Regional Research Partnership
Currently Funded Research
1. Title
Alterations of gene expression and the development of brain metastases
2. Principal Investigators
|
Didier Dréau, Ph.D. Tel: 704 687 8314 |
Anthony Asher, M.D. Tel: 704 376 1605 (o) |
Richard L. White, M.D. Tel: 704 355 3809 |
3. Co-investigators
|
James Dollar, M.D. (Pathology) Tel: 704-355-2884 |
Stuart Burri, M.D. (Radiation Oncology) Tel: 704-355-2884 |
Kayvan Najarian, Ph.D. Tel: 704 687 8573 |
4. Responsible Chairperson and Safety Monitor:
Frederic L. Greene, MD
Chairman
Dept. General Surgery
Tel: (704) 355-Incomplete
Fax: (704) 355-Incomplete
Email
Personnel
Heather Wyan, Jonathan Adams
5. Purpose of the study:
5.1 Hypothesis
The goal of this application is to determine the key gene expressed in lung carcinoma tumors leading to brain metastases. T he hypothesis is that primary lung tumor associated with the development of brain metastases have altered expression of multiple genes compared to primary tumor not associated with the development of brain metastases. The overall benefit of this study is the definition of key cancer markers associated with the development of brain metastases following the resection of lung carcinoma. These markers will be critical in the development of refined classifications of primary lung carcinoma, and potentially may become treatment targets.
5.2 Specific aimsThe specific aims are to:
- Compare the gene expression in paired primary and metastases to the brain of lung cancer.
- Analyze the presence of specific markers within primary lung tumor and their association with the development of brain metastases.
- Confirm the alterations in gene/pathway- and protein- expressions most significant to the development of brain metastases in lung cancer patients.
- Determine whether the expression of these genes is also altered in primary breast, melanoma, renal, and colon cancers associated with brain metastases.
5.3 Purpose
The purpose of this application is to identify gene and/or protein associated with the development of brain tumor in lung carcinoma patients.
5.4 Rationale
The identification of pathway alteration and/or gene or protein expression alterations in lung cancer patients will allow the development of new diagnosis and/or treatment and potentially limit or eradicate the development of brain metastases.
5.5 Value/significance of the study
This study is innovative because it uses both paired and primary lung tumor to elucidate the differences in gene and protein expression associated with the development of brain tumor in lung cancer patients. These analyses will provide cancer marker targets, as well as possible new therapeutic approaches. Moreover, it is expected that such studies will provide a basis for developing standard diagnostic tools that define the aggressiveness of a given lung cancer and facilitate the selection of the most appropriate treatment.
6. Background and significance
6.1 Background
Lung cancers
Worldwide, lung cancer is the most common cause of cancer-related death. In 2005, 173,000 new cases and 163,000 deaths from lung cancer (small cell and non-small cell combined) were recorded in the United States.[1] Untreated, patients with small cell carcinoma of the lung have a median survival from diagnosis time of only 2 to 4 months.[2, 3] The overwhelming majority of lung tumors are carcinomas, i.e. cancers of epithelial origin. Lung cancers are generally heterogeneous, consisting frequently of cells of different histological subtypes.[4] Based on histology and clinical behavior, lung tumors are categorized as either small cell carcinoma (SCLC 20-25%) or non-small cell lung cancer (NSCLC 70-80%).[5] NSCLC are further identified as either squamous cell carcinoma (30%) adeno-carcinoma (30%) or large cell carcinoma (10%). In the early stages of disease, lung cancer tends to be asymptomatic and only 20% of patients are diagnosed with resectable, localized, disease (stages I-IIIA).[3, 6] Advanced stage at diagnosis and the relative resistance of the disease to currently available anti-cancer drugs leads to a high mortality rate (5-year survival ~ 10 and 15%). The 3-year survival of SCLC patients with limited or extensive disease is 5-10% and <1%, respectively. The prognosis for 5-year survival following NSCLC diagnosis is of 60-70% for stage I disease and <1% for stage IV disease.[3]
The primary cause of lung cancer is smoking tobacco and the stage of the cancer is correlated to the level of tobacco exposure.[4] Additionally, genetic factors, diet or other environmental factors appear to be associated with increased disease risks for lung cancers.[4] Lung cancer is caused by a chronic exposure of the bronchial epithelium to multiple carcinogenic agents.[4] In both SCLC (~ 100%) and NSCLC (~ 90%) a loss of coding potential from the short arm of chromosome 3 occurs prior to morphological alterations in the early stages of the neoplastic transformation.[4] Lung cancers have been associated with alterations in the gene associated with cell repairs including p53.[7] The loss of p53 function (mutation) is detected in the majority of lung carcinomas. The mutation of K-Ras2 also occurs in approximately 20% of NSCLC lesions.[4] Microarray analyses have shown that many other genes show dramatic differences in expression between lung tumors and normal lung tissue. [8]
Brain metastasis
The incidence of brain metastasis is rising with improved survival of cancer patients with 40% of intracranial metastatic tumors.[9] In the US, 170,000 cancer patients develop brain metastases annually. Intracranial metastases are seen in approximately 24% of patients that die from cancer.[10] Autopsy series suggest that lung cancers are the most common primary tumors to metastasize to the brain.[10] Approximately one third of patients with lung carcinoma develop intracranial metastases eventually, and 50% (or 85,000) of brain metastases result from this type of cancer.[11] Multiple tumors are observed in more than 50% of the patients depending on the type of primary tumor. Male and female patients develop metastases mostly from lung and breast cancer, respectively. Incidence of brain metastases parallels that of primary systemic tumors.[9] Metastases are generally round well-demarcated lesions with leaky tumor vessels resulting in an extensive zone of edema surrounding the tumor.[12]
About two thirds of brain metastases become symptomatic due to increased intracranial pressure and focal irritation or destruction of neurons.[12] Surgical resection can be used in patients with one apparent metastasis whereas radio-surgery is an effective treatment for surgically inaccessible lesions.[13] The median latent interval between the initial diagnosis of lung cancer and diagnosis of brain metastases is 6-9 months. In 20% of patients, metastases are detected during diagnosis of the primary tumor, and in 50% of patients, they are detected within 1 year following diagnosis. Patients with the best prognostic indicators usually survive 18 to 24 months.[10]
Brain metastases from lung cancers
Lung cancer metastasis to the brain results in dismal prognosis for the patients. Molecular observations have characterized the differences in gene expression between normal lung tissues and primary lung cancer and suggest that p53 in particular as well as K-ras may be part of the gene altered during the neoplastic progression.[4] Whether key differences exist between primary lung cancer and brain metastatic tumor remains unclear. Also whether the primary tumors can be segregated in 2 groups based on their gene expression and their propensity to develop brain metastases would be of tremendous interest to clinicians and the patients affected by lung cancer. Recently, neutral formalin-fixed and paraffin embedded biopsy specimens were used to retrieve RNA and the RNA was used in microarray analyses.[14] Also, observations that the primary tumors included tumor cells with features associated with the host tissue for the metastases strongly suggest that an identification of genes associated with the development of brain tumor in lung cancer patients will provide new gene protein targets.[15]
Brain metastases from other cancers
In addition to lung, breast, melanoma, renal and coon cancers are the most common to result in brain metastases.[10] Whether these primary tumors present a common specific gene expression signature is currently unknown. Because of the poor prognosis associated with the development of brain metastases from any primary tumor, a better understanding and the identification of a gene expression profile associated with the development of brain metastases will provide an additional tool in the diagnosis and individualization of treatment.
New research tools
Microarrays are a relatively new method to ascertain the presence and to a lesser degree quantify the expression level of all the genes expressed in a sample. Previous studies have shown that the microarrays associated with microdissection were powerful instigation tools.[15, 16] Indeed, micro-dissection allows the isolation and analysis of specific tissues and can even isolate individual cells for analysis. Such detailed analyses have identified in primary tumor the presence of tumor cells with features specific to metastases to the brain. Additionally, although RNA is degraded by fixation in formalin and paraffin embedding, the most commonly used methods to prepare histological samples, recent research suggests that RNA may be isolated from archived tissues and provide enough quality RNA for microarray analysis.[14]
Therefore, here we propose to use such methods to determine differences in gene expression between primary and metastatic tumors in paired samples. Based on these observations, a gene expression profile (list of genes with the most alterations) will be associated with the development of brain tumor in lung cancer patients. In another subset of patients, tumor developing in organs other than the brain will be assessed and provide a refining list of genes directly associated with brain metastases. Finally, the detection of the expression of specific genes on blinded samples (NSCLC) will allow the evaluation of the specificity and sensitivity as an independent prognosis factor of the likelihood of a given lung primary tumor to develop brain metastases. Confirmations of the expression gene set, real time PCR and immunohistochemistry analyses will be conducted.
6.2 Significance
This study is significant because it will identify gene and protein pathways associated with the development of brain metastases in lung cancer patients. The long-term goal of the present application is to
- identify the mechanisms involved in brain metastasis development and to
- develop a molecular diagnostic tool to identify primary lung tumor likely to generate metastases to the brain.
6.3 Preliminary studies
No preliminary studies have been conducted prior to this pilot study. However, one of the investigators (DD) has expertise in microarray analyses from RNA isolation to the interpretation of the data on both melanoma and hepato-cellular carcinoma. Although unlikely, if the analysis of the results does require the development of new software, the help of Dr. K. Najaryan will be enrolled. Additionally, Drs. Asher, White, Burri, and Dollars have years of experience in the clinical area including the development of research protocols involving patients specimens. Furthermore, Dr. Dollars is a trained pathologist who specializes in lung carcinoma and who will be key in identifying the appropriate patient cohort to be analyzed in the present study.
7. Study Design
7.1 Study design
7.1.1. Type and description of the study
7.1.1.1 Type of study
This is an experimental pilot study.
7.1.1.2 Description of the study
One type of sample will be collected.
1) Tumor specimens (paraffin embedded and neutral formalin fixed) with either primary or metastatic tumor mass from patients treated at Carolinas Medical Center. Prior to use, the paraffin block and resulting slides will be coded in the Pathology department. The paraffin blocks and slides will be stored at room temperature in a dedicated area and slide/block tray.
7.1.2 Study population
7.1.2.1 Sample
see § 7.1.1 above
7.1.2.2 Method for selecting the samples
Primary lung tumor and brain metastatic tumor specimens (paraffin embedded and neutral formalin fixed) will be obtained through the Pathology department at Carolinas Medical Center. No selection criteria other than the nature of cancer (lung) and the patient general health history will be used.
7.1.2.3 Representativity of the samples
Because of the nature of the study (pilot and experimental) and the limited number of cancer patients available, there is only a reasonable expectation for a close representation to the entire lung cancer patient population. However, the number (n=50) of biopsy specimens used is expected to allow for the analysis of many potential cancer markers.
7.1.2.4 Exclusion criteria
Based on experimental requirements, only biopsy specimens in good conditions (with sufficient tumor tissue left, well conserved) will provide for adequate RNA and proteins.
7.1.3 Definition of the variables
The biopsy specimens of primary and metastatic to the brain tumors will be used to analyze potential early cancer markers associated with the development of brain metastases in patients with lung carcinoma.
The variables collected include the type of cancer and stage of the primary tumor, the time interval to brain metastasis, the tumor size (primary and metastatic). The rate of tumor progression, the type of treatment received after diagnosis with lung cancer and the overall survival. In addition, gender, age, smoking and drinking status, and socio-economic status will be collected.
7.1.4 Monitoring of the study
The safety monitor (Dr. F. Greene, MD) will monitor the safety of the study. Specifically, one aspect will be monitored: the confidentiality of the data and samples. Each tumor biopsy specimen will be assigned a unique identifier. The unique identifier number will be associated with the patient name or history/hospital number and kept under lock key in a drawer (a backup of the notebook will also be made on a computer disk kept locked in a different location). Only the investigators and their designees will have access to the notebook and disk. In the unlikely event that a breach of confidentiality occurs the study safety monitor and the IRB will be notified. In addition, it is the prerogative of the safety officer to verify at any time that the confidentiality is kept. Indeed, the safety officer can, for example, request to see where the notebook and disk are kept and verify the lock.
7.1.5 Project duration
The selection of the biopsy specimens will be completed prior to the RNA/protein extraction. The estimated duration of this project is 24 months.
7.2 Material & Methods
After coding, archived biopsy specimens will be examined for 1-fixative used (only neutral formalin fixed paraffin embedded specimens will be used), 2- molds (only samples without mold or other pathogens will be used) or 3- any degradation of the tissue. The presence of a tumor mass on the available paraffin blocks will be assessed by one slide 4-5um stained using H&E. If the tumor mass is sufficient (1 x 1 x 1 mm in extreme cases 0.1 x 1 x 1 mm), additional serially cut slides will be generated (3-5 serially cut 8-20 um slides). All slides will be labeled using the code defined by the pathologist.
Each tissue slide will undergo micro-dissection to isolate the tumor tissue from the connective and surrounding tissue. This step will be completed using the Arcturus apparatus at the University of South Carolina at Columbia. Post micro-dissections, RNA will be extracted from the isolated cells using the Arcturus Paradise reagents. The amplification step required for a low number of cells will be conducted using the Arcturus Paradise RNA amplification kit. For some samples, when enough tissue is available, the expression of specific protein associated with key pathways will be assessed by immunohistochemistry.
Following an evaluation of the RNA quality (Arcturus assessment kit), labeled cRNA will be obtained following the Affymetrix protocol. Labeled samples will be assessed for gene expression at the Charlotte Genomic consortium. The difference is gene expression will be collected.
Biopsy specimen numbers will be used to protect the confidentiality of each subject entered in the study. However, should a subject request the destruction of the serum and cells isolated from his/her blood, a log book (computerized or not) will be accessible to the investigator and his designees to correlate to number assigned and the patient identity. This log will be kept in a locked drawer.
8. Definitive study
Not Applicable
9. Use of the data
The samples collected in the present experimental pilot study will be analyzed and will provide information on the brain tumors in lung cancer patients.
10. Statistical analyses
Statistical analyses conducted on this project will include the paired comparison of both primary and metastatic to the brain gene and selected protein profiles using Affymetrix statistical software as well as non-parametric and parametric tests. Additional variables to be considered are the stage of the primary tumor, the time interval to brain metastasis, the tumor size (primary and metastatic), the rate of tumor progression, the type of treatment received after diagnosis with lung cancer and the overall survival. Gender, age, smoking and drinking status, and socio-economic status may allow be considered.
11. Funding
Funding of the study ($100,000) will be supported by the brain tumor fund (50%) and the University of North Carolina – Charlotte (50%).
12. Budget (in $)
Supplies:
| 1- Preliminary methodological testing: RNA preparation and amplification testing | $1,250 | ||
| 2- Aim 1: | Histology | $ 50 x 20 | 1,000 |
| Microdissection | $ 250 x 20 | 5,000 | |
| RNA isolation | $250 (4 samples) x 20/4 | 1,250 | |
| Micro-array (array 450-500 each)+ hybridization | $650 x 20 | 13,000 | |
| subtotal | $20,250 | ||
| 3- Aim 2: | Histology | $ 50 x 30 | 1,500 |
| Microdissection | $ 250 x 30 | 7,500 | |
| RNA isolation | $250 (4 samples) x 30/4 | 1,8750 | |
| Micro-array (array 450-500 each)+ hybridization | $650x30 | 19,500 | |
| subtotal | $30,375 | ||
| 4- Aim 3*: | Additional Microdissection | $ 250 x 30 | 7,500 |
| RNA, protein isolations (n=30 samples) (reagents, tubes) | 2,000 | ||
| RNA expression by RT-PCR (primers, kits, gels) | 2,500 | ||
| Protein expression - Immunoblots (antibodies, buffers, membranes) | 3,500 | ||
| - Immunohistochemistry (antibodies, buffers) | 3,000 | ||
| subtotal | $18,500 | ||
| 5- Aim 4* | Additional Microdissection | $ 250 x 12 | 3,000 |
| RNA, protein isolations (n=12 samples) (reagents, tubes) | 800 | ||
| RNA expression by RT-PCR (primers, kits, gels) | 1,000 | ||
| Protein expression - Immunohistochemistry (antibodies, buffers) | 1,000 | ||
| subtotal | $5,800 | ||
| *Estimates based on the testing of 10-12 genes/proteins of interest and 30 biopsy samples | |||
| Total supplies | $74,925 | ||
Personnel (fringes/benefits included in hourly wage):
| 1- Preliminary methodological testing: | 3 weeks @ $25.00/hr | $3,000 |
| 2- Aim 1 | Histology 0.8 week @ $25.00/hr | 800 |
| Microdissection 1 week @ $25.00/hr + travel time ($200) | 1,200 | |
| RNA isolation for 20 samples 1.5 weeks @ $25.00/hr | 1,500 | |
| Preparation for microarray (3 days for up to 6 samples) 3days x20/6= 2 wks @ 25.00/hr |
2,000 | |
| subtotal | $5,500 | |
| 3- Aim 2 | Histology 1 week @ $25.00/hr | 1,000 |
| Microdissection 1 week @ $25.00/hr + travel time ($200) | 1,200 | |
| RNA isolation for 30 samples 2 weeks @ $25.00/hr | 2,000 | |
| Preparation for microarray (3 days for up to 6 samples) 3days x30/6= 3 wks @ 25.00/hr |
3,000 | |
| subtotal | $7,200 | |
| 4- Aim 3 and 4* | Microdissection 1 week @ $25.00/hr + travel time ($200) | 1,375 |
| RNA and Protein isolation (n=30 samples) 1 week @ $25.00/hr | 1,300 | |
| RT-PCR reactions for 30 samples and 12 genes 2 weeks @ $25.00/hr | 2,200 | |
| Protein expression (30 samples and 12 proteins) - Immunoblots 2 weeks @ $25.00/hr |
2,000 | |
| - Immunohistochemistry 2.5 weeks @ $25.00/hr | 2,500 | |
| subtotal | $9,375 | |
| *Estimates based on the testing of 10-12 genes/proteins of interest and 30 biopsy samples | ||
| Total personnel | $25,075 | |
Total cost: $100,000
**Budget notes:
- the budget proposed here is tentative and represents an estimate on the cost of the complete analysis.
- the cohort selection is extremely critical to the success of this project
- the number of tumor specimens proposed per group (at least 10) because of intrinsic and extrinsic heterogeneity is the minimum number of arrays likely to render meaningful (significant) results.
- the publication of microarray observations alone is very difficult. Therefore, post-microarray analyses of RNA and/or protein of the genes/proteins/pathways found to be significantly different between the patient cohorts were added.
13. References
- American Cancer Society: Cancer facts and Figures 2005. In.: American Cancer Society; 2005.
- Jackman DM, Johnson BE: Small-cell lung cancer. Lancet 2005, 366:1385-1396.
- Birim O, Kappetein AP, van Klaveren RJ, Bogers AJ: Prognostic factors in non-small cell lung cancer surgery. Eur J Surg Oncol 2006, 32:12-23.
- Liu G, Zhou W, Christiani DC: Molecular epidemiology of non-small cell lung cancer. Semin Respir Crit Care Med 2005, 26:265-272.
- Turrisi AT, Crowley J, Albain K, Gaspar L, Gandara D: Southwest Oncology Group: two decades of experience in non-small cell lung cancer. Semin Oncol 2005, 32:S119-121.
- Christodoulou C, Skarlos DV: Treatment of small cell lung cancer. Semin Respir Crit Care Med 2005, 26:333-341.
- Sun SY, Yue P, Wu GS, El-Deiry WS, Shroot B, Hong WK, Lotan R: Implication of p53 in growth arrest and apoptosis induced by the synthetic retinoid CD437 in human lung cancer cells. Cancer Res 1999 , 59:2829-2833.
- Margalit O, Eisenbach L, Amariglio N, Kaminski N, Harmelin A, Pfeffer R, Shohat M, Rechavi G, Berger R: Overexpression of a set of genes, including WISP-1, common to pulmonary metastases of both mouse D122 Lewis lung carcinoma and B16-F10.9 melanoma cell lines. Br J Cancer 2003, 89:314-319.
- Norden AD, Wen PY, Kesari S: Brain metastases. Curr Opin Neurol 2005, 18:654-661.
- Kirsch DG, Loeffler JS: Treating brain metastases: current approaches and future directions. Expert Rev Neurother 2004, 4:1015-1022.
- Reck M: Current approaches in chemotherapy of advanced and metastatic non-small cell lung cancer (NSCLC). Anticancer Res 2005, 25:1501-1506.
- Gavrilovic IT, Posner JB: Brain metastases: epidemiology and pathophysiology. J Neurooncol 2005, 75:5-14.
- Langer CJ, Mehta MP: Current management of brain metastases, with a focus on systemic options. J Clin Oncol 2005, 23:6207-6219.
- Bibikova M, Yeakley JM, Chudin E, Chen J, Wickham E, Wang-Rodriguez J, Fan JB: Gene expression profiles in formalin-fixed, paraffin-embedded tissues obtained with a novel assay for microarray analysis. Clin Chem 2004, 50:2384-2386.
- Mimori K, Kataoka A, Yoshinaga K, Ohta M, Sagara Y, Yoshikawa Y, Ohno S, Barnard GF, Mori M: Identification of molecular markers for metastasis-related genes in primary breast cancer cells. Clin Exp Metastasis 2005, 22:59-67.
- Nishidate T, Katagiri T, Lin ML, Mano Y, Miki Y, Kasumi F, Yoshimoto M, Tsunoda T, Hirata K, Nakamura Y: Genome-wide gene-expression profiles of breast-cancer cells purified with laser microbeam microdissection: identification of genes associated with progression and metastasis. Int J Oncol 2004, 25:797-819.
14. Abstract:
Background: Lung carcinoma is associated with the development of brain metastases in a sizeable number of patients. Currently, these metastases remain difficult to treat and result in high morbidity. Although great progress has been made in the understanding of the development of metastases, the key features associated with the homing of tumor cells within the brain remain poorly understood. Scattered evidence suggests that the “seed / soil” hypothesis, which associates specific cell surface molecules on the tumor cells with defined extracellular matrix components also applies to brain metastases. Additionally, extensive research has been conducted on the ability of tumor cells to evade the immune system and on the important role of the vascular system in the development of those metastases. Taken together these observations confirm that the development of brain metastases involves alteration of gene expressions in multiple cellular pathways. Recent technical breakthroughs now allow analysis of RNA for formalin-fixed samples. This combined with the use of microarray technology will permit detailed analyses of the mRNA expression in both primary lung tumors and brain metastases. Hypothesis: Primary tumors leading to the development of brain metastases have altered expression of multiple genes compared to primary tumors not associated with the development of brain metastases. Specific aims: 1- the presence of specific markers associated with the development of brain metastases will be analyzed in paired samples comparing primary tumor and the brain tumor 2- the presence of specific markers associated with the development of brain metastases will be studied in primary tumors, either non-metastatic, metastatic but not to the brain or metastatic to the brain, 3- the presence of gene/pathway- and protein- expressions associated with the most significant difference (i.e. susceptible to be used as markers) will be analyzed. Methods: Cohorts of patients will be selected and archival biopsy specimens retrieved and mRNA isolated. Cohorts of patients used in aims 1 and 2 will require a very close matching of parameters associated with the tumor (type, stage, vascularization, size, location…), the patient (gender, age…), and the treatment/medication received. Additionally, the quality of the archival biopsy specimens will be assessed and only unaltered biopsy samples with sufficient tumor mass will be considered. For aim 3, randomly selected good quality samples will be used to validate the gene, protein expression (RT-PCR, immunohistology). Expected results: the experiments should provide detailed information on potential markers associated with the development of brain metastases. Furthermore, it should contribute to the development of guidelines or even a test to reliably diagnose the likelihood of the development of brain tumors in a given patient. Such crucial information would allow clinicians to better tailor post-surgical treatments in patients with cancers likely to metastasize to the brain.
