Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Abstract

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification ( Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor 1,2 , and some of these sub-clones give rise to metastatic (and therefore lethal) disease.Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension . A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue 5 , providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra-and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system 6 . Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index 7 .To d...

HER2 expression in ovarian carcinoma: caution and complexity in biomarker analysis

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“…Four (20%) cases with HER2-positive areas showed marked heterogeneity in expression by IHC, an example of which is shown in figure 1. This marked intratumoural heterogeneity has been noted previously,5 and we have recently demonstrated variable protein expression of HER2 in ovarian cancer using quantitative immunofluorescence 8…”

Quantum dots for quantitative imaging: from single molecules to tissue

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Tumour Heterogeneity of Breast Cancer: From Morphology to Personalised Medicine

“…The choice of the appropriate methodology depends on 3 factors: (i) the importance given to rare cell populations versus abundant cell populations, (ii) whether similarities exist across different cell populations, and (iii) the spatial scale(s) at which heterogeneity is quantified. Ecologists have studied diversity within biological systems for decades, resulting in the development of a number of diversity measures; these measures were adapted to quantify genetic [21], molecular [99,101,102], and microenvironmental [45] heterogeneity within breast tumours. The most cited measures are the Shannon entropy [103], the Simpson index [104], and the Rao quadratic entropy [105].…”