Prognostic Correlation Between Neutrophilic Leukocytosis, VEGF, VEGFR and ERCC1 in Advanced Non-Small Cell Lung Cancer (NSCLC)

Background:: Leukocytosis is a known paraneoplastic syndrome that confers poor prognosis in patients with advanced NSCLC. Several studies in the past have proven the association between the increased production of white blood cells (WBC) and neutrophils to the tumor cell production of hematopoietic growth factors such as granulocyte-colonystimulating factor (G-CSF). Angiogenesis plays a vital role in tumorigenesis and has attracted interest as a potential target in cancer therapy. Expression of vascular endothelial growth factor (VEGF) in lung cancer tumor tissue of patients was associated with shorter survival. Patients with tumors expressing VEGF or VEGF receptor KDR had poorer outcomes. Also, VEGF expression and KDR expression were positively correlated. Tumor tissue expression of the excision repair cross-complementation group 1 (ERCC1) was found to be a useful predictive marker of resistance to Cisplatin and its analogues. The protein product of this gene repairs DNA damage caused by Cisplatin through an excision repair mechanism. When treated with Cisplatin compounds, longer survival was noted in patients with negative expression of ERCC1 in tumor tissue compared to patients whose tumors had positive expression. Aim of the study: To study the role of leukocytosis and neutrophilia as clinical prognostic markers along with pharmacogenomic markers, VEGF, VEGFR and ERCC1 in relation to the overall survival in patients with advanced NSCLC. Patients and methods: From 1998–2007, 29 patients with advanced NSCLC were included in our retrospective study. Chart review revealed their baseline WBC count and clinical course. Tumor histology was reviewed and selected for immunohistochemical analysis of various pharmacogenomic markers. The levels of expression of VEGF, VEGFR2/KDR and ERCC1 were measured and correlated with overall survival using Cox proportional hazards model. The rabbit polyclonal VEGF antibody (A-20) was used at 1:100 dilution from Santacruz Biotechnology, while the VEGFR2/KDR (55B11) rabbit monoclonal antibody was used at 1:200 dilution from Cell Signaling. The mouse monoclonal ERCC1 antibody (8F1) was used at 1:400 dilution from Thermo Scientific. The interpretation of the VEGF, VEGFR and ERCC1 staining was done by two pathologists independently. Results: Out of the 29 studied patients, the mean age was 64 (42–84). There were 7 females (24%) and 22 males (76%). Five patients (17%) had leukocytosis with WBC more than 11,000/microliter (11,400-18,600). Twenty one patients (72%) had documented differential white cell counts on presentation. Among those 21 patients, six patients (21%) had absolute neutrophil count (ANC) of more than 8,000/microliter (8,170-16,160). Five patients had neutrophilia in addition to the leukocytosis and one patient had neutrophilia without leukocytosis. Leukocytosis and neutrophilia were felt to be tumor related. Four patients had thrombocytosis (14%) with platelet count more than 400,000/ microliter. Eosinophilia was present in two patients with eosinophils more than 5% (5,800–6,800). All six patients with leukocytosis and/or ANC >8,000 had positive VEGF and VEGFR2/ KDR expression where 67% of those patients had ERCC-1 positive expression. On statistical analysis using Cox proportional hazards model, among the 29 patients, female gender was found to be significantly associated with better overall survival (p=0.048). Higher levels of expression of ERCC1 was significantly associated with shorter survival as shown in figure 1 (p=0.018). All patients with leukocytosis had positive expression of VEGF and VEGFR expression. The level of expression of those markers did not reach statistical significance in relation to the overall survival, possibly due to the small number of patients (p=0.58, 0.28 and 0.33 respectively)., Thrombocytosis was associated with poor survival (p=0.091). For the 21 patients with documented differential white cell counts, neutrophilia at presentation had a statistical trend towards shorter survival as shown in figure 2 (p=0.16). Conclusion: Neutrophilic leukocytosis at presentation correlates with high levels of expression of VEGF, VEGFR and ERCC1 in tumor tissue and is an important prognostic marker associated with shorter survival in patients with advanced NSCLC.