Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen.

Pre-analytical factors, such as fixation time, influence morphology of diagnostic and predictive immunohistochemical staining, which are increasingly used in the evaluation of lung cancer. Our aim was to investigate if variations in fixation time influence the outcome of immunohistochemical staining in lung cancer. From lung resections, specimen with tumor size bigger than 4 cm, 10 samples were obtained: 2 were put through the standard fixation protocol, 5 through the delayed, and 3 through the prolonged fixation protocol. After paraffin embedding, tissue microarrays (TMAs) were made. They were stained with 20 antibodies and scored for quality and intensity of staining. Samples with delay in fixation showed loss of TMA cores on glass slides and deterioration of tissue quality leading to reduction in the expression of CK 7, Keratin MNF116, CAM 5.2, CK 5/6, TTF-1, C-MET, Napsin A, D2-40, and PD-L1. Prolonged fixation had no influence on the performance of immunohistochemical stains. Delay of fixation negatively affects the expression of different immunohistochemical markers, influencing diagnostic (cytokeratins) and predictive (PD-L1) testing. These results emphasize the need for adequate fixation of resection specimen.

Independent Validation of EarlyR Gene Signature in BIG 1-98: A Randomized, Double-Blind, Phase III Trial Comparing Letrozole and Tamoxifen as Adjuvant Endocrine Therapy for Postmenopausal Women With Hormone Receptor-Positive, Early Breast Cancer.

BACKGROUND: EarlyR gene signature in estrogen receptor-positive (ER+) breast cancer is computed from the expression values of ESPL1, SPAG5, MKI67, PLK1, and PGR. EarlyR has been validated in multiple cohorts profiled using microarrays. This study sought to verify the prognostic features of EarlyR in a case-cohort sample from BIG 1-98, a randomized clinical trial of ER+ postmenopausal breast cancer patients treated with adjuvant endocrine therapy (letrozole or tamoxifen). METHODS: Expression of EarlyR gene signature was estimated by Illumina cDNA-mediated Annealing, Selection, and Ligation assay of RNA from formalin-fixed, paraffin-embedded primary breast cancer tissues in a case-cohort subset of ER+ women (N = 1174; 216 cases of recurrence within 8 years) from BIG 1-98. EarlyR score and prespecified risk strata (≤25 = low, 26-75 = intermediate, >75 = high) were "blindly" computed. Analysis endpoints included distant recurrence-free interval and breast cancer-free interval at 8 years after randomization. Hazard ratios (HRs) and test statistics were estimated with weighted analysis methods. RESULTS: The distribution of the EarlyR risk groups was 67% low, 19% intermediate, and 14% high risk in this ER+ cohort. EarlyR was prognostic for distant recurrence-free interval; EarlyR high-risk patients had statistically increased risk of distant recurrence within 8 years (HR = 1.73, 95% confidence interval = 1.14 to 2.64) compared with EarlyR low-risk patients. EarlyR was also prognostic of breast cancer-free interval (HR = 1.74, 95% confidence interval = 1.21 to 2.62). CONCLUSIONS: This study confirmed the prognostic significance of EarlyR using RNA from formalin-fixed, paraffin-embedded tissues from a case-cohort sample of BIG 1-98. EarlyR identifies a set of high-risk patients with relatively poor prognosis who may be considered for additional treatment. Further studies will focus on analyzing the predictive value of EarlyR signature.

The Genomic Grade Assay Compared With Ki67 to Determine Risk of Distant Breast Cancer Recurrence.

IMPORTANCE: The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay was developed and named the Genomic Grade (GG). The GG assay has the potential to increase the clinical application of the GGI, but robust demonstration of the clinical validity of the GG assay is required. OBJECTIVE: To evaluate the prognostic ability of the GG assay to detect breast cancer recurrence compared with centrally reviewed immunohistochemical testing of Ki67 antigen proliferation. DESIGN, SETTING, AND PARTICIPANTS: This is an internationally collaborative substudy of a large phase 3 4-arm adjuvant trial. Patients had endocrine receptor-positive, node-positive, or node-negative nonmetastatic primary breast cancer. Patients included in this study had available formalin-fixed, paraffin-embedded samples of their primary tumors and were randomized to either a 5-year tamoxifen monotherapy arm or a 5-year letrozole monotherapy arm. Associations between either GG assay results or log2-transformed Ki67 data and survival end points were evaluated using Cox regression models stratified for chemotherapy use; the 2 vs 4 arm randomization option; and endocrine therapy assignment with and without adjustment for clinicopathological parameters, including centrally reviewed histological grade, hormone receptors, and ERBB2 (formerly HER2 or HER2/neu). The likelihood ratio statistic was used to assess the added prognostic value. INTERVENTIONS: Central evaluation and comparison, blinded for clinical information, of the GG assay, breast cancer histological grade, and Ki67. MAIN OUTCOMES AND MEASURES: Distant recurrence-free interval (DRFI). RESULTS: Genomic Grade assay data were obtained in 883 breast cancer samples (62%). At a median follow-up of 8.1 years, 84 (10%) had distant recurrences. Increasing GG or Ki67 were both significantly associated with lower DRFI and added independent prognostic information to the clinicopathological prognostic factors. In patients with early node-negative breast cancer who were endocrine-only treated, 38% were GG1 with a 10-year DRFI of 99% (95% CI, 97%-100%), and 18% were histological grade 1 with a 10-year DRFI of 100% (95% CI, 100%-100%). For GG equivocal patients, the 10-year DRFI was 94% (95% CI, 90%-98%), and for GG3 patients, the 10-year DRFI was 87% (95% CI, 80%-94%). CONCLUSIONS AND RELEVANCE: Either the GG assay or centrally reviewed Ki67 significantly improves clinicopathological models to determine distant recurrence of breast cancer. Compared with the histological grade, the GG assay can identify a higher proportion of endocrine-only treated patients with very low risk of distant recurrence at 10 years. TRIAL REGISTRATION: clinicaltrials.gov Identifiers: NCT00004205 and NCT00004205.