00019606-200903000-00002ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2009 Lippincott Williams & Wilkins, Inc.18March 2009
p 11-21A Novel Proximity Assay for the Detection of Proteins and Protein Complexes: Quantitation of HER1 and HER2 Total Protein Expression and Homodimerization in Formalin-fixed, Paraffin-Embedded Cell Lines and
Breast Cancer TissueOriginal ArticlesShi, Yining PhD; Huang, Weidong MD; Tan, Yuping MS; Jin, Xueguang MS; Dua, Rajiv PhD; Penuel, Elicia PhD; Mukherjee, Ali PhD; Sperinde, Jeff PhD; Pannu, Herjit MD; Chenna, Ahmed PhD; DeFazio-Eli, Lisa PhD; Pidaparthi, Sailaja PhD; Badal, Youssouf PhD; Wallweber, Gerald PhD; Chen, Lili MD; Williams, Steve PhD; Tahir, Hasan PhD; Larson, Jeff PhD; Goodman, Laurie PhD; Whitcomb, Jeannette PhD; Petropoulos, Christos PhD; Winslow, John PhDDepartments of Research and Development, Clinical Research, and Operations, Monogram Biosciences Inc, South San Francisco, CASupported by Monogram Biosciences Inc.Reprints: John W. Winslow, PhD, Research and Development (Oncology), Monogram Bioscience Inc, 345 Oyster Point Boulevard, South San Francisco, CA 94080 (e-mail:
[email protected]).AbstractThe availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.ArticlePlusClick on the links below to access all the ArticlePlus for this article.Please note that ArticlePlus files may launch a viewer application outside of your web browser.http://links.lww.com/PDM/A6http://links.lww.com/PDM/A7http://links.lww.com/PDM/A8http://links.lww.com/PDM/A9http://links.lww.com/PDM/A10http://links.lww.com/PDM/A11The epidermal growth factor receptor (EGFR), or HER/erbB family of receptor tyrosine kinases, is widely recognized as a component of signal transduction networks that are dysregulated in several major cancers.1–4 EGFR/HER1/erbB1 and HER2/neu/erbB2 are members of the EGFR family, which are genetically amplified or overexpressed in approximately 40% of nonsmall cell lung cancers (NSCLCs) and approximately 30% of breast cancers, respectively.5–7 Several antibody-based and small molecule-based inhibitors of the HER kinases have been developed as therapeutics and widely tested. Although responses to these agents have been encouraging in some cases, their effectiveness has not been as widespread as predicted. In this respect, only approximately 50% of patients with HER2-positive metastatic
breast cancer receiving the anti-HER2 antibody Herceptin (trastuzumab) derive significant clinical benefit.8–10 A similar picture has emerged for the prediction of responses to drugs targeted at EGFR in NSCLC.11–14 There remains a need for new diagnostic tests that are able to identify patient subgroups that may respond to HER1-targeted and HER2-targeted drugs.Currently, 2 commercially available testing methods are used in the clinical evaluation of HER1 and HER2 status in NSCLC and
breast cancer patient samples, respectively. Immunohistochemistry (IHC) analysis detects HER protein expression levels and fluorescence in situ hybridization (FISH) detects the amplification status of the HER genes.15,16 IHC provides a semiquantitative measure of protein levels, whereas FISH, a quantitative measure of gene amplification, is not always representative of protein expression. Neither test provides quantitative data that reflect the activation state of signaling pathways in tumors, which may limit their utility in patient selection.To determine whether continuous and quantitative measures of signaling protein expression levels and activation states can better correlate and predict patient responses to targeted therapies, we developed the VeraTag assay. This assay format is based on the proximity and light-dependent release of antibody-conjugated fluorescent reporter tags, each with unique migration properties for capillary electrophoresis (CE). The enhanced specificity and sensitivity resulting from the required proximal binding of 2 antibodies, coupled with the analytical power of CE, provide the capability needed to detect molecular markers in samples such as formalin-fixed, paraffin-embedded (FFPE) tumor specimens. Furthermore, the proximity requirement for the assay signal enables the quantitative measure of protein-protein interactions as surrogate markers of signaling pathway activation. Specifically, HER receptor dimerization is documented upon ligand activation or as a result of gene amplification, overexpression, or mutation,2–4,17 and may potentially indicate the dependence of tumor growth on HER signaling pathways. In this paper, we characterize VeraTag assays for HER1 and HER2 protein expression and homodimerization in FFPE human cancer cell lines, and compare quantitative HER2 protein levels in breast tumor tissue with those measured by standard IHC methods. In a companion paper,18 the quantitative measure of HER2 protein expression or HER2-HER2 dimer levels determined by VeraTag assays of patient samples may correlate with outcomes after Herceptin treatment (HER1-HER1 and HER2-HER2 homodimerization is defined in this assay as 2 HER1 or HER2 receptors located within close proximity of one another, including true HER1-HER1 or HER2-HER2 homodimers that interact along a defined protein-protein interface, and HER1 or HER2 receptors in dense arrays (30 to 100 nm) as a result of overexpression.)MATERIALS AND METHODSAntibodies, VeraTag-antibody, Biotin-antibody Conjugates, and “Molecular Scissors”Monoclonal antibodies Ab15 and Ab10 directed against HER1 intracellular and extracellular domains, respectively, and Ab8 and Ab15 directed against the intracellular domain of HER2 (product inserts) were purchased from Lab Vision. Mouse monoclonal antihuman IgG1 isotype control antibody was purchased from Sigma, biotinylated mouse antihuman IgG1 isotype control antibody from BD Biosciences, and antiphosphotyrosine antibody 4G10 from Upstate Biotechnology. Fluorescent reporter group “tags” (Pro11, Pro14, Pro201) and streptavidin-conjugated methylene blue (SA-MB: “molecular scissors”) were synthesized and purified according to protocol described earlier (US Patent 7,105,308). Antibody-fluorescent tag and antibody-biotin conjugates, Ab10-biotin, Ab15-Pro11, Ab8-Pro11, Ab15-biotin, and 4G10-Pro201, were made using sulfo-NHS-LC-LC-biotin (Pierce) according to the manufacturer's protocol, and the conjugation products were purified by high performance liquid chromatography (Agilent). The substitution ratio is generally 2 to 4 mole/mole.Cell Culture, Fixation, Processing, and Paraffin EmbeddingAll cancer cell lines were purchased from American Type Culture Collection and were maintained at 37°C and 5% CO2 in Dulbecco modified Eagle medium: F12 (50:50), 10% fetal bovine serum, 1% Pen-Strep, and 2 mM L-glutamine. To generate FFPE tissue blocks, ten 150-mm culture plates per cell line were washed and fixed overnight with 10% neutral-buffered formalin at 4°C. Cells were made into FFPE blocks by scraping, centrifugation, packing the cell pellet into a plastic O-ring, and processed using a Tissue-Tek processor and standard protocols, and paraffin-embedded. Frozen breast tissues with different HER2 expression levels were purchased from Biooptions, and were fixed, processed, and paraffin-embedded as above without O-ring packing. Sections of 5 μm thickness for both cell line and tumor tissue FFPE blocks were placed on positively charged glass slides (VWR), air-dried for 30 minutes, and then baked at 60°C for 1 hour. All sample slides were stored at 4°C until assay and generally used within 3 to 4 weeks.VeraTag HER Assays in FFPE Cell Lines and Breast TissuesFFPE samples were deparaffinized/rehydrated by a standard sequence of increasingly polar solvents. Heat-induced epitope retrieval used citrate buffer (pH 6.0) (Lab Vision) boiled in a microwave oven (Spacemaker II, GE). After cooling for 1 hour and rinsing with water, a hydrophobic circle was drawn on the slide to retain reagents in a defined area. Sections were blocked for 1 hour [1% mouse serum, 1.5% bovine serum albumin, Roche protease, and phosphatase inhibitors in 1× phosphate-buffered saline (PBS)], followed by incubation with reporter tag-conjugated and biotin-conjugated antibodies (1 to 4 μg/mL) overnight in a humidified chamber at 4°C. Samples were aspirated and rinsed with 0.25% Triton X-100 in 1× PBS. SA-MB (2.5 μg/mL in 1× PBS) was added to sections and incubated for 1 hour, aspirated, and the samples were washed with Triton buffer and deionized water. Illumination buffer (IB) containing 3 pM fluorescein and 2 CE markers in 0.01× PBS was added to sample sections. Reporter tags were released at approximately 4°C by a customized LED array illuminator equipped with a chiller block (Torrey Pine Scientific). Samples were collected from the slides and the tags were separated and detected on an ABI3100 CE instrument under CE injection conditions of 6 kV, 50 s at 30°C (22-cm array; Applied Biosystems).IHC and Hematoxylin and Eosin StainingHER2 IHC was performed on the Ventana Discovery XT system according to the manufacturer's instructions for the CB11 antibody. For HER1 IHC, the same protocol was used except blocking with 1% bovine serum albumin in 1× PBS for 1 hour, followed by HER1 Ab15 (Lab Vision) at 2 μg/mL overnight at 4°C.Data AnalysisThe identification and quantification of VeraTag assay reporter tags are performed by Monogram developed software that recognizes fluorescent peaks from a CE electropherogram through the relative elution time of 2 internal markers. Fluorescein included in the illumination recovery buffer at a specified concentration (3 pM) is used to correct for recovery. The CE fluorescence signal or peak area of each HER reporter tag is calculated as the peak height integrated over the peak elution time. The HER reporter tag peak area is then normalized to the peak area of the internal standard fluorescein, resulting in the relative peak area (RPA), which is proportional to the concentration of the HER analyte. The final quantification unit is RPA×IB/TA [RPA multiplied by the IB volume added onto the sample section, divided by the tumor area (TA) in mm2, and with units of pmol/mm2].Titration of Sample Section Size and Estimation of TACell line sections (5 μm in thickness; ∼50 mm2, containing 180,000 to 200,000 cells) of a decreasing area were trimmed with a razor blade. After the assay, the sections were air-dried and scanned using a commercial flatbed scanner. Section area (mm2) was measured using ImageJ software.19 For titration of TA, multiples of microtome-cut adjacent sections of breast tumor tissues were captured on a single slide. Post-VeraTag assay, slides were stained with hematoxylin and eosin (H&E), and the TA was circled and calculated by a certified pathologist using standard criteria for invasive carcinoma. Tumor samples with significant tumor necrosis or ductal carcinoma in situ were excluded.VeraTag and IHC Assay Crossvalidation in Human Breast TumorsOne hundred and seventy tumors were processed by FFPE, and 5-μm sections were evaluated for tumor content by H&E staining. Samples were assayed by VeraTag assay as described, and compared with HER2 IHC staining intensity quantified by standard test categories and by using a histologic score (H-score) that corrects for the relative percentages of different staining test categories (H-score=%IHC3+×3+%IHC2+×2+%IHC1+×1+%IHC0×0). All HER2 VeraTag measurements were adjusted for the IB initial volume, and normalized to TA (RPA×IB/TA).HER1-HER1 and HER2-HER2 Homodimer/Proximer Assays, Chemical Crosslinking, and ImmunoprecipitationBT-20 cells were grown in 150 mm plates, serum-starved overnight, and treated with 0, 1, 5, and 50 nM EGF for 10 minutes at 37°C. Cells were washed and either prepared for FFPE blocks for VeraTag assays as described above, or crosslinked with the aqueous-soluble agent BS3.20 After crosslinking, cells were lysed with Triton lysis buffer and centrifuged, and 250 to 500 μg of lysate protein was immunoprecipitated with either HER1 Ab15 or HER2 Ab8 antibodies.20 Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblotting was applied.RESULTSDevelopment of VeraTag HER Expression Assays for FFPE Tumor CellsAn outline of the FFPE VeraTag assay and the individual steps are shown for HER receptor assays in Figure 1 and described in detail in the Methods section. The main steps of the assay are (1) binding of the fluorescent reporter tag-conjugated and biotin-conjugated antibody pair to their analytes in tissue sections; (2) incubation with streptavidin conjugated with the photosensitizer methylene blue (SA-MB); and (3) illumination with 670 nm light during which the photosensitizer bound to the biotin antibody converts dissolved oxygen to a more reactive, singlet state oxygen (1O2) in the buffer solution. The 1O2 molecules are short-lived,21 and their limited diffusion results in the proximity-based cleavage of a thioether bond and release of the fluorescent tag from the tissue-bound antibody (Fig. 1B, C); (4) applied to conventional CE instruments, (5) the released tags are separated and detected as a fluorescence peak in an electropherogram (Figs. 1A, 2A, C). The 2 VeraTag proximity assay formats characterized in this study measure HER protein expression (Fig. 1B) or HER-HER homodimer levels (Fig. 1C) (To estimate the proximal selectivity of 1O2-mediated cleavage, a simple model of the competition between quenching and diffusion of 1O2 can be conceived as concentration ofJOURNAL/dimp/04.03/00019606-200903000-00002/math_2MMU1/v/2021-02-17T200000Z/r/image-tiff; where k=quenching rate, D=1O2 diffusion rate, and r=radial distance from 1O2 generating source (Sperinde J and Williams S, manuscript in preparation). This equation can then be used to predict the fall-off in Csinglet O2 relative to the approximate distance between antibodies bound to dimerized targets of approximately 30 nm. Using the quenching rate of k=3×105/s and diffusion constant of DO2=1.2×10−5 cm2/s, Csinglet O2 is approximately 10% and 0.14% of the level 30 nm from the source, at 100 nm and 300 nm, respectively. It is possible that the resolution resulting in significant thioether cleavage may be largely within the 30 to 100 nm radius from the 1O2 source).JOURNAL/dimp/04.03/00019606-200903000-00002/figure1-2/v/2021-02-17T200000Z/r/image-jpeg
A to C, VeraTag assay. A, Assay steps and workflow. The figure represents the 5 main steps of the VeraTag assay as described in the Results. B, VeraTag HER protein expression assay represented by HER2. The figure depicts the proximal antibody binding relationship between conjugated HER2 Ab8 and Ab15, and subsequent light-dependent formation of singlet oxygen and chemical cleavage of the fluorescent reporter tag (Pro11) from Ab8. The released product is a benzyl-fluorescein carboxylic acid quantified by CE. The singlet oxygen sensitizer SA-MB, bound to biotinylated Ab15, is represented as an “S.” C, VeraTag HER homodimer assay represented by HER2. The figure depicts the binding relationship between HER2 Ab8 conjugated with the Pro11 reporter group or biotin to a single epitope on HER2, resulting in a proximal antibody pair as a consequence of HER2 homodimerization. The remaining assay steps are the same as in part B. CE indicates capillary electrophoresis; SA-MB, streptavidin-conjugated methylene blue.JOURNAL/dimp/04.03/00019606-200903000-00002/figure2-2/v/2021-02-17T200000Z/r/image-jpeg
A and B, VeraTag HER1 expression assay. A, Assay signal is shown as a CE electropherogram fluorescent peak of the Pro11 tag released from HER1 bound Ab15 (HER1), and a CE marker (fluorescein). Shown below in parallel are HER1 IHC signals for tumor cell lines expressing increasing levels of HER1. Corresponding IHC staining category for each cell line is presented on the bottom. B, Quantification of VeraTag HER1 expression assay shown in A. Signal is represented as relative peak area (RPA) of Pro11-tag fluorescent units to that of 3 pM fluorescein internal standard. C and D, VeraTag HER2 expression assay. Description of the data is the same as in parts A and B except for HER2. Both assays performed with 4 μg/mL antibody concentrations. CE indicates capillary electrophoresis; IHC, immunohistochemistry.HER Antibody and VeraTag Assay Characterization, Optimization, and SpecificityMultiple HER1 and HER2 antibodies were conjugated with either fluorescent reporter tags or biotin, and specific combinations that generated CE fluorescent signals paralleling expected HER IHC results were identified (Figs. 2A-D; Refs. 22,23). The final HER1 and HER2 antibody pairs selected for assay development bound with KD of 2 to 4 μg/mL, and approached saturation at 4 to 6 μg/mL (supplemental Figure 1). Optimized antibody concentrations that produced the highest signal to background ratios near saturation were chosen. HER2 protein levels measured under these conditions are linearly proportional to nondenatured HER2 receptor levels quantified by fluorescence-activated cell sorting or enzyme-linked immunosorbent assay (Table 1; data not shown). HER antibodies were specific for their targeted analytes, were recognized as distinct, nonoverlapping epitopes in competition experiments, and exhibited 5% to 20% background at the high and low end of the assay dynamic range, respectively (supplemental Figure 2). These results indicate the specific antibody recognition and signal generation of the VeraTag assay over a broad range of HER receptor levels.JOURNAL/dimp/04.03/00019606-200903000-00002/table1-2/v/2021-02-17T200000Z/r/image-tiff Summary of FACS Analysis of HER1 and HER2 Expression in Cell Lines and HER2 VeraTag Assay DataVeraTag HER1-HER1 and HER2-HER2 Homodimer AssaysHER2 weakly associates to form homodimers and can be stabilized by Hsp90 when overexpressed in gene-amplified breast tumor cells such as SKBR-3 cells.24 HER1 homodimers can be stabilized at much lower receptor levels with activating ligands such as EGF and transforming growth factor-α.17,20,24,25 An antibody proximity format similar to the VeraTag HER total protein assay was used to develop assays compatible with the measure of HER1-HER1 and HER2-HER2 proximity and homodimerization (Fig. 1C). Optimization of an antibody proximity pair consisting of equal concentrations of HER1 Ab15 conjugated with Pro11 fluorescent reporter tag, or biotin “molecular scissors” (Fig. 3A), resulted in an EGF dose-dependent increase in HER1-HER1 homodimerization (3-fold to 4-fold) and autophosphorylation (5-fold to 8-fold) levels in BT-20 cells whereas HER1 total levels remain constant (Fig. 3C). Similar results are observed in intact formalin-fixed BT-20 cells (data not shown). VeraTag assay homodimer levels parallel the increase in homodimerization detected by chemical crosslinking and immunoprecipitation (Fig. 3B). These results show the ability of the VeraTag assay to measure changes in receptor interactions resulting from activation.JOURNAL/dimp/04.03/00019606-200903000-00002/figure3-2/v/2021-02-17T200000Z/r/image-jpeg
A to C, VeraTag HER1-HER1 homodimer assay. Assay signal is presented as relative peak area (RPA) of Pro11 reporter group (Methods). A, Titration of HER1 Ab15-Pro11 (reporter group) and Ab15-biotin (scissors) in a VeraTag HER1-HER1 homodimer assay using EGF-stimulated (0, 5, 50 nM) and FFPE-processed BT-20 cells. B, HER1-HER1 homodimer crosslinking assay. BT-20 cells were EGF stimulated, BS3 crosslinked, and lysed, immunoprecipitated, and Western blotted to measure homodimerization and autophosphorylation. C, VeraTag FFPE cell assays of HER1 homodimerization, autophosphorylation, and total expression performed with cells stimulated and generated in part B. EGF indicates epidermal growth factor; FFPE, formalin-fixed, paraffin-embedded.A similar VeraTag assay format was applied to the analysis of HER2-HER2 homodimerization in FFPE sections of breast tumor cell lines. Antibody titration experiments performed with a HER2 Ab8 proximity pair (Fig. 1C) resulted in concentration-dependent and saturating assay signals (Fig. 4A). Although lacking an activating ligand that promotes homodimerization, the relative level of HER2-HER2 homodimers is consistent with the expected levels based on total HER2 expression and monomer-dimer equilibrium13,17,20,24,26–30 (ie SKBR-3≅BT-474>MDA-MB-453). Similar relative HER2 homodimer levels are detected in the same cell lines under optimized signal-to-background conditions (2 μg/mL Ab8), and by crosslinking and immunoprecipitation (Figs. 4B, C). Parallel results were observed with an independent titration of a HER2 Ab15 proximity pair (data not shown). Replacing Ab8-biotin with biotinylated control antibodies (mouse antihuman IgG1, cytokeratin, HER1, PTEN) indicated a background of less than 10% of HER2-HER2 homodimer signal in cell lines and tumors, consistent with the expected assay proximity and specificity (data not shown).JOURNAL/dimp/04.03/00019606-200903000-00002/figure4-2/v/2021-02-17T200000Z/r/image-jpeg
A to C, VeraTag HER2-HER2 homodimer assay. A, Optimization of HER2-HER2 homodimer assay signal to background of Ab8-Pro11 (fluorescent tag) and Ab8-biotin (scissors) in FFPE cell lines expressing HER2. Assay signal is presented as relative peak area (RPA—Methods). B, HER2-HER2 homodimer crosslinking assay. After crosslinking and lysis, equivalent amounts of HER2 protein from each cell line (∼20 μg) were immunoprecipitated and Western blotted with the indicated antibodies. C, VeraTag FFPE cell assays of HER2-HER2 homodimers and HER2 expression performed with cells generated in part B. The section size (cell number) for SKBR-3 and BT-474 was adjusted to match the total HER2 expression in approximately 50 mm2 sections of MDA-MB-453 cells, allowing direct comparison of the relative level of HER2-HER2 homodimers. The assay signal is presented as the normalized relative peak area of the reporter tag described in the Methods. FFPE indicates formalin-fixed, paraffin-embedded.Comparison of VeraTag Assay Measurement of HER2 Protein and HER2-HER2 Homodimerization With IHC in Human Breast TumorsThe HER2 protein level measured in FFPE breast tumor tissue sections can vary as a consequence of the tumor content, as well as differences in the receptor level per cell. Therefore, it is necessary to normalize the HER2 protein and homodimer values to a measure of the tumor content: TA stained with H&E. TA normalization was technically verified by showing that VeraTag HER2 protein signal increases linearly with sectional area (5 to 250 mm2) for breast tumors and cell lines (supplemental Figures 3 and 4). To evaluate the accuracy of the VeraTag assay, VeraTag HER2 protein and homodimer levels normalized to TA (range, 10 to 140 mm2) were compared with HER2 IHC in 170 human
breast cancer samples. In contrast to conventional IHC test categories, the HER2 protein levels determined by VeraTag assay represent a continuous measurement over a dynamic range >2 log10 (Fig. 5A). The correlation is significant (P<0.001), but the VeraTag HER2 expression levels overlapped with adjacent IHC categories indicating increased sample discrimination. The IHC histoscore, a measure that corrects for the percentage of HER2-positive cells, correlates well with the lower range of VeraTag levels, but displays a plateau at high levels whereas the VeraTag assay extends the HER2 measurement (Fig. 5B). A correlation is observed between levels of HER2 expression and HER2-HER2 homodimers measured by VeraTag assays (r2=0.7, P<0.001; data not shown). These results show that HER2 expression and HER2-HER2 homodimer levels in FFPE human
breast cancer tissue samples can be reliably measured when compared with a reference assay, yet differ from HER2 IHC scoring in several respects.JOURNAL/dimp/04.03/00019606-200903000-00002/figure5-2/v/2021-02-17T200000Z/r/image-jpeg
A and B, Comparison of VeraTag HER2 expression levels with HER2 IHC. A, Comparison with IHC test categories. Adjacent microtome sections from 170
breast cancer samples were either assayed by VeraTag HER2 protein expression assay or by HER2 IHC. The distribution of the VeraTag HER2 expression assay signal, presented as the normalized relative peak area (RPA) of Pro11 tag (Methods) is plotted within each HER2 IHC category. B, The HER2 IHC histoscore (H-score; Methods) is compared with the corresponding VeraTag HER2 expression assay signal. IHC indicates immunohistochemistry.VeraTag HER2 Assay Reproducibility and PerformanceReproducibility studies were performed to assess assay variability and develop methods for the comparison of VeraTag data from multiple studies. To summarize, the observed variability within replicate samples was generally 10% to 20% for a single operator performing 10 separate VeraTag HER2 protein and HER2-HER2 homodimer assays (supplemental Figure 5A). This level of variation allows for the correction of each assay relative to a reference set of results, using HER2-expressing cell lines as controls. The normalization method was applied to large-scale testing by multiple operators, resulting in coefficients of variation ranging from 11% to 29% (supplemental Figure 5B). A similar performance is observed for the HER2-HER2 homodimer assay (data not shown).The assay normalization method allowed initial characterization of assay properties such as precision and upper and lower limits of detection. A standard curve of VeraTag assay levels of HER2 expression and HER2-HER2 homodimers was generated from a panel of 12 cell lines, and was proportional to a range of fluorescence-activated cell sorting-determined receptor levels of approximately 600,000 to 1,000,000 HER2/cell to approximately 10,000 to 20,000 HER2/cell (Table 1; Fig. 6A). This proportionality with the native receptor is linear (R2=0.9), as is HER2 VeraTag FFPE measurement compared with HER2 ELISA of cell lysates (R2=0.94; Table 1 and data not shown). A significant signal is observed down to 40,000 to 60,000 MCF-7 cells (supplemental Figure 3), whereas signals for MDA-MB-453 and SKBR-3 are well above the background when measured on approximately 5 to 10 mm2 sections (20,000 to 40,000 cells).JOURNAL/dimp/04.03/00019606-200903000-00002/figure6-2/v/2021-02-17T200000Z/r/image-jpeg
A and B, FFPE cell line standard curve: VeraTag HER2 proximity assay comparison with HER-2 receptor levels. A, HER2 expression. VeraTag HER2 protein expression assays were performed on multiple sections for each cell line (n=14 assays). Signal is represented as the mean and standard deviation (y-error bars) of the relative peak area normalized to tumor area and corrected for systematic assay variation (Methods; supplemental Figure 5). The VeraTag HER2 levels are plotted against the mean and standard deviation (x-error bars) of the HER2 receptor number per cell determined by FACS (n=3). B, VeraTag HER2-HER2 homodimer assays were performed on multiple sections for each cell line; each section adjacent to the section assayed for HER2 expression at the same time (n=14 assays). VeraTag assay signal and FACS receptor numbers are represented as in A. All sections are approximately 50 mm2, containing 180,000 to 200,000 cells. FACS indicates fluorescence-activated cell sorting; FFPE, formalin-fixed, paraffin-embedded.The HER2--HER2 homodimer signal is detectable at receptor levels of approximately 60,000 to 100,000 HER2/cell, respectively (180,000 to 200,000 cells). The limit of detection for IHC category 2+ MDA-MB-453 cell line is a sectional area of approximately 20 mm2 (80,000 to 100,000 cells; Fig. 6B; data not shown). High HER2-expressing cell lines such as SKBR-3 and BT-474 have a significant signal down to the tested levels of approximately 5 to 10 mm2 section size (20,000 to 40,000 cells). Taken together, these studies show that the VeraTag assays can sensitively and accurately measure a broad range of HER2 expression, HER2--HER2 homodimerization, and tumor content.DISCUSSIONIn this paper, we present a novel technology for the quantitation of HER protein expression levels in FFPE tumor cell lines and tissues, and also a measure consistent with their functional status through the quantitation of receptor homodimerization. The basis of the technology is a chemically modified antibody pair that is required to bind to their analyte(s) in close proximity to release a small fluorescent reporter group in a light-dependent and singlet oxygen-dependent reaction. This requirement results in an assay with high specificity, sensitivity, and quantitation over a broad dynamic range that is proportional to HER2 receptor levels. Antibody proximity binding also enables detection of protein-protein interactions such as HER1-HER1 and HER2-HER2 homodimers when configured with the same antibody conjugated with fluorescent reporter groups or biotin plus SA-MB “molecular scissors.” That clinically meaningful information regarding the response to HER2-targeted therapies may be gained from quantitative HER2 expression and the HER2-HER2 homodimer measurements are supported by recent VeraTag assay studies on HER2-positive metastatic
breast cancer patients treated with Herceptin.18,31–33HER2 protein levels measured by the VeraTag proximity assay and HER2 IHC correlate in human breast tumor samples assayed in parallel; however, several differences were observed. These include the overlapping distribution of HER2 expression levels detected by the VeraTag assay when compared with IHC test categories (Fig. 5A). Furthermore, although VeraTag HER2 expression levels are consistent with an HER2 histoscore at low HER2 levels, at higher levels the histoscore plateaus, while VeraTag HER2 expression levels extend the dynamic range of the HER2 measurement (Fig. 5B). Similar results were obtained in different clinical cohorts comprising nearly 200
breast cancer tumor samples.18,31–32 In parallel studies, we have also observed that HER2 gene amplification measured by FISH (HER2/CEP17 ratio and gene copy number) loosely correlates with IHC categories and VeraTag HER2 expression levels, suggesting that HER protein levels may not always strongly correspond with gene amplification.18,31 These results indicate the accuracy of the VeraTag HER2 assay measurement when compared with accepted methods, but also support the idea that novel information may be gained.In addition to being quantitative and sensitive, the VeraTag technology also offers the unique ability to measure protein-protein interactions in FFPE tumor tissue, notably the functional responses resulting from HER1 and HER2 homodimerization, and HER2 heteroassociation with HER1 or HER3. VeraTag technology is capable of tracking EGF-dependent increases in HER1-HER1 homodimers and autophosphorylation both in fixed cells and FFPE format (Fig. 3). Similar validation for a VeraTag HER2 homodimer assay is challenging. HER2, being an orphan receptor, does not form homodimers in response to HER family ligands, but is thought to form weak homodimers in equilibrium with monomers that are dependent on protein expression levels.3,4,17,24,29 Furthermore, HER2 has been proposed to cluster within secondary domains such as receptor patches or rafts26,27 when overexpressed in
breast cancer cells, or localized in specialized membrane protrusions.30 A significant fraction of highly expressed HER2 may exist as homodimers stabilized by Hsp90.24 In this study, we show that a single HER2 antibody, when conjugated with a fluorescent tag or biotin to form a VeraTag assay proximity pair, generates a titratable and saturable signal in
breast cancer cell lines, which parallels crosslinked levels of HER2 homodimers. Interestingly, the VeraTag HER2-HER2 homodimer signal becomes significant only in cell lines expressing above 105 receptors/cell, a level above which correlates with cell transformation because of the proposed spontaneous homodimerization upon transfection and overexpression.28,34 The VeraTag HER2-HER2 homodimer assay was also shown to be proximity-specific with respect to the biotinylated antibody partner, as control antibodies result in low signals near background levels (data not shown). Although we cannot at this time definitively distinguish between detection of HER2 homodimers and receptors in close proximity, our results are consistent with independent studies supporting a model of overexpressed HER2 existing as associations of weak homodimers.13,17,20,24,26–29,35 Studies are in progress to better characterize the proximity resolution and association state of HER1 and HER2 detected by the VeraTag assays.Aside from the HER1 and HER2 assays described here, VeraTag assays measuring the status of all HER protein levels, including heterodimerization of HER2 and p95-HER2 truncated forms, could potentially be of significant use as biomarkers for drug responsiveness, such as for breast cancers coexpressing multiple HER family members.22,36–38 Such assays could predict the HER functional signaling pathways in tumor samples and contribute to the identification of drug resistance mechanisms.ACKNOWLEDGMENTSThe authors thank Drs Michael Bates and Gordon Parry for important input and critical reading of the manuscript, Drs Linda Kiss, Agnes Paquet, and Colombe Chappey for data analysis and statistical methods development, and Jolly Bose and Kristi Frankson for excellent technical assistance with some of the experiments.REFERENCES1. Hunter T. Signalling-2000 and beyond. Cell. 2000;100:113–127.[Context Link][CrossRef][Medline Link]2. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000;103:211–215.[Context Link][CrossRef][Medline Link]3. Yarden Y, Sliwkowski MX. Untangling the ErbB signaling network. Nat Rev Mo Cell Biol. 2001;2:127–137.[Context Link]4. Hynes NE, Lane HA. ERRB receptors and cancer: the complexity of targeted inhibitors. Nature Rev Cancer. 2005;5:341–354.[Context Link]5. Hirsh FR, Varella-Garcia M, Bunn PA, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol. 2003;21:3798–3807.[Context Link][Full Text][CrossRef][Medline Link]6. Pegram MD, Slamon DJ. Biological rational for HER-2/neu (c-erbB2) as a target for monoclonal antibody therapy. Semin Oncol. 2000;27:13–19.[Context Link][Medline Link]7. Slamon DJ, Clark DM, Wong SG, et al. Human
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Clin Cancer Res. 2006;12:424–431.[Context Link][Medline Link]Supplementary dataSupplemental Figures 1–4 can be viewed online at http://www.molecularpathology.com.JOURNAL/dimp/04.03/00019606-200903000-00002/figure7-2/v/2021-02-17T200000Z/r/image-jpegsupplementary Figure 1.JOURNAL/dimp/04.03/00019606-200903000-00002/figure8-2/v/2021-02-17T200000Z/r/image-jpegsupplementary Figure 2.JOURNAL/dimp/04.03/00019606-200903000-00002/figure9-2/v/2021-02-17T200000Z/r/image-jpegsupplementary Figure 3.JOURNAL/dimp/04.03/00019606-200903000-00002/figure10-2/v/2021-02-17T200000Z/r/image-jpegsupplementary Figure 4AB.JOURNAL/dimp/04.03/00019606-200903000-00002/figure11-2/v/2021-02-17T200000Z/r/image-jpegsupplementary Figure 4C.JOURNAL/dimp/04.03/00019606-200903000-00002/figure12-2/v/2021-02-17T200000Z/r/image-jpegsupplementary Figure 5.HER/erbB receptors; HER1; HER2; protein quantitation; receptor dimerization; FFPE tumors; proximity immunoassay; singlet oxygen; capillary electrophoresis; breast 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A to C, VeraTag assay. A, Assay steps and workflow. The figure represents the 5 main steps of the VeraTag assay as described in the Results. B, VeraTag HER protein expression assay represented by HER2. The figure depicts the proximal antibody binding relationship between conjugated HER2 Ab8 and Ab15, and subsequent light-dependent formation of singlet oxygen and chemical cleavage of the fluorescent reporter tag (Pro11) from Ab8. The released product is a benzyl-fluorescein carboxylic acid quantified by CE. The singlet oxygen sensitizer SA-MB, bound to biotinylated Ab15, is represented as an “S.” C, VeraTag HER homodimer assay represented by HER2. The figure depicts the binding relationship between HER2 Ab8 conjugated with the Pro11 reporter group or biotin to a single epitope on HER2, resulting in a proximal antibody pair as a consequence of HER2 homodimerization. The remaining assay steps are the same as in part B. CE indicates capillary electrophoresis; SA-MB, streptavidin-conjugated methylene blue.
A and B, VeraTag HER1 expression assay. A, Assay signal is shown as a CE electropherogram fluorescent peak of the Pro11 tag released from HER1 bound Ab15 (HER1), and a CE marker (fluorescein). Shown below in parallel are HER1 IHC signals for tumor cell lines expressing increasing levels of HER1. Corresponding IHC staining category for each cell line is presented on the bottom. B, Quantification of VeraTag HER1 expression assay shown in A. Signal is represented as relative peak area (RPA) of Pro11-tag fluorescent units to that of 3 pM fluorescein internal standard. C and D, VeraTag HER2 expression assay. Description of the data is the same as in parts A and B except for HER2. Both assays performed with 4 μg/mL antibody concentrations. CE indicates capillary electrophoresis; IHC, immunohistochemistry. Summary of FACS Analysis of HER1 and HER2 Expression in Cell Lines and HER2 VeraTag Assay Data
A to C, VeraTag HER1-HER1 homodimer assay. Assay signal is presented as relative peak area (RPA) of Pro11 reporter group (Methods). A, Titration of HER1 Ab15-Pro11 (reporter group) and Ab15-biotin (scissors) in a VeraTag HER1-HER1 homodimer assay using EGF-stimulated (0, 5, 50 nM) and FFPE-processed BT-20 cells. B, HER1-HER1 homodimer crosslinking assay. BT-20 cells were EGF stimulated, BS3 crosslinked, and lysed, immunoprecipitated, and Western blotted to measure homodimerization and autophosphorylation. C, VeraTag FFPE cell assays of HER1 homodimerization, autophosphorylation, and total expression performed with cells stimulated and generated in part B. EGF indicates epidermal growth factor; FFPE, formalin-fixed, paraffin-embedded.
A to C, VeraTag HER2-HER2 homodimer assay. A, Optimization of HER2-HER2 homodimer assay signal to background of Ab8-Pro11 (fluorescent tag) and Ab8-biotin (scissors) in FFPE cell lines expressing HER2. Assay signal is presented as relative peak area (RPA—Methods). B, HER2-HER2 homodimer crosslinking assay. After crosslinking and lysis, equivalent amounts of HER2 protein from each cell line (∼20 μg) were immunoprecipitated and Western blotted with the indicated antibodies. C, VeraTag FFPE cell assays of HER2-HER2 homodimers and HER2 expression performed with cells generated in part B. The section size (cell number) for SKBR-3 and BT-474 was adjusted to match the total HER2 expression in approximately 50 mm2 sections of MDA-MB-453 cells, allowing direct comparison of the relative level of HER2-HER2 homodimers. The assay signal is presented as the normalized relative peak area of the reporter tag described in the Methods. FFPE indicates formalin-fixed, paraffin-embedded.
A and B, Comparison of VeraTag HER2 expression levels with HER2 IHC. A, Comparison with IHC test categories. Adjacent microtome sections from 170
breast cancer samples were either assayed by VeraTag HER2 protein expression assay or by HER2 IHC. The distribution of the VeraTag HER2 expression assay signal, presented as the normalized relative peak area (RPA) of Pro11 tag (Methods) is plotted within each HER2 IHC category. B, The HER2 IHC histoscore (H-score; Methods) is compared with the corresponding VeraTag HER2 expression assay signal. IHC indicates immunohistochemistry.
A and B, FFPE cell line standard curve: VeraTag HER2 proximity assay comparison with HER-2 receptor levels. A, HER2 expression. VeraTag HER2 protein expression assays were performed on multiple sections for each cell line (n=14 assays). Signal is represented as the mean and standard deviation (y-error bars) of the relative peak area normalized to tumor area and corrected for systematic assay variation (Methods; supplemental Figure 5). The VeraTag HER2 levels are plotted against the mean and standard deviation (x-error bars) of the HER2 receptor number per cell determined by FACS (n=3). B, VeraTag HER2-HER2 homodimer assays were performed on multiple sections for each cell line; each section adjacent to the section assayed for HER2 expression at the same time (n=14 assays). VeraTag assay signal and FACS receptor numbers are represented as in A. All sections are approximately 50 mm2, containing 180,000 to 200,000 cells. FACS indicates fluorescence-activated cell sorting; FFPE, formalin-fixed, paraffin-embedded.supplementary Figure 1.supplementary Figure 2.supplementary Figure 3.supplementary Figure 4AB.supplementary Figure 4C.supplementary Figure 5.A Novel Proximity Assay for the Detection of Proteins and Protein Complexes: Quantitation of HER1 and HER2 Total Protein Expression and Homodimerization in Formalin-fixed, Paraffin-Embedded Cell Lines and
Breast Cancer TissueShi Yining PhD; Huang, Weidong MD; Tan, Yuping MS; Jin, Xueguang MS; Dua, Rajiv PhD; Penuel, Elicia PhD; Mukherjee, Ali PhD; Sperinde, Jeff PhD; Pannu, Herjit MD; Chenna, Ahmed PhD; DeFazio-Eli, Lisa PhD; Pidaparthi, Sailaja PhD; Badal, Youssouf PhD; Wallweber, Gerald PhD; Chen, Lili MD; Williams, Steve PhD; Tahir, Hasan PhD; Larson, Jeff PhD; Goodman, Laurie PhD; Whitcomb, Jeannette PhD; Petropoulos, Christos PhD; Winslow, John PhDOriginal ArticlesOriginal Articles118p 11-21
