Stability of Phosphoprotein as a Biological Marker of Tumor Signaling

Baker, Amanda F.; Dragovich, Tomislav; Ihle, Nathan T.; Williams, Ryan; Fenoglio‐Preiser, Cecilia M.; Powis, Garth

doi:10.1158/1078-0432.ccr-05-0422

Cited by 147 publications

(136 citation statements)

References 19 publications

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“…The immunogenicity of the epitopes being measured may be subject to uncontrollable pre-analytical factors which artifactually alter their expression across the tissue section (such as cold ischemia time, or the length of fixation, and edge artifact). This is particularly a problem for phospho-epitopes, which are notoriously labile 14,15 . Therefore the technique might be better suited to resection specimens rather than small biopsies, and performed on multiple sections rather than just one.…”

Section: Discussionmentioning

confidence: 99%

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Faratian

Christiansen

Gustavson

et al. 2011

JoVE

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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...

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Section: Discussionmentioning

confidence: 99%

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Faratian

Christiansen

Gustavson

et al. 2011

JoVE

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“…Immunohistochemistry, on the other hand, is a widely accepted technique of assessing protein expression with much lower costs than microarrays, although validation and standardisation are also critical issues. However, it is also known that prognostic markers based on IHC can provide inconsistent or contradictory results, owing to the use of different antibodies and processing methods (Atkins et al, 2004;Baker et al, 2005), as well as different scoring and categorisation systems. All these issues emphasise the need for standardised processing and scoring procedures.…”

Section: Discussionmentioning

confidence: 99%

Combined assessment of EGFR pathway-related molecular markers and prognosis of NSCLC patients

et al. 2008

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The purpose of this study is to evaluate the prognostic value of the combined assessment of multiple molecular markers related to the epidermal growth factor receptor (EGFR) pathway in resected non-small cell lung cancer (NSCLC) patients. Tumour specimens of 178 NSCLC patients were collected and analysed for EGFR and KRAS mutation status by DNA sequencing, and for EGFR copy number by fluorescent in situ hybridisation. Tissue microarrays were generated and used to determine the expression of multiple EGFR pathway-related proteins by immunohistochemistry. We analysed the association between each marker and patient prognosis. Univariate analyses for each clinical variable and each molecular marker were performed using Kaplan -Meier curves and log-rank tests. From these results, we selected the variables KRAS mutations and expression of cytoplasmic EGFR, granular pERK, nuclear pSTAT3, cytoplasmic E-cadherin and cytoplasmic pCMET to enter into a Cox proportional hazards model, along with stage as the strongest clinical variable related with prognosis. Of the EGFR-related markers evaluated here, the markers EGFR, pERK, pSTAT3, E-cadherin, pCMET and mutations in KRAS were associated with survival when analysed in combination in our patient cohort, with P ¼ 0.00015 as the P-value for a test of the additional impact of markers on prognosis, after taking stage into consideration. Confirmation of the impact of these markers in independent studies will be necessary.

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“…HER2 phosphorylation was not assayed on this study and the three patients with HER2-overexpressing tumors that were enrolled on this study did not have on-treatment tumor biopsies for analysis. It should be noted that the use of immunohistochemical techniques on paraffin-embedded tissues using phosphospecific antibodies is fraught with technical problems that limits their reliability, and until newer techniques emerge, such studies should be interpreted with caution (Baker et al, 2005). Despite the technical problems associated with phosphoprotein immunostaining and the fact that these two studies were not designed to specifically determine target inactivation in HER2-overexpressing tumors at maximal drug dosage, they do appear to suggest that the drugs do reach their tumor targets and at least partially inactivate them.…”

Section: Evidence That Her Tkis Inhibit Her2 Function In Patientsmentioning

confidence: 99%