Cold Ischemia Time: Effect on HER2 Detection by In-Situ Hybridization and Immunohistochemistry.
Bryce P Portier, Erinn Downs-Kelly, Jordi J Rowe, Deepa Patil, George T Budd, Chris Lanigan, Zhen Wang, David Hicks, David L Rimm, Raymond R Tubbs. Cleveland Clinic, OH; Taussig Cancer Institute, Cleveland, OH; University of Rochester, NY; Yale University, New Haven, CT
Background: The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 testing guidelines address pre-analytical variables (type and length of fixation) known to effect HER2 assay results. The guidelines suggest time to tissue fixation (cold ischemia time; CIT) be as short as possible, however little has been published on CIT. To that end, we evaluated HER2 status by two different ISH methods and via IHC in a cohort of breast carcinomas with varying CIT.
Design: CIT was tracked in minutes for 84 invasive mammary carcinomas; all tissues were fixed in 10% neutral buffered formalin for ≥6 hours and ≤48 hours. A tissue microarray was constructed and HER2 status was assessed via fluorescence in situ hybridization (FISH; PathVysion, Abbott-Vysis, Chicago, IL) and dual in situ hybridization (Dual ISH) method under development (Ventana, Tucson, AZ). HER2/CEP 17 ratios were recorded per ASCO/CAP guidelines and signal strength in stromal and neoplastic nuclei was recorded (0 absent to 3 strong). HER2 IHC (PATHWAY anti-HER2 (4B5) Ventana, Tucson, AZ) was also performed, scored per ASCO/CAP.
Results: CIT was stratified into <1hr (n=45), 1:00-1:59 (n=27), 2:00-2:59 (n=6), and >3 hrs (n=6). Detectable endogenous stromal signals, an excellent internal control, were seen with both ISH methodologies at all CIT points. Greater than 75% of cases had signal strength of 2 or 3 in neoplastic and stromal cells over all CIT points. FISH and dual ISH were equivalent within the same CIT with a 100% concordance in HER2 status determination. Complete agreement between ISH methodologies and IHC was seen in 77% (65/84) of cases; % per CIT categories were 76, 89, 67, and 50%, respectively. Minor discordances (ie FISH non-amplified and IHC equivocal) were identified in 18% (15/84) of cases with major discordances (ie FISH amplified and IHC equivocal or negative) identified in 5% (4/84) of cases.
Conclusions: In this series which is the largest to date evaluating CIT while controlling for fixation type and length, no discernible effect on HER2 signal was detected in CIT up to and greater than 3 hours. In addition, 77% of cases were concordant between IHC and ISH with only 5% (4 cases) having a major discordance; well within the expected “biologic” discordance seen when employing ISH versus IHC based testing.
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 27, Monday Morning