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Determination of HER2/neu status: a pilot study comparing HER2/neu dual in situ hybridization DNA probe cocktail assay performed on cell blocks to immunohistochemisty and fluorescence in situ hybridization performed on histologic specimens.

Author(s): Hartman AK, Gorman BK, Chakraborty S, Mody DR, Schwartz MR

Publication: Arch Pathol Lab Med, 2014, Vol. 138, Page 553-8

PubMed ID: 24678687 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of fixative type (CytoLyte, neutral buffered formalin, and RPMI), pretreatment protease and protocol, and slide staining system on the ability to determine Her2 status by ISH on cell blocks and investigated if Her2 status by ISH of cell blocks was concordant with that in resection specimens by IHC.  Cell scrapings from 18 surgical (mastectomy, lumpectomy, and a single lung wedge resection) specimens were collected in CytoLyte, 10% neutral buffered formalin, and Roswell Park Memorial Institute medium (RPMI). Specimens collected in CytoLyte were directly embedded to make Cellient blocks and sectioned at 4µm. Specimens collected in formalin were fixed for 1 h, centrifuged, and wrapped in lens paper.  Specimens in RPMI were centrifuged, mixed with plasma and thrombin, and the thrombin cell pellet was wrapped in lens paper. Thrombin and formalin-fixed cell pellets were fixed in 10% formalin for 6-48 h, embedded in paraffin, and sectioned at 4µm. The first, fifth, and tenth section of each block was H&E stained and the remaining sections were used for dual-color ISH (Her2 and Chromosome 17) with pretreatment of Cellient blocks with 2 different proteases (Protease 2 and Protease 3) for 4 min and pretreatment of all three block types with Protease 3 or 4, 12, or 20 min. Results were compared to IHC status in the surgical specimen and FISH was performed in cases where Her2 status by IHC was equivocal.

   Summary of Findings:

   Initial FISH results stained with a Ventana BenchMark ST from nine of the specimens were enumerable in a comparable percentage of sections from Cellient (4 of 11), FFPE (2 of 13), and thrombin (4 of 11) blocks and the authors report no consistent effect of protease type or digestion duration. When stained with the Ventana BenchMark ULTRA, enumerable signals were obtained from a higher percentage of sections of FFPE and thrombin cell blocks than Cellient cell blocks (92% and 89% versus 35%, P=0.002 and P=0.02; respectively), but a specific protocol yielding optimal results was not identified.  A higher percentage of FFPE and thrombin block sections were enumerable when stained using the Ventana BenchMark Ultra rather than the Ventana BenchMark ST (P<0.001 and P<0.03, respectively), but there was no effect of slide staining system on the percentage of sections from Cellient blocks that were enumerable. When Her2 status was determinable by dual-ISH, it was concordant with the status determined by IHC.

   Biospecimens

   • Cell - Breast
   • Tissue - Breast

   Preservative Types

   • Other Preservative
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	FISH
   Protein	Immunohistochemistry
   DNA	In situ hybridization

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of cell acquisition	Scraping
   In situ hybridization Specific	Technology platform	Ventana BenchMark ST Ventana BenchMark ULTRA IHC
   Analyte Extraction and Purification	Protein digestion	Protease 2 Protease 3 4 min 12 min 20 min
   Biospecimen Acquisition	Method of tissue acquisition	Mastectomy Lumpectomy Surgical resection

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