Diagnostic reproducibility of tumour budding in colorectal cancer: a multicentre,multinational study using virtual microscopy

Puppa G, Senore C, Sheahan K, Vieth M, Lugli A, Zlobec I, Pecori S, Wang L M, Langner C, Mitomi H, Nakamura T, Watanabe M, Ueno H, Chasle J, Conley S A, Herlin P, Lauwers G Y & Risio M
(2012) Histopathology  61, 562–575 Diagnostic reproducibility of tumour budding in colorectal cancer: a multicentre, multinational study using virtual microscopy Aims: Despite the established prognostic relevance of tumour budding in colorectal cancer, the reproducibility of the methods reported for its assessment has not yet been determined, limiting its use and reporting in routine pathology practice. Methods and results: A morphometric system within telepathology was devised to evaluate the reproducibility of the various methods published for the assessment of tumour budding in colorectal cancer. Five methods were selected to evaluate the diagnostic reproducibility among 10 investigators, using haematoxylin and eosin (H&E) and AE1‐3 cytokeratin‐immunostained, whole‐slide digital scans from 50 pT1–pT4 colorectal cancers. The overall interobserver agreement was fair for all methods, and increased to moderate for pT1 cancers. The intraobserver agreement was also fair for all methods and moderate for pT1 cancers. Agreement was dependent on the participants’ experience with tumour budding reporting and performance time. Cytokeratin immunohistochemistry detected a higher percentage of tumour budding‐positive cases with all methods compared to H&E‐stained slides, but did not influence agreement levels. Conclusions: An overall fair level of diagnostic agreement for tumour budding in colorectal cancer was demonstrated, which was significantly higher in early cancer and among experienced gastrointestinal pathologists. Cytokeratin immunostaining facilitated detection of budding cancer cells, but did not result in improved interobserver agreement.     

Pyrosequencing: applicability for studying DNA damage‐induced mutagenesis

Site‐specifically modified DNAs are routinely used in the study of DNA damage‐induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a pre‐determined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively labeled probes, and verification of mutations by Sanger sequencing. In the search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site‐specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N⁶‐(deoxy‐D‐erythro‐pentofuranosyl)‐2,6‐diamino‐3,4‐dihydro‐4‐oxo‐5‐N‐methylformamidopyrimidine (MeFapy‐dG)‐containing vectors in primate cells, with the lesion being positioned in the 5′‐GCNGG‐3′ sequence context. Pyrosequencing detected ～8% G to T transversions and ～3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure (Earley LF et al. [2013]: Chem Res Toxicol 26:1108–1114). However, ～3.5% G to C transversions and ～2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ～1–2% for A and T and ～5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy‐dG‐containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage‐induced mutagenesis as an effective complementary experimental approach to current protocols. Environ. Mol. Mutagen. 55:601–608, 2014. © 2014 Wiley Periodicals, Inc.     

Consequences of redefining Alzheimer's disease in terms of amyloid burden without regard to cognitive decline

正Alzheimer’s disease(AD)redefined:For the past century,AD has been defined as a disease of progressive cognitive decline paired with a burden of amyloid-β(Aβ)plaques and pathologic tau tangles in the hippocampus and forebrain.However,a recent Framework paper jointly sponsored by the National Institute on Aging and the Alzheimer’s Association(Jack et al.,2018)proposes new classification guidelines for AD,which,if adopted,will have profound     

Flow cytometric analysis of Pig‐a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD‐1 mice

Procarbazine is a genotoxic carcinogen whose DNA‐damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD‐1 mice using a multi‐endpoint study design that scored micronucleated reticulocyte (MN‐RET) frequency and gene mutation at the Pig‐a locus. CD‐1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN‐RETs, with the high dose group exhibiting a mean 29‐fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig‐a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Environ. Mol. Mutagen. 54:294–298, 2013. © 2013 Wiley Periodicals, Inc.