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Storage Conditions and Immunoreactivity of Breast Cancer Subtyping Markers in Tissue Microarray Sections.

ABSTRACT: Loss of immunoreactivity in tissue sections has been shown to occur when slide sections are stored at room temperature for prolonged periods of time. We conducted a systematic investigation to determine the extent of staining loss in various storage conditions to determine an optimal storage method. We investigated 6 antibodies that are commonly used for breast cancer subtyping in research studies with immunohistochemistry (ER, PR, HER2, CK5/6, EGFR, and Ki67) in formalin-fixed paraffin-embedded breast tissue microarrays consisting of 148 patients. Tissue microarrays were sectioned at various time points: fresh, 1 week, 1 month, 6 months, and 12 months before staining. Slides sectioned at each time point were stored in 5 storage conditions: desiccator, paraffin dipped, 4°C, -20°C, and -80°C. Immunohistochemistry scores were assessed over time with McNemar Test and Bowker Test of Symmetry. Desiccator storage was the only storage condition that did not show any loss in immunoreactivity for any antibody or time point in our study. Paraffin coated slides were the most difficult storage method operationally and also showed the most loss in immunoreactivity. Storing sections in a desiccator was the most effective method for minimizing immunoreactivity loss. Cold storage at 4°C is an intermediate option that is not as protective as a desiccator, but offers the advantage of being accessible to virtually all research labs.

SUBMITTER: Omilian AR

PROVIDER: S-EPMC6906262 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.

Project description:BackgroundAlthough the prognostic value of Ki67 in breast cancer is well documented, using optimal cut-points for patient stratification, reproducibility of the scoring and interpretation of the results remains a matter of debate particularly when using tissue microarrays (TMAs). This study aims to assess Ki67 expression assessed on TMAs and their matched whole tissue sections (WTS). Moreover, whether the cut-off used for WTS is reproducible on TMA in BC molecular classes and the association between Ki67 expression cut-off, assessed on TMAs and WTS, and clinicopathological parameters and patient outcome were tested.MethodA large series (n = 707) of primary invasive breast tumours were immunostained for Ki67 using both TMA and WTS and assessed as percentage staining and correlated with each other, clinicopathological parameters and patient outcome. In addition, MKI67 mRNA expression was correlated with Ki67 protein levels on WTS and TMAs in a subset of cases included in the METABRIC study.ResultsThere was moderate concordance in Ki67 expression between WTS and TMA when analysed as a continuous variable (Intraclass correlation coefficient = 0.61) and low concordance when dichotomised (kappa value = 0.3). TMA showed low levels of Ki67 with mean percentage of expression of 35 and 22% on WTS and TMA, respectively. MKI67 mRNA expression was significantly correlated with protein expression determined on WTS (Spearman Correlation, r = 0.52) and to a lesser extent on TMA (r = 0.34) (p < 0.001). Regarding prediction of patient outcome, statistically significant differences were detected upon stratification of patients with tumours expressing Ki67 at 10, 15, 20, 25 or 30% in TMA. Using TMA, ≥20% Ki67 provided the best prognostic cut-off particularly in triple-negative and HER2-positive classes.ConclusionKi67 expression in breast cancer can be evaluated using TMA although different cut-points are required to emulate results from WTS. A cut-off of ≥20% for Ki67 expression in BC provides the best prognostic correlations when TMAs are used.

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Storage Conditions and Immunoreactivity of Breast Cancer Subtyping Markers in Tissue Microarray Sections.

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