{'fraction_non_alphanumeric': 0.038791208791208794, 'fraction_numerical': 0.028021978021978023, 'mean_word_length': 5.028817489234846, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Correlation of genes associated with drug response to prognosis of large cell lung carcinoma

Platinum-based chemotherapy remains the main treatment of advanced lung cancer. However, platinum resistance has become a major treatment obstacle. Novel therapies, particularly tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKI) and agents that target vascular endothelial growth factor (VEGF), have improved the treatment. Both chemotherapy and targeted therapy have their molecular mechanisms. This study aimed to determine the mutation, amplification, or expression status and interrelationships of the epidermal growth factor receptor (EGFR), K-Ras proto-oncogene, excision repair cross-complementation group 1 (ERCC1), and VEGF genes as well as their correlations to prognosis of large cell lung carcinoma (LCLC) after EGFR-targeted therapy, chemotherapy, and anti-VEGF therapy. EGFR and K-Ras mutations in 60 specimens of LCLC were detected by direct DNA sequencing. EGFR, ERCC1, and VEGF protein expression was detected by immunohistochemistry (IHC). EGFR gene copy number was detected by fluorescence in situ hybridization (FISH). One (1.7%) patient had an EGFR L858M point mutation in exon 21, 3 (5.0%) had K-Ras mutations, and 10 (19.6%) had EGFR amplification (FISH positive). Positive rates of EGFR, ERCC1, and VEGF proteins were 38.3%, 56.7%, and 70.0%, respectively. EGFR amplification was positively correlated to EGFR protein expression (r = 0.390, P = 0.005). The positive rate of VEGF protein was significantly higher in patients with lymph node metastasis than in those without (84.6% vs. 58.8%, P = 0.046). No significant correlations were observed among the EGFR, K-Ras, ERCC1, and VEGF genes. EGFR gene amplification and the low rate of EGFR mutation suggest that patients with LCLC are likely to obtain little benefit from anti-EGFR therapies.

Lung cancer is the number one cause of deaths among all cancers [1] . Platinumbased chemotherapy, one of main treatments for this disease, often fails due to drug resistance of tumors. The excision repair crosscomplementing 1 (ERCC1) protein plays a key role in the nucleotide excision repair system, which is primarily responsible for cancer cell resistance to platinumbased drugs. The expression level of the ERCC1 protein is an important marker in predicting platinum drug resistance in tumors; ERCC1positive patients are refractory to platinumbased chemotherapy [24] .
When the efficacy of chem otherapy reaches to a plateaus [5] , lung cancertargeting drugs, such as tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFRTKIs) [69] and antivascular endothelial growth factor (VEGF) drugs [10,11] , have achieved good outcomes. Increasing evidence suggests that potential biological markers, including gene mutation, amplification, or protein expression, are associated with EGFRTKI drug efficacy [69] , whereas mutations are closely related with drug resistance [12,13] . VEGF is the most important regulatory factor for tumor angiogenesis , and its protein expression is associated with poor prognosis [14,15] and better efficacy of antiangiogenic drugs [ 16 ] on nonsmall cell lung cancer (NSCLC). Therefore, targeted therapy and chemotherapy for cancer are based on different molecular mechanisms.
In the future, treatment options for NSCLC patients will be more dependent on precise molecular characterization. The National Comprehensive Cancer Network (NCCN) guidelines state that NSCLC patients with mutated and wildtype should be treated with EGFRTKIs in clinic. However, the majority of patients treated with EGFRTKIs were nonsmoking female patients with adenocarcinoma. Moreover, molecular studies of EGFRTKI treatment have been mainly focused on squamous cell carcinoma and adenocarcinoma [79] . Large cell lung carcinoma (LCLC), a pathologic type of NSCLC, has been rarely studied. How targeted therapy and chemotherapy are correlated with their associated genes, and how patients with optimal outcomes for targeted therapy are related to those with optimal outcomes for chemotherapy remain unknown. In addition, the correlations of these drugassociated genes to the prognosis after targeted therapy and chemotherapy are still unclear. In this study, we correlated the gene statuses of , , , and in 60 biopsies from LCLC patients with their prognoses, explored the molecular basis of targeted therapy, and discussed relationship between targeted therapy and chemotherapy.

Patients
Sixty paraffinembedded tissue samples were collected from patients with LCLC treated between February 1993 and July 2009 at the Tianjin Medical University Cancer Institute and Hospital. The pathology of each patient was rereviewed. No patient received targeted drug therapy. Patients consisted of 44 men and 16 women who were 30 to 76 years old with a median age of 58 years . Fortyfive ( 75.0% ) patients had a smoking history, which was defined as smoking one or more cigarettes per day continuously for 3 or more months.

DNA extraction and mutation analysis
DNA was isolated from the paraffinembedded tissues using an E.Z.N.A. TM Tissue DNA kit (OMEGA, US) according to the manufacturer爷s instructions. Nested polymerase chain reaction (PCR) was used to amplify exons 1821 and exon 2 [17] . The PCR was run at 94益 for 60 s, 35 cycles of 58益 for 30 s and 72益 for 30 s, followed by an extension step at 72 益 for 10 min. After purification, DNA was directly sequenced using the ABI Prism 3730 DNA sequencer (Applied Biosystems, Foster City, CA).

FISH detection of EGFR gene amplification
The LSI SpectrumOrange/chromosome 7 (CEP7) SpectrumGreen probe (Vysis; Abbott Laboratories, Downers Grove, IL) was used according to the manufacturer爷s instructions. Paraffinembedded LCLC sections (4 滋 m) were baked, deparaffined by washing in xylene, and dehydrated in 100% ethanol, and then incubated in pretreatment liquid (paraffin pretreatment kit) for 20 to 25 min at 80益. Next, sections were digested with proteinase K (0.25 mg/mL in 2 伊 SSC; pH7.0) for 15 to 25 min at 37 益 , after which EGFR/CEP7 probes were placed at the tumor regions for hybridization in the instrument. The hybridization was performed as denaturation at 73益 for 10 min, incubation at 37 益 16 h, followed by DAPI staining (0.15 mg/mL, Vector Laboratories, Burlingame, CA). Evaluation was made according to reported literature [7] . Specifically, samples with high copy number (more than 40% of cells containing 4 or more gene copies) and gene amplification (cells containing gene clusters and with a ratio of gene and chromosomes 逸2, or more than 10% of cells containing 15 or more gene copies) were regarded as FISHpositive.

Immunohistochemical detection of protein expression
The 4滋 m sections were baked, dewaxed, and rehydrated with gradient ethanol followed by overnight incubation at 4 益 with primary mouse antihuman EGFR monoclonal antibody (31G7, Zymed Laboratories, South San Francisco, CA), mouse antihuman VEGF monoclonal antibody (1:100 dilution, VG1, abcam, Hong Kong), and mouse antihuman ERCC1 monoclonal antibody (1:50 dilution, 8F1, abcam, Hong Kong), respectively, in a final volume of 100 滋 L antibody dilution solution. Next, the samples were incubated with 100 滋 L antimouse or antirabbit secondary antibody (ChemMate TM EnVision TM Detection Kit, Dako, USA) for 30 min at 37益. After staining, the samples were sealed. EGFR [8] , ERCC1 [18] , and VEGF [14] expression were evaluated according to reported literature. A sample which had more than 10% of cells with membrane EGFR staining was regarded as EGFRpositive.

Statistical methods
Pearson爷s Chisquare or Fisher爷s method in SPSS 13.0 software was used to examine relationship between biological markers and clinicopathologic features. The KaplanMeier method was used for survival analysis, and the logrank method was used to test difference. The Cox proportional regression model was used for multivariate analysis, and the Spearman rank correlation coefficient analysis was used to analyze marker correlation.

Figure 1.
A, the overall survival rate of patients with lymph node metastasis is lower than that of patients without lymph node metastasis; B, the overall survival rate of patients with early TNM stage disease is higher than that of patients with advanced stage disease.

Clinicopathologic features and biomarkers of LCLC and their correlations with prognoses
A bette r prognosis trend was found in the patients with smoking history ( = 0.067), tumor diameter < 3 cm ( = 0.083), or low VEGF expression ( = 0.098), though these findings were not statistically significant. Univariate analysis showed that lymph node metastasis ( = 0.049) and advanced stage ( < 0.001) were associated with poor prognosis (Table 1, Figure 1). Multivariate analysis showed that TNM stage (HR = 3.347, 95% CI = 1.7916.254, < 0.001) was the only independent prognosis factor. The biomarker features had no correlation with prognosis ( Table 1).
The relationship among markers amplification was positively correlated with EGFR protein expression ( = 0.390, = 0.005). The rate of amplification was higher in ERCC1positive samples than in ERCC1negative samples (23.3% vs. 14.2% ). The positive rate of ERCC1 protein expression in samples with amplification was 70%, suggesting a copositivity trend of the two markers, but this result was not statistically significant ( = 0.433). In addition, mutations were often observed in ERCC1negative samples ( = 0.411), VEGFpositive samples ( = 0.252), and samples without amplification ( = 0.486), though, similarly, these data  were not statistically significant. The positive rate of VEGF in ERCC1negative samples was as high as 65.3% , but this result was not statistically significant. A meaningful analysis could not be completed for the low rate of mutations.

Discussion
LCLC , which has an incidence of about 3 % , is associated with poor prognosis [19] . Currently, there is no effective treatment available for this disease. This study showed that LCLC occurred more frequently in male smokers, but sex and smoking history were not correlated with the prognosis. In addition, smokers had longer median overall survival (OS) than did nonsmoker patients, presumably because of the small sample size and unevenly collected data. The impact of amplification and EGFR protein expression on the prognosis of patients with NSCLC is still unclear. A previous metaanalysis www.cjcsysu.com Cheng Chen et al.
Genes status of large cell lung carcinoma indicated that EGFRpositive patients had worse prognosis than EGFRnegative patients (HR = 1.14, 95% CI = 0.941.39) [20] , and Jeon . [21] reported that nonadenocarcinoma patients with amplification had poor prognosis. Our study indicated that amplification and protein expression were not correlated with prognosis of LCLC, consistent with other reports [22,23] . Mutations of and protein expression of ERCC1/VEGF had no impact on the prognosis of patients. Only lymph node metastasis ( = 0.014) and TNM stage ( < 0.001) were found to affect prognosis, and multivariate analysis showed that only TNM stage ( < 0.001) affected prognosis. Suzuki . [23] reported that EGFR expression was related to lymph node metastasis ( = 0.028) and pathologic stage ( = 0.0046). Cappuzzo . [7] Figure 2.
A, positive immunoreactivity of EGFR (in yellow) is evident in the membrane of tumor cells (伊400). B, positive expression of ERCC1 protein was detected in the nuclei of cancer cells (伊400). C, immunoreactivity for VEGF was detected predominantly in the cytoplasm of tumor cells (伊400). D, EGFR amplification was detected by dual鄄 color FISH assay with probes for EGFR (red) and chromosome 7 (CEP7, green). FISH鄄 negative tumor cells show two red and two green signals (伊1000). E, cells with amplification show an excess of red signals (伊1000). reported that high gene copy numbers were often observed in females ( = 0.04) and nonsmoking patients ( = 0.001). In consistence with most reports [6,7,22] , however, we did not find that copy number or protein expression level were correlated with sex, smoking history, TNM stage, and lymph node metastasis, suggesting that the pathologic relationship of gene copy number and expression level is not as prominent as mutations mostly found in Asia, women, nonsmoking or adenocarcinoma patients [8,18,24] . We found that mutations and ERCC1 protein expression were not statistically correlated with clinicopathologic features of LCLC. However, we found that VEGF protein expression occurred more commonly in patients with lymph node metastasis ( = 0.052) and poor prognosis ( = 0.098), suggesting that it may be a negative prognostic factor of LCLC.
We further found that the rates of mutation, amplification, and protein expression were 1.7% , 19.6%, and 38.3% , respectively, and mutation rate was 5.0% . Previous studies showed that the rates of mutation, amplification, and protein expression in patients with lung adenocarcinoma ranged from 15% to 55 % , from 32 . 8 % to 47 .7 % , and from 50 . 0 % to 59.1% [68,18,25,26] , whereas mutation rate ranged from 15.2% to 27.7% [12,13] . In contrast, mutation and amplification rates of in LCLC patients were far lower than those in patients with lung adenocarcinoma. This may be ascribed to the high percentage of male patients (73.3% ) and smokers (75.0%) in our LCLC cohorts because mutations primarily occur in female nonsmoking patients with adenocarcinoma [8,18,24] .
After statistical analysis of 1335 patients with NSCLC, Mitsudomi . [27] found that 70% of patients with mutations were responsive to EGFRTKIs, whereas only 10% of patients with wildtype were responsive to EGFRTKIs, 35% of 663 patients with high gene copy number were responsive to EGFRTKIs, whereas only 9% of patients with low copy number were responsive to the same treatment. High responsiveness of patients with mutations and amplification to EGFRTKIs has been confirmed in other related studies [6,7,25] . Our study found that only a few patients with LCLC contained mutations or high gene copy number, suggesting that only a few patients can benefit from EGFRTKI treatment.
The positive rate of EGFR protein in NSCLC was reported in the range of 40% to 80% [28] . However, we observed EGFR protein in only 38.3% of LCLC patients. This may be due to low mutation rate or gene copy number, or to protein degradation during long storage time. Few studies suggest that EGFR protein expression is correlated with high patient responsiveness to EGFR TKIs [6] , most results failed to support this conclusion [29,30] . Although this supposition is still being debated, low EGFR protein expression rate give hint that most LCLC patients can not benefit from the EGFRTKI treatment.
High level of ERCC1 protein expression is a marker of resistance to platinum chemotherapy in NSCLC patients [3,4] . Furthermore, VEGFpositive patients generally have higher responsiveness to antiVEGF drugs than VEGFnegative patients [16] . Correlation of gene mutation, expression, or amplification status and ERCC1 protein expression have seldom been studied. Andrieux . [31] reported that EGFR induced ERCC1 expression via MAPK pathway in human hepatoma cells, and Kim . [32] showed that EGFR protein expression was positively correlated with ERCC1 expression in gastric cancer. Nevertheless, we did not find any correlation among EGFR expression, amplification, and ERCC1 protein expression.
amplification was more frequently observed in patients with ERCC1 expression, suggesting that higher copy number may induce protein expression of ERCC1 in LCLC patients. In contrast, Lee . [18] reported that mutations were more common in ERCC1negative NSCLC patients with squamous cell carcinomas or adenocarcinomas. These inconsistent results suggest a complicated relationship between EGFR and ERCC1 protein expression in lung cancer. Different pathologic types and statuses may result in distinct patterns of ERCC1 protein expression. Further study of larger scale is required to uncover their intrinsic correlations. Samples with amplification and without ERCC1 expression accounted for only 5.8% (3/51) of all studied cases, suggesting that only a few patients can benefit from both EGFRTKI and platinum therapy. The fact that amplification was more commonly observed in ERCC1positive samples implies that there is no overlap between LCLC patients with optimal response to EGFRTKIs and those with optimal response to platinumbased chemotherapy. Nevertheless, the significance of this hypothesis was limited statistically by the prevalence of few samples with mutations. Studying patient samples on a larger scale may provide further insight into the two groups of patients and their optimal treatments.
The positive rate of VEGF in ERCC1negative patients was as high as 65.3% , suggesting a major overlap between LCLC patients with optimal outcomes for platinumbased chemotherapy and those with optimal outcomes for antiVEGF treatment. Combination of these two treatments may provide a greater benefit for the patients than single agents. However, it should be noted that the correlation of gene and VEGF protein expression is statistically meaningless, suggesting that the impact of combination therapy on clinical outcomes of the patients is still unclear.
In conclusion, low mutation and amplification rates suggest only a few patients with LCLC can benefit from EGFRTKI treatment. Although correlation analysis did not find significant relationships among , , , and genes or protein expression status, this study suggests that patients with optimal outcomes for platinumbased chemotherapy overlapped with patients with optimal outcomes for antiVEGF treatment rather than those for EGFRTKI treatment. Thus, it may be useful to treat patients with lower ERCC1 expression with the combination of antiangiogenic therapy and chemotherapy, and treat patients with higher ERCC1 expression with EGFRTKItargeted therapy, although this suggestion still needs supportive data from large scale clinical trials. In addition, we did not find biomarkers that affect the prognosis of LCLC, and TNM stage was the only factor that affect prognosis.