Biomarker Imaging Research Lab (BIRL), Sunnybrook Research Institute, 2075 Bayview Avenue, Toronto, ON, M4N 3M5, Canada.
Biosciences, GE Research (GER), Niskayuna, NY, USA.
Breast Cancer Res. 2021 Dec 18;23(1):114. doi: 10.1186/s13058-021-01475-y.
The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification.
Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures.
On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment.
Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.
乳腺癌细胞异质性的程度可能对诊断和长期预后有潜在影响。然而，病理学评估仅限于生物标志物免疫组织化学染色和肿瘤的形态学。细胞间生物标志物异质性通常未得到评估。作为对乳腺癌细胞异质性程度的初步评估，我们对代表圣加仑替代分类定义的亚型的组织微阵列（TMA）进行了雌激素受体（ER）、孕激素受体（PR）、表皮生长因子受体 2（HER2）、Ki67、TP53、CDKN1A（P21/WAF1）、CDKN2A（P16INK4A）、CD8 和 CD20 的定量和空间成像。
使用免疫荧光蛋白多重化平台（MxIF）进行单细胞定量成像，研究蛋白质共表达特征及其空间定位模式。将每个蛋白质标志物的 MxIF 强度值范围与 TMA 核心的相应 IHC 评分进行比较。使用共现矩阵和多样性指数测量来分析空间邻域中异质性的程度。
在研究的 59 例 101 个核心中，在激素受体阳性肿瘤核心中，ER 和 PR 的 MxIF 测量显示出不同的表达水平和分布。如预期的那样，Luminal A 样癌症表现出更高比例的共表达 ER 和 PR 的细胞群，而 Luminal B 样（HER2-阴性）癌症则由 ER+、PR- 组成。由 Ki67 阳性定义的增殖细胞主要存在于 PR 阴性细胞的细胞群中。三阴性乳腺癌（TNBC）表现出最高的增殖分数和异常 P53 和 P16 表达的发生率。在免疫组化显示 P53 过表达的肿瘤中，发现一组 TNBC 的 MxIF 测量的 P53 信号强度明显高于 HER2+、Luminal B 样和其他 TNBC 病例。CD8 和 CD20 细胞的密度在 HER2+癌症中最高。空间分析表明，在癌症和肿瘤微环境中，细胞邻域的异质性存在可变性。
蛋白质标志物多重化和定量图像分析显示，每种乳腺癌亚型的蛋白质共表达特征和细胞排列存在明显异质性。这些生物标志物表达和空间模式的精细描述符可能有助于开发更具信息量的工具，以指导诊断和治疗。
