Tumor Cell Expression of Vascular Endothelial Growth Factor Receptor 2 Is an Adverse Prognostic Factor in Patients with Squamous Cell Carcinoma of the Lung

<div><p>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 (<i>p</i> = 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, <i>p</i> = 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.</p></div

VEGFR2 IHC is specific and selective against other VEGFR family members.

<p>The anti-VEGFR2 55B11 antibody specifically recognizes a unique epitope in the sequence HSDDTDTTVYSSEEA that is harbored within the c-terminal region. (A) HIS-tagged recombinant proteins corresponding to the intracellular domains of VEGFR1, VEGFR2, and VEGFR3 were incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of ductal carcinoma of the breast. VEGFR2 and not VEGFR1 or VEGFR3 pre-absorbed the antibody leading to negative immunoreactivity. (B) Recombinant 21 or 22-mer peptides corresponding to contiguous stretches of the immunogenic sequence of 55B11 were generated as indicated in the schematic (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080292#pone.0080292.s002" target="_blank">Table S2</a>). Each peptide was incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of pulmonary SCC. Pre-incubation with peptides 6 and 12 abolished immunoreactivity, while pre-incubation with all other peptides had no effect. The results shown for peptides 5 and 13 are representative of peptides 1–5, 7–11, and 13. (C) Recombinant 10 or 11-mer peptides corresponding to contiguous stretches of the consensus sequence of peptides 6 and 12 were generated as indicated in the schematic. Each peptide was incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of pulmonary SCC, a unique patient sample than in panel B. Pre-incubation with peptides 6 (data not shown), 12 and 15 abolished all immunoreactivity, while pre-incubation with peptides 14, 17 and 18 had no effect. Pre-incubation with peptide 16 showed an attenuated effect. All slides were counterstained with hematoxylin. Original magnification, ×200. Scale bars: 50 µm.</p

Histopathological subtypes and VEGFR2 IHC immunoreactivity in a multi-tissue screening TMA cohort, n = 50.

<p>Pos, immunopositivity.</p

Refinement of IHC assay parameters was integral to detect subcellular immunoreactivity in tumor cells.

<p>Representative immunoreactivity is shown on serial sections of a pulmonary SCC. Slides were subjected to heat induced epitope retrieval (HIER) conditions using buffers of differing pH, followed by an optimized IHC protocol using 55B11: primary antibody reagent negative control (A), Tris buffer (B), citrate buffer (C), and EDTA buffer (D). VEGFR2 immunoreactivity in endothelial cells lining blood vessels (black arrows) and membranous (black arrowheads), cytoplasmic, and nuclear compartments of malignant tumor cells was found to be most optimal with EDTA buffer (panel D). Slides were counterstained with hematoxylin. Original magnification ×200. Scale bar: 50 µm, applicable to all panels.</p

VEGFR2 immunoreactivity in areas of squamous differentiation.

<p>(A) Tumor cell-derived cytoplasmic and nuclear immunoreactivity (black arrowhead) in a focus of squamous differentiation on a background of pulmonary ADC. (B) tumor cell-derived cytoplasmic and nuclear immunopositivity in SCC of the cervix. Keratin pearls (open arrowhead) are not immunoreactive. Slides were counterstained with hematoxylin. Original magnification ×200. Scale bar: 50 µm, applicable to both panels.</p

Vascular endothelial cell and tumor cell-derived VEGFR2 immunoreactivity on representative cases on a multi-tumor survey.

<p>Left panels, H? right panels, VEGFR2 IHC. (A) VEGFR2 IHC on renal cell carcinoma of the kidney showed endothelial cell immunoreactivity (×400). (B) VEGFR2 IHC on ADC of the colon showed endothelial cell immunoreactivity in the stromal mucosa. Tumor cells were negative for VEGFR2 (×400). (C) VEGFR2 IHC on SCC of the lung showed endothelial cell and a range of tumor cell-derived nuclear, cytoplasmic, and membranous immunoreactivity (×200). (D) VEGFR2 IHC showed vascular endothelial cell immunoreactivity and a range of tumor cell cytoplasmic and nuclear immunoreactivity on SCC of the cervix (×200). Immunoreactivity in endothelial cells lining vessels (white and black arrows). Slides were counterstained with hematoxylin. Scale bars: 50 µm.</p

Characterization of VEGFR2 pathological scores in NSCLC patient cohort.

<p>(A) percent of patients with VEGFR2 vessel positivity per histological subtype. (B) VEGFR2 tumor cell positivity per histological subtype. (C) VEGFR2 tumor cell positivity per vessel immunoreactivity status. Light grey lines show median values for each group. <i>p</i>-values for associations are shown with statistically significant observations indicated with an asterisk. Kaplan-Meier survival curves were grouped by disease stage I or stages II–IV for: whole patient population (D), ADC (E), and SCC (F). Survival curves were also grouped by vascular endothelial cell expression of VEGFR2 for: whole patient population (G), ADC (H), and SCC (I). Additional survival curves were grouped by tumor cell-derived VEGFR2 immunoreactivity for: whole patient population (J), ADC (K), and SCC (L). Statistically optimal VEGFR2 expression cutpoints for dividing patients into groups based on survival were identified as H-scores of 10, 0, and 10, respectively. <i>p</i>-values for each curve are shown with statistically significant observations indicated with an asterisk. Points on the curves represent censored data (patients alive at follow-up time). For analysis involving VEGFR2 expression, only tumor-cell derived VEGFR2 immunopositivity in SCC patients showed a statistically significant associate (<i>p</i> = 0.0134) in median overall survival between VEGFR2 high (24.7 mo) and VEGFR2 low populations (92.8 mo).</p

An optimized IHC assay sensitively detects VEGFR2 protein levels manipulated <i>in vitro</i>.

<p>siRNA harboring VEGFR2 sequences were used to manipulate VEGFR2 protein levels in H441 cells to inform IHC assay development. Orthogonal techniques were used to detect VEGFR2. VEGFR2 mRNA abundance was reduced 77.7% as detected by qRT-PCR analysis (A). Protein levels for VEGFR2 were determined by three methods: western blot analysis (B), IA-MS analysis (C), and positive pixel counts of immunoreactivity of IHC on FFPE cells (D). VEGFR2 protein levels were reduced by 78.5% and by 32.9% for IA-MS and IHC, respectively. VEGFR1 siRNAs were included as controls in each panel. Error bars show standard deviation of three technical replicates for panels A and C, and the standard deviation of 2 histological spots of approximately 1000 cells each for panel D. Representative VEGFR2 IHC immunoreactivity patterns exhibiting membranous and cytoplasmic immunoreactivity in trypsinized, processed, and sectioned FFPE cells (E). Original magnification, ×1000. Scale bar: 10 µm.</p