The Predict And Prognosis Studies Of Uncommon Genes BCL11A And ROS1 In Non-small Cell Lung Cancer

BackgroundLung cancer is the leading cause of cancer death worldwide, accounting for more deaths than breast, prostate and colon cancer combined. Non-small-cell lung cancer (NSCLC) accounts for approximately 80% -85% of primary lung cancers. According to the lung cancer classification published by World Health Organization (WHO) in 2004, there were 4 types for NSCLC. Adenocarcinoma (31.5%), Squamous cell carcinoma (29.4%), Small cell lung cancer (17.8%), Large cell carcinoma (9.2%). With the study and explore of targeted therapy, non small cell lung cancer (NSCLC) has been treated as a single disease. The paper published by Dr. William D. Travis on Journal of Clinical Oncology told us that personalized medicine in advanced lung cancer is determined not by histology but also by genetics. EGFR (epidermal growth factor receptor), EML4-ALK (echinoderm microtubule-associated protein like 4, EML4), c-MET(mesenchymal-epithelial transition factor, c-MET) and ROS1 (c-ros oncogene 1) are the candidate genes for adenocarcinoma to detect. However, according to the clinical data and research results, squamous cell carcinoma patients have low positive rate, even positive they will not get long survival to targeted therapy. So, it is important to find more and more useful driver genes for squamous cell carcinoma.According to the data, the squamous patients have low EGFR mutation rate and there is only 7% patient can receive EGFR-TKIs. Unfortunately, the clinical data told us the lung adenocarcinoma patients who received EGFT-TKIs have a better response than non-adenocarcinoma patients. The response rate of non-adenocarcinoma patients and adenocarcinoma patients is 27% and 66%, the mPFS is 3.0m and 9.4m, respectively. In order to tell the molecular difference between the adenocarcinoma and non-adenocarcinoma patients, The Cancer Genome Atlas (TCGA) use the high-throughput techniques analyzing the genetic and epigenetic basis of cancers systematically and comprehensively. Using simplified description, they derived a hierarchical classification of 3,299 TCGA tumors from 12 cancer types. The top classes are dominated by either mutations (M class) or copy number changes (C class). For the non-small cell lung, adenocarcinoma is classified as M and squamous cell carcinoma is C. Maybe this result can tell us some reasons of why the squamous carcinoma patients didn’t have the same response rate to EGFR-TKI as well as the adenocarcinoma patients. Also the Cancer Genome Atlas Research Network provide a comprehensive landscape of genomic and epigenomic alterations of lung squamous cell carcinoma. The tumor type is characterized by complex genomic alterations, with a mean of 360 exonic mutations,165 genomic rearrangements, and 323 segments of copy number alteration per tumor. Recurrent mutations in 11 genes were fund, including the TP53, CDKN2A, PTEN, PIKK3CA, KEAP1, MLL2, HLA-A,NEF2L2,NOTCH1 and RBI.Many genes are associated with lung squamous cell carcinoma, such as FGFR1 gene amplification, DDR2 muation, PI3CA amplification and mutation, PTEN mutation and so on. The drugs are ongoing research and maybe in the future we could have some good news. At the same time, more research work should be done in order to find more useful genes for cancer therapy.Previous studies of our lab examined B-cell lymphoma/leukemia 11A (BCL11A) expression at the protein and mRNA levels in a cohort of NSCLC patients. Compared to adjacent non-cancerous lung tissues, BCL11A expression levels were upregulated in NSCLC tissues at both the protein and mRNA levels. Intrestingly, BCL11A protein levels were higher in patients with squamous histology. Also, we found that BCL11A activation regulated by miR-30a and gene amplification may be a potential diagnostic and prognostic biomarker for effective management of NSCLC. The proto-oncogene BCL11A, also called Evi9 (ecotropic viral integration site 9) and CTIP1 (chicken ovalbumin upstream promoter transcription factors), belongs to the BCL11 Gene family. Bcllla locates at the human 2p16.1 and is expressed in several hematopoietic tissues including bone marrow, splenic B and T cells, monocytes and megakaryocytes, encodes a Kriippel zinc-finger transcription factor, which has been shown to be essential for pre-B-cell development, lymphocyte maturation, and goblin switching. Aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and considered as a proto-oncogene. The article published on Nature 9 Jan 2015 shows us BCL11A gene is a proto-oncogene of triple-negative breast cancer. The results give us more evidence and confidence to analyze BCL11A gene in solid tumors. According to our previous result, we know that the BCL11A expression level was much higher in SCC and LCC samples with amplifications than in other histology types. Also, BCL11A was an independent prognostic factor for disease-free survival and overall survival. The Alternative pre-mRNA splicing of human BCL11A leads to three transcripts predicted to yield protein isoforms designated as eXtra-Long (XL; 5.9 kb/125 kD), Long (L; 3.8 kb/100 kD) and Short (S,2.4 kb/35kD). BCL11A isoforms have different characters and different functions. First, the three isoforms have different structures and function areas. Second, they have different sub-cellular localization patterns. The XL and L isoforms locate in the nucleus and interact with BCL6. The S isoform locates in the cytoplasmic and couldn’t interact with BCL6. Through the upper results, if we want to find more functions about BCL11A, we may analyze the isoforms and do further research on the isoforms. In order to find more functions of BCL11A in lung cancer, I did the following experiments to analyze the isoforms in NSCLC.ROS1 receptor tyrosine kinase rearrangements define a subset of NSCLC and were found in only 1.2-1.7% NSCLCs, but seemingly only in triple negative (EGFR and KRAS non-mutated and ALK non-rearranged) adenocarcinomas and preferentially in non-or light smokers. The ROS1-positive patients sensitive to the small-molecule tyrosine kinase inhibitor crizotinib, approved for the treatment of advanced ALK-positive NSCLC. Updated efficacy and safety data are presented for crizotinib in patients with advanced ROS1-rearranged NSCLC. Some clinical trail showed the objective response rate (ORR) was about 56%, the disease control rate (DCR) was about 85%. The clinical data showed ROS1 fusion gene can be used to guide the treatment and predict the prognosis of non-small cell lung cancer patients, especially in the treatment of patients with lung adenocarcinoma.ObjectiveThis study is mainly about the effect and prognosis of two rare oncogenes in patients with non-small cell lung cancer. On the basis of our previous result, I use the IHC method to analyze the isoforms of BCL11A in NSCLC. First, select three antibodies for three isoforms and choose the BCL11A positive samples that previously detected. Then, do the immunohistochemistry experiment and according the isoform result, analyze the relationship between isoforms and the prognosis of patients. Finally, reports the efficacy of crizotinib in ROS1-positive non-small cell lung cancer patients and points out the prognosis of ROS1 gene in lung cancer patients especially in lung adenocarcinoma patients.Chapter 1 The BCL11A Isoforms In Non-small Cell Lung Cancer Methods1. Select the tissue samplesAccording to our previous results, the BCL11A expression level was much higher in SCC and LCC samples with amplifications than in other histology types. So I choose 40 BCL11Ahigh expression samples,27 Squamous-Cell carcinomas (SCC) account for 67.5%,8 Large-Cell lung carcinomas (LCC) account for 20.0% and 5 Adenocarcinomas account for 12.5%.2. Select the antibodies for immunohistochemistryBase to the different structures and different sub-cellular localization of the BCL11A isoforms, I choose three monoclonal primary antibodies to identify them. (1) BCL11 A/123 antibody (Active Motif) which was raised against a recombinant protein corresponding to amino acids 637-835 of BCL11A-XL. (2) Ab 19487 (Abeam) antibody which epitope is in core of animo acids 172-434 can identify the BCL11A-XL and L isoforms. We can use the exclusive method to distinguish the two isoforms. (3) Ab18688 (Abeam) which epitope is in core of animo acids 1-171 can identify all the three isoforms. On the base of sub-cellular localization, the S isoform locates in cytoplasm while the XL and L are both in the nucleus. So in theory we can distinguish the S isoform. (4) The second antibodies were Mouse IgG labeled by enzyme horseradish peroxidase and labeled by GFP (GeneTex).3. Protocol of the Immunohistochemistry3.1 Sample PreparationUsing the paraffin-embedded tissues to make sections, every section is about 4-6μm. Every sample prepares 7 sections, one section for pathological assessment, the other 3 for negative control.3.2 Procedure of IHC(1) Bake the sections:65℃ for 2-3 hours. It is important to bake the sections to avoiding the falling and make sure the result.(2) Deparaffinization and rehydration:Incubate sections in three washes of xylene for 10 minutes each. Then incubate them in gradient ethanol concentrations liquid for 5 minutes, concentrations are 100%,95%,90%,80% and 70%.(3) Antigen retrieval:Bring slides to a boil in 0.01M sodium citrate buffer PH 6.0 then maintain at 95℃ for 3 minutes. Cool slides for 30 minutes and incubate sections in three washes of PBS for 5 minutes each.(4) Remove the endogenous peroxidase:Incubate sections in 3% hydrogen peroxide for 10 minutes at room temperature. Then wash them three washes of PBS for 5 minutes each.(5) Blocking:Block each section with appropriate blocking solution for 30 minutes and remove it.(6) Incubate primary antibody:Add 100μl primary antibody (1:100) to each section and incubate overnight at 4℃. Then rewarm at room temperature for 30 minutes and wash sections in PBS three times for 5 minutes each.(7) Incubate second antibody:Add 100μl horseradish peroxidase labelled second antibody to each section and incubate for 30 minutes at room temperature. Also add the second antibody to the negative control sections. After that remove it and wash sections in PBS three times for 5 minutes each.(8) Staining:Add suitable DAB to each section and monitor staining closely. Immerse slides in water as soon as the sections develop.(9) Counterstain and block sections:Counterstain sections in hematoxylin per manufacturers’instructions for 5 minutes and then dehydrate sections and block them.4. Protocol of the Immunofluoresence4.1 Cell line preparationAll lung cancer cell lines were cultivated in flasks. When the cells grew well to occupy 80%～90% of the flask, they would be appropriated to use.4.2 Procedure of the IHC-P(2) Fixed the cells:Wash the cells with PBS for twice slightly and fix them with paraformaldehyde solubilized in PBS for 10 minutes. Then wash the cell with PBS in 10 minutes for twice.(3) Blocking:Block each section with appropriate blocking solution for 30 minutes and remove it.(4) Incubate primary antibody:Add the primary antibody (1:100) which diluted by Triton-X100 to each section and incubate overnight at 4℃. Triton-X100 can damage cell membrance so that the antibody can easily through and combine with the antigen. Remove the antibody and wash the cells in PBS three times for 5 minutes each.(5) Incubate second antibody:Add GFP labelled second antibody to each section and incubate for one hour at room temperature. After that remove it and wash cells in PBS three times for 5 minutes each.(6) Staining:Add suitable DAPI to each section for 3 minutes and then wash in PBS three times for 5 minutes each.(7) Blocking sections:Add the anti-fading agent to cover the cells and block them.5. Results1. The BCL11A protein isoforms results of total 40 NSCLC cases. (1) The BCL11A-XL isoform especially expressed in SCC and LCC and the subcellular location is nucleus, which is accordance with other research results. There are 19 (19/40,47.5%) cases positive for BCL11A-XL, SCC cases account for 63.2%(12/19), LCC for 36.8%(7/19) and no positive case for AC. (2) All cases are BCL11 A-L and S isoforms positive and the subcellular locations are nucleus and cytoplasm. 2. The IHC-P test showed the BCL11 A-L protein expressed in both nucleus and cytoplasm in A549 lung cancer cell.Chapter 2 Relationship of BCL11A-XL overexpression and the prognosis of Non-small Cell Lung CancerMethods1. Analyses objectsAccording to the isoforms result, the BCL11 A-XL isoform was histological differentially expressed. So the correlation analysis was performed to explore whether BCL11 A-XL expression was related to clinicopathological variables in patients with NSCLC. A consecutive cohort of patients diagnosed with the TNM stage Ⅰ to ⅢA non-small cell lung cancer received operation at Guangdong General Hospital between January 2003 and July 2008 were enrolled in this retrospective analyses. Survival data were collected from the Guangdong General Hospital medical record system or by telephone follow-up. DFS was defined as the time from operation to relapse for metastasis, OS was defined as the time from initial diagnosis to death or the last follow-up. DFS from operation to the last follow-up for normal or loss to follow-up were censored data, OS from initial diagnosis to the last follow-up for being alive or loss to follow-up were censored data.2. MethodsAll analyses were performed using SPSS 13.0 software. The chi-square test was used to compare qualitative variables, and those with an expected frequency of<5 were analyzed by Fisher’s exact test. A non-parametric test was used to analyze quantitative data. Chi-square tests were used to assess the association of BCL11A-XL level with clinical variables. Survival curves between subgroups divided according to BCL11A-XL expression level were analyzed using the Kaplan-Meier method, and significant differences among subgroups were compared by log-rank test. A multivariate analysis was performed using the stepwise method. Hazards ratios and 95% confidence intervals were calculated using Cox proportional hazards models. P values< 0.05 were considered statistically significant.Results1. Baseline analysesThe result showed that BCL11A-XL expression had no relationship with age, gender, smoking status, histology, clinical stage, tumor size and lymph node status, but it did correlate with histology (p=0.006) and smoking status (p=0.049). BCL11A-XL isoform especially expressed in SCC and LCC and it expression showed a negative correlation with smoking status.2. Prognosis analysesIn all patients, the median disease free survival and median overall survival were 54.4 months and 73.9 months. The survival analysis showed DFS and OS were both no statistically significant difference between BCL11A-XL positive and negative groups (p>0.1). But in the subgroup of patients with SCC and LCC, BCL11A-XL expression was predictive of better DFS (x2=5.32, P=0.021).The median DFS of subgroup patients without BCL11A-XL expression was 20.8 months, but in the BCL11A-XL expression group, only 36.8% of patients relapsed at the endpoint of follow-up. Although no relationship between BCL11A-XL expression and overall survival (OS) was found in SCC and LCC patients, there was a tendency towards decreased survival in patients whose tumors lack of BCL11A-XL (p>0.1).3. Multivariate stepwise Cox regression analysisTo estimate the risk of DFS and OS in the cohort of 40 patients, theage, gender, smoking status, TNM stage, histology type and BCL11A-XL expression were used as covariates in a Cox regression model. The cox regression survival analysis indicated that BCL11A-XL expression was an independent marker of DFS (hazards ratio [HR] 0.246; 95% confidence interval [CI] 0.065-0.939,p=0.040) in patients with NSCLC, but not for OS.Chapter 3 The predict and prognosis role of ROS1 gene in NSCLCMethod1. Analyses objectsCollected the ROS1-positive non-small cell lung cancer patients from Guangdong lung cancer institute, all patients were accepted crizotinib treatment. Survival data were collected from the Guangdong General Hospital medical record system or by telephone follow-up.2. MethodThe ROS1 status was determined by RT-PCR assays, and patients were received crizotinib 250 mg BID. Six patients were evaluable for response. The objective response rate (ORR) was determined based on RECIST 1.1. The disease control rate (DCR; stable disease [SD]+partial response [PR]+complete response [CR]) was evaluated at 8 weeks.Results1. Basic informationA total of 710 cases non-small cell lung cancer patients detected the ROS1 gene from January 1,2014 to December 31,2014, and the positive rate is 2.0%(14/710). There were 6 patients received crizotinib treatment, the median age was 48.5 (range 33-57), all patients were never-smokers,83.3%(5/6) patients had adenocarcinoma histology. All patients were tested for ALK rearrangement and all were negative.2. Clinical efficacyThe median number of prior treatments for advanced disease was 1 (range 0-1). To date, the ORR is 50%(3/6),3 partial responses achieved by the first restaging scan at 4 weeks. The DCR at 8 weeks was 100%(6/6), with 3 PRs and 3 SDs. Median duration of treatment was 22 weeks (range 16-56). Two patients had disease progression and were discontinued from the study. Responses are ongoing and 4 patients continue on study.Conclusions1. Using IHC we have detected the BCL1A-XL and L two isoforms in non-small cell lung cancer, and the IHC-P test showed the BCL11A-L protein expressed in both nucleus and cytoplasm in A549 lung cancer cell.2. The BCL11A-XL isoform differently expressed in squamous cell carcinoma and large cell carcinoma and it expression was negatively related to smoking status.3. BCL11A-XL protein predicts the relapse in early stage SCC and LCC patients and can be an independent prognostic factor for disease-free survival of non-small cell lung cancer.4. ROS1 fusion gene can be used as the targeted gene of patients with adenocarcinoma and predict the clinical efficacy.