Projects and Cores
Project 1: Intersection of Estrogen Receptor Signaling and Epidermal Growth Factor Receptor Signaling in Lung Cancer
Jill M. Siegfried, PhD Co-Project Leader
Jennifer R. Grandis, MD, Co-Project Leader
Lung cancer incidence is increasing in women worldwide and it is apparent from epidemiological studies that sex differences exist in the presentation of lung cancer. The proportion of patients diagnosed with lung cancer under age 50 is significantly higher for women compared to men (1). Women also are diagnosed to a greater extent than men with adenocarcinoma and small cell carcinoma (2, 3), both of which are secretory-type tumors. Never smokers diagnosed with lung cancer are also predominantly female (4). These differences in presentation suggest there are sex differences in the development of lung cancer. We hypothesize that one component of these sex differences is related to estrogen and its receptors. Evidence from our laboratory obtained during the first SPORE grant period shows that both known estrogen receptors (ERs), ER alpha (ERα) and ER beta (ERβ), are commonly expressed in non-small cell lung cancers (NSCLCs) of different histologic types. ERα appears to be mainly present as a variant protein of smaller molecular weight than full-length ERα, while ERβ protein is present at the expected size. These receptors are localized in both the nucleus and the cytoplasm, as well as some membrane localization, in NSCLC tissues and in normal lung. Genomic signaling through ERβ has been clearly demonstrated by us in NSCLC, as well as non-genomic signaling involving activation of the epidermal growth factor receptor (EGFR). The non-genomic signaling may involve both ERα and ERβ. We have evidence that combined targeting of the ER and the EGFR produces enhanced anti-proliferative effects in preclinical models. We also discovered that tumors from males contain ERs and can respond to estrogens. Many male lung tumors also appear to contain aromatase. This suggests that although some of the hormonal effects due to estrogen may be more pronounced in women compared to men due to a greater lifetime production of ligand, the ER pathway might also be targeted in males with NSCLC, especially if local estrogen production in lung tissues is present via aromatase, or if ligand-independent signaling plays a role in ER action in the lung. The hypothesis under investigation in Project One of the UPCI Lung Cancer SPORE renewal is that ER expression and signaling have functional significance in NSCLC. Based on results obtained in the first SPORE grant period, we hypothesize that ER and EGFR both activate proliferative signaling pathways in NSCLC; these pathways overlap and interact. Co-inhibition of ER and EGFR may show greater anti-tumor activity in NSCLC than inhibition of either pathway alone.
The Specific Aims are:
- Determine signaling molecules involved in ER-EGFR pathway interactions in NSCLC cell lines. We will ask the following questions in this aim: (a) Does EGFR cause ligand–independent activation of ERα and ERβ in NSCLC cells, resulting in genomic signaling in the absence of estrogen? (b) Does the ligand-dependent activation of ER that causes EGFR activation (non-genomic signaling) depend on Src, and can EGFR activation by ER be interrupted by Src inhibitors? We will examine cell lines that have both wild-type EGFR and EGFR with mutation in the tyrosine kinase domain.
- Examine effectiveness of joint inhibition of the ER-EGFR pathways on tumor growth in NSCLC compared to single therapy in NSCLC, using clinically relevant agents. We will ask the following question in this aim: Is the anti-tumor efficacy of combined ER/EGFR and/or Src targeting using clinically relevant agents superior to targeting individual pathways in preclinical models?
- Determine if aromatase is present and functional in normal and malignant lung cells and if an aromatase inhibitor has anti-tumor activity. We will ask the following questions in this aim: (a) How common is the expression of aromatase, the enzyme that synthesizes estrogen, in normal and malignant cells from the human airway? (b) Does aromatase function sufficiently in cultured normal and malignant cells from human airway to result in measurable production of estrogen? (c) Do primary NSCLC tissues from males and females contain aromatase, which cell types within tumors express it, and is aromatase related to other parameters such as ER expression, sex, histology, or outcome? (d) Does an aromatase inhibitor have anti-tumor effects against NSCLC in preclinical models?
- Analysis of ER and EGFR status in tissues obtained from NSCLC patients treated on clinical trials of combination therapy (anti-estrogen and EGFR TKI). We will ask the following questions in this aim: (a) Does combination therapy produce anti-tumor effects against late-stage NSCLC? (b) Is combination therapy superior to targeting the EGFR pathway alone? (c) What is the relationship between response to therapy and ER and EGFR pathway signaling in NSCLC patients? (d) Are variations in the EGFR gene observed in association with therapeutic response to combination therapy, and what are the characteristics of patients who show clinical benefit?
Translational Value of Project One
- Targeting the EGFR through small molecule tyrosine kinase inhibitors (TKIs) to date is of limited utility in the absence of an EGFR mutation, which occurs in a minority of lung cancer patients. Even with an EGFR mutation, duration of response to EGFR TKIs may be short and resistance also develops through selection of further EGFR mutation (T790M) (5). Understanding how ER and EGFR interact in NSCLC will provide a rational basis for therapies targeting these two pathways to increase the effectiveness of EGFR targeted therapy. Combination therapy may increase duration of response in the presence of mutant EGFR, as well as improve therapeutic response in patients with wild-type EGFR.
- Results to date of a Phase I clinical trial being carried out in the first SPORE grant period (described below) targeting both pathways together with a gefitinib/fulvestrant combination showed that combination therapy is safe and has anti-tumor activity in Stage IIIB/IV female post-menopausal NSCLC patients. Further testing of combination therapy (erlotinib/fulvestrant) compared to single therapy in animals and humans, proposed in this project, will determine whether combination therapy gives superior anti-tumor effects compared to EGFR TKI monotherapy. Since erlotinib increased survival in NSCLC patients in a Phase III trial vs. placebo/best supportive care, while gefitinib did not, erlotinib will be substituted for gefitinib in this project.
- Increased understanding of the role of estrogen, estrogen synthesis, and ER in lung cancer will provide a rationale for future targeting of this pathway for therapy earlier in the course of disease as well as for future testing of a role for anti-estrogens in lung cancer prevention.
Role of Inter-SPORE Collaborations: Aims 3 and 4 of this project are collaborations with the UCLA Lung Cancer SPORE (Steven Dubinett, MD, PI). Regarding Aim 3, the UCLA SPORE is also examining aromatase in NSCLC and finds expression of this enzyme in both lung tumor cell lines and in tissues from lung cancer patients. The UCLA SPORE has also documented conversion of testosterone to estrogen in NSCLC cell lines and has shown anti-tumor effects of the aromatase inhibitor, anastrazole, in the NSCLC cell lines A549 and H23. We plan in our Aim 3 to utilize cell lines (including NSCLC cell lines, normal lung fibroblasts and human bronchial epithelial cells) and NSCLC tissues not being studied at UCLA, to demonstrate how widely aromatase protein and function is present in lung cancer. We will also examine a different set of NSCLC patients’ tumor and normal tissues for aromatase activity by immunohistochemistry. Since the role of aromatase in NSCLC is an entirely novel area of investigation, this joint endeavor will help to increase new knowledge and to confirm each SPORE’s findings. We plan to share our preclinical data and compare results with different aromatase antibodies in Aim 3 as well as to share the data from the other aims with the UCLA group, thus allowing the work to proceed at a faster pace. In Aim 4, the Phase II clinical trial involving erlotinib alone versus erlotinib plus the anti-estrogen fulvestrant will be conducted jointly at UCLA and Pittsburgh. We intend to enroll patients at each institution and to carry out the analysis of clinical response and signaling molecules in the patients’ paraffin-embedded tumors jointly. We will analyze the data from the clinical trial together and present and publish it together.
Aims 1 and 2 are also collaborations with the University of Pittsburgh Head and Neck Cancer SPORE (Jennifer Grandis, M.D., PI). Drs. Siegfried, Stabile, and Grandis are undertaking a developmental research project in the Pittsburgh Head and Neck Cancer SPORE to determine the extent of ER expression in head and neck cancers, and the signaling pathways activated by estrogen. The preliminary data suggest that ERβ is expressed by all head and neck cancer cell lines examined, and estrogens increase cell proliferation and ERE transcriptional activity in head and neck cancer cell lines, while the anti-estrogen fulvestrant can inhibit these responses. Knowledge from each tumor type will be shared between our two SPOREs, and if the preclinical data from head and neck cancers provides a rationale for treatment with anti-estrogens (alone or in combination with an anti-EGFR agent), Drs. Grandis and Siegfried will propose a clinical trial for head and neck cancer patients in the Head and Neck SPORE.
Translational Value of Project One
Targeting the EGFR through small molecule tyrosine kinase inhibitors (TKIs) to date is of limited utility in the absence of an EGFR mutation, which occurs in a minority of lung cancer patients. Even with an EGFR mutation, duration of response to EGFR TKIs may be short and resistance also develops through selection of further EGFR mutation (T790M) (5). Understanding how ER and EGFR interact in NSCLC will provide a rational basis for therapies targeting these two pathways to increase the effectiveness of EGFR targeted therapy. Combination therapy may increase duration of response in the presence of mutant EGFR, as well as improve therapeutic response in patients with wild-type EGFR.
Results to date of a Phase I clinical trial being carried out in the first SPORE grant period (described below) targeting both pathways together with a gefitinib/fulvestrant combination showed that combination therapy is safe and has anti-tumor activity in Stage IIIB/IV female post-menopausal NSCLC patients. Further testing of combination therapy (erlotinib/fulvestrant) compared to single therapy in animals and humans, proposed in this project, will determine whether combination therapy gives superior anti-tumor effects compared to EGFR TKI monotherapy. Since erlotinib increased survival in NSCLC patients in a Phase III trial vs. placebo/best supportive care, while gefitinib did not, erlotinib will be substituted for gefitinib in this project.
Increased understanding of the role of estrogen, estrogen synthesis, and ER in lung cancer will provide a rationale for future targeting of this pathway for therapy earlier in the course of disease as well as for future
PROJECT 2: CYCLIN B1 IMMUNOTHERAPY
PROGRESS REPORT
Co-Project Leaders: Olivera J. Finn, PhD
Athanassios Argiris, MD
Specific aims
This project is a continuation of a project carried out in the first 5-year grant period that validated Cyclin B1 (CB1) as a lung tumor antigen. The first hypothesis that we proposed to test in the continuation of this project is that vaccines will elicit or boost CB1-specific immunity in lung cancer patients and this will result in an anti-tumor effect. The second hypothesis we proposed to test is that the immune response against CB1 could be a biomarker of risk for development of future lung cancer in subjects with a positive smoking history, or for recurrence among early-stage patients newly diagnosed with lung cancer. To test these hypotheses we proposed three specific aims.
SPECIFIC AIM 1. Test in phase I/II clinical trials toxicity and immunogenicity of cancer vaccines composed of CB1 peptides and proteins processed and presented by dendritic cells (DC), in combination with novel delivery of adjuvants, and evaluate immune effector mechanisms generated. Patients in these trials will be those with resectable stage I and II lung cancer. The first trial will be carried out in HLA-A2+ patients and will test a vaccine composed of DC loaded with two CB1 peptides known to elicit HLA-A2 restricted CTL, and transdermal adjuvant. The second trial will test DC loaded with recombinant CB1 protein plus transdermal adjuvant, and thus it will be open to patients of all HLA types. Vaccinated patients will be observed for signs of toxicity or adverse reactions to the vaccine, and examined pre and post vaccination for the following immune responses: CB1 specific antibodies (IgM, IgG, IgA) by ELISA; CB1 specific CD4 and CD8 T cells (IFN- or IL-4), by ELISPOT and intracellular cytokine staining; for general immune responsiveness that may reveal existence of T regulatory cells. Future trials will be designed based on the analysis of data from these two trials and availability of new adjuvants and methods to increase vaccine efficacy.
SPECIFIC AIM 2. Perform detailed quantitative and qualitative analysis of spontaneously occurring CB1 specific antibodies and T cells in lung cancer patients at different stages of disease and in the PLuSS High Risk Sub-Cohort (described in Clinical Core). We will analyze antibody isotype, titer, and affinity. T cells will be studied for their phenotype (nave, effector memory, regulatory T cells) and cytokine production (Type I versus Type II). Fine antigen specificity will also be analyzed using a CB1 peptide library composed of overlapping 15-mer peptides, to look for evidence of immunodominant versus sub-dominant epitopes, which may influence effectiveness of the immune response. This information will be analyzed in the context of clinical outcome, in an attempt to define immune correlates of protection. This information will also be important for defining certain immune responses as surrogate end-points for monitoring efficacy of CB1 vaccines.
SPECIFIC AIM 3. Assay for the presence or absence of CB1 specific antibodies in individuals at high risk for developing lung cancer (3,600 PluSS subjects) and in two retrospective sets of lung cancer cases, in order to evaluate the potential of the immune response to be a biomarker of risk for future lung cancer and/or a useful prognostic indicator. We will perform semi-automated high-throughput ELISA assays for detection of anti-CB1 antibodies that we have developed. ELISA will be designed to be isotype specific. Isotype switching is a T cell mediated event and different isotypes are promoted by the action of Th1 versus Th2 cells. The antibody data will be added to the information about other biomarkers defined elsewhere in the SPORE. Lung nodules that are resected as part of the diagnostic procedures for PLuSS participants with CT screening results of high suspicion will be stained for CB1 and immunohistology data correlated with ELISA data. A long-term follow up of antibody positive and antibody negative groups will test if the presence of antibody correlates with a higher or lower rate of lung cancer development in the entire PluSS cohort, and whether presence of antibody correlates with extent of CB1 expression in the resulting lung tumor. We will also determine whether CB1 immunity is associated with protection from recurrence by monitoring outcome of PLuSS participants as well as patients from Retrospective NSCLC Cohorts A and B. Data from this aim will be contributed to the data set on biomarkers being generated by Projects 3 and 4 using the PLuSS High-Risk Sub-Cohort, and the data on estrogen receptors and aromatase expression as it relates to clinical outcome in Project 1. The value of the immune response as a biomarker will be evaluated relative to the other markers.
Our aims have not changed and in this first year of our project we have focused on Aims 1 and 2.
PROJECT 3: SERUM PROTEOMIC BIOMARKERS FOR LUNG CANCER DETECTION AND PROGNOSIS
PROGRESS REPORT
Co-Project Leaders: William L. Bigbee, PhD
James D. Luketich, MD
Overview
Our ongoing effort in the second year of the new Project 3 has been focused in six areas: 1) the final analysis of the preliminary Luminex and SELDI-TOF-MS data and manuscript preparation; 2) database development and clinical annotation of the SPORE Lung Cancer Registry case/control patient cohort and standardized blood sample collection resources; 3) optimization and refinement of the initial Luminex multianalyte serum panels (xMAP) to include new candidate lung cancer markers and independent verification of preliminary results on the Searchlight multiplexed immunoassay platform; 4) development and finalization of new workflows for the depletion and fractionation of plasma/serum specimens for MALDI-T0F-MS comparative proteomic profiling and, most recently, LC-MS based label-free peptide quantitation and protein identification profiling for biomarker discovery; 5) final study design and case/control sample pooling strategy for analysis using the parallel MS discovery workflows; and 6) development and funding of complementary collaborative biomarker studies.
PROJECT 4: NUCLEOTIDE EXCISION REPAIR/CELL CYCLE CONTROL HAPLOTYPES AND LUNG CANCER RISK AND PROGNOSIS
PROGRESS REPORT
Co-Project Leaders: Marjorie Romkes, PhD, Joel Weissfeld, MD, M.P.H., Emanuela Taioli, MD, PhD
Specific Aims
Project 4 of the UPCI Lung Cancer SPORE is investigating the hypothesis that nucleotide excision repair and cell cycle control gene haplotypes may not only predict lung cancer risk, but also drug resistance and survival. The ability to identify individuals with the highest risk of developing tobacco-related cancers, most importantly lung cancer, has important public health and clinical implications for screening, early detection, prevention and treatment. In addition to variability in activation and detoxification pathways of mutagenic agents, there is a very strong biologic rationale to also study the variability in the capacity to repair smoking induced DNA damage as another major family of susceptibility biomarkers. The nucleotide excision repair (NER) pathway is important in the repair of chemical carcinogen induced genotoxic damage. The XPD protein is a key member of this pathway and mutations in the XPD gene, including the common A35931C (Lys751Gln) variant allele, result in reduced repair capacity. Furthermore, regulation of the cell cycle control mechanism can influence the potential for increased cell proliferation and the promotion of genetic instability. Cyclin D1 (CCND1) is an essential cell cycle regulatory protein and is involved in the regulation of proliferation and differentiation. The CCND1 G870A single nucleotide polymorphism (SNP) has been reported to enhance alternate splicing and increase cyclin D1 protein half-life. Our initial case/control studies, partially supported by the previous SPORE Genomics Core, of which Dr. Romkes was the Director, have demonstrated a significant association between elevated risk of upper aerodigestive tract cancer among individuals who carried both the CCND1 G870A variant allele and XPD Gln allele (OR=7.1, 95%CI 4.0-12.5).
We proposed to further investigate these preliminary results and to conduct a genetic epidemiological haplotype association study by evaluating polymorphisms of genes in the NER and cell cycle control pathways in a series of NSCLC cases and controls. The following Specific Aims are designed to validate these initial observations in a larger patient population and to also extend the model to a prospective study to evaluate the prognostic significance of the “at risk” haplotypes.
Specific Aim 1. To select tagSNPs identifying haplotypes for genes of the NER and cell cycle control pathways.
Specific Aim 2. To develop a model relating NER and cell cycle control pathway gene haplotypes to lung cancer risk.
Specific Aim 3. To test the prognostic significance of the NER and cell cycle control pathway gene haplotypes by genotyping the PLuSS and Moffitt Cancer Center High-Risk sub-cohorts.
Specific Aim 4. To develop a final predictive model by combining the datasets from Specific Aims 2 and 3 for purposes of external validation.
Specific Aim 5. To evaluate whether the NER pathway haplotypes are associated with platinum drug resistance and survival.
BIOINFORMATICS/BIOSTATISTICS CORE
Core members have participated in the regular meetings of the Pittsburgh Lung Cancer SPORE. Dr. Land serves on the SPORE Tissue Utilization and Prioritization Committee evaluating requests for sample use. Another major contribution of the Core in Year 2 is the development of an Access database for data from the Carinal Registry Study. This database includes patient characteristics and clinical follow-up for patients whose tissues have been banked. Those samples are a rich repository for the experimental studies related to Projects 1 and 2 of the SPORE. The use of the Core by SPORE projects was as follows:
Project 1 — Intersection of Estrogen Receptor Signaling and Epidermal Growth Factor Receptor Signaling in Lung Cancer.
Several studies have been conducted that provide additional pilot information for Project 1. In one study, tumor tissue from Carinal Registry patients was analyzed for ER-alpha, ER-beta, PR, aromatase, and EGFR markers. Statisticians Land and Shuai analyzed the associations between these markers and patient and tumor characteristics. In addition, overall survival and progression free survival were modeled based on marker expression, age at tissue collection, smoking, gender, histology, and disease stage. Analytic techniques included tree-based methods, Cox regression and Fisher’s exact tests. A manuscript is in preparation. Dr. Land also provided sample size calculations for a xenograft experiment for Project 1. Dr. Land is providing support for the dissertation project of Ji Young Song, student of Dr. Weissfeld. That study will be a comparison of ER expression in Carinal Registry Study lung tissue with tissue from non-cancer controls, and an examination of the relevance of ER expression in normal lung tissue to lung cancer outcomes in Carinal patients. Dr. Land also provided a statistical analysis plan and sample size calculations for proposed work by Dr. H. Srinivas. In that project, we will test newly standardized ER-alpha and ER-beta antibodies on paraffin-embedded lung tumor sections of patients, which we have obtained from Lung SPORE tissue bank. We will perform statistical analysis of the associations between ER immunostaining and clinical and histopathological variables.
Project 2 — Cyclin B1 in Immunotherapy, Diagnosis and Prognosis of Lung Cancer
Statisticians Land and Shuai examined laboratory assays of cyclin B1 antibody levels. We estimated the effect of each plate (assay) using linear regression in order to develop a normalization procedure, which is performed by subtracting the relevant plate effect from the value for each test sample. We also compared healthy controls with cancer patients (Carinal Registry), and tested the effect of age on antibody levels. The cyclin-B1 antibody levels were significantly lower among cancer patients (estimated difference in medians 0.053; p=0.0017). There was no significant difference by age (p=0.81). In a third analysis, we analyzed the association of survival time and progression free survival time with cyclin B1 antibody levels in tissue from non-small cell lung cancer patients from the Carinal Registry study. These analyses were performed with Cox proportional hazards regression.
Project 3 — Serum-Based Proteomics for Lung Cancer Detection and Prognosis
Bioinformaticians Gopalakrishnan and Hauskrecht have been integral to the work of Project 3. Our goals for this year were to perform extensive analyses of the preliminary data generated in Project 3 by proteomic profiling of lung cancer in order to be able to (a) validate previous analyses of the same data; and (b) suggest protein identities for putative disease-specific biomarkers. To achieve these goals, we performed multiple analyses on the UPCI and Vanderbilt data that include and extend the machine learning analysis and validation conducted previously. We measured sensitivity, specificity and the achieved classification error across multiple techniques. The putative biomarker discovery phase utilized a rule learning algorithm, RL and two of the datasets used in the analyses.
CLINICAL CORE
A number of other clinical trials are currently ongoing at UPCI and most are expected to be completed in 2008. These studies include the following:
- Phase II Trial of RAD001 (Everolimus) in Previously Treated Small Cell Lung Cancer (Investigator-initiated. PI: Argiris. UPCI #06-049)
- A Phase II Study of Cetuximab in Combination with External Beam Radiation Followed by Consolidation Chemotherapy for Patients with Locally Advanced Non-Small Cell Lung Cancer (NSCLC) (Investigator-initiated. PI: Argiris. UPCI #05-106)
- Phase II Study of Bortezomib (PS-341) for Patients with Advanced Bronchiolo-Alveolar Carcinoma (BAC) or Adenocarcinoma with BAC Features (NCI 7003, CCC-P PhII-57) (UPCI PI: Argiris. UPCI #04-163)
- Phase II Study of C225 (Cetuximab) for the Treatment of Patients with Advanced Bronchioalveolar Carcinoma (BAC) or Adenocarcinoma with BAC Features (ECOG PI: Ramalingam. ECOG 1504) (UPCI PI Argiris. UPCI #05-093)
TISSUE AND BLOOD BANK CORE
The Tissue Resource has been involved in three major areas of support. The principal areas of activity for the tissue resource are:
- Collection of tissue and biological materials.
- Histology and immunohistochemical support.
- Paraffin Tissue Array construction and use.