4th Palermo Conference on INNOVATIVE THERAPIES FOR LYMPHOID MALIGNANCIES
Published online 22 September 2009
Haematologica, Vol 95, Issue 2, 247-252 doi:10.3324/haematol.2009.011635
Copyright © 2010 by Ferrata Storti Foundation
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Malignant Lymphomas

Double-staining chromogenic in situ hybridization as a useful alternative to split-signal fluorescence in situ hybridization in lymphoma diagnostics

Anke van Rijk1, Tim Svenstroup-Poulsen2, Margaret Jones3, José Cabeçadas4, Juan Cruz Cigudosa5, Lorenzo Leoncini6, Anja Mottok7, Christiane Copie Bergman8, Evi Pouliou9, Stephen Hamilton Dutoit10, Han J. van Krieken1

1 Radboud University Nijmegen Medical Centre, Department of Pathology, Nijmegen, The Netherlands
2 Copenhagen University Hospital Herlev, Department of Pathology, Herlev, Denmark
3 LRF Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
4 Serviço de Anatomia Patológica, Instituto Português de Oncologia, Lisboa, Portugal
5 Cytogenetics Unit, Spanish National Cancer Centre, (CNIO), Madrid, Spain
6 Department of Human Pathology and Oncology, University of Siena, Siena, Italy
7 Department of Pathology, University of Wuerzburg, Wuerzburg, Germany
8 APHP, Hôpital Henri Mondor, Département de Pathologie, Université Paris, Faculté de Médecine, Créteil, France
9 Hematopathology Department. Evaggelismos Hospital, Athens, Greece
10 Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark

Correspondence: Anke van Rijk, Radboud University Nijmegen Medical Centre, Department of Pathology-824, P.O.Box 9101, 6500 HB Nijmegen, The Netherlands., E-mail: a.vanrijk{at}pathol.umcn.nl

Background: Malignant lymphomas are classified based on morphology, immunophenotype, genetics and clinical features. The pathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in specific entities, their detection is an important adjunct for increasing the reliability of the diagnosis. Recently, split-signal fluorescence in situ hybridization has become available as a robust method to detect chromosomal breaks in paraffin-embedded formalin-fixed tissues. A bright field approach would bring this technology within the reach of every pathology laboratory.

Design and Methods: Our study was initiated to determine the consistency between chromogenic in situ hybridization and fluorescence in situ hybridization, both using split-signal probes developed for the detection of chromosomal breaks. Five hundred and forty cases of 11 lymphoma entities and reactive, benign lymphoid tissues, collected from eight different pathology laboratories, placed on 15 fluorescence in situ hybridization pre-stained tissue microarray slides, were double stained for the chromogenic hybridization. For each core morphology and actual signal were compared to the original fluorescence hybridization results. In addition, hematoxylin background staining intensity and signal intensity of the double-staining chromogenic in situ hybridization procedure were analyzed.

Results: With respect to the presence or absence of chromosomal breaks, 97% concordance was found between the results of the two techniques. Hematoxylin background staining intensity and signal intensity were found to correspond. The overall morphology after double-staining chromogenic in situ hybridization had decreased compared to the initial morphology scored after split-signal fluorescence in situ hybridization staining.

Conclusions: We conclude that double-staining chromogenic in situ hybridization is equally reliable as fluorescence in situ hybridization in detecting chromosomal breaks in lymphoid tissue. Although differences in morphology, hematoxylin staining and chromogenic signal intensity vary between the tumor entities none of the entities appeared more easy or difficult to score.

Key words: double staining, CISH, split-signal, lymphoma diagnostics.