Integrin Alpha V Beta 5 And EGFR As Biomarkers For The Assessment Of Non-Small-Cell Lung Cancer Metastasis And Overall Survival

ObjectiveIt is reported by WHO in Jan 2013 that Cancer is still coming on top causing mortality in the world. There are 760 million of people died from Cancer in 2008, it is predicted the cancer death population would reach to 1300 million in 2030. Lung Cancer is number one among all high incident cancers in China since 2000, it has been growing fastest compare with any other cancers.Lung cancer metastasis is the main cause of the death so recognized as the main challenge in the field. 90% of cancer caused death relates to primary cancer metastasis. Finding the predictive biomarkers will support high risk patient stratification and help for lung cancer patients to improve the treatment response and choose for more suitable therapy.There are 80-85% are Non-Small Cell Lung Cancer(NSCLC) among all lung cancer type.60-70% NSCLC patient are in IIIB or IV when diagnosed, almost lost the opportunity to cure, only treat with Chemotherapy to release the symptom and prolong the overall survival, however it always comes with low efficacy and side toxicity is severe. Therefore targeted therapy is gradually becoming NSCLC patients preferred treatment, is the drug discovery new strategy and challenge for anti-cancer drug development.Current targeted therapy in lung cancer are tyrosine kinase inhibitors (TKIs), it decreases the NSCLC tumor volume, prolong the patient survival and have less side effects. If we may develop targeted therapy to treat metastasis, it may well combine with TKIs to treat the lung cancer patients in the future.In tumor cells, cooperation between integrin and members of the Endothelial growth factor (EGF) receptor family affect many aspects of tumor progression, including tumor initiation, proliferation, migration and invasion. The EGF pathway is often hyperactivated in cancer, which potentiates the tumor cell migration and metastasis of this aggressive disease. EGF stimulates tumor cell migration in vitro and metastasis in vivo, and these effects require integrin avβ5. This research design is to approve this hypothesis in clinical study and potentially may apply to many cancer indications.There is no publication on integrin avβ5 Immunohistochemistry (IHC) staining, showing this platform is specific for detection it’s expression on human NSCLC tissue. Further this method could be developed for targeted therapy patient recruitment. At the same time, there is no clinical study to learn if this biomarker relates to any clinical outcomes such as overall survival or metastasis. No clinical research to determine the both EGFR and integrin av(35 positive patients ratio, this may help health professionals to design the treatments for NSCLC and metastasis patients.MethodsPatient recruitmentThis experiment recruited 147 NSCLC in patient undertaken surgical procedures in Fudan University Zhongshan Hospital during 2005-2007. The selected patients require full clinical information on: patient number, age, gender, NSCLC cancer type such as Squamous Cell Carcinomas (SCC), Adenocarcinoma (AC) or others, Clinical pathological grade (high, mid, low), Overall survival, TNM staging and Metastasis status. All patient cancer tissue were collected the further research use.32 NSCLC patient was recruited for a different study, however the adjacent normal (non-tumor) tissue were stored under same procedures. The clinical pathological information for the tumor tissue is collected same factors as 147 cases.IHC assav establishmentTwo sets of tissue collected above will be used into IHC staining in order to set up IHC staining protocol, assessment and compare the expression level in tumor and non-tumor tissue.IHC staining protocol was established and tested on DAKO autostainer with finalized standard operational protocols (SOPs) and kept the assay with repeatable staining for same sample. Anti-integrin avβ5 is a Rabbit monoclonal antibody with 1:300 dilutions for experimental slides. Each experimental run will include positive control sample form previous experiments and negative IgG controls.To guarantee reproducibility of the IHC evaluation by making consensus among two Pathologists (concordance rates≥ 85% refer to FDA requirements) who participate this study results evaluation.EGFR mutation147 patients samples participated in EGFR mutation testing. Haematoxylin & Eosin (H& E) staining for each case were used to identify the cancer tissue and adjacent normal tissue. Thin sections of Formalin Fixed Paraffin Embedded (FFPE) samples are subjected to this analysis should be used within a week after cutting and remaining paraffin-tissue blocks are usually archived. Standard DNA isolation procedures often face challenges in low DNA yield or poor performance in downstream applications (e.g., PCR). To ensure the quality of the DNA extraction, the procedures of chemical modification by formaldehyde and fragmentation of the DNA during tissue processing (sampling, fixing, embedding) and storage (humidity, time, temperature) of the samples are restricted.PCR amplification is carried out with primers that flank the target. To estimate the concentration and purity of DNA samples, it is recommend that the luL Plasmid or 3-5μL PCR product be electrophoresed on an agarose gel (≤0.75%) and the intensities of the ethidium bromide bands of DNA samples be compared against that of the bands present in a ladder of mass standards.PCR steps is carried out with the primers binding (55-60℃) and the polymerase extension (50-72℃), it is recommend to join into one step as 68-72℃. This step includes primer binding the target and polymerase extension at once; the recommended time for this step is 1 second for each 100 bases of PCR product. A positive control can also be included (amplification of a plasmid which is known to work) and Negative controls have designed to guard against gross error.DNA sequencing will be carried out in order to report the mutation status of EGFR in each case at exon 18,19,20 and 21. After three decades of improvement, the Sanger biochemistry can be applied to achieve read-lengths of up to-1,000 bp, and per-base’raw’accuracies as high as 99.999%.Statistical analvsisThe R.version2.15.2(2012-10-26) statistical package was applied for data analysis. Clinicopathological features in integrin avβ5 positive and negative patients, or cytoplasmic membrane-positive and negative patients, were compared using the Pearson chi-square tests for categorical variables. Integrin avβ5 expression levels in tumor tissue and non-tumor tissue were compared using the Wilcoxon signed rank test on the presence or absence of integrin avβ5 expression.Kaplan-Meier plots and log-rank tests were performed for survival analysis. Overall survival time was calculated from the date of surgery to death or last follow-up date. Cox regression was completed in the univariate and multivariate survival analysis to determine the association of individual Clinicopathological variables with overall survival. All variables with P< 0.05 in addition to age, gender and the identified prognostic factors for this cohort (ie, tumor size, tumor stage), and EGFR mutation were subsequently calculated to find positive ratio that relates to integrin avβ5 positive ratioResults147 patients median of follow up time is 42 months (1-84 months total) joined this study. This current study provides first time integrin αvβ5 IHC staining expression and prevalence data relates to NSCLC.This research has proved the hypothesis that integrin αvβ5 is a predictive marker for NSCLC metastasis (P<0.011). At the same time, integrin expression positive is significant different when compare disease stages (P<0.027) and cancer type (P<0.007). However, the expression level of integrin αvβ5 is not significant different when compare age, gender and pathology grade.32 cases of non-tumor tissue were applied into IHC staining in order to identify the significant expression level between tumor and non-tomor tissues. It was compared using the Wilcoxon signed rank test on the presence or absence of integrin αvβ5 expression and concluded significant expression differences (P<0.0023).Apply Kaplan-Meier to analyze patient overall survival relates to integrin αvβ5 expression. Expression positive (2+/3+) patient overall living time is significant shorter than negative (0,1+) patients (P<0.029), it is concluded integrin αvβ5 as a prognostic biomarker for NSCLC patients.Overall survival significantly relates to integrin αvβ5 positive/negative value (P<0.0317); nodal stage (P<0.0001); pathology grade (P<0.0065); disease stage (P<0.0001) and tumor size (P<0.0001) while it does not significantly relate to gender, age, Cancer type according to Cox regression analysis (Univariate analysis). When apply Cox regression analysis (Multivariate analysis), there is no significant relationship between overall survival relates to clinical factors except lymph node metastasis (P<0.0064).EGFR mutation patients in AC but not in SCC or other subtype for Chinese patients have been reported, this study shows the same results. Male patients with EGFR mutation is higher than Female patients is same as published data, this experiement shows male is 34.0% of total patients and female is 7%.Apply Kaplan-Meier to analyze patient overall survival significantly relates to both EGFR and integrin αvβ5 two biomarkers (P<0.0257). When we look at both EGFR and integrin αvβ5 positive patients, it with most significant poor survival compare with other three groups; the patients who have less poor survival is integrin αvβ5 positive and EGFR negative patients. When integrin αvβ5 is not present, EGFR positive and negative have made no significant differences especially survival rate continuously be same till experiment completed.With all the experimental results above, we would like to consider on treatment and drug development strategy. This study has approved the hypothesis at the beginning of this study:when EGF pathway is hyper-activated, which potentiates the tumor cell migration and metastasis. EGF stimulates tumor cell migration and metastasis, and these effects require integrin av|35. It is calculated that there are 12.3% patients are both EGFR positive and integrin avβ5positive, this may apply to study design on patient recruitment and further target therapy treatment.Conclusions1. This research has proved the hypothesis that integrin avβ5 is a predictive marker for NSCLC metastasis and prognostic biomarker for NSCLC patients.2. It is approved integrin avβ5 could be developed as a targeted therapy for NSCLC metastasis and more cancer indications.3. Integrin avβ5 IHC testing platform could be applied for biomarker screening for patient recrutment.4. This experiment proves the publication data on:EGFR mutation patients in AC but not in SCC or other subtype for Chinese patients. Male patients with EGFR mutation is higher than Female patients male is 19.7% and female is 15.0% total patients.5. When we look at both EGFR and integrin avβ5 positive patients, it with most significant poor survival compare with other three groups; the patients who have less poor survival is integrin avβ5 positive and EGFR negative patients. When integrin avβ5 is not present, EGFR positive and negative have made no significant differences especially survival rate continuously be same till experiment completed.6.12.3% patients are both EGFR and integrin avβ5 positive, it is expected to combine EGFR TKIs amd integrin av(35 antagonists to treat the NSCLC and metastasis.