X-Ray of Excised Cancerous Breast Tissue Does Not Affect Clinical Biomarker Expression.

CONTEXT: College of American Pathologists (CAP) and the American Society of Clinical Oncology have emphasized the need to reduce preanalytic variables for evaluating predictive biomarker expression in breast cancer. Postoperative x-ray of excised breast tissue is commonplace, yet is a variable that has not been investigated previously. We asked whether such radiation affects expression of relevant biomarkers. DESIGN: A previous study found that human breast cancers grown in mice demonstrate the same immunohistochemical and molecular profiles as the original tumors. Thirteen patient-derived xenografts were harvested fresh and divided for specimen radiography and a matched nonirradiated control, while following CAP/ASCO guidelines for cold ischemia time and fixation. Samples were processed in a tissue microarray for immunohistochemistry. Estrogen receptor (ER), progesterone receptor (PR), p53, and Ki67 staining was evaluated using an optimized scoring algorithm performed on digitally scanned slides. Samples were also scored manually by a blinded pathologist using the H-score method, and HER2 by the CAP/ASCO 2013 protocol. Histologic scores were compared by analysis of variance. RESULTS: There was no significant difference in quantity or intensity of staining between irradiated and nonirradiated samples for estrogen receptor (P=0.28), p53 (P=0.96), and Ki67 (P=0.94). A small but statistically significant difference was observed for PR (P=0.0058). HER2 staining was similarly unchanged in the 1 tumor exhibiting 3+ staining. CONCLUSIONS: Our study demonstrates that x-ray of breast carcinomas does not significantly affect the expression of predictive biomarkers, with the exception of PR for unclear reasons. It also highlights the utility of the patient-derived xenograft model for biomarker studies.

Cervical Cytology Specimen Stability in Surepath Preservative and Analytical Sensitivity for HPV Testing with the cobas and Hybrid Capture 2 Tests.

None of the commercial HPV tests are U.S. FDA-approved for testing of cervical cytology specimens in SurePath preservative. Still, ~30% of HPV testing is performed on specimens in this formalin-containing preservative. Formalin-induced DNA fragmentation and cross-linking may interfere with HPV detection. We evaluated analytical sensitivity and specimen stability of the cobas 4800 HPV (Roche) and Hybrid Capture 2 HPV (HC2, Qiagen) tests with residual cervical cytology samples in SurePath preservative available within 1 week of collection. Cobas testing was performed with and without heating samples at 120°C for 20 min diluted 1:1 in an alkaline environment (pretreatment) to revert DNA crosslinking. Stability was tested after 2 weeks of storage at ambient temperature followed by ≤10 weeks at 4°C. Analytical sensitivity and positivity rates (HC2, 18%; cobas pretreated, 46%; cobas untreated, 47%) were greater for cobas than HC2 (n = 682). After 6 weeks of storage, mean HC2 ratios were lower (mean 0.9, SD 6.3) but high variability limited statistical power to detect trends. Cobas threshold cycles (Ct's) increased in untreated (mean 2.1) but not pretreated samples (mean 0.3; n = 110; p≤0.0001). Overall, cobas had greater analytical sensitivity for samples in SurePath preservative. Although pretreatment introduced a manual sample transfer step and 30 min of incubation times, it improved stability without negatively affecting analytical sensitivity. While awaiting results of large trials to evaluate the clinical performance of cobas, the addition of the pretreatment step may improve the detection of HPV, especially after prolonged sample storage.

Performance Characteristics of Commercial Immunoassays for the Detection of IgG and IgM Antibodies to ß2 Glycoprotein I and an Initial Assessment of Newly Developed Reference Materials for Assay Calibration.

OBJECTIVES: To investigate the performance characteristics and impact of newly developed reference calibrators on the commutability between anti-ß2 glycoprotein I (anti-ß2 GPI) immunoassays in antiphospholipid syndrome (APS) and/or systemic lupus erythematosus (SLE). METHODS: Immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-ß2 GPI immunoassays from four manufacturers were evaluated. Serum samples from 269 patients (APS only, n = 31; SLE and APS, n = 83; SLE only, n = 129; pregnancy-related clinical manifestations without APS, n = 26) and 162 women with histories of successful pregnancies were tested. Results were expressed in kit-specific arbitrary units and in the calibrator reference units (RUs) based on 99th percentile cutoff values. Diagnostic accuracies, correlation between kits, and specific clinical manifestations in APS were investigated. RESULTS: The sensitivities of the assays ranged from 15.8% to 27.2% (IgG) and 12.3% to 15.8% (IgM) while specificities ranged from 79.4% to 86.5% (IgG) and 80.6% to 84.5% (IgM). There was moderate to almost perfect interassay reliability (Cohen κ, 0.69-0.98), and Spearman correlation coefficients were generally improved when results of the IgG determinations were expressed in RUs. CONCLUSIONS: Although qualitative agreements between immunoassays for both antibody isotypes are acceptable, correlations with APS clinical manifestations were kit dependent. Only the use of IgG reference material improved quantitative correlations between assays.

The use of circulating donor specific antibody to predict biopsy diagnosis of antibody-mediated rejection and to provide prognostic value after heart transplantation in children.

BACKGROUND: Antibody-mediated rejection (AMR) is a significant cause of mortality after heart transplantation (HT). Although the presence of donor specific antibody (DSA) is a risk factor for developing AMR, serial DSA testing is not widely performed. We aimed to investigate the predictive values and prognostic implications of circulating DSA using endomyocardial biopsy as the gold standard for AMR diagnosis in pediatric recipients of HT. METHODS: We performed a retrospective study in pediatric recipients of HT followed during the period 2009-2013 with at least 1 biopsy paired with DSA testing. Positive DSA was defined at mean fluorescent intensity (MFI) ≥2,000 using single antigen bead testing. Statistical analyses included 2 × 2 contingency tables, receiver operating characteristic analysis for optimal MFI cutoffs, Spearman correlation of MFI strength to AMR grade, and Kaplan-Meier analysis of event-free survival. RESULTS: Of 66 children included, 27 (41%) had ≥1 DSA positive test. DSA testing had a sensitivity of 92.6%, specificity of 62.2%, positive predictive value of 24.0%, and negative predictive value of 98.5% for biopsy diagnosis of AMR at our institution. There was a statistically significant correlation between higher MFI and higher AMR grade. Patients with positive DSA and AMR had similar survival early after DSA detection but trended toward lower cardiovascular event-free survival later compared with patients without DSA and a negative biopsy. CONCLUSIONS: The results of DSA testing in this cohort showed excellent sensitivity and negative predictive value for biopsy-diagnosed AMR, suggesting that DSA testing may aid in the non-invasive prediction of AMR absence in HT. The correlation of DSA MFI strength with higher AMR biopsy grade and the trend toward differences in longer term cardiovascular outcomes provide evidence for routine DSA monitoring after pediatric HT.

Ethnic Differences in Autoantibody Diversity and Hierarchy: More Clues from a US Cohort of Patients with Systemic Sclerosis.

OBJECTIVE: To determine the autoantibody repertoire and clinical associations in a multiethnic cohort of American patients with systemic sclerosis (SSc). METHODS: There were 1000 patients with SSc (196 Hispanic, 228 African American, 555 white, and 21 other) who were screened for antinuclear antibodies (ANA), including anticentromere antibodies (ACA) by indirect immunofluorescence assay, antitopoisomerase-1 (topo-1/Scl-70) by immunodiffusion, and anti-RNA polymerase III (RNAP III) by ELISA. Sera from 160 patients with mainly nucleolar and/or speckled ANA pattern, but negative for ACA, Scl-70, and RNAP III, were further characterized by immunoprecipitation for SSc-specific antibodies. RESULTS: The prevalence of antibodies against RNAP III, Th/To, and PM/Scl did not differ significantly among the ethnic groups. The frequency of anti-Scl-70 was lowest in whites (18.0%) compared with 24.0% and 26.8% in Hispanics and African Americans (p = 0.01), respectively. Compared with African American patients, Hispanic and white subjects had a higher frequency of ACA (p < 0.0001) and lower frequency of U3-RNP (p < 0.0001). U3-RNP antibodies were uniquely higher in African American patients, independent of clinical subset, while Th/To autoantibodies were associated with limited cutaneous SSc in white subjects. Overall, Hispanic and African American patients had an earlier age of onset and a predominance of diffuse cutaneous SSc compared with their white counterparts. CONCLUSION: SSc-specific antibodies may predict disease subset; however, the hierarchy of their prevalence differs across ethnic groups. This study provides the most extensive analysis to date on the relevance of autoantibodies in the diagnosis and clinical manifestations of SSc in Hispanic American patients.

Digitally guided microdissection aids somatic mutation detection in difficult to dissect tumors.

Molecular genetic testing on formalin fixed, paraffin embedded (FFPE) tumors frequently requires dissection of tumor from tissue sections mounted on glass slides. In a process referred to as "manual macrodissection," the pathologist reviews an H&E stained slide at the light microscope and marks areas for dissection, and then the laboratory performs manual dissection from adjacent sections without the aid of a microscope, using the marked reference H&E slide as a guide. Manual macrodissection may be inadequate for tissue sections with low tumor content. We compared manual macrodissection to a new method, digitally guided microdissection, on a series of 32 FFPE pancreatic cancer samples. KRAS hotspot mutation profiling was performed using the Sequenom MassARRAY system (Agena Bioscience). Digitally guided microdissection was performed on multiple smaller areas of high tumor content selected from within the larger areas marked for manual macrodissection. The KRAS mutant allele fraction and estimated neoplastic cellularity were significantly higher in samples obtained by digitally guided microdissection (p < 0.01), and 7 of the 32 samples (22%) showed a detectable mutation only with digitally guided microdissection. DNA quality and yield per cubic millimeter of dissected tissue were similar for both dissection methods. These results indicate a significant improvement in tumor content achievable with digitally guided microdissection.

Multigene and Drug Interaction Approach for Tamoxifen Metabolite Patterns Reveals Possible Involvement of CYP2C9, CYP2C19, and ABCB1.

Tamoxifen is metabolically activated to 4-hydroxytamoxifen and endoxifen by cytochrome P450 (CYP). CYP phenotypes have been correlated to tamoxifen outcomes, but few have considered drug interactions or combinations of genes. Fewer still have considered ABCB1, which encodes P-glycoprotein and transports active tamoxifen metabolites. We compared the concentrations of tamoxifen and metabolites in 116 breast cancer patients with predicted phenotypes for CYP2D6, CYP3A4, CYP3A5, CYP2C9, CYP2C19, and ABCB1 genotypes. A significant correlation between CYP2D6 phenotypes and tamoxifen metabolites was seen, strongest for endoxifen (P < .0001). Statistical fit of the data improved when using gene activity scores adjusted for known drug interactions. Concentration of tamoxifen was significantly higher (P = .02) for patients taking a CYP2C19 inhibitor. No significant relationships were found for other genes unless patients were subgrouped according to CYP2D6 phenotypes or ABCB1 genotypes. Lower concentrations of endoxifen and endoxifen/4-hydroxytamoxifen ratios were seen with impaired CYP2C9 (P = .05 and P = .03, respectively) if patients had the same CYP2D6 phenotype and were not taking a CYP2D6 or CYP2C19 inhibitor. Lower concentrations of 4-hydroxytamoxifen were seen for impaired CYP2C19 when ABCB1 SNP3435 was nonvariant (P = .04). With 3 impaired CYP phenotypes, endoxifen concentrations were lower than if only CYP2D6 was impaired (P = .05). When CYP2D6 was impaired, ABCB1 3435 CC (rs1045642) was associated with significantly higher endoxifen (P = .03). Thus, impairment in CYP2C9, CYP2C19, or ABCB1 contributes to a lower steady-state endoxifen concentration at the dose studied. These studies represent an improved way of examining relationships between pharmacogenetics, drug concentrations, and clinical outcomes and warrants study in larger populations.

Online information resources of toxicology in Sweden.

This article presents toxicological information resources in Sweden available to the public on the Internet. The main focus is on websites of organizations and universities with toxicological information in English. For example, the National Chemicals Inspectorate (KemI) has several databases with information on chemical substances on their website as well as regulatory information about chemicals. The Swedish Environmental Protection Agency has a database containing environmental information and on the website of the National Institute of Working Life a database on occupational health is available. An important part of toxicological research is carried out at the Institute of Environmental Medicine at the Karolinska Institute. Several universities and colleges are responsible for research, education and training in toxicology and ecotoxicology.