Abstract P1-02-01: Pre-analytical setting is critical for an assessment of the Ki-67 labeling index for breast cancer
Authors: Arima N , Toyozumi Y , Nishimura R , Osako T , Nishiyama Y , Nakano M , Fujisue M , Tashima R , Moriya T 
Published: August 2014

Abstract
Background: The Ki-67 labeling index (LI) is useful in determining the efficacy of chemotherapy for patients with ER-positive and HER2-negative breast cancer. However, standardization of Ki-67 assessment has not yet been established. In terms of the factors that may affect Ki-67 LI, recent studies have focused on inter-observer variability in the interpretation of these values and insufficient attention has been given to the importance of handling the tissue. Therefore, this study focused on the effect of tissue fixation on Ki-67 LI.
Methods:
The effects of several pre-analytical conditions on the Ki-67 LI were studied as follows;
(1) Type of fixation: Each 173 surgically excised tumors were fixed with either 10% neutral buffered formalin or 15% formalin on the same condition, followed by assessment of Ki-67 LI.
(2) Time of fixation: Each study was repeated more than three times to confirm the result.
A: Time to fixation: A part of tumor, sliced from the surgically excised tumor, was kept in 4° for several hours to overnight before formalin fixation. Ki-67 LI was compared between the tumor with immediate and delayed fixation.
B: Time of fixation:
Insufficient fixation: A part of tumor, sliced from the surgically excised tumor, was fixed with formalin for 3 hours, followed by preparation of a paraffin-embedded block. Ki-67 LI was compared between the tumor with proper and shorter fixation.
Prolonged fixation: Some pieces of slice were prepared from the surgically excised tumor, followed by each preparation of a paraffin-embedded block at a designated fixation period. Ki-67 LI was compared among each sample fixed in a different period of time.
(3) Surgically excised tumors on Ki-67 LI:
A: Effect of cutting onto the tumor before fixation: Ki-67 LI in each 595 surgically excised tumor with or without cutting onto the tumor before fixation, was analyzed.
B: Comparison between core needle biopsy and surgically excised tissue: Ki-67 LI in each 136 pairs of core needle biopsy and surgically excised tissue originated from the same tumor was compared.
Results:
(1) Ki-67 LI was significantly higher when 10% neutral buffered formalin was used as a fixative compared to 15% formalin (p = 0.018).
(2) Ki-67 LI was reduced in some degree when time to fixation was delayed, while insufficient fixation caused dramatic reduction of Ki-67 LI compared to that seen in an either ER or HER2. A prolonged period of fixation caused gradual reduction of Ki-67 LI associated with diminished nuclear labeling.
(3) Ki-67 LI was significantly higher in the tumor cut onto before fixation (p = 0.018). There was no significant difference in Ki-67 LI between core needle and surgically excised specimens (p = 0.107), when the tumor was properly fixed.
Conclusion: Our results suggest that pre-analytical setting critically influences the Ki-67 labeling. In order to standardize Ki-67 LI assessment in breast cancer, the care in handling of the pre-analytical tissue is crucial in determining accurate Ki-67 values.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-01.