A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, n = 118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0–300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (p = 0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, n = 25, p = 0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.

The Vascular Endothelial Growth Factor (VEGF) pathway plays an important role in the genesis, growth and progression of human cancer, including colorectal carcinomas (CRC). The key mediators of VEGF signaling are VEGFR1, VEGFR2, and VEGFR3, part of a family of related receptor tyrosine kinases. The relative expression, activity, or interplay among these receptors may determine the response of CRC patients to anti-angiogenic therapies. Using high-affinity, specific monoclonal primary anti-VEGFR1, 2 3 antibodies, we have developed robust imunohistochemical assays in our laboratory to quantify VEGFR1, 2 and 3 in archival human tissues. Using a well-annotated CRC tissue microarray (TMA), we carried out comprehensive comparative evaluation of immunohistochemical (IHC) expression of the three VEGFRs in archival primary CRC tissues (n=84). VEGFR1 immunoreactivity was reported as H-score (range 0-300); VEGFR2 positive vessels were counted and normalized to the number of CD34-positive vessels and reported as vascular positivity index (VEGFR2-VPI) and VEGFR3-positive vessels were counted in each core by the same solid tumor immunopathologist, who was blinded to the clinico-pathologic details. Based on immunoreactivity for VEGFRs, each case was scored as negative, low, medium or high. Thresholds were selected based on the overall range of expression of each receptor in the CRC tissues examined: 0, 1-50, 51-100, >100 (VEGFR1 H-scores); 0, 1-25, 26-49, 50-100 (VEGFR2-VPI); 0, 1-5, 6-10, >10 (VEGFR3+ vascular count). Based on VEGFR (1,2,3) expression, a set of eight VEGFR staining profiles were noted: Triple VEGFR positive (n=9, 11%), VEGFR1 predominant (17, 20%), VEGFR2 predominant (7, 8%), VEGFR3 predominant (1, 1%), VEGFR1/2 predominant (42, 50%), VEGFR1/3 predominant (2, 2%), VEGFR2/3 predominant (3, 4%), and triple VEGFR negative (3, 4%). These new data provide original insights on the distribution, subcellular localization and heterogeneity of expression of VEGFRs in human CRC stromal vessels and tumor cells. The proposed human CRC sub-classification, based on the observed differential VEGFR 1, 2, 3 expression profiles, has identified various subsets of human CRCs. Clinical trials incorporating IHC profiling would be required to test whether these subsets show differential responsiveness to targeted agents in the VEGF/VEGFR2 family. Citation Format: Timothy R. Holzer, Leslie A. O'Neill, Drew M. Nedderman, Angie D. Fulford, Beverly L. Falcon, Mark T. Uhlik, Laura E. Benjamin, Andrew E. Schade, Aejaz Nasir. Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in primary human colorectal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3007. doi:10.1158/1538-7445.AM2014-3007

VEGFR3 plays a key role in the regulation of lymphangiogenesis in adults, and is also important for tumor angiogenesis and metastasis. In order to characterize human cancer tissues for imunohistochemical (IHC) localization and prevalence of VEGFR3 protein, we developed a robust IHC assay in our lab using a mouse monoclonal primary antibody (Millipore). To optimize the various assay parameters, we utilized high-quality VEGFR3 positive (and internal negative) control tissues (Kaposi's sarcoma, angiosarcoma, lymph node). Reagent negative controls were satisfactory. Specificity of the primary anti-VEGFR3-antibody was supported by a discrete band at the expected molecular weight on western blots using a VEGFR3 transfected cell lysate and negative control cell lines. We also demonstrated lack of specific vascular VEGFR3 staining in pre-absorption experiments with VEGFR3 (but not VEGFR1 or 2) recombinant protein, supporting selectivity of the primary antibody for VEGFR3. Using the fully optimized assay, we stained a human multi-tumor screening tissue microarray (TMA) to demonstrate full range of specific vascular VEGFR3 staining in glioblastoma and carcinomas of the colon, breast, ovary, pancreas, lung, larynx, kidney, cervix, bladder, extrahepatic cholangiocarcinoma and malignant melanoma. Specific, unequivocal VEGFR3 immunoreactivity was interpreted qualitatively (VEGFR3 positive/negative) in tumor blood and lymphatic vessels. No tumor cell staining was seen. Some colon cancer tissues in multi-tumor TMA were VEGFR3+, while others were VEGFR3-. The observed variation in VEGFR3 expression and vascular distribution on screening TMA were further evaluated in two independent cohorts of well-characterized human colorectal cancer tissues (organ-specific TMAs) by a Board-certified, subspecialty GI pathologist (AN), with overall VEGFR3 prevalence rates of 62% (36 of 58 cases) and 56% (55 of 98 cases). Using CD34 and D2-40 IHC assays from a CLIA-certified lab, we confirmed IHC localization of VEGFR3 protein both in the stromal blood vessels and lymphatics within the invasive colorectal cancer stroma. In conclusion, following well-established IHC assay development and standardization protocols and efficient workflow paradigm, we have used a technically robust IHC assay to characterize routinely processed archival human cancer tissues and have demonstrated specific VEGFR3 expression patterns, tissue localization and prevalence in human colorectal cancer tissues. Based on its performance in our hands, this assay can be used to further evaluate patterns of VEGFR3 expression in areas of tumor angiogenesis in other human cancer tissues. Data-driven hypotheses generated from such investigations will be relevant to corroborate VEGF receptor biology and emerging clinical experience with anti-VEGF/anti-VEGFR therapies. Citation Format: Timothy R. Holzer, Drew M. Nedderman, Aejaz Nasir. Robust immunohistochemical assay to characterize human cancer tissues for prevalence of vascular endothelial growth factor receptor 3 (VEGFR3). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2015-4175

The Vascular Endothelial Growth Factor (VEGF) pathway plays a prominent role in the growth and progression of human cancers, including non-small cell lung carcinoma (NSCLC). The key mediators of VEGF signaling are a family of related receptor tyrosine kinases that include VEGFR1, VEGFR2, and VEGFR3. The relative expression levels, activity and cross-talk among these receptors may contribute to clinical response of NSCLC patients to anti-angiogenic therapies. Using a well-annotated tissue microarray (TMA) and robust immunohistochemical (IHC) assays developed, standardized and implemented in our laboratory, we comparatively evaluated expression of the three VEGFRs in archival primary NSCLC tissues (n = 97). VEGFR1 and VEGFR2 were localized both in tumor vessels and cells, while VEGFR3 was only localized in tumor vessels. VEGFR1 immunoreactivity was reported as negative/low, medium, or high, based on intensity and proportion of stained tumor cell by an experienced solid tumor immunopathologist (AN), who was blinded to clinico-pathologic details. VEGFR2 and VEGFR3 positive vessels were counted by manual assessment of each core by the same immunopathologist. For systematic comparative analysis of VEGFR data, IHC expression thresholds were selected based on the 25% and 75% quartiles around the median of the range of counts for VEGFR2 and VEGFR3: 0-2, 3-10 and >10 (VEGFR2+ vascular count) respectively; and 0-1, 2-9 and >9 (VEGFR3+ vascular count) respectively. Based on VEGFR (1,2 and3) expression levels defined above, a set of eight VEGFR staining profiles were identified: Triple VEGFR positive (n = 11, 11.3%), VEGFR1 predominant (22, 22.7%), VEGFR2 predominant (9, 9.3%), VEGFR3 predominant (3, 3.1%), VEGFR1/2 predominant (13, 13.4%), VEGFR1/3 predominant (2, 2.1%), VEGFR2/3 predominant (9, 9.3%), and triple VEGFR negative (28, 28.9%). These new data provide original insights on the tissue distribution, subcellular localization and heterogeneity of expression of VEGFRs in human NSCLC cells and stromal vessels. The proposed human NSCLC sub-classification, based on the observed differential VEGFR 1, 2, 3 expression profiles, has identified various subsets of human NSCLC, especially triple VEGFR +, triple VEGFR -, VEGFR1 predominant and VEGFR 1 / 2 predominant. These profiles are distinct from the VEGF receptor profiles that we reported in a prior study on colorectal carcinomas (Holzer, Nasir et al., AACR 2014), of which VEGFR1/2 predominant subset was 50% and triple VEGFR-negative subset was only 4%. This work suggests distinct patterns of heterogeneity of VEGF receptor profiles in human NSCLC and CRCs. These data also support further evaluation of whether the reported VEGFR profiles correlate with differential sensitivity to therapeutic VEGF/VEGFR pathway inhibition. Citation Format: Timothy R. Holzer, Angie D. Fulford, Leslie O'Neill Reising, Drew M. Nedderman, Laura E. Benjamin, Andrew E. Schade, Aejaz Nasir. Heterogeneity of vascular endothelial growth factor receptors 1, 2, and 3 in human non-small cell lung carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2015-4157