The survival of patients with non–small-cell lung cancer (NSCLC), even when resectable, remains poor. Several small studies suggest that occult metastases (OMs) in pleura, bone marrow (BM), or lymph nodes (LNs) are present in early-stage NSCLC and are associated with a poor outcome. We investigated the prevalence of OMs in resectable NSCLC and their relationship with survival.
Patients and Methods
Eligible patients had previously untreated, potentially resectable NSCLC. Saline lavage of the pleural space, performed before and after pulmonary resection, was examined cytologically. Rib BM and all histologically negative LNs (N0) were examined for OM, diagnosed by cytokeratin immunohistochemistry (IHC). Survival probabilities were estimated using the Kaplan-Meier method. The log-rank test and Cox proportional hazards regression model were used to compare survival of groups of patients. P < .05 was considered significant.
From July 1999 to March 2004, 1,047 eligible patients (538 men and 509 women; median age, 67.2 years) were entered onto the study, of whom 50% had adenocarcinoma and 66% had stage I NSCLC. Pleural lavage was cytologically positive in only 29 patients. OMs were identified in 66 (8.0%) of 821 BM specimens and 130 (22.4%) of 580 LN specimens. In univariate and multivariable analyses OMs in LN but not BM were associated with significantly worse disease-free survival (hazard ratio [HR], 1.50; P = .031) and overall survival (HR, 1.58; P = .009).
In early-stage NSCLC, LN OMs detected by IHC identify patients with a worse prognosis. Future clinical trials should test the role of IHC in identifying patients for adjuvant therapy.
A number of staging systems have been proposed for malignant pleural mesothelioma (MPM) in the past, but few have utilized a TNM (tumor, node, metastasis) system. The International Association for the Study of Lung Cancer (IASLC) and the International Mesothelioma Interest Group (IMIG) previously developed a TNM-staging system which has been accepted by the International Union Against Cancer (UICC) and the American Joint Commission on Cancer (AJCC). The present study examines this staging system by analysing the updated IASLC database for patients with MPM.
De-identified data from participating centres dated from 1995 to 2009 were submitted to the IASLC Statistical Center. Surgical procedures included those with a curative or palliative intent. Survival was measured from the date of pathologic diagnosis to the most recent contact or death. Endpoints included overall survival and analysis of potential prognostic factors.
Data was available for 3,101 patients from 15 centers, mostly from North America and Europe. After a median follow-up of 15 months, a number of clinicopathological and treatment-related prognostic factors were found to significantly influence overall survival. These included overall tumor stage based on the proposed TNM staging system, T category, N category, tumor histology, gender, age, and type of operation.
The IASLC database represents the largest, multicenter and international database on MPM to date. Analyses demonstrate that the proposed TNM staging system effectively distinguishes the T and N categories, but also highlight areas for potential revision in the future.
Pleural mesothelioma; extrapleural pneumonectomy; pleurectomy; decortication; trimodality therapy; multi-institutional database; survival; staging
This is a step-by-step video demonstration of a left extrapleural pneumonectomy for malignant pleural mesothelioma, including thoracotomy incision, extrapleural mobilization of tumor, resection of diaphragm, mediastinal nodal dissection and division of hilar vessels followed by reconstruction of diaphragm and closure of the thoracotomy.
Extrapleural pneumonectomy (EPP); malignant pleural mesothelioma
Mesothelin (MSLN) is a tumor-associated antigen, being investigated as a biomarker and therapeutic target in malignant pleural mesothelioma (MPM). The biological function of MSLN overexpression in MPM is unknown. We hypothesized that MSLN may promote tumor invasion in MPM, a tumor characterized primarily by regional aggressiveness and rare distant metastases.
Human and murine MPM cells with MSLN forced expression and shRNA knockdown were examined for proliferation, invasion, and matrix metalloproteinase (MMP) secretion. The influence of MSLN overexpression on MPM cell invasion was assessed in an orthotopic mouse model and in patient samples.
MSLN expression promotes MPM cell invasion and MMP secretion in both human and murine MPM cells. In an orthotopic MPM mouse model characterized by our laboratory, MPM cells with MSLN overexpression preferentially localized to the tumor invading edge, co-localized with MMP-9 expression, and promoted decreased survival without an increase in tumor burden progression. In a tissue microarray from epithelioid MPM patients (n=139, 729 cores), MSLN overexpression correlated with higher MMP-9 expression at individual core level. Among stage III MPM patients (n=72), high MSLN expression was observed in 26% of T2 tumors and 51% of T3 tumors.
Our data provide evidence elucidating a biological role for MSLN as a factor promoting tumor invasion and MMP-9 expression in MSLN-expressing MPM. As regional invasion is the characteristic feature in MSLN-expressing solid cancers (MPM, pancreas, and ovarian), our observations add rationale to studies investigating MSLN as a therapeutic target.
Mesothelin; mesothelioma; matrix metalloproteinase; tumor invasion; locoregional aggressiveness
We have recently proposed to reclassify the pleomorphic subtype of epithelioid malignant pleural mesothelioma (MPM) as non-epithelioid (biphasic/sarcomatoid) histology due to its similarly poor prognosis. We sought to investigate whether preoperative maximum standardized uptake value (SUVmax) on 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) correlates with histologic subtype in MPM.
Clinical data was collected for 78 patients with MPM who underwent preoperative FDG-PET. We retrospectively classified the epithelioid tumors into five subtypes: trabecular, tubulopapillary, micropapillary, solid and pleomorphic. Tumors were categorized by SUVmax into two groups: low (<10.0) and high (≥10.0).
The median overall survival of epithelioid tumors with high-SUVmax (n=12) was significantly shorter (7.1 months) than that of epithelioid tumors with low-SUVmax (n=54, 18.9 months, p<0.001) and comparable to non-epithelioid tumors (n=12, 7.2 months). Epithelioid tumors with pleomorphic subtype (n=9) had marginally higher SUVmax (mean±SD: 10.6±5.9) than epithelioid non-pleomorphic subtype (n=57, 6.5±3.2, p=0.050), and were comparable to that of non-epithelioid tumors (n=12, 9.1±4.8). Among the epithelioid tumors with high-SUVmax (n=12), 50% (n=6) showed pleomorphic subtype. In contrast, among epithelioid tumors with low-SUVmax (n=54), 6% (n=3) showed epithelioid pleomorphic subtypes (p=0.001). A positive correlation between mitotic count and SUVmax was observed (r=0.30, p=0.010).
Pleomorphic subtype of epithelioid MPM showed higher SUVmax than epithelioid non-pleomorphic subtype and was similar to non-epithelioid histology. Preoperative SUVmax on FDG-PET in epithelioid MPM can indicate patients with pleomorphic subtype with poor prognosis, supporting their reclassification as non-epithelioid.
Mesothelioma; Pleural neoplasm; Positron emission tomography; Pleomorphic
The purpose of this study was to prospectively compare the adequacy of core needle biopsy specimens with the adequacy of specimens from resected tissue, the histologic reference standard, for mutational analysis of malignant tumors of the lung.
SUBJECTS AND METHODS
The first 18 patients enrolled in a phase 2 study of gefitinib for lung cancer in July 2004 through August 2005 underwent CT- or fluoroscopy-guided lung biopsy before the start of gefitinib therapy. Three weeks after gefitinib therapy, the patients underwent lung tumor resection. The results of EGFR and KRAS mutational analysis of the core needle biopsy specimens were compared with those of EGFR and KRAS mutational analysis of the surgical specimens.
Two specimens were unsatisfactory for mutational analysis. The results of mutational assay results of the other 16 specimens were the same as those of analysis of the surgical specimens obtained an average of 31 days after biopsy.
Biopsy with small (18- to 20-gauge) core needles can yield sufficient and reliable samples for mutational analysis. This technique is likely to become an important tool with the increasing use of pharmacotherapy based on the genetics of specific tumors in individual patients.
biopsy; lung cancer; molecular typing; personalized medicine; targeted therapy
Mesothelin is overexpressed in several malignancies and is purportedly a specific marker of malignant transformation. In this pilot study, we investigated whether tissue and serum mesothelin are potential markers of neoplastic progression in Barrett’s esophagus (BE) and in esophageal adenocarcinoma (EAC).
Mesothelin expression was retrospectively evaluated in normal, BE, and EAC tissue from surgically resected esophageal specimens (n = 125). In addition, soluble mesothelin-related peptide (SMRP) levels were measured in serum.
Normal esophageal mucosa did not express mesothelin. BE tissue with high-grade dysplasia specifically expressed mesothelin, whereas BE tissue with low-grade or without dysplasia did not. Fifty-seven (46%) EAC tumors were positive for mesothelin. EAC tumors with BE expressed mesothelin more often than those without BE (58% vs 35%, P = 0.01). SMRP levels were elevated in 70% of EAC patients (mean, 0.89 nM; range, 0.03-3.77 nM), but not in patients with acid reflux and/or BE.
Mesothelin is commonly expressed in BE-associated esophageal adenocarcinoma. Based on this pilot study, a prospective study is under way to evaluate tissue and serum mesothelin are potential markers of neoplastic progression in BE and in EAC (NCT01393483).
Current surveillance methods in Barrett’s esophagus are invasive and neither cost-effective nor sensitive. This pilot study suggests that serum mesothelin is a marker of neoplastic transformation in BE and may provide a noninvasive method to improve identification of malignant transformation.
Mesothelin; SMRP; Barrett’s esophagus; esophageal cancer; screening
EGFR mutations underlie the sensitivity of lung cancers to erlotinib and gefitinib and can occur in any patient with this illness. Here we examine the frequency of EGFR mutations in smokers and men.
We determined the frequency of EGFR mutations and characterized their association with cigarette smoking status and male sex.
We tested 2,142 lung adenocarcinoma specimens for the presence of EGFR exon 19 deletions and L858R. EGFR mutations were found in 15% of tumors from former smokers (181 of 1,218; 95% CI, 13% to 17%), 6% from current smokers (20 of 344; 95% CI, 4% to 9%), and 52% from never smokers (302 of 580; 95% CI, 48% to 56%; P < .001 for ever v never smokers). EGFR mutations in former or current smokers represented 40% of all those detected (201 of 503; 95% CI, 36% to 44%). EGFR mutations were found in 19% (157 of 827; 95% CI, 16% to 22%) of tumors from men and 26% (346 of 1,315; 95% CI, 24% to 29%) of tumors from women (P < .001). EGFR mutations in men represented 31% (157 of 503; 95% CI, 27% to 35%) of all those detected.
A large number of EGFR mutations are found in adenocarcinoma tumor specimens from men and people who smoked cigarettes. If only women who were never smokers were tested, 57% of all EGFR mutations would be missed. Testing for EGFR mutations should be considered for all patients with adenocarcinoma of the lung at diagnosis, regardless of clinical characteristics. This strategy can extend the use of EGFR tyrosine kinase inhibitors to the greatest number individuals with the potential for substantial benefit.
The detection of mutations in the epidermal growth factor receptor (EGFR) gene, which predict sensitivity to treatment with EGFR tyrosine kinase inhibitors (TKIs), represents a major advance in the treatment of lung adenocarcinoma. KRAS mutations confer resistance to EGFR -TKIs. The prevalence of these mutations in African-American patients has not been thoroughly investigated.
We collected formalin-fixed, paraffin-embedded material from resected lung adenocarcinomas from African-American patients at three institutions for DNA extraction. The frequencies of EGFR exon 19 deletions, exon 21 L858R substitutions and KRAS mutations in tumor specimens from African-American patients were compared to data in Caucasian patients (n=476).
EGFR mutations were detected in 23 of the 121 specimens from African-American patients (19%, 95% CI 13–27%), while KRAS mutations were found in 21 (17%, 95% CI 12−25%). There was no significant difference between frequencies of EGFR mutations comparing African-American and Caucasian patients, 19% vs. 13% (61/476, 95% CI 10–16%) (p=0.11). KRAS mutations were more likely among Caucasians, 26% (125/476, 95% CI 23−30%) (p=0.04).
This is the largest study to date examining the frequency of mutations in lung adenocarcinomas in African-Americans. Although KRAS mutations were somewhat less likely, there was no difference between the frequencies of EGFR mutations in African-American patients as compared to Caucasians. These results suggest that all patients with advanced lung adenocarcinomas should undergo mutational analysis prior to initiation of therapy.
EGFR mutation; KRAS; African-Americans; racial differences
The prognosis for patients with large-cell neuroendocrine carcinoma (LCNEC) of the lung is extremely poor, and an optimal treatment has not yet been established. It has been recently reported that molecular-targeted therapies, such as tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR), are effective in patients with lung carcinoma. In efforts to improve the prognosis of patients with LCNEC, we analyzed gene expression, gene mutations and immunohistochemical (IHC) expression of known molecular targets in LCNECs, and compared the expression to that of lung adenocarcinomas (ACs). Thirteen patients with primary LCNEC and 14 patients with AC were analyzed. We evaluated IHC expression for c-KIT, human epidermal growth factor receptor type 2 (HER2) and vascular endothelial growth factor (VEGF), gene mutations for EGFR, K-ras and c-kit, and gene expression using fluorescence in situ hybridization for EGFR. In cases with LCNEC, the IHC expression of c-KIT, HER2 and VEGF was 76.9, 30.8 and 100%, respectively. There was a significant difference in the IHC expression of c-KIT and HER2 between the LCNEC and AC cases. Two cases of LCNEC had overexpression of HER2, and the frequency of EGFR gene mutations was higher in the the AC group, with only a single EGFR mutation (exon 18) identified in the LCNEC group. Although LCNEC had a higher rate of expression of c-KIT by IHC, no c-kit gene mutations were found. These findings suggest a potential role for anti-VEGF-, anti-c-KIT- and possibly anti-HER2-targeted agents in the treatment of LCNEC.
large-cell neuroendocrine carcinoma; molecular-targeted therapy; vascular endothelial growth factor; c-KIT; human epidermal growth factor receptor type 2
Cytological analysis of body fluids is currently used for detecting cancer. The objective of this study was to determine if the herpes virus carrying an enhanced green fluorescent protein (EGFP) could detect rare cancer cells in body fluids against millions of normal cells. Human cancer cells suspended with normal murine cells were infected with NV1066 at a multiplicity of infection (MOI) of 0.5 and 1.0 for 18 h. Fluorescent microscopy and flow cytometry were used for EGFP detection of cancer cells. EGFP-expressing cells were confirmed as cancer cells with specific markers by immunohistochemistry staining. Limits of detection of cancer cells in body fluid were measured by serial dilutions. Applicability of technique was confirmed with samples from patients with malignant pleural effusions. NV1066 expressed EGFP in 111 human cancer cell lines detected by fluorescent microscopy at an MOI of 0.5. NV1066 selectively infected cancer cells and spared normal cells as confirmed by immunohistochemistry. Sensitivity of detecting fluorescent green cells was 92% (confidence interval [CI] 83% to 97%) at a ratio of 1 cancer cell to 1 million normal cells. EGFP-positive cells were detected by fluorescent microscopy in patients’ malignant pleural effusion samples. Our data show proof of the concept that NV1066-induced EGFP expression allows detection of a single cancer cell against a background of 1 million normal cells. This method was demonstrated to be a reliable screening tool for human cancer cells in a suspension of normal murine cells as well as clinical specimens of malignant pleural effusions.
Thymomas and thymic carcinomas are rare intrathoracic malignancies that can be invasive and refractory to conventional treatment. Because these tumors both originate from the thymus, they are often grouped together clinically. However, whether the underlying biology of these tumors warrants such clustering is unclear, and the optimum treatment of either entity is unknown.
All thymic tumors were profiled for mutations in genes encoding components of the EGFR and KIT signaling pathways, assessed for EGFR and KIT expression by immunohistochemistry (IHC), and analyzed by array-based comparative genomic hybridization (aCGH). Previously untreated tumors were subjected to global gene expression arrays.
We analyzed 45 thymic tumors (thymoma n=38 (type A: n=8, type B2: n=22, type B3: n=8), and thymic carcinoma n=7). One thymoma and one thymic carcinoma harbored KRAS mutations (G12A and G12V, respectively), and one thymoma had a G13V HRAS mutation. Three tumors displayed strong KIT staining. Two thymic carcinomas harbored somatic KIT mutations (V560del and H697Y). In cell viability assays, the V560del mutant was associated with similar sensitivities to imatinib and sunitinib, while the H697Y mutant displayed greater sensitivity to sunitinib. Genomic profiling revealed distinct differences between type A-B2 thymomas vs. type B3 and thymic carcinomas. Moreover, aCGH could readily distinguish squamous cell carcinomas of the thymus vs. the lung, which can often present a diagnostic challenge.
Comprehensive genomic analysis suggests that thymic carcinomas are molecularly distinct from thymomas. These data have clinical, pathological, and therapeutic implications for the treatment of thymic malignancies.
Thymoma; Thymic Carcinoma; EGFR, RAS mutations; KIT mutations; mutational profiling; genomic analysis
Neoadjuvant pemetrexed plus cisplatin was administered, followed by extrapleural pneumonectomy (EPP) and hemithoracic radiation (RT), to assess the feasibility and efficacy of trimodality therapy in stage I to III malignant pleural mesothelioma.
Patients and Methods
Requirements included stage T1-3 N0-2 disease, no prior surgical resection, adequate organ function (including predicted postoperative forced expiratory volume in 1 second ≥ 35%), and performance status 0 to 1. Patients received pemetrexed 500 mg/m2 plus cisplatin 75 mg/m2 for four cycles. Patients without disease progression underwent EPP followed by RT (54 Gy). The primary end point was pathologic complete response (pCR) rate.
Seventy-seven patients received chemotherapy. All four cycles were administered to 83% of patients. The radiologic response rate was 32.5% (95% CI, 22.2 to 44.1). Fifty-seven patients proceeded to EPP, which was completed in 54 patients. Three pCRs were observed (5% of EPP). Forty of 44 patients completed irradiation. Median survival in the overall population was 16.8 months (95% CI, 13.6 to 23.2 months; censorship, 33.8%). Patients completing all therapy had a median survival of 29.1 months and a 2-year survival rate of 61.2%. Radiologic response of complete or partial response was associated with a median survival of 26.0 months compared with 13.9 months for patients with stable disease or progressive disease (P = .05).
This multicenter trial showed that trimodality therapy with neoadjuvant pemetrexed plus cisplatin is feasible with a reasonable long-term survival rate, particularly for patients who completed all therapy. Radiologic response to chemotherapy, but not sex, histology, disease stage, or nodal status, was associated with improved survival.
Malignant pleural mesothelioma (MPM) is a fatal disease with a median survival of less than 14 months. For the first time, a genetically engineered vaccinia virus is shown to produce efficient infection, replication, and oncolytic effect against MPM. GLV-1h68 is a replication-competent engineered vaccinia virus carrying transgenes encoding Renilla luciferase, green fluorescent protein (both inserted at the F14.5L locus), β-galactosidase (inserted at the J2R locus, which encodes thymidine kinase), and β-glucuronidase (at the A56R locus, which encodes hemagglutinin). This virus was tested in six human MPM cell lines (MSTO-211H, VAMT, JMN, H-2373, H-2452, and H-2052). GLV-1h68 successfully infected all cell lines. For the most sensitive line, MSTO-211H, expression of green fluorescent protein (GFP) started within 4 hr with increasing intensity over time until nearly 100% of cells expressed GFP at 24 hr. All cell lines were sensitive to killing by GLV-1h68, with the degree of sensitivity predictable by infectivity assay. Even the most resistant cell line exhibited 44 ± 3.8% cell survival by day 7 when infected at a multiplicity of infection of 1.0. Viral proliferation assays demonstrated 2-to 4-fold logarithmic replication of GLV-1h68 in the cell lines tested. In an orthotopic model, GLV-1h68 effectively prevented development of cachexia and tumor-related morbidity, reduced tumor burden, and cured MPM in both early and late treatment groups. GLV-1h68 was successfully used to treat MPM in vitro and in an orthotopic model (in vivo). These promising results warrant clinical investigation of GLV-1h68 as a novel agent in the treatment of MPM.
Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non–small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal–regulated kinase (ERK)-1/2 in human 293T cells and to growth factor–independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.
Background: Replication-competent, tumor specific herpes simplex virus NV1066 expresses green fluorescent protein (GFP) in infected cancer cells. We sought to determine the feasibility of GFP-guided imaging technology in the intraoperative detection of small tumor nodules.
Methods: Human cancer cell lines were infected with NV1066 at multiplicities of infection of 0.01, 0.1 and 1. Cancer cell specific infectivity, vector spread and GFP signal intensity were measured by flow cytometry and time-lapse digital imaging (in vitro); and by use of a stereomicroscope and endoscope equipped with a fluorescent filter (in vivo).
Results: NV1066 infected all cancer cell lines and expressed GFP at all MOIs. GFP signal was significantly higher than the autofluorescence of normal cells. One single dose of NV1066 spread within and across body cavities and selectively infected tumor nodules sparing normal tissue. Tumor nodules undetectable by conventional thoracoscopy and laparoscopy were identified by GFP fluorescence.
Conclusion: Virally-directed fluorescent imaging (VFI) is a real-time novel molecular imaging technology that has the potential to enhance the intraoperative detection of endoluminal or endocavitary tumor nodules.
Fluorescent detection; Oncolytic virus; Gene therapy; Fluorescent laparoscopy; Fluorescent thoracoscopy; Herpes simplex virus
Purpose: Current efforts on expanding minimally-invasive techniques into the realm of oncological surgery are hindered by lack of accurate visualization of tumor margins and failure to detect micro metastases in real time. We used a systemic delivery of a herpes viral vector with cancer selective infection and replication to precisely differentiate between normal and malignant tissue.
Procedures: NV1066 is a genetically modified, replication-competent herpes simplex virus carrying a transgene for enhanced green fluorescent protein (GFP). We tested the potential of NV1066 in delineating tumor tissue in vitro and in vivo in a wide range of cancers and whether NV1066-induced GFP expression can detect small foci of tumors and metastases in in vivo models using an operating endoscope with fluorescent filters.
Findings: Our findings indicate that NV1066 can be used for real-time intraoperative imaging and enhanced detection of early cancers and metastases. We demonstrate that a single dose of NV1066, administered either locally (intratumoral or intracavitary) or systemically, will detect loco-regional and distant disease throughout the body. Such cancer selectivity is confirmed in 110 types of cancer cells from 16 different primary organs. Fluorescence-aided minimally-invasive endoscopy revealed microscopic tumor deposits unrecognized by conventional laparoscopy/thoracoscopy. Furthermore, NV1066 ability to transit and infect tumor and metastases is proven in syngenic and transplanted tumors in different animal models, both immunocompetent and immunodeficient. Cancer selective GFP expression is confirmed by histology, immunohistochemistry and qRT-PCR.
Conclusion: These studies form the basis for real-time, intraoperative diagnostic imaging of tumor and metastases by minimally-invasive endoscopic technology.
herpes simplex virus; oncolytic viral therapy; gene therapy; HSV; endoscopy; minimally-invasive surgery
Background: NV1066, a replication-competent oncolytic herpes simplex virus type 1 (HSV-1) attenuated by a deletion in the gene γ134.5, preferentially replicates in and kills malignant cells. γ134.5 encodes ICP34.5, a viral protein essential for productive replication, which has homology with mammalian stress response induced GADD34 (Growth Arrest and DNA Damage-Inducible Protein). We hypothesized that cisplatin upregulates GADD34 expression, which enhances NV1066 replication and oncolysis.
Methods: Ten human malignant pleural mesothelioma (MPM) cell lines were infected with NV1066 at multiplicities of infection (MOI; ratio of viral particles per tumor cell) 0.005 to 0.8 in vitro, with and without cisplatin (1 to 4 μM). In the MPM cell line VAMT, viral replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 induction by quantitative RT-PCR and Western blot. Synergistic efficacy was confirmed by the isobologram and combination index methods of Chou-Talalay. GADD34 upregulation by cisplatin was inhibited with GADD34 siRNA to further confirm the synergistic efficacy dependence with GADD34.
Results: Combination therapy with NV1066 and cisplatin showed strong synergism in epithelioid (H-2452, H-Meso), sarcomatoid (H-2373, H-28), and biphasic (JMN, Meso-9, MSTO-211H) MPM cell lines, and an additive effect in others. In VAMT cells combination therapy enhanced viral replication 4 to 11-fold (p < 0.01) and cell kill 2 to 3-fold (p < 0.01). Significant dose reductions for both agents (2 to 600-fold) were achieved over a wide range of therapeutic-effect levels (LD50 – LD99) without compromising cell kill. Synergistic cytotoxicity correlated with GADD34 upregulation (2 to 4-fold, p < 0.01) and was eliminated following transfection with GADD34 siRNA.
Conclusion: Cisplatin-induced GADD34 expression selectively enhanced the cytotoxicity of the γ134.5-deficient oncolytic virus, NV1066. This provides a cellular basis for combination therapy with cisplatin and NV1066 to treat MPM and achieve synergistic efficacy, while minimizing dosage and toxicity.
Chemotherapy; Gene therapy; HSV; Combination therapy
Replication-competent oncolytic herpes simplex viruses (HSV) with deletion of the γ134.5 gene preferentially replicate in and kill malignant cells. γ134.5 codes for ICP 34.5, a protein that enhances viral replication, and is homologous to Growth Arrest and DNA damage Protein 34 (GADD34), a radiation-inducible DNA repair gene. We hypothesized that radiation therapy may potentiate efficacy of oncolytic viral therapy by up-regulating GADD 34 and promoting viral replication.
A549 and H1299 lung cancer cell lines were infected with NV1066, an oncolytic HSV at multiplicities of infection (MOI; number of viral particles per tumor cell) of 0.1 to 0.5 in vitro with radiation (2 to 10 Gy) or without radiation. Viral replication was determined by plaque assay, cell-to cell spread by flow cytometry, cell kill by lactate dehydrogenase assay and GADD 34 induction by real time RT-PCR and western blot method. Evidence of synergistic cytotoxicity dependence with GADD34 induction is further confirmed by siRNA inhibition of GADD34 expression.
Using both the isobologram method and combination-index method of Chou-Talalay, significant synergism was demonstrated between radiation therapy and NV1066 both in vitro and in vivo. As a result of such synergism, a dose-reduction for each agent (2 to 6000-fold) can be accomplished over a wide range of therapeutic-effect levels without sacrificing tumor cell kill. This effect is correlated with increased GADD34 expression and inhibited by transfection of siRNA directed against GADD34.
These data provide the cellular basis for the clinical investigation of combined use of radiation therapy with oncolytic HSV therapy in the treatment of lung cancer to achieve synergistic efficacy while minimizing dosage and toxicity.
Ionizing radiation; Gene therapy; Viruses; Non small cell lung cancer; 7-AAD 7-amino-actinomycin D; DRI Dose-reduction index; EGFP Enhanced green fluorescent protein; Fa Fraction affected; GADD34 Growth arrest and DNA damage repair gene 34; HSV Herpes simplex virus; ICP 34.5 Infectious cell protein 34.5; LDH Lactate dehydrogenase; MOI Multiplicity of infection; NSCLC Non-small cell lung cancer; PFU Plaque forming unit; RT Radiation therapy; siRNA Small inhibitory RNA
Herpes simplex virus-one (HSV-1) oncolytic therapy and gene therapy are promising treatment modalities against cancer. NV1066, one such HSV-1 virus carries a marker gene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine whether NV1066 is cytotoxic to lung cancer and whether EGFP is a detectable marker of viral infection in vitro and in vivo. We further investigated whether EGFP expression in infected cells can be used to localize the virus and to identify small metastatic tumor foci (< 1 mm.) in vivo by means of minimally invasive endoscopic systems equipped with fluorescent filters.
In A549 human lung cancer cells, in vitro viral replication was determined by plaque assay, cell kill by LDH release assay, and EGFP expression by flow cytometry. In vivo, A549 cells were injected into the pleural cavity of athymic mice. Mice were treated with intrapleural injection of NV1066 or saline and examined for EGFP expression in tumor deposits using a stereomicroscope or a fluorescent thoracoscopic system.
NV1066 replicated in, expressed EGFP in infected cells and killed tumor cells in vitro. In vivo, treatment with intrapleural NV1066 decreased pleural disease burden, as measured by chest wall nodule counts and organ weights. EGFP was easily visualized in tumor deposits, including microscopic foci, by fluorescent thoracoscopy.
NV1066 has significant oncolytic activity against a human NSCLC cell line and is effective in limiting the progression of metastatic disease in an in vivo orthotopic model. By incorporating fluorescent filters into endoscopic systems, a minimally-invasive means for diagnosing small metastatic pleural deposits and localization of viral therapy for thoracic malignancies may be developed using the EGFP marker gene inserted in oncolytic herpes simplex viruses.
Gene therapy; Green fluorescent protein; Lung neoplasm; Minimally-invasive; Targeted therapy; Thoracoscopy; Bronchoscopy; CMV: cytomegalovirus; EGFP: enhanced green fluorescent protein; HSV-1: herpes simplex virus-1; LDH: lactate dehydrogenase; MIS: minimally-invasive surgical
Thoracotomy is associated with severe pain that may persist for years. Acupuncture is a complementary therapy with a proven role in pain control. A randomized trial showed that acupuncture was effective in controlling pain after abdominal surgery, but the efficacy of this technique for the treatment of thoracotomy pain has not been established. We developed a novel technique for convenient application of acupuncture to patients undergoing thoracotomy, and in a Phase II trial evaluated the safety of this intervention and the feasibility of doing a randomized trial.
Adult patients scheduled for unilateral thoracotomy with preoperative epidural catheter placement received acupuncture immediately prior to surgery. Eighteen semi-permanent intradermal needles were inserted on either side of the spine, and four were inserted in the legs and auricles. Needles were removed after four weeks. Using a numerical rating scale, pain was measured on the first five postoperative days. After discharge, pain was assessed using the Brief Pain Inventory at 7, 30, 60 and 90 days.
Thirty-six patients were treated with acupuncture. Of these, 25, 23, and 22 patients provided data at 30, 60, and 90 days, respectively. The intervention was well tolerated by patients with only one minor and transient adverse event of skin ulceration.
The rate of data completion met our predefined criterion for determining a randomized trial to be feasible (at least 75% of patients tolerated the intervention and provided evaluable data). This novel intervention is acceptable to patients undergoing thoracotomy and does not interfere with standard preoperative care. There was no evidence of important adverse events. We are now testing the hypothesis that acupuncture significantly adds to standard perioperative pain management in a randomized trial.
Tumor biomarkers provide a quantitative tool for following tumor progression and response to therapy. However, investigations of clinically useful tumor biomarkers are time-consuming, costly, and limited by patient and tumor heterogeneity. In addition, assessment of biomarkers as indicators of therapy response is confounded by the concomitant use of multiple therapeutic interventions. Herein we report our use of a clinically relevant orthotopic animal model of malignant pleural mesothelioma for investigating tumor biomarkers. Utilizing multi-modality imaging with correlative histopathology, we demonstrate the utility and accuracy of the mouse model in investigating tumor biomarkers – serum soluble mesothelin-related peptide (SMRP) and osteopontin (OPN). This model revealed percentage change in SMRP level to be an accurate biomarker of tumor progression and therapeutic response – a finding consistent with recent clinical studies. This in vivo platform demonstrates the advantages of a validated mouse model for the timely and cost-effective acceleration of human biomarker translational research.
Malignant pleural mesothelioma (MPM) is an aggressive cancer that is refractory to current treatment modalities. Oncolytic herpes simplex viruses (HSV) used for gene therapy are genetically engineered, replication-competent viruses that selectively target tumor cells while sparing normal host tissue. The localized nature, the potential accessibility and the relative lack of distant metastasis, make MPM a particularly suitable disease for oncolytic viral therapy.
The infectivity, selective replication, vector spread and cytotoxic ability of three oncolytic HSV: G207, NV1020 and NV1066 were tested against eleven pathological types of MPM cell lines including those that are resistant to radiation therapy, gemcitabine or cisplatin. The therapeutic efficacy and the effect on survival of NV1066 were confirmed in a murine MPM model.
All three oncolytic HSV were highly effective against all the MPM cell lines tested. Even at very low concentrations of MOI 0.01 (MOI: multiplicity of viral infection, ratio of viral particles per cancer cell), HSV were highly effective against MPM cells that are resistant to radiation, gemcitabine and cisplatin. NV1066, an oncolytic HSV that expresses green fluorescent protein (GFP) was able to delineate the extent of the disease in a murine model of MPM due to selective infection and expression of GFP in tumor cells. Furthermore, NV1066 was able to reduce the tumor burden and prolong survival even when treated at an advanced stage of the disease.
These findings support the continued investigation of oncolytic HSV as potential therapy for patients with therapy resistant malignant pleural mesothelioma.
Gene therapy; Herpes simplex virus; NV1066