title: A comparative study of quantitative immunohistochemistry and quantum dot immunohistochemistry for mutation carrier identification in Lynch syndrome.
abstract: OBJECTIVE
  Lynch Syndrome is caused by mutations in DNA mismatch repair (MMR) genes. Mutation carrier identification is facilitated by immunohistochemical detection of the MMR proteins MHL1 and MSH2 in tumour tissue and is desirable as colonoscopic screening reduces mortality. However, protein detection by conventional immunohistochemistry (IHC) is subjective, and quantitative techniques are required. Quantum dots (QDs) are novel fluorescent labels that enable quantitative multiplex staining. This study compared their use with quantitative 3,3'-diaminobenzidine (DAB) IHC for the diagnosis of Lynch Syndrome.
  
  
  METHODS
  Tumour sections from 36 mutation carriers and six controls were obtained. These were stained with DAB on an automated platform using antibodies against MLH1 and MSH2. Multiplex QD immunofluorescent staining of the sections was performed using antibodies against MLH1, MSH2 and smooth muscle actin (SMA). Multispectral analysis of the slides was performed. The staining intensity of DAB and QDs was measured in multiple colonic crypts, and the mean intensity scores calculated. Receiver operating characteristic (ROC) curves of staining performance for the identification of mutation carriers were evaluated.
  
  
  RESULTS
  For quantitative DAB IHC, the area under the MLH1 ROC curve was 0.872 (95% CI 0.763 to 0.981), and the area under the MSH2 ROC curve was 0.832 (95% CI 0.704 to 0.960). For quantitative QD IHC, the area under the MLH1 ROC curve was 0.812 (95% CI 0.681 to 0.943), and the area under the MSH2 ROC curve was 0.598 (95% CI 0.418 to 0.777).
  
  
  CONCLUSIONS
  Despite the advantage of QD staining to enable several markers to be measured simultaneously, it is of lower utility than DAB IHC for the identification of MMR mutation carriers. Automated DAB IHC staining and quantitative slide analysis may enable high-throughput IHC.
authors.affiliation: Department of General Surgery, Manchester Royal Infirmary, Oxford Road, Manchester, UK. emma.barrow@doctors.org.uk
authors.forename: Emma
authors.initials: E
authors.lastname: Barrow
authors.forename: D Gareth
authors.initials: DG
authors.lastname: Evans
authors.forename: Ray
authors.initials: R
authors.lastname: McMahon
authors.forename: James
authors.initials: J
authors.lastname: Hill
authors.forename: Richard
authors.initials: R
authors.lastname: Byers
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000368:Aged; D014408:Biomarkers, Tumor / Q000378:metabolism; D016022:Case-Control Studies; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000175:diagnosis* / Q000235:genetics; D053843:DNA Mismatch Repair / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005455:Fluorescent Antibody Technique / Q000379:methods; D006580:Genetic Carrier Screening / Q000379:methods*; D018095:Germ-Line Mutation; D006579:Heterozygote; D006801:Humans; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D045663:Quantum Dots
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein