Gene Therapy for Lung Cancer.

Gene therapy was originally conceived to treat monogenic diseases. The replacement of a defective gene with a functional gene can theoretically cure the disease. In cancer, multiple genetic defects are present and the molecular profile changes during the course of the disease, making the replacement of all defective genes impossible. To overcome these difficulties, various gene therapy strategies have been adopted, including immune stimulation, transfer of suicide genes, inhibition of driver oncogenes, replacement of tumor-suppressor genes that could mediate apoptosis or anti-angiogenesis, and transfer of genes that enhance conventional treatments such as radiotherapy and chemotherapy. Some of these strategies have been tested successfully in non-small-cell lung cancer patients and the results of laboratory studies and clinical trials are reviewed herein.

A window of opportunity study of potential tumor and soluble biomarkers of response to preoperative erlotinib in early stage non-small cell lung cancer.

BACKGROUND: Erlotinib is highly active in EGFR mutant NSCLC, but may benefit some with wild-type tumors. We examined pre-operative erlotinib in early stage NSCLC to assess response and correlation with potential biomarkers. RESULTS: Twenty-five patients were enrolled; 22 received erlotinib treatment and were evaluable (median follow-up 4.4 years). Histology was predominantly adenocarcinoma although 31% had squamous carcinoma. PET response was observed in 2 patients (9%), both with squamous carcinoma. Most (20/22) had stable disease (RECIST), with frequent minor radiographic regression and histologic findings of fibrosis/necrosis including in squamous histology. Only two had EGFR mutations identified, one with minor radiographic response and the other stable disease after 4 weeks of EGFR TKI. High pre-treatment serum levels of TGF-α correlated with primary resistance to erlotinib (p = 0.02), whereas high post-treatment soluble EGFR levels correlated with response (p = 0.03). EGFR, PTEN, cMET and AXL expression did not correlate with tumor response. METHODS: Clinical stage IA-IIB NSCLC patients received erlotinib 150 mg daily for 4 weeks followed by resection. Tumor response was assessed using CT, PET and pathological response. Tumor genotype was established using Sequenom Mass ARRAY; EGFR, PTEN, cMET and AXL expression was assessed by immunohistochemistry, circulating markers of EGFR activation (TGF-α, amphiregulin, epiregulin, EGFR ECD) by ELISA and EGFR, MET copy number by FISH. CONCLUSIONS: Erlotinib appears to demonstrate activity in EGFR wild-type tumors including squamous carcinoma. Further research is needed to characterize those wild-type patients that may benefit from EGFR TKI and predictive biomarkers including TGF-α, EGFR copy and others.

The right drugs at the right time for the right patient: the MD Anderson precision oncology decision support platform.

The development of resources for clinical interpretation of cancer-associated genetic alterations has significantly lagged behind the technical developments enabling their detection in a time- and cost-efficient manner. The lack of scientific and informatics decision support for oncologists can lead to no action being taken or suboptimal therapeutic choices being made, which could affect the clinical outcome of a patient as well as convoluting research findings from clinical trials. In this article, we describe the precision oncology decision support (PODS) platform developed within The Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy (IPCT) at MD Anderson Cancer Center; the platform aims to bridge the gap between molecular alteration detection and identification of appropriate treatments.

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy.

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.

Using Ontology Fingerprints to disambiguate gene name entities in the biomedical literature.

Ambiguous gene names in the biomedical literature are a barrier to accurate information extraction. To overcome this hurdle, we generated Ontology Fingerprints for selected genes that are relevant for personalized cancer therapy. These Ontology Fingerprints were used to evaluate the association between genes and biomedical literature to disambiguate gene names. We obtained 93.6% precision for the test gene set and 80.4% for the area under a receiver-operating characteristics curve for gene and article association. The core algorithm was implemented using a graphics processing unit-based MapReduce framework to handle big data and to improve performance. We conclude that Ontology Fingerprints can help disambiguate gene names mentioned in text and analyse the association between genes and articles. Database URL: http://www.ontologyfingerprint.org

Lung adenocarcinoma with concurrent KRAS mutation and ALK rearrangement responding to crizotinib: case report.

Chromosomal translocation resulting in the fusion between the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene was recently identified as a novel genetic alteration in a subset of non-small cell lung cancer (NSCLC). EML4-ALK translocations are rare events associated with specific clinicopathological features, such as never or light smoking history, young age and adenocarcinoma with signet ring or acinar histology. Reports suggest ALK gene arrangements are mutually exclusive with EGFR and KRAS mutations. To the best of to our knowledge, this is the first case report of a patient with concurrent KRAS mutation and ALK translocation. This patient had an excellent response to crizotinib, suggesting that the ALK translocation was the oncogenic driver.

Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue.

BACKGROUND: Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. METHODS: A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. RESULTS: The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/µg to 12,860amol/µg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/µg. CONCLUSIONS: This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.

Histopathological and immunohistochemical features associated with clinical response to neoadjuvant gefitinib therapy in early stage non-small cell lung cancer.

To define the pathological features associated with response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in NSCLC, we have evaluated tumor histopathological features and immunohistochemical markers of proliferation (Ki-67) and epithelial mesenchymal transition (EMT) in 36 resected early stage NSCLC from patients treated preoperatively with gefitinib for 28 days. Tumors studied included 7 squamous cell carcinoma, 27 adenocarcinoma (ADC), one adenosquamous carcinoma, and one large cell carcinoma. Six of the ADC harboured an EGFR tyrosine kinase domain (TKD) mutation; five were the sensitizing type. Five ADC with TKD mutation demonstrated non-mucinous lepidic growth pattern as the dominant histological feature. Post-gefitinib treated EGFR TKD mutant tumors demonstrated lower tumor cellularity and proliferative index compared to wild type ADC and non-ADC cases, features correlating with clinical response. Responding tumors also showed large areas of fibrosis, within which focal residual viable tumor cells were noted. However, there was no significant correlation between the degree of fibrosis and radiological changes in tumor size. Expression of EMT markers was not associated with significant change in tumor size. The results suggest that radiologically assessed response to EGFR TKI in NSCLC is related to loss of tumor cellularity and reduced tumor cell proliferation, but residual viable tumor cells may persist even after prolonged treatment. Neoadjuvant studies in early stage NSCLC offer a unique opportunity to evaluate pathological and biomarker changes induced by targeted drugs.

Phase II study of preoperative gefitinib in clinical stage I non-small-cell lung cancer.

PURPOSE: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have proven efficacy in advanced non-small-cell lung cancer (NSCLC). Their role in early-stage NSCLC has not been established. Our purpose was to explore the use of preoperative gefitinib in clinical stage I NSCLC to assess tumor response, toxicity, and clinical and molecular predictors of response. PATIENTS AND METHODS: Patients received gefitinib 250 mg/d for up to 28 days, followed by mediastinoscopy and surgical resection in an open-label, single-arm study. Tumor response was evaluated by Response Evaluation Criteria in Solid Tumors. Blood samples and tumor biopsies were collected and analyzed for transforming growth factor alpha level, EGFR protein expression, EGFR gene copy number, and EGFR (exon 19 to 21) and KRAS mutations. RESULTS: Thirty-six patients completed preoperative treatment (median duration, 28 days; range, 27 to 30 days). Median follow-up time is 2.1 years (range, 0.86 to 3.46 years). Three patients experienced grade 3 toxicities (rash, diarrhea, and elevated ALT). Tumors demonstrated EGFR-positive protein expression in 83%, high gene copy number in 59%, EGFR mutations in 17%, and KRAS mutations in 17%. Tumor shrinkage was more frequent among women and nonsmokers. Partial response was seen in four patients (11%), and disease progression was seen in three patients (9%). The strongest predictor of response was EGFR mutation. CONCLUSION: Preoperative window therapy with gefitinib is a safe and feasible regimen in early NSCLC and provides a trial design that may better inform predictors of treatment response or sensitivity.

The role of intrapulmonary de novo lymphoid tissue in obliterative bronchiolitis after lung transplantation.

Chronic rejection after lung transplantation is manifested as obliterative bronchiolitis (OB). The development of de novo lymphoid tissue (lymphoid neogenesis) may contribute to local immune responses in small airways. Compared with normal lungs, the lung tissue of 13 lung transplant recipients who developed OB demonstrated a significantly larger number of small, airway-associated, peripheral node addressin-positive (PNAd(+)) high endothelial venules (HEVs) unique to lymphoid tissue (p < 0.001). HEVs were most abundant in lesions of lymphocytic bronchiolitis and "active" OB infiltrated by lymphocytes compared with those of "inactive" OB. T cells in lymphocytic bronchiolitis and active OB were predominantly of the CD45RO(+)CCR7(-) effector memory phenotype. Similar lymphoid tissue was also observed in the rat lung after intrapulmonary transplantation of allograft trachea (Brown Norway (BN) to Lewis), but not after isograft transplantation. Subsequent orthotopic transplantation of the recipient Lewis lung containing a BN trachea into an F(1) (Lewis x BN) rat demonstrated stable homing of Lewis-derived T cells in the lung and their Ag-specific effector function against the secondary intrapulmonary BN trachea. In conclusion, we found de novo lymphoid tissue in the lung composed of effector memory T cells and HEVs but lacking delineated T cell and B cell zones. This de novo lymphoid tissue may play a critical role in chronic local immune responses after lung transplantation.

Tissue heterogeneity of EGFR mutation in lung adenocarcinoma.

Tissue heterogeneity of EGFR gene mutation was studied in 10 formalin-fixed paraffin-embedded (FFPE) samples from four cases that demonstrated EGFR mutations in snap-frozen samples. EGFR mutations identical to those in frozen sample were demonstrated in 8 of 10 FFPE samples by direct sequencing and in 9 of 10 by fragment length analysis, but an exon-19 deletion mutation could not be identified in one FFPE sample analyzed by both techniques, despite multiple repeated assays. This suggests that some tumors may demonstrate intratumoral heterogeneity for the occurrence of EGFR mutation.

Tomographic comparison of ventilation techniques for CT-guided thoracoscopic staple excision of subcentimeter lung nodules.

This study was planned to compare the computed tomographic detectability of lung nodules in three ventilatory conditions: total lung capacity, high-frequency ventilation, and total lung deflation. In an ex vivo lung model, 44 nodules were simulated. Using computed tomography (CT) scans, nodules were detected and compared to the actual number and excised under CT guidance. Simulated nodules measured 6.2 +/- 2.1 mm and demonstrated an attenuation of 175 +/- 14 HU. Observer confidence was highest at total lung capacity (5.00 +/- 0.00), in comparison to high-frequency ventilation and total lung deflation (4.69 +/- 0.78, 4.94 +/- 0.27, p = .24). The kappa score for total lung capacity, high-frequency ventilation, and total lung deflation was 1.00, 0.96, and 0.98, respectively, indicating a very high interrater reliability. Although surgical devices generated a substantial artifact, 90% of nodules were excised. Thus, although total lung capacity produces the highest confidence level, all three of the ventilatory techniques examined have similar detection of subcentimeter pulmonary nodules using computed tomography scans.

Peripheral lung nodules: fluoroscopically guided video-assisted thoracoscopic resection after computed tomography-guided localization using platinum microcoils.

OBJECTIVES: We sought to test the safety and efficacy of fluoroscopically guided, video-assisted, thoracoscopic resection after computed tomography (CT)-guided localization using platinum microcoils. SUMMARY BACKGROUND DATA: Video-assisted thoracoscopic (VATS) resection of small pulmonary nodules >5 mm deep to the visceral pleura fails to locate the nodule and requires conversion to open thoracotomy in two thirds of cases. Therefore, we developed a new technique for intraoperative localization of these nodules using CT-guided placement of platinum microcoils. This study tests the safety and efficacy of this technique in a Phase I human study. METHODS: Twelve patients with undiagnosed growing pulmonary nodules <20 mm were marked preoperatively using percutaneously placed CT-guided platinum microcoils. The coil was deployed adjacent to the nodule with the distal end of the coil placed deep to the nodule and the superficial end coiled on the pleural surface. The nodule and coil were excised using endostaplers guided by VATS and fluoroscopy. Histopathologic diagnosis was performed immediately after resection. RESULTS: CT-guided microcoil localization was successful in all patients. A small hemothorax and a pneumothorax requiring a chest tube occurred in 2 patients. Mean distance from visceral pleura to the deep edge of the nodule was 30.9 +/- 15.4 mm. VATS resection of the nodules (size = 11.8 +/- 3.2 mm) was successful in all patients. Mean microcoil localization, fluoroscopy, and operative times were 42 +/- 14, 3.1 +/- 2.0, and 67 +/- 27 minutes. A diagnosis of primary nonsmall cell bronchogenic carcinoma was made in 6 patients who then received a completion lobectomy. Six patients (hamartoma: 2, reactive lymph node: 1, bronchoalveolar cell carcinoma: 2, metastatic sarcoma: 1) did not receive further resections. CONCLUSIONS: Preoperative localization of pulmonary nodules using percutaneous CT-guided platinum microcoil insertion combined with operative fluoroscopic visualization is a safe, effective technique that increases the success rate of VATS excision.

[Evaluation of the use of bovine pericardium in non-anatomical lung resections in dogs]. / Evaluación de la utilidad del pericardio bovino en resecciones pulmonares no anatómicas en perros.

UNLABELLED: In this study we assessed the usefulness, healing, as well as the integration to lung tissue of glutaraldehyde preserved at 0.5% bovine pericardium GPBP and lyophilized (GPBPL), after reinforced resection of lung tissue in dogs by thoracotomy or thoracoscopy. MATERIAL AND METHODS: GPBP and GPBPL were prepared and used to reinforce the suture line of lung resection in 30 mongrel dogs: Group I (n = 6): The GPBP were fixed on the lung with 4-0 polypropylene by thoracotomy. Group II (n = 6): The resection and fixed of the GPBP were performed with an linear stapler by thoracotomy. Group III (n = 6): The resection and fixed of the GPBPL were performed with an linear stapler by thoracotomy. Group IV (n = 6): The resection and fixed of the GPBP strips were performed with a linear stapler by thoracoscopy. Group V: The resection and fixed of the GPBPL strips were performed with a linear stapler by thoracoscopy. Clinico-radiological evaluation was done until euthanasia of all animals at week 8 postop. Progressive insufflation up to 40 cm H2O of airway pressure was done to evaluated resistance of the heal in the suture line reinforced. Macroscopic, and microscopic examination of the GPBP, GPBPL and lung were evaluated. RESULTS: All animals survived the surgical procedure and study time (8 weeks). No airleaks were evident at any time during the study including the insufflation test. Macroscopic examination of the GPBP and GPBPL showed good adaptation to the lung tissue. Microscopically all animals presented good healing with deposition of fibrotic tissue layer on the GPBP and GPBPL. CONCLUSION: GPBP and GPBPL are an adequate materials to reinforce lung staple line, when resection of lung tissue was performed in dogs by thoracotomy or thoracoscopy.

Evaluación de la utilidad del pericardio bovino en resecciones pulmonares no anatómicas en perros / Evaluation of the use of bovine pericardium in non-anatomical lung resections in dogs

In this study we assessed the usefulness, healing, as well as the integration to lung tissue of glutaraldehyde preserved at 0.5 bovine pericardium GPBP and lyophilized (GPBPL), after reinforced resection of lung tissue in dogs by thoracotomy or thoracoscopy. MATERIAL AND METHODS: GPBP and GPBPL were prepared and used to reinforce the suture line of lung resection in 30 mongrel dogs: Group I (n = 6): The GPBP were fixed on the lung with 4-0 polypropylene by thoracotomy. Group II (n = 6): The resection and fixed of the GPBP were performed with an linear stapler by thoracotomy. Group III (n = 6): The resection and fixed of the GPBPL were performed with an linear stapler by thoracotomy. Group IV (n = 6): The resection and fixed of the GPBP strips were performed with a linear stapler by thoracoscopy. Group V: The resection and fixed of the GPBPL strips were performed with a linear stapler by thoracoscopy. Clinico-radiological evaluation was done until euthanasia of all animals at week 8 postop. Progressive insufflation up to 40 cm H2O of airway pressure was done to evaluated resistance of the heal in the suture line reinforced. Macroscopic, and microscopic examination of the GPBP, GPBPL and lung were evaluated. RESULTS: All animals survived the surgical procedure and study time (8 weeks). No airleaks were evident at any time during the study including the insufflation test. Macroscopic examination of the GPBP and GPBPL showed good adaptation to the lung tissue. Microscopically all animals presented good healing with deposition of fibrotic tissue layer on the GPBP and GPBPL. CONCLUSION: GPBP and GPBPL are an adequate materials to reinforce lung staple line, when resection of lung tissue was performed in dogs by thoracotomy or thoracoscopy.