always presents with proteinuria or nephrotic syndrome. Although hyperlipidemia is not always seen, most patients have type III hyperlipoproteinemia with apolipoprotein (apo) E2/3 phenotyping. Although the clinical feature of LPG is rarely described, LPG associated with other glomerulopathy, including IgA nephropathy, membranous nephropathy, and lupus nephritis, has been documented. Until now, there have been no reports of psoriasis vulgaris associated with LPG. The authors present 2 cases of LPG with apo E3/3 genotyping associated with psoriasis vulgaris. The first patient was a 65-year-old woman who presented with nephrotic syndrome with daily urinary protein loss of 9.05 g and itchy erythematous scaly plaques on her trunk and lower limbs for 1 year. The renal biopsy results showed LPG, and the skin biopsy results showed psoriasis. The second patient was a 50-year-old man with history of psoriasis over his trunk and 4 limbs for 30 years. He also presented with nephrotic syndrome with daily urinary protein loss of 7.55 g. The renal biopsy results also showed LPG. The genotype of apo E showed E3/3, and lipoprotein electrophoresis showed a type III hyperlipoproteinemia-like pattern in both cases. The authors suggest that presence of apo E3/3 genotype cannot rule out the diagnosis of type III hyperlipoproteinemia and LPG. Besides, LPG should be included in the differential diagnosis of psoriatic patients with nephrotic syndrome, especially in Asian patients who show poor response to traditional therapy. Renal biopsy should be performed to make the definitive diagnosis.

A novel genetic variant in the apolipoprotein A5 gene is associated with hypertriglyceridemia.

The apolipoprotein A5 gene (APOA5 ) has been shown to play an important role in determining plasma triglyceride concentrations in humans. We describe here a novel variant, c.553G>T, in the apolipoprotein A5 gene that is associated with hypertriglyceridemia. In contrast to some other polymorphisms, which occur in non-coding regions of the gene, this variant occurs within the coding region and causes the change of amino acid sequence (a substitution of a cysteine for a glycine residue). The minor allele frequencies were 0.042 and 0.27 (P<0.001) for control and hypertriglyceridemic patients, respectively. The serum triglyceride level was significantly different among the genotypic groups (G/G 92.5+/-37.8 mg/dl, G/T 106.6+/-34.8 mg/dl, T/T 183.0 mg/dl, P=0.014) in control subjects. Multiple logistic regression revealed individuals carrying the minor allele had age, gender and BMI (body mass index)-adjusted odds ratio of 11.73 (95% confidence interval of 6.617-20.793; P<0.0001) for developing hypertriglyceridemia in comparison to individuals without that allele. These findings suggest the possible use of c.553G>T polymorphisms in APOA5 as prognostic indicators for hypertriglyceridemia susceptibility in Chinese.

Effects of air bubbles and tube transportation on blood oxygen tension in arterial blood gas analysis.

BACKGROUND AND PURPOSE: Pneumatic tube transport has been reported to aggravate the error in partial pressure of oxygen (PO(2)) measurements caused by air bubbles. The aim of this study was to clarify the effect of manual and pneumatic tube methods of sample transportation and different amounts of air bubbles on arterial blood gas analysis. METHODS: Blood gas samples from 15 patients and a pooled wasted blood mixture with 3 different levels of PO(2) were analyzed to determine the effects of air bubbles and manual versus pneumatic tube transportation on PO(2) levels. RESULTS: PO(2) increased significantly in samples containing 10% air bubbles and was exaggerated by pneumatic tube transport (from 115.63 +/- 9.31 mm Hg to 180.51 +/- 11.29 mm Hg, p < 0.001). In samples with low PO(2) ( approximately 30 mm Hg), the measurement was not aberrant regardless of the method of transportation or the amount of air bubbles contained in the specimen. However, in samples with medium and high PO(2) (> 70 mm Hg), aberrances in measurements were noted even with only 0.5% air bubbles and regardless of whether the sample was transported by manual methods or pressurized tube. The increments of PO(2) correlated positively with the amount of air introduced into the specimens. Thus, the measured PO(2) increased 8.13 and 31.77 mm Hg when 0.5% and 10% air bubbles were introduced, respectively, to samples with medium PO(2) (p < 0.05). The interaction between the amount of air bubbles and the method of transportation was significant (p < 0.001). CONCLUSIONS: Trapped air in the syringe should be expelled as thoroughly as possible, since the presence of only 1% air bubbles can result in aberrance in PO(2) measurement. Samples for blood gas analysis should be carried in ambient pressure to the laboratory because pneumatic tube delivery systems significantly aggravate the air bubble-related aberrance in PO(2) measurement.