Development and validation of a protocol for optimizing the use of paraffin blocks in molecular epidemiological studies: The example from the HPV-AHEAD study

PLoS One. 2017 Oct 16;12(10):e0184520. doi: 10.1371/journal.pone.0184520. eCollection 2017.

Abstract

Worldwide use of formalin-fixed paraffin-embedded blocks (FFPE) is extensive in diagnosis and research. Yet, there is a lack of optimized/standardized protocols to process the blocks and verify the quality and presence of the targeted tissue. In the context of an international study on head and neck cancer (HNC)-HPV-AHEAD, a standardized protocol for optimizing the use of FFPEs in molecular epidemiology was developed and validated. First, a protocol for sectioning the FFPE was developed to prevent cross-contamination and distributed between participating centers. Before processing blocks, all sectioning centers underwent a quality control to guarantee a satisfactory training process. The first and last sections of the FFPEs were used for histopathological assessment. A consensus histopathology evaluation form was developed by an international panel of pathologists and evaluated for four indicators in a pilot analysis in order to validate it: 1) presence/type of tumor tissue, 2) identification of other tissue components that could affect the molecular diagnosis and 3) quality of the tissue. No HPV DNA was found in sections from empty FFPE generated in any histology laboratories of HPV-AHEAD consortium and all centers passed quality assurance for processing after quality control. The pilot analysis to validate the histopathology form included 355 HNC cases. The form was filled by six pathologists and each case was randomly assigned to two of them. Most samples (86%) were considered satisfactory. Presence of >50% of invasive carcinoma was observed in all sections of 66% of cases. Substantial necrosis (>50%) was present in <2% of samples. The concordance for the indicators targeted to validate the histopathology form was very high (kappa > 0.85) between first and last sections and fair to high between pathologists (kappa/pabak 0.21-0.72). The protocol allowed to correctly process without signs of contamination all FFPE of the study. The histopathology evaluation of the cases assured the presence of the targeted tissue, identified the presence of other tissues that could disturb the molecular diagnosis and allowed the assessment of tissue quality.

Publication types

  • Multicenter Study
  • Validation Study

MeSH terms

  • Carcinoma / epidemiology
  • Carcinoma / pathology
  • Europe
  • Head and Neck Neoplasms / epidemiology
  • Head and Neck Neoplasms / pathology
  • Humans
  • India
  • Molecular Diagnostic Techniques / standards
  • Molecular Epidemiology / methods*
  • Molecular Epidemiology / standards*
  • Necrosis / epidemiology
  • Necrosis / pathology
  • Paraffin
  • Paraffin Embedding / standards*
  • Pilot Projects
  • Random Allocation

Substances

  • Paraffin

Grant support

This study was supported by the European Commission, grant HPV-AHEAD (FP7-HEALTH-2011-282562), URLs: http://cordis.europa.eu/project/rcn/100268_es.html, All authors, Coordinator MT; Instituto de Salud Carlos III-ISCIII (Spanish Government) cofunded by FEDER funds / European Regional Development Fund (ERDF) - a way to build Europe (Reference: RD12/0036/0056), URLs: http://www.rticc.org/epidemiologia-prevencion-grupos.php#tech, MM, ST, BL, Coordinator Francesc Xavier Bosch.; Instituto de Salud Carlos III-ISCIII (Spanish Government) cofunded by FEDER funds / European Regional Development Fund (ERDF) - a way to build Europe (CIBERESP Group 36 CB06/02/0073), URLs: http://www.ciberesp.es/grupos/grupo-de-investigacion?id=12515, XC and LA, Coordinator SdS; Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR 2014SGR1077), URLs: http://agaur.gencat.cat/en/inici/index.html, XC, LA and ST, Coordinator Francesc Xavier Bosch.; Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR 2014SGR756), URLs: http://agaur.gencat.cat/en/inici/index.html, MM, Coordinator SdS.