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Ras-related protein 1 and the insulin-like growth factor type I receptor are associated with risk of progression in patients diagnosed with carcinoma in situ

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Abstract

Currently, there are no applied molecular markers to aid in predicting risk of carcinoma in situ (CIS) progression to invasive cancer, and therefore, all women diagnosed with CIS undergo surgery. Standard assessment of protein expression in fixed tissue by immunohistochemistry (IHC) is not quantitative and hence is not well suited for measuring biomarkers. In this study, we developed an original analytical method for IHC quantification. Using our novel image-based uniplex (IBU) method, quantitative protein profiling was performed on 90 samples of the breast (17 histologically normal tissues, 16 benign lesions, 15 CIS, and 42 invasive carcinomas). Differences between groups were assessed using analysis of variance (ANOVA) and mixed effects models. Measuring protein expression on a continuous scale revealed a significant increase in Ras-related protein 1 (Rap1) and the insulin-like growth factor type I receptor (IGF-IR) in conjunction with the presence of cancer invasion. Women with invasive cancers were four times more likely to have increased levels of Rap1 [odds ratio (OR) = 3.91; P = 0.0002] and IGF-IR (OR = 4.33; P < 0.0001) than women with non-invasive lesions. Furthermore, expression of both proteins was also increased significantly in CIS adjacent to invasive tumors compared with non-cancerous tissue. These novel findings of a significant up-regulation of Rap1 and IGF-IR in CIS progressing to invasive cancers warrant further investigation of Rap1 and IGF-IR together as a dual biomarker to aid in predicting risk of progression and ultimately providing non-surgical treatment options to those at lower risk.

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Acknowledgments

We thank Indira Prabakaran for excellent help with IHC and Robin Noel for excellent help with figure editing. Grant Support: American Cancer Society IRG# 78-002026 to MAG, Roy and Diana Vagelos Foundation to DKF; the Pennsylvania Department of Health to MDF.

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Authors and Affiliations

  1. The Division of Endocrine and Oncologic Surgery, Department of Surgery, School of Medicine, University of Pennsylvania, 313 Stemmler Hall, 37th and Hamilton Walk, Philadelphia, PA, 19104, USA

    Dana K. Furstenau, Robert Lewis, Douglas L. Fraker & Marina A. Guvakova

  2. Department of Biostatistics and Epidemiology, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Nandita Mitra & Fei Wan

  3. Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA

    Michael D. Feldman

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  1. Dana K. Furstenau
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  2. Nandita Mitra
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  3. Fei Wan
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  4. Robert Lewis
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  5. Michael D. Feldman
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  6. Douglas L. Fraker
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  7. Marina A. Guvakova
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Correspondence to Marina A. Guvakova.

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10549_2010_1227_MOESM1_ESM.pdf

Figure 1S. Validation of Rap1 (121) rabbit polyclonal antibody from Santa Cruz Biotechnology (sc-65). Immunoperoxidase staining of formalin-fixed, paraffin-embedded human tissue using Rap1 (121) rabbit polyclonal antibody from Santa Cruz Biotechnology (sc-65). Magnification ×400. a Normal breast epithelium (left) adjacent to invasive ductal carcinoma (right). b Normal breast epithelium (arrows) adjacent to invasive lobular breast cancer. Staining pattern in the breast: cytoplasmic and membranous staining. Quantity: >75% cells were stained. Quality: weak staining in normal breast glandular cells; moderate to high in breast carcinomas. Background staining is negligible. c Four human gliomas obtained from CHTN, Philadelphia, PA were negative. The representative human glioma section stained with Rap1 antibody is shown. d Antibody recognized a single major band of Rap1 (arrow) corresponding to the predicted size in kDa (±5%). A specific 2.2-fold increase in the level of active Rap1 (Rap1GTP) was detected in a pull down assay with Rap1 effector RalGDS. To activate Rap1, serum-starved MCF-derived human breast carcinoma cells were treated with 10 μM forskolin for 5 min. The total levels of Rap1 in the same samples were detected in the whole lysates. (PDF 174 kb)

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Furstenau, D.K., Mitra, N., Wan, F. et al. Ras-related protein 1 and the insulin-like growth factor type I receptor are associated with risk of progression in patients diagnosed with carcinoma in situ. Breast Cancer Res Treat 129, 361–372 (2011). https://doi.org/10.1007/s10549-010-1227-y

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