Research ArticleExperimental Studies

GLI1, CTNNB1 and NOTCH1 Protein Expression in a Thymic Epithelial Malignancy Tissue Microarray

JONATHAN W. RIESS, ROBERT WEST, MICHELLE DEAN, ALEX C. KLIMOWICZ, JOEL W. NEAL, CHUONG HOANG and HEATHER A. WAKELEE
Anticancer Research February 2015, 35 (2) 669-676;

Abstract

Background/Aim: Thymic epithelial tumors (TET) are rare. Wingless and INT (WNT), NOTCH and sonic hedgehog pathway interactions between thymocytes and thymic stroma are important to thymus and T-cell development. We analyzed a thymoma tissue microarray (TMA) for glioma associated oncogene homolog 1 (GLI1), NOTCH1 and catenin (cadherin-associated protein, beta 1) (CTNNB1) expression as surrogate markers of sonic hedgehog, NOTCH and WNT pathway activity. Materials and Methods: GLI1, NOTCH1 and CTNNB1 expression were assayed in a tissue microarray of 68 TET and eight benign thymus by fluorescent immunohistochemistry (AQUA) as surrogates for activity of the sonic hedgehog, NOTCH and WNT pathways respectively. Results: No difference in tumor GLI1 (mean 201 vs. 211, p=0.31), CTNNB1 (mean 222 vs. 306, p=0.66) or NOTCH1 expression (mean 317 vs. 325, p=0.82) was noted between thymic tumor and benign thymus. Conclusion: No evidence for preferential expression of GLI1, NOTCH1 or CTNNB1 was noted. High-throughput immunofluorescence using AQUA technology can help overcome limitations of small sample size and tissue heterogeneity when analyzing protein expression in thymic tumors.

Thymoma and thymic carcinoma are rare malignancies, with an annual incidence of 0.15 cases per 100,000 persons, but represent the third most common type of primary mediastinal tumor (after lymphoma and germ cell tumors) (1, 2). There is a paucity of data on the biology as well as therapeutics, especially for patients with refractory disease requiring treatment on a second-line or subsequent basis.

Research into targeted-therapeutics is lacking for thymic tumors. The rarity of thymic tumors, the high-percentage of curable, early-staged patients and the lack of established thymoma cell lines have hindered development of targeted-therapeutics for the subset of patients with incurable, metastatic disease (3).

The NOTCH, sonic hedgehog (SHh) and wingless and INT (WNT/β-catenin) pathways are important in vertebrate and invertebrate development and have also been shown to be re-activated in a wide range of malignancies (4). Constitutive SHh signaling has been implicated in the pathogenesis of multiple malignancies, particularly where it is involved in development of the organ in which the tumor arises (5). Binding of Hh to a transmembrane receptor patched (PTCH1), releases its inhibition of smoothened (SMO). Release of SMO results in activation of the transcription factors GLI1, GLI2 and GLI3. These transcription factors are thought to mediate most of the effects of the hedgehog pathway (6). Develop mental pathways have been implicated in thymus development and the complex interaction between thymic stroma and hematopoietic cells. Thymic development involves paracrine signaling between thymic stroma and hematopoietic pro genitors (7) with hedgehog ligands produced in the thymic stroma, where PTCH (the receptor) and SMO are also expressed (8, 9). One study looked at immunohistochemistry (IHC) staining of hedgehog pathway-related proteins and found increased GLI1 expression in 14 out of 26 thymoma samples, with GLI1 expression being higher in thymoma tissue than in normal thymic epithelium (10). However, rigorous quantification of GLI1 levels by quantitative IHC was not performed.

View this table:
Table I.

Demographics of patients whose samples were included in thymic malignancy tissue microarray (N=68).

The NOTCH pathway is involved in embryogenesis and cellular differentiation (11). It has also been shown to be involved in both hematological malignancies and solid tumors. Its activity may be context-dependent, acting as an oncogene or tumor suppressor or even affecting angiogenesis, depending on the malignancy (12). After a notch ligand binds to the NOTCH receptor, the receptor is cleaved into its active intracellular domain that is then translocated to the nucleus, initiating a transcription cascade and activating the hairy and enhancer of split-1 (HES1) family of transcription factors (12). Within the thymus, NOTCH has been shown to be important in T-cell development (13). The role of NOTCH in normal thymic epithelium is less established; although NOTCH pathway activation has been shown in thymic epithelial cells (14). To our knowledge, activation of the NOTCH pathway in thymic malignancies has not been examined.

The WNT pathway directs cell fate at various stages of development in several organ systems (15). In the canonical pathway, β-catenin accumulates in the nucleus and transcribes target genes (16). Aberrant WNT signaling has been reported in several tumor types (17). The WNT pathway is involved in thymus development. In particular, stabilizing mutations of β-catenin in the mouse thymic epithelium blocks thymus development and function (18). Thymic epithelial cells have also been shown to provide WNT signals to developing thymocytes (19). A Japanese group examined 21 thymoma tissue samples for β-catenin by IHC and found WNT to be present in certain histological subtypes (WHO B1-B3) (20, 21).

View this table:
Table II.

Mean and standard deviation of AQUA score for GLI1 (glioma associated oncogene homolog 1), NOTCH1 and CTNNB1 (cadherin-associated protein, beta 1) in thymic tumors and benign thymic controls.

Multiple promising drugs at different stages of development exist for each of these stem-cell pathways (16, 22, 23). Given the importance of the SHh, WNT and NOTCH pathways in thymus and T-cell development and the lack of examination of these pathways in thymic malignancies, we undertook this study to quantifying GLI1, NOTCH1 and CTNNB1 protein expression and as surrogates of sonic hedgehog, notch and WNT pathways, respectively.

Materials and Methods

Study population. After obtaining approval from the Institutional Review Board of Stanford University School of Medicine and the Scientific Research Committee at Stanford Cancer Institute (#21319), 68 patients with a pathological diagnosis of thymic carcinoma or thymoma and eight benign thymus controls with pathology at Stanford were identified. Clinical and pathologic data was abstracted from the medical record in the patients identified by Stanford Cancer Center and Stanford Pathology Databases.

Tissue microarray (TMA) construction. Formalin-fixed paraffin-embedded (FFPE) tissue blocks were retrieved from the surgical pathology archives at Stanford University Hospital and Clinics. A TMA was constructed from the FFPE blocks of thymic malignancy from 68 individual patient samples and eight benign thymic controls by using a tissue arrayer (Beecher Instruments, Silver Spring, MD, USA) to create one new paraffin block from representative 0.6 mm cores taken in triplicate from the blocks of tumor. Each tissue sample was represented in triplicate in the TMA. Eleven patients with thymic tumor also had non-tumoral thymus tissue included as a paired matched control. Positive and negative cell line controls for GLI1, NOTCH1 and CTNNB1 included as noted in Figures 1, 2 and 3.

Fluorescent immunohistochemistry for GLI1, NOTCH1 and CTNNB1. TMA sections (4μm) were de-paraffinized in xylene, rinsed in ethanol and rehydrated as previously described (25). Heat-induced epitope retrieval for GLI1 and CTNNB1 targets was performed by heating slides to 121°C for 3 min (GLI1) or 6 min (CTNNB1) in a citrate-based buffer (pH6, Dako) using a decloaking chamber (Biocare Medical, Concord, CA, USA). A Tris/EDTA-based buffer (pH 9.0) Target Retrieval Solution (Dako) at 121°C for 6 min was used for NOTCH1 staining. Using a Dako Autostainer, endogenous peroxidase activity was quenched with a 10-min incubation of peroxidase block (Dako) followed by a 15-min protein block (Signal Stain; Cell Signaling, Danvers, MA, USA) to eliminate non-specific antibody binding.

Figure 1.
Figure 1.

Representative samples of GLI1 expression by AQUA. DAPI staining for all cells, PCK stain for epithelial cells, vimentin stain for stromal cells. Single marker staining noted in greyscale and merged images in color.

For GLI1, slides were washed with Tris-Buffered Saline and 0.05% Tween-20 (TBST) wash buffer (Dako) and then incubated at room temperature for 60 min with Signal Stain protein block (Cell Signaling) containing a 1:2,000 dilution of mouse monoclonal antibody vimentin, clone V9 (Dako). For NOTCH1, slides were washed with TBST wash buffer (Dako)and then incubated at room temperature for 60 minutes with Signal Stain protein block (Cell Signaling) containing a 1:250 dilution of rat monoclonal antibody to vimentin clone 280618 (R&D Systems, Minneapolis, MN, USA). For CTNNB1, slides were washed with TBST wash buffer (Dako) and then incubated at room temperature for 60 minutes with Signal Stain protein block (Cell Signaling) containing a 1:200 dilution of rabbit monoclonal antibody to CD45, clone EP322Y (Epitomics, Burlingame, CA, USA).

Secondary reagents were incubated with slides at room temperature for 60 min: pre-diluted goat anti-mouse (vimentin clone V9) or anti-rabbit (CD45) antibodies conjugated to a horseradish peroxidase (HRP)-decorated dextran polymer backbone from the DAKO EnVision+ system (Dako). Signal Stain protein block (Cell Signaling) containing a 1:200 dilution of Rat HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to compliment vimentin clone 280618.

The slides were washed with TBST wash buffer (Dako) and incubated for 5 min with the TSA-Plus Cy3 tyramide signal amplification reagent (PerkinElmer, Woodbridge, ON, Canada), then incubated off-line for 10 minutes in 0.1% Azide TBST buffer to stop enzyme activity. After two washes in TBST wash buffer (DAKO), slides were placed back onto the autostainer.

Figure 2.
Figure 2.

Representative samples of NOTCH1 expression by AQUA. DAPI staining for all cells, PCK stain for epithelial cells, Single markers staining noted in greyscale and merged images in color.

A second set of primary antibodies was applied at room temperature for 60 minutes in Signal Stain protein block (Cell Signaling), including, a 1:100 dilution of guinea pig monoclonal antibody anti-pan-cytokeratin (Acris, San Diego, CA, USA) (to identify tumor cells), combined with a 1:200 dilution of rabbit monoclonal antibody to GLI1, clone EPR4523 (Epitomics, Burlingame, CA, USA), a 1:200 dilution of rabbit monoclonal antibody to NOTCH1, clone EP1238Y (Epitomics, Burlingame, CA, USA) or a 1:1,000 dilution of mouse monoclonal antibody to CTNNB1, clone β-Catenin-1 (Dako).

Slides were washed with TBST wash buffer (Dako) and corresponding secondary antibodies were applied for 60 minutes at room temperature: a 1:200 dilution of Alexa-488 conjugated goat anti-guinea pig antibody (Invitrogen, Burlington, ON, Canada) in pre-diluted goat anti-rabbit (GLI1, NOTCH1) or anti-mouse (CTNNB1) antibodies conjugated to a HRP-decorated dextran polymer backbone from the DAKO EnVision+ system (Dako). The slides were washed with TBST wash buffer (Dako) and incubated for 5 min with the TSA-Plus Cy5 tyramide signal amplification reagent (PerkinElmer, Woodbridge, ON, Canada). After three washes in TBST wash buffer, the TMA slides were mounted with ProLong® Gold anti-fade mounting medium containing diamidino-2-phenylindole (DAPI) (Invitrogen) and stored at 4°C.

Figure 3.
Figure 3.

Representative samples of CTNNB1 expression by AQUA. DAPI staining for all cells, PCK stain for epithelial cells, CD45 for lymphocytes, Single markers staining noted in greyscale and merged images in color.

Control cell lines and tissues. Briefly, ten million cells in a tissue culture vessel were rinsed in PBS, dissociated and pelleted by centrifugation. Cells were fixed in 1mL of 10% neutral- buffered formalin for 30 min, pelleted and resuspended in 250 μL of liquified Histogel (Thermo Scientific, USA) on ice until the gel hardens. The solidified Histogel pellet was transferred to a histology cassette and placed into a 10% neutral-buffered formalin for at least 8 h prior to processing and paraffin embedding by Calgary Laboratory Services. Cores (0.6 mm diameter) were taken from each cell line block using a Beecher Manual Tissue Microarrayer (Beecher Instruments Inc. Sun Prairie, WI, USA). These were placed alongside cores from various normal and cancer FFPE tissue blocks into TMAs that served as assay controls for each stain. Cell lines that served as positive and negative controls were previously identified during each antibody's optimization and confirmed during experimental runs.

The following cell lines were used as protein expression controls: A549 (ATCC, Virginia, USA), human epithelial lung adenocarcinoma; OPM2 (DSMZ, Braunchweig, Germany), human multiple myeloma; 786-0 (ATCC), human epithelial renal cell adenocarcinoma; C35ABR (BT) human lymphoblastoid cells, kindly provided by Dr. S.P. Lees-Miller (University of Calgary, Canada); H226 (ATCC), human epithelial lung squamous cell carcinoma; and H522 (ATCC), human epithelial lung adenocarcinoma.

Figure 4.
Figure 4.

Box-plot with mean, median and 1.5× interquartile range of AQUA score for GLI1 in thymic tumors and thymic controls. Diamond, mean; line, median, p=0.32.

Figure 5.
Figure 5.

Box-plot with mean, median and 1.5× interquartile range of AQUA score for Notch1 in thymic tumor and thymic controls. Diamond, mean; line, median, p=0.66.

Figure 6.
Figure 6.

Box-plot with mean, median and 1.5× interquartile range of AQUA score for CTNNB1 in thymic tumor and thymic controls. Diamond, mean; line, median, p=0.82.

For each stain, images of tissues that normally express the target marker were taken from the antibody-treated and non-treated samples and shown with cell line images in Figures 1, 2 and 3.

Automated image acquisition and analysis. Automated image acquisition was performed using an Aperio Scanscope FL (Aperio Inc., Vista, CA, USA). Seamless high-resolution slide images were acquired using the Scanscope FL 10-bit monochrome TDI line-image capture camera using filters specific for DAPI to define the nuclear compartment, fluoroscein isothiocyanate (FITC) to define cytokeratin for the tumor cytosolic compartment, Cy3 to define vimentin-positive non-malignant stromal cells and Cy5 to define target antibodies.

Images were then analyzed using the HistoRX AQUAnalysis® program, version 2.3.4.1 as previously described (24). Briefly, a tumor-specific mask and a tumor cytoplasmic mask were generated to distinguish the thymoma and thymic carcinoma cells from surrounding stromal tissue by thresholding the pan-cytokeratin images. Thresholding created a binary mask that identified the presence or absence of tumor cells by the presence of a pixel that was ‘on’ or ‘off’, respectively. A similar thresholding technique was applied to generate a non-malignant stroma-specific mask from the pan-cytokeratin negative regions of the vimentin images, a proven superior method for identifying stromal regions (25). A tumor nuclear mask was generated by inverting the cytoplasmic mask within the tumor mask. Thresholding levels were verified and adjusted, if necessary, by spot-checking a small sample of images to determine an optimal threshold value. All images were then processed using this optimal threshold value and all subsequent image manipulations involved only image information from the masked area. Images were validated according to the following: i) >10% of the tissue area was pan-cytokeratin-positive, ii) >50% of the image was usable (i.e. not compromised due to overlapping or out of focus tissue). Unusable areas within each image were manually cropped so that they were excluded from the final analysis.

Statistical analysis. Tissue expression of GLI1, NOTCH1 and CTNNB1 was quantified as a continuous variable of expression by automated quantitative analysis (AQUA). Expression of GLI1, NOTCH1 and CTNNB1 for each tumor sample representing each patient was averaged and this average was used for further statistical analysis. Case and unpaired control values were compared by a two-tailed unpaired t-test. One-way ANOVA was performed to detect any differences between control tissue and WHO histological subtypes. All statistical analyses were performed using SAS Enterprise Guide version 5.0 (Cary, NC, USA).

Results

Pertinent demographics of patients whose thymic tumors were included in the tissue microarray are highlighted in Table I. No significant differences in expression were noted between controls and tumor tissues for GLI1, NOTCH1 and CTNNB1 (Table II, Figures 4, 5 and 6).

To determine whether specific histological subsets of thymoma had increased expression of GLI1, NOTCH1 and CTNNB1 ANOVA by WHO Classification was performed. ANOVA for expression by AQUA: GLI1 F=0.27, NOTCH1 F=0.0047, CTNNB1 p=0.11.

Since ANOVA was significant for NOTCH1, Dunnett's t-test was performed with benign thymic controls as the reference value to compare whether any thymic tumor histological subtypes by WHO classification were significantly different from controls and the results were non-significant.

Discussion

Using AQUA and fluorescent IHC to examine biomarker expression offers several advantages in studying rare tumors, such as thymic epithelial malignancies. This includes reporting continuous variable of quantitative immunofluorescence to reduce the effect of a small sample size in order to achieve statistically significant differences, as well as overcoming difficulties in examining thymomas that have substantial admixture of lymphocytes.

Another benefit of this approach is the higher discriminatory power between cancer sub-types, which is especially useful when cut-off points are not well-described (26).

We hypothesized that GLI1, CTNNB1 and NOTCH1 would have increased expression in our thymic TMA based on the importance of the SHh, WNT and NOTCH pathways in thymic development. However, in our thymic tissue microarray, GLI1, CTNNB1 and NOTCH1 were not significantly increased compared to benign thymic controls. Thymic tumors are histologically-heterogeneous, so we examined differences in GLI1, NOTCH1 and CTNNB1 expression by WHO pathological subtype compared to thymic controls. We also found no clinically significant differences by WHO histology. We also examined values for nucleur localization of GLI1, CTNNB1 and NOTCH1 and these were also not different from benign thymic controls (data not shown).

NOTCH1 mutations and overexpression have been noted in squamous lung and head and neck cancer. We hypothesized that thymic carcinoma in particular (many of which have squamous differentiation) would overexpress NOTCH1 (27, 28). However, it was only possible to include three thymic carcinomas in our TMA, precluding definitive analysis. In thymoma microarrays, high gene expression of NOTCH signaling proteins was associated with metastatic disease (29). We only examined protein expression for NOTCH1, so it is possible other notch family proteins, such as NOTCH2 and NOTCH3 are more involved than NOTCH1 in NOTCH signaling in thymoma.

In conclusion, no increase in GLI1, NOTCH1 or CTNNB1 expression was noted in thymoma. AQUA is a useful tool for examining protein expression in thymoma as it can help overcome issues complicating analysis due to tumor hetererogeneity and small samples sizes for this uncommon tumor type.

Acknowledgements

We express special thanks to Ray Balise, Ph.D. and Kristen Sainani, Ph.D. for statistical assistance. This project was supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through grant 5 KL2 RR025743 (JWR). The content is solely the responsibility of the Authors and does not necessarily represent the official views of the National Institutes of Health. This project was also supported by a Stanford University School of Medicine Translational and Applied Medicine Pilot Grant (JWR).

  • Received May 7, 2014.
  • Revision received November 4, 2014.
  • Accepted November 7, 2014.

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GLI1, CTNNB1 and NOTCH1 Protein Expression in a Thymic Epithelial Malignancy Tissue Microarray
JONATHAN W. RIESS, ROBERT WEST, MICHELLE DEAN, ALEX C. KLIMOWICZ, JOEL W. NEAL, CHUONG HOANG, HEATHER A. WAKELEE
Anticancer Research Feb 2015, 35 (2) 669-676;
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