The local immune phenotype influences prognosis in patients with nodal-positive rectal cancer after neoadjuvant chemoradiation

International Journal of Colorectal Disease - The local immune contexture in patients with locally advanced rectal cancer (LARC) has important prognostic value after neoadjuvant chemoradiation and...     

Comparative evaluation of the VERSANT HCV RNA 3.0, QUANTIPLEX HCV RNA 2.0, and COBAS AMPLICOR HCV MONITOR version 2.0 Assays for quantification of hepatitis C virus RNA in serum

A comparison of quantitative results expressed in hepatitis C virus (HCV) international units per milliliter, obtained from the VERSANT HCV RNA 3.0 (bDNA-3.0) assay, the QUANTIPLEX HCV RNA 2.0 (bDNA-2.0) assay, and the COBAS AMPLICOR HCV MONITOR version 2.0 (HCM-2.0) test was performed. A total of 168 patient specimens submitted to the Mayo Clinic Molecular Microbiology Laboratory for HCV quantification or HCV genotyping were studied. Of the specimens tested, 97, 88, and 79% yielded quantitative results within the dynamic range of the bDNA-3.0, bDNA-2.0, and HCM-2.0 assays, respectively. Overall, there was substantial agreement between the results generated by all three assays. A total of 15 out of 29 (52%) of the specimens determined to contain viral loads of <31,746 IU/ml by the bDNA-3.0 assay were categorized as containing viral loads within the range of 31,746 to 500,000 IU/ml by the bDNA-2.0 assay. Although substantial agreement was noted between the results generated by the bDNA-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-2.0 assay was noted (P = 0.001). Likewise, although substantial agreement was noted between the results generated by the HCM-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-3.0 assay was noted (P < or = 0.001). The discrepancy between the HCM-2.0 and bDNA-3.0 results was more pronounced when viral loads were >500,000 IU/ml and resulted in statistically significant differences (P < or = 0.001) in determining whether viral loads were above or below 800,000 IU/ml of HCV RNA, the proposed threshold value for tailoring the duration of combination therapy. The expression of quantitative values in HCV international units per milliliter was a strength of both the bDNA-3.0 and HCM-2.0 assays.     

Evaluation of the MagNA Pure LC used with the TRUGENE HBV Genotyping Kit.

BACKGROUND: The current manual sample processing method recommended for use with the TRUGENE HBV Genotyping Kit (TRUGENE HBV; Bayer HealthCare LLC, Tarrytown, NY) is labor-intensive and may be prone to specimen cross-contamination. Recent evaluations of the MagNA Pure LC (MP; Roche Applied Science, Indianapolis, IN) suggest that it is suitable for automated, contamination-free extraction and purification of viral nucleic acids from large-volume (1.0 mL) serum or plasma specimens. OBJECTIVES: We evaluated the MP Total Nucleic Acid Isolation Kit--Large Volume (Roche Applied Science) in conjunction with TRUGENE HBV to establish the analytical sensitivity (threshold titer) of the assay, in HBV DNA International Units (IU)/mL, for obtaining consistent, interpretable sequence data from TRUGENE HBV. STUDY DESIGN: HBV analytical standards, prepared as 10 replicates (1.0 mL each) at each of the following concentrations: 200, 1000, 5000, and 10,000 IU/mL, were processed by MP and analyzed by TRUGENE HBV according to manufacturer's instructions. Performance of TRUGENE HBV used in conjunction with MP sample processing was evaluated further using 22 clinical serum specimens containing low titers of HBV DNA. RESULTS: All replicates of HBV analytical standards at 1000, 5000, and 10,000 IU/mL yielded interpretable TRUGENE HBV sequences, whereas interpretable sequences were obtained in 90% (9 of 10) of the replicates at 200 IU/mL. TRUGENE HBV sequences were interpretable in 86% (19 of 22) of the clinical specimens studied. CONCLUSIONS: MP sample processing is efficient and suitable for use with TRUGENE HBV. When combined with MP sample processing, TRUGENE HBV yielded interpretable sequences from HBV analytical standards and clinical serum specimens with HBV DNA titers of > or =200 IU/mL.     

High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR

We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r(2) = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.     

Noninvasive visualization of tumor growth in a human colorectal liver metastases xenograft model using bioluminescence in vivo imaging

Background

Bioluminescence imaging (BLI) is an ideal tool for noninvasive, quantitative monitoring of tumor progression/regression in animal models. The effectiveness of different treatment strategies is displayed by an altered intensity of bioluminescence, demonstrating a change of the tumor burden. The aim of this study was to establish a reliable, reproducible colorectal hepatic metastases cancer animal model.

Methods

Cells of the human colon carcinoma cell line HCT-116 Luc^pos expressing the firefly luciferase enzyme gene were used. HCT-116 Luc^pos cells (2.5 × 10⁶) were injected through the portal vein into the liver of immunoincompetent nude mice. BLI was used to analyze intrahepatic tumor burden and growth kinetic.

Results

HCT-116 Luc^pos cells demonstrated a progressive and reproducible growth in the liver after intraportal injection. Four days after injection, the animals were analyzed for tumor growth by BLI, and mice without or too low bioluminescence signals were excluded (between 10% and 20% animals). HCT-116 Luc^pos intrahepatic tumors responded successfully to different dosages (5 and 10 mg/kg) of 5-fluorouracil.

Conclusions

BLI is an important tool with many potential advantages for investigators. The measurement of intrahepatic tumor growth by imaging luciferase activity noninvasively provides valuable information on tumor burden and effectiveness of therapy. Thus, the presented intrahepatic metastases model based on the growth of HCT-116 Luc^pos cells is suitable for in vivo testing of different cancer therapy strategies.     

Importance of recurrence rating,morphology, hernial gap size,and risk factors in ventral and incisional hernia classification

Background

There is limited evidence on the natural course of ventral and incisional hernias and the results of hernia repair, what might partially be explained by the lack of an accepted classification system. The aim of the present study is to investigate the association of the criteria included in the Wuerzburg classification system of ventral and incisional hernias with postoperative complications and long-term recurrence.

Methods

In a retrospective cohort study, the data on 330 consecutive patients who underwent surgery to repair ventral and incisional hernias were analyzed. The following four classification criteria were applied: (a) recurrence rating (ventral, incisional or incisional recurrent); (b) morphology (location); (c) size of the hernial gap; and (d) risk factors. The primary endpoint was the occurrence of a recurrence during follow-up. Secondary endpoints were incidence of postoperative complications. Independent association between classification criteria, type of surgical procedures and postoperative complications was calculated by multivariate logistic regression analysis and between classification criteria, type of surgical procedures and risk of long-term recurrence by Cox regression analysis.

Results

Follow-up lasted a mean 47.7 ± 23.53 months (median 45 months) or 3.9 ± 1.96 years. The criterion “recurrence rating” was found as predictive factor for postoperative complications in the multivariate analysis (OR 2.04; 95 % CI 1.09–3.84; incisional vs. ventral hernia). The criterion “morphology” had influence neither on the incidence of the critical event “recurrence during follow-up” nor on the incidence of postoperative complications. Hernial gap “width” predicted postoperative complications in the multivariate analysis (OR 1.98; 95 % CI 1.19–3.29; ≤5 vs. >5 cm). Length of the hernial gap was found to be an independent prognostic factor for the critical event “recurrence during follow-up” (HR 2.05; 95 % CI 1.25–3.37; ≤5 vs. >5 cm). The presence of 3 or more risk factors was a consistent predictor for “recurrence during follow-up” (HR 2.25; 95 % CI 1.28–9.92). Mesh repair was an independent protective factor for “recurrence during follow-up” compared to suture (HR 0.53; 95 % CI 0.32–0.86).

Conclusions

The ventral and incisional hernia classification of Dietz et al. employs a clinically proven terminology and has an open classification structure. Hernial gap size and the number of risk factors are independent predictors for “recurrence during follow-up”, whereas recurrence rating and hernial gap size correlated significantly with the incidence of postoperative complications. We propose the application of these criteria for future clinical research, as larger patient numbers will be needed to refine the results.