| BackgroundMost patients with esophageal squamous cell carcinoma (ESCC) have locally advanced disease at their initiation diagnosis. Bedenne et al. showed that surgical resection after neoadjuvant chemoradiation provided no benefit for overall survival (OS) compared with treatment with additional chemoradiation, especially in patients with locally advanced squamous cell cancer (SCC) who experienced response to initial chemoradiation therapy and achieved a similar prognosis and disease-free and overall survival rates after definitive chemoradiation compared with patients with comparable tumor stages treated by neoadjuvant chemoradiation plus surgery. So definitive chemoradiation therapy is also an appropriate option for non-surgical candidates, with technically resectable cancer, proposed by National Comprehensive Cancer Networks (NCCN) Clinical Practice Guidelines in Oncology (the edition in 2009). Although multiple modality combination therapy for esophageal cancer,5-year OS still fluctuates about 10%. Studies have shown that adding anti-epidermal growth factor receptor (EGFR) or anti-vascular endothelial growth factor (VEGF) monoclonal antibodies, such as cetuximab and bevacizumab, targeted therapy into chemotherapy provided additional survival benefit compared with single chemotherapy in non-small cell lung cancer, gastric cancer or colorectal cancer. It would be important to set up a method to differentiate responders from nonresponders for non-surgical esophageal cancer with rationally multiple modality combination therapy in order to change therapy scheme or intensify surveillance to improve poor 5-year OS.About 70% proportion of non-small cell lung cancer (NSCLC) patients have attained more than stageⅢat their initial examination,5-year overall survival rate of these patients remains poor approximately 20%. Combined chemoradiotherapy (CRT) or surgical resection are might been used for stageⅢNSCLC at the discretion of doctor. For non-surgical patients with stageⅢA NSCLC, combined CRT is proposed more than single radiation or chemotherapy. It might be wonderful that therapy response and outcome of combined CRT are anticipated prior to therapy so as to administrate therapy individually.The glucose metabolic derangement is also an inherent character of malignant tumor because of the increase of glucose transporter protein-1 (Glut-1) and hexokinase activity, which can be measured quantitatively in vivo by fluorine-18 flurodeoxyglucose positron emission tomography combined computed tomography (F-18 FDG PET/CT). F-18 FDG PET/CT has been shown to identify primary tumors, regional lymph nodes, and distant metastases with considerable diagnostic accuracy. 18F-FDG uptake is correlated with the growth rate and proliferation capacity of tumors, which has shown the more metabolically active the tumor, the worse the outcome. FDG uptake in ESCC has been increased markedly reported in several studies. The higher sensitivity and specificity to differentiate responders from nonresponders by detecting the decrease of FDG uptake before and after the completion of neoadjuvant therapy also have been shown in several reports. However, no information is available describing the role of FDG-PET/CT for the noninvasive assessment of response to definitive chemoradiation therapy in ESCC. F-18 FDG uptake in NSCLC has also been identified as an independent prognostic factor correlated with tumor progression and survival. The need for noninvasive methods to differentiate responders and nonresponders during definitive chemoradiation therapy for ESCC is potentially significant.The epidermal growth factor receptor (EGFR) is over-expressed in many human cancers, including NSCLC, glioblastomas, head and neck squamous cell carcinomas, and cancers of the colon and breast, and is correlated with disease progression, poor response to cytotoxic agents, and short overall survival. HIF-1αand EGFR are two generously promising tumor prognostic markers for NSCLC verified by many investigations, whose specific indicatory usage for therapy administration need to be explored further.Hypoxia is one of basic biological characters of tumor cell, which brings about a more malignant phenotype, a poor prognosis, an increased likelihood of metastasis, and a transmission resistance to both radiotherapy and various types of chemotherapy. Under hypoxia conditions, hypoxia-inducible factor-1α(HIF-1α) molecular accumulates in tumor cytoplasm and then transits into nucleus because of the ubiquitination of HIF-1αstepping down or even ceasing. The stabilized HIF-1αdimerizes with HIF-1βand the heterodimers binding to the hypoxia response elements (HRE) of more than one hundred kinds of target genes promoter, such as carbonic anhydraseⅨ(CAⅨ), Glut-1, VEGF, erythropoietin, CXCR7 and P53 et al., makes tumor biological behavior more aggressive, leading to treatment failure. HIF-1αmolecular expression is constantly regulated by multiple growth factors binding to their ligands through phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. A variety of methods for measuring oxygen levels in tumor tissue was developed. At present, the standard method of measurement of oxygen tension was developed by an oxygen electrode, which has been validated as a predictor of radiation response and successfully used to evaluate hypoxia in human cancers. The disadvantages of the oxygen electrode are the invasive nature of the procedure in routine practice, bigger heterogeneity for multi-center sample and the application being restricted to accessible tumors. Therefore, measuring tumor hypoxia through detecting HIF-1αmolecule expression in surgically excised sample or in biopsy by immunohistochemistry (IHC) might be highly desirable.Purpose1. The purpose of the first study was to use F-18 FDG-PET/CT imaging in non-surgical patients with esophageal squamous cell carcinoma (ESCC) to assess the relationship between the decrease of FDG uptake and progression-free survival (PFS) treated by definitive chemoradiation. The correlation between clinicalpathological parameters in different responders grouped by different PFS and survival was analysed.2. The aim of the second study was to investigate the difference of expression of EGFR in primary and recurrence of Nasopharyngeal Cancer (NPC) to supply a micro-evidence of anti-EGFR targeted maintenance therapy for NPC; The second section of this contents was to compare the prognostic values of the maximum standardized uptake value (SUVmax) measured by 18F-FDG PET/CT and EGFR or HIF-1αmolecular expression detected by IHC in patients with stageⅢA NSCLC treated with definitive chemoradiotherapy.3. The purpose of this study was to investigate the dynamic expression of HIF-1αand EGFR during tumor growth and after radiotherapy. It is necessary to clarify the relationship of the expression between HIF-1αand EGFR after radiotherapy to supply the basic information of usage of anti-EGFR target therapy.Contents1. F-18 FDG-PET/CT imaging in patients with ESCC to assess the relationship between FDG uptake, treatment response and progression-free survival (PFS) treated by definitive chemoradiation.Patients with ESCC accepted definitive chemoradiation were eligible for this investigation, included the presence of biopsy-proven ESCC with or without metastases in local lymph nodes (tumor with clinical stage T1-4, N0/1, and M0, according to AJCC TNM Classification of Carcinoma of the Esophagus), baseline and during therapy F-18 FDG-PET/CT scans. The relationship of FDG uptake and the effect of chemoradiotherapy was explored.2. EGFR or HIF-1αmolecular expression detected by immunohistochemistry in patients with stageⅢA NSCLC treated with definitive chemoradiotherapy (CRT) and the comparison of their prognostic values to the maximum standardized uptake value (SUVmax) measured by F-18 FDG PET/CTPatients with stageⅢA NSCLC (tumor with clinical stage T1-3, N1/2, and M0) underwent definitive CRT were included in the study. All patients have finished punctures of lung tumor biopsy, guided by compute tomography (CT) and F-18 FDG PET/CT scans at their initial examination. The prognostic value of FDG uptake and the expression of HIF-1αor EGFR were compared in assessing the outcome of NSCLC treated with definitive CRT.3. The expression of EGFR in primary and relapse NPCA retrospective review of 40 patients with clinical stageⅠtoⅣb was performed. All patients were verified tumor locoregional relapse with/or without distant metastasis by CT or MRI after combined CRT by primary and recurrence biopsies. The correlation between EGFR and Ki67 expression inspected by immunohistochemistry was analysed.4. To explore the relationship of the expression between HIF-1αand EGFR after radiotherapyXenograft tumor samples of male BALB/c nude mice were obtained after single large dose radiotherapy. The expressions of EGFR, HIF-1α, VEGFR and p-Akt were inspected at protein level. The expressions of EGFR and HIF-1αwere also detected at RNA level in order to clarify the dynamic expression of HIF-1αand EGFR during tumor growth and after radiotherapy.Methods1. PET/CT scanningPET scan was performed by a combined PET/CT scanner (Discovery LS, GE Healthcare, USA) with a multislice helical CT. The standardized uptake value (SUV) was calculated. For the quantitative evaluation of regional FDG uptake, regions of interest (ROIs) were manually placed overall tumors at the site of FDG uptake in the baseline scan and fusion PET/CT to minimize partial volume effects. FDG standardize uptakes normalized to patients’ body weight were calculated from the average activity values in the ROI, resulting in parametric images representing regional standardized uptake values (SUVs).2. Statistical AnalysisLogistic regression, Kaplan-Meier method, Cox proportional hazards model and a receiver operating characteristic curve (ROC), et al. were applied in this study.3. Detection of HIF-1αand EGFR protein expression by immunohistochemistry in patients sample of biopsy.4. Cell culture and 2-dimensional radiotherapy.5. Detection of HIF-1αand EGFR protein expression by Western Blot and immunohistochemistry in animal xenograft sample. 6. Detection of HIF-1αand EGFR RNA expression by real-time RT-PCR in animal xenograft sample.Results1. F-18 FDG-PET/CT imaging in patients with ESCC to assess the relationship between FDG uptake, treatment response and progression-free survival (PFS) treated by definitive chemoradiation61 patients referred to simultaneous radiotherapy and chemotherapy. Median total dose of radiotherapy was 6000 cGy. The cycle number of chemotherapy was 5 weeks for 45 patients and 6 weeks for 16 patients.5 patients had a complete remission (CR), 28 patients had a partial remission (PR),24 patients had a minor remission (SD), and 4 patients showed no change in primary tumor, but mediastinum lymph nodes relapse (PD). All patients underwent imaging before and during treatment. The average of SUVmean-before was 8.26±2.14 and the averaged of SUVmean-after decreased significantly to 4.56±1.68 during treatment (P=0.001), with a decrease in FDG uptake in responders (CR+PR) of 49.4%±2.37 versus 37.9%±2.69 in nonresponders (SD+PD). Patients with 5-years PFS (n=13) had a decrease in FDG uptake of 59.4%±13.4, whereas those with progressive survival (n=48) showed a decrease of only 40.2%±12.5 (P=0.000). ROC analysis for assessment of cancer PFS by FDG uptake revealed that applying a cutoff value of a 51% decrease of baseline FDG uptake would provide the highest result of sensitivity plus specificity, with a sensitivity of 76.9% and a specificity of 79.2%. The PPV and NPV was 50% and 95%, respectively. Cox proportional hazards regression analysis identified both pretherapy T stage and metabolic response assessed by decrease of SUV as independent prognostic factors for PFS, CSS and OS.2. EGFR or HIF-1αmolecular expression detected by immunohistochemistry in patients with stageⅢA NSCLC treated with definitive chemoradiotherapy (CRT) and the comparison of their prognostic values to the maximum standardized uptake value (SUVmax) measured by F-18 FDG PET/CT64 patients referred to definitive chemoradiotherapy.9 patients had a complete response (CR), whereas other 55 patients, including 73.4% incomplete response (PR+SD, n=47) and 12.5% progression disease (PD, n=8) to definitive CRT, evaluated by RECIST at the 4th to 6th weekend.37 patients experienced both locoregional and/or distant relapse within 1-year.2-year overall survival (OS) was 26.0%. Expression of HIF-1αwas found within the tumor cells in 57.8%(37/64) of patients, expressed in 60% of adenocarcinoma (ADC), which was not significantly higher than in 56.4% of squamous cell cancer (SCC) (p>0.05). Expression of EGFR was found within the tumor cells in 60.9% of patients, expressed in 60% of ADC, which was not significantly higher than in 61.5% of SCC (p>0.05). Coexpression of HIF-1αand EGFR molecules was found in 40.6%(26/64) of the cases,66.7% of these SCC cases exhibited positive coexpression of HIF-1αand EGFR, similar results shown in 66.7% of ADC. A strong significant correlation between HIF-1αand EGFR molecules expression was obtained (r=0.627, p=0.000). F-18 FDG uptake was observed in all 64 patients, with an average of SUVmax of 9.26±2.40, There was also a strong correlation between HIF-1αexpression and FDG uptake (r=0.586, p=0.000), and a weak correlation between EGFR expression and FDG uptake (r=0.281, p=0.024). Factors including SUVmax, HIF-1αexpression and coexpression of two molecules retained their significance for predicting therapy response in multivariate logistic regression analysis. This study demonstrated a significantly worse 1-year locoregional failure (LRF) free survival,1-year distant metastasis failure (DMF) free survival and 2-year OS (p=0.025 in patients with positive expression of HIF-1αor EGFR. Any significant relation between FDG uptake and survival was not found. Coexpression of HIF-1αand EGFR molecules was a significant independent predictor of 1-year LRF free survival [p=0.031, hazard ratio 6.148 (1.177-32.111)],1-year DMF free survival [p=0.003, hazard ratio 13.835 (2.507-76.339)]) and 2-year OS [p=0.047, hazard ratio 2.052 (1.011-4.265)] in multivariate analysis, FDG uptake excluded.3. The expression of EGFR in primary and relapse NPCThe difference of time to recurrence grouped by different expression of EGFR and Ki67 was compared by Log-rank test. EGFR in primary was not significantly different with recurrent focus. A strong significant correlation between EGFR and Ki67 molecules expression was obtained in primary (r=0.573, p=0.001) and in recurrent focus (r=0.698; p=0.000). A significantly shorter time to loco-regional relapse in patients with positive expression of EGFR than patients with negative EGFR expression in primary (p=0.010) and in relapse (p=0.022).4. The relationship of the expression between EGFR and HIF-1αafter radiotherapyTumor samples of A549 cell line transplanted subcutaneously in 21 male BALB/c nude mice were irradiated a dose of 2000cGy and then obtained. The expression of EGFR protein continues high before and after radiotherapy. The expression of HIF-1αprotein begins to ascend at 2 hours after radiotherapy and continues to ascend at 6 hours,24 hours and 48 hours after radiotherapy. The RNA expressions of EGFR and HIF-1αhad no change before and after radiotherapy. The trend of the expression of VEGFR is similar with the trend of HIF-1αprotein. The correlation between RNA expression of EGFR and HIF HIF-1αwas not found.Conclusions1. A cutoff value of a 51% decrease of baseline FDG uptake, which has higher NPV, is important for patients who do not respond to treatment could be identified and a change in the therapy regimen might be considered. Patients with pretreatment clinical stage T3-4 though achieving more than 51% decrease of baseline FDG uptake should consider to accept intensified therapy or consolidation therapy (such as anti-EGFR or anti-VEGF targeted therapy), or to ask for strict surveillance after chemoradiation completion.2. A significant correlation was found between HIF-1αand EGFR expression, their positive rate of coexpression is 40.6%. A correlation between FDG uptake and HIF-1αor EGFR expression was found. Both F-18 FDG uptake and expression of HIF-1αor EGFR were shown valuable prediction in treatment response. However, more potential of coexpression of HIF-1αand EGFR measured by IHC than noninvasive FDG PET/CT imaging before treatment was shown to prognose outcome of stageⅢA NSCLC treated with definitive CRT.3. The recurrent NPC cells that survive after radiotherapy express a chemoradiation induced cytoprotective phenotype that is over-expression of EGFR and Ki67. In our opinion, these findings have clinical significance and provide further support for carrying out clinical trials to apply the use of maintenance cetuximab after chemoradiotherapy to increase curability and prolong the disease-free interval in patients with tumors that are dependent on EGFR.4. The modulation of expression of HIF-1αwas at the protein level after hypo-fraction radiotherapy, which was related with the expression of VEGFR and p-Akt. The modulation of expression of EGFR and HIF-1αat the RNA level after radiotherapy was not found, which might related with their corresponding protein expression before and after radiotherapy. The persistent level of EGFR protein might demonstrate an evidence of anti-EGFR target maintenance therapy during and after radiotherapy, similarly, the gradually ascending expression of HIF-1αprotein may be another target molecule. |
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