Effect of fixation time on breast biomarker expression: a controlled study using cell line-derived xenografted (CDX) tumours.
OBJECTIVE
Altering the length of time specimens are placed in fixative without compromising analytical testing accuracy is a continuous challenge in the anatomical pathology lab. The aim of this study was to determine under controlled conditions the effects of variable fixation time on breast biomarker expression in human breast cancer cell line-derived xenografted (CDX) tumours.


METHODS
CDX tumours using strong oestrogen receptor (ER)-positive, Her2-negative (MCF7) and weak ER-positive, Her2 equivocal (T47D) breast cancer cell lines were fixed for various times ranging from 1 to 336 hours in 10% neutral buffered formalin. CDX tumours were processed according to routine biomarker testing protocols and stained for ER and Her2 immunohistochemistry (IHC) and processed forHER2 fluorescence in situ hybridisation (FISH). The tumours were evaluated using Allred scoring for ER and current ASCO/CAP guidelines for Her2, and by objective cell counting methodology.


RESULTS
No differences were found in expression of ER in either MCF7 or T47D CDX tumours under variable fixation. T47D tumours displayed equivocal Her2 staining when fixed for 24 hours, but fixation for ≤8 hours resulted in consistently negative staining while tumours fixed for >72 hours demonstrated consistent equivocal staining (p<0.01). Cell counting assays revealed only a significant increase in sensitivity in tumours fixed for >72 hours (p<0.01). As expected, FISH results were unaffected by variable fixation.


CONCLUSIONS
Neither shortened nor prolonged fixation affects ER expression, consistent with previous findings. In equivocal Her2-expressing tumours, however, increasing fixation increased the sensitivity of Her2 IHC reporting while not affecting FISH.
Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John's, Newfoundland and Labrador, Canada.|Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, St. John's, Newfoundland and Labrador, Canada.
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Kao
Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John's, Newfoundland and Labrador, Canada.
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Hasan
Department of Cancer Cytogenetics, Laboratory Medicine Program, University Health Network, Toronto General Hospital, Toronto, Ontario, Canada.|Instituto de Pesquisa Pelé Pequeno Princípe, Curitiba, Paraná, Brazil.
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Baptista
Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John's, Newfoundland and Labrador, Canada.
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Truong
Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, St. John's, Newfoundland and Labrador, Canada.
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Gai
Department of Cancer Cytogenetics, Laboratory Medicine Program, University Health Network, Toronto General Hospital, Toronto, Ontario, Canada.|Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Canada.
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Smith
Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John's, Newfoundland and Labrador, Canada.
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Li
Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John's, Newfoundland and Labrador, Canada.
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Gonzales
Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, St. John's, Newfoundland and Labrador, Canada.
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Voisey
Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, St. John's, Newfoundland and Labrador, Canada.
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Eriwvo
Division of Biomedical Sciences, Faculty of Medicine, Memorial University, St. John's, Newfoundland and Labrador, Canada.|Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, St. John's, Newfoundland and Labrador, Canada.
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Power
Division of Anatomical Pathology, Laboratory Medicine Program, Eastern Health, St. John's, Newfoundland and Labrador, Canada.
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Denic
D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis*; D005260:Female; D064593:Heterografts; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis*; D011960:Receptors, Estrogen / Q000032:analysis*; D013997:Time Factors; D046888:Tissue Array Analysis; D016707:Tissue Fixation / Q000379:methods*
D014408:Biomarkers, Tumor; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; C508053:ERBB2 protein, human
BREAST CANCER; FIXATION; IMMUNOHISTOCHEMISTRY; IN SITU HYBRIDISATION