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New therapeutic strategies are needed for malignant pleural mesothelioma (MPM). We conducted a single-center, open-label, nonrandomized, pilot and feasibility trial using two intrapleural doses of an adenoviral vector encoding human IFN-α (Ad.IFN-α2b). Nine subjects were enrolled at two dose levels. The first three subjects had very high pleural and systemic IFN-α concentrations resulting in severe "flu-like" symptoms necessitating dose de-escalation. The next six patients had reduced (but still significant) pleural and serum IFN-α levels, but with tolerable symptoms. Repeated vector administration appeared to prolong IFN-α expression levels. Anti-tumor humoral immune responses against mesothelioma cell lines were seen in seven of the eight subjects evaluated. No clinical responses were seen in the four subjects with advanced disease. However, evidence of disease stability or tumor regression was seen in the remaining five patients, including one dramatic example of partial tumor regression at sites not in contiguity with vector infusion. These data show that Ad.IFN-α2b has potential therapeutic benefit in MPM and that it generates anti-tumor immune responses that may induce anatomic and/or metabolic reductions in distant tumor. Clinical trial registered with www.clinicaltrials.gov (NCT 01212367). [ABSTRACT FROM AUTHOR]

Context.—: Mesothelioma is an uncommon tumor that can be difficult to diagnose., Objective.—: To provide updated, practical guidelines for the pathologic diagnosis of mesothelioma., Data Sources.—: Pathologists involved in the International Mesothelioma Interest Group and others with expertise in mesothelioma contributed to this update. Reference material includes peer-reviewed publications and textbooks., Conclusions.—: There was consensus opinion regarding guidelines for (1) histomorphologic diagnosis of mesothelial tumors, including distinction of epithelioid, biphasic, and sarcomatoid mesothelioma; recognition of morphologic variants and patterns; and recognition of common morphologic pitfalls; (2) molecular pathogenesis of mesothelioma; (3) application of immunohistochemical markers to establish mesothelial lineage and distinguish mesothelioma from common morphologic differentials; (4) application of ancillary studies to distinguish benign from malignant mesothelial proliferations, including BAP1 and MTAP immunostains; novel immunomarkers such as Merlin and p53; fluorescence in situ hybridization (FISH) for homozygous deletion of CDKN2A; and novel molecular assays; (5) practical recommendations for routine reporting of mesothelioma, including grading epithelioid mesothelioma and other prognostic parameters; (6) diagnosis of mesothelioma in situ; (7) cytologic diagnosis of mesothelioma, including use of immunostains and molecular assays; and (8) features of nonmalignant peritoneal mesothelial lesions., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Background: Pulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs., Methods: Lung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were H&E stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1., Results: In lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs., Conclusion: Vascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment., (© 2022. The Author(s).)

Background: Macroscopic complete resection can improve survival in a select group of patients with malignant pleural mesothelioma. During resection, differentiating residual tumor from inflammation or scar can be challenging. This trial evaluated near-infrared (NIR) intraoperative imaging using TumorGlow (a novel NIR imaging approach utilizing high-dose indocyanine green and delayed imaging) technology to improve detection of macroscopic residual disease., Methods: Twenty subjects were enrolled in an open-label clinical trial of NIR intraoperative imaging with TumorGlow (Indocyanine Green for Solid Tumors [NCT02280954]). Twenty-four hours before pleural biopsy or pleurectomy and decortication (P/D), patients received intravenous indocyanine green. All specimens identified during standard-of-care surgical resection and with NIR imaging underwent histopathologic profiling and correlative microscopic fluorescent tomographic evaluation. For subjects undergoing P/D (n = 13), the hemithorax was evaluated with NIR imaging during P/D to assess for residual disease. When possible, additional fluorescent lesions were resected., Results: Of 203 resected specimens submitted for evaluation, indocyanine green accumulated within 113 of 113 of resected mesothelioma specimens, with a mean signal-to-background fluorescence ratio of 3.1 (SD, 2.2 to 4.8). The mean signal-to-background fluorescence ratio of benign tissues was 2.2 (SD, 1.4 to 2.4), which was significantly lower than in malignant specimens (p = 0.001). NIR imaging identified occult macroscopic residual disease in 10 of 13 subjects. A median of 5.6 resectable residual deposits per patient (range, 0 to 11 deposits per patient), with a mean size of 0.3 cm (range, 0.1 to 1.5 cm), were identified., Conclusions: TumorGlow for malignant pleural mesothelioma is safe and feasible. Excellent sensitivity allows for to reliable detection of macroscopic residual disease during cytoreductive surgical procedures., (Copyright © 2019 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Purpose: Intraoperative localization and resection of ill-defined pulmonary ground-glass opacities (GGOs) during minimally invasive pulmonary resection is technically challenging. Current preoperative techniques to facilitate localization of GGOs include microcoil and hook wire placement, both of which have logistic limitations, carry safety concerns, and do not help with margin assessment. In this clinical trial, we explored an alternative method involving near-infrared molecular imaging with a folate receptor-targeted agent, OTL38, to improve localization of GGOs and confirmation of resection margins., Methods: In a human trial, 20 subjects with pulmonary GGOs who were eligible for video-assisted thoracoscopic surgery (VATS) resection received 0.025 mg/kg of OTL38 before the resection. The primary objectives were to (1) determine whether use of OTL38 allows safe localization of GGOs and assessment of margins during VATS and (2) determine patient, radiographic, and histopathologic variables that predict the amount of fluorescence during near-infrared imaging., Results: We observed no toxicity. Of the 21 GGOs, 20 accumulated OTL38 and displayed fluorescence upon in situ or back table evaluation. Intraoperatively, near-infrared imaging localized 15 of 21 lesions whereas VATS alone localized 10 of 21 (p = 0.05). The addition of molecular imaging affected care of nine of 21 subjects by improving intraoperative localization (n = 6) and identifying close margins (n = 3). This approach was most effective for subpleural lesions measuring less than 2 cm. For lesions deeper than 1.5 cm from the pleural surface, intraoperative localization using fluorescent feedback was limited., Conclusions: This approach provides a safe alternative for intraoperative localization of small, peripherally located pulmonary lesions. In contrast to alternative localization techniques, use of OTL38 also allows confirmation of adequate margins. Future studies will compare this approach to alternative localization techniques in a clinical trial., (Copyright © 2018 International Association for the Study of Lung Cancer. All rights reserved.)

Background: Clinical applicability of folate receptor-targeted intraoperative molecular imaging (FR-IMI) has been established for surgically resectable pulmonary adenocarcinoma. A role for FR-IMI in other lung cancer histologies has not been studied. In this study, we evaluate feasibility of FR-IMI in patients undergoing pulmonary resection for squamous cell carcinomas (SCCs)., Methods: In a human clinical trial (NCT02602119), twelve subjects with pulmonary SCCs underwent FR-IMI with a near-infrared contrast agent that targets the folate receptor-α (FRα), OTL38. Near-infrared signal from tumors and benign lung was quantified to calculate tumor-to-background ratios (TBR). Folate receptor-alpha expression was characterized, and histopathologic correlative analyses were performed to evaluate patterns of OTL38 accumulation. An exploratory analysis was performed to determine patient and histopathologic variables that predict tumor fluorescence., Results: 9 of 13 SCCs (in 9 of 12 of subjects) displayed intraoperative fluorescence upon NIR evaluation (median TBR, 3.9). OTL38 accumulated within SCCs in a FRα-dependent manner. FR-IMI was reliable in localizing nodules as small as 1.1 cm, and prevented conversion to thoracotomy for nodule localization in three subjects. Upon evaluation of patient and histopathologic variables, in situ fluorescence was associated with distance from the pleural surface, and was independent of alternative variables including tumor size and metabolic activity., Conclusions: This work demonstrates that FR-IMI is potentially feasible in 70% of SCC patients, and that molecular imaging can improve localization during minimally invasive pulmonary resection. These findings complement previous data demonstrating that ∼98% of pulmonary adenocarcinomas are localized during FR-IMI and suggest broad applicability for NSCLC patients undergoing resection., Competing Interests: CONFLICTS OF INTEREST SAK and PL are a Board Members of On Target Laboratories, producers of the study drug (OTL38).

Purpose: "In situ vaccination" using immunogene therapy has the ability to induce polyclonal antitumor responses directed by the patient's immune system., Experimental Design: Patients with unresectable malignant pleural mesothelioma (MPM) received two intrapleural doses of a replication-defective adenoviral vector containing the human IFNα2b gene (Ad.IFN) concomitant with a 14-day course of celecoxib followed by chemotherapy. Primary outcomes were safety, toxicity, and objective response rate; secondary outcomes included progression-free and overall survival. Biocorrelates on blood and tumor were measured., Results: Forty subjects were treated: 18 received first-line pemetrexed-based chemotherapy, 22 received second-line chemotherapy with pemetrexed (n = 7) or gemcitabine (n = 15). Treatment was generally well tolerated. The overall response rate was 25%, and the disease control rate was 88%. Median overall survival (MOS) for all patients with epithelial histology was 21 months versus 7 months for patients with nonepithelial histology. MOS in the first-line cohort was 12.5 months, whereas MOS for the second-line cohort was 21.5 months, with 32% of patients alive at 2 years. No biologic parameters were found to correlate with response, including numbers of activated blood T cells or NK cells, regulatory T cells in blood, peak levels of IFNα in blood or pleural fluid, induction of antitumor antibodies, nor an immune-gene signature in pretreatment biopsies., Conclusions: The combination of intrapleural Ad.IFN, celecoxib, and chemotherapy proved safe in patients with MPM. OS rate was significantly higher than historical controls in the second-line group. Results of this study support proceeding with a multicenter randomized clinical trial of chemo-immunogene therapy versus standard chemotherapy alone. Clin Cancer Res; 22(15); 3791-800. ©2016 AACR., (©2016 American Association for Cancer Research.)

Based on studies in mouse tumor models, granulocytes appear to play a tumor-promoting role. However, there are limited data about the phenotype and function of tumor-associated neutrophils (TANs) in humans. Here, we identify a subset of TANs that exhibited characteristics of both neutrophils and antigen-presenting cells (APCs) in early-stage human lung cancer. These APC-like "hybrid neutrophils," which originate from CD11b(+)CD15(hi)CD10(-)CD16(low) immature progenitors, are able to cross-present antigens, as well as trigger and augment anti-tumor T cell responses. Interferon-γ and granulocyte-macrophage colony-stimulating factor are requisite factors in the tumor that, working through the Ikaros transcription factor, synergistically exert their APC-promoting effects on the progenitors. Overall, these data demonstrate the existence of a specialized TAN subset with anti-tumor capabilities in human cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01-restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.

Context: Malignant mesothelioma (MM) is an uncommon tumor that can be difficult to diagnose., Objective: To provide updated practical guidelines for the pathologic diagnosis of MM., Data Sources: Pathologists involved in the International Mesothelioma Interest Group and others with an interest in the field contributed to this update. Reference material includes peer-reviewed publications and textbooks., Conclusions: There was consensus opinion regarding (1) distinction of benign from malignant mesothelial proliferations (both epithelioid and spindle cell lesions), (2) cytologic diagnosis of MM, (3) key histologic features of pleural and peritoneal MM, (4) use of histochemical and immunohistochemical stains in the diagnosis and differential diagnosis of MM, (5) differentiation of epithelioid MM from various carcinomas (lung, breast, ovarian, and colonic adenocarcinomas, and squamous cell and renal cell carcinomas), (6) diagnosis of sarcomatoid mesothelioma, (7) use of molecular markers in the diagnosis of MM, (8) electron microscopy in the diagnosis of MM, and (9) some caveats and pitfalls in the diagnosis of MM. Immunohistochemical panels are integral to the diagnosis of MM, but the exact makeup of panels used is dependent on the differential diagnosis and on the antibodies available in a given laboratory. Immunohistochemical panels should contain both positive and negative markers. It is recommended that immunohistochemical markers have either sensitivity or specificity greater than 80% for the lesions in question. Interpretation of positivity generally should take into account the localization of the stain (eg, nuclear versus cytoplasmic) and the percentage of cells staining (>10% is suggested for cytoplasmic membranous markers). These guidelines are meant to be a practical reference for the pathologist.

Purpose: Thymidylate synthase (TS) is a potential predictor of outcome after pemetrexed (Pem) in patients with malignant pleural mesothelioma (MPM), and assays measuring TS levels are commercially marketed. The goal of this study was to further evaluate the value of TS and to study another potential biomarker of response, the enzyme, folyl-polyglutamate synthase (FPGS), which activates Pem intracellularly., Methods: Levels of TS and FPGS were semi-quantitatively determined immunohistochemically using H-scores on tissue samples from 85 MPM patients receiving Pem as primary therapy. H-score was correlated with radiographic disease control rate (DCR), time to progression (TTP) and overall survival (OS). In addition, expression levels of TS and FPGS in MPM cell lines were determined using immunoblotting and correlated with their sensitivity to Pem-induced cell death., Results: H-scores from patients with disease control versus progressive disease showed extensive overlap. There were no significant correlations of DCR, TTP, or OS to either TS levels (p = 0.73, 0.93, and 0.59, respectively), FPGS levels (p = 0.95, 0.77, and 0.43, respectively) or the ratio of FPGS/TS using the median scores of each test as cutoffs. There was no correlation between TS or FPGS expression and chemosensitivity of mesothelioma cells to Pem in vitro., Conclusions: Although previous retrospective data suggest that TS and FPGS expression might be potential markers of Pem efficacy in MPM, our data indicate these markers lack sufficient predictive value in individual patients and should not be used to guide therapeutic decisions in the absence of prospective studies.

We previously showed that a single intrapleural dose of an adenoviral vector expressing interferon-beta (Ad.IFN-beta) in patients with malignant pleural mesothelioma (MPM) or malignant pleural effusions (MPE) resulted in gene transfer, humoral antitumor immune responses, and anecdotal clinical responses manifested by modified Response Evaluation Criteria in Solid Tumors (RECIST) disease stability in 3 of 10 patients at 2 months and an additional patient with significant metabolic response on positron emission tomography (PET) imaging. This phase I trial was conducted to determine whether using two doses of Ad.IFN-beta vector would be superior. Ten patients with MPM and seven with MPE received two doses of Ad.IFN-beta through an indwelling pleural catheter. Repeated doses were generally well tolerated. High levels of IFN-beta were detected in pleural fluid after the first dose; however, only minimal levels were seen after the second dose of vector. Lack of expression correlated with the rapid induction of neutralizing Ad antibodies (Nabs). Antibody responses against tumor antigens were induced in most patients. At 2 months, modified RECIST responses were as follows: one partial response, two stable disease, nine progressive disease, and two nonmeasurable disease. One patient died after 1 month. By PET scanning, 2 patients had mixed responses and 11 had stable disease. There were seven patients with survival times longer than 18 months. This approach was safe, induced immune responses and disease stability. However, rapid development of Nabs prevented effective gene transfer after the second dose, even with a dose interval as short as 7 days.

Abstract Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-beta (IFN-beta) gene (VSV.IFN-beta). We conducted this study to determine the ability of VSV.IFN-beta to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-beta did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-beta. In the eight resistant lines, pretreatment with IFN-beta prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-beta protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2',5'-oligo-adenylate-synthetase (2'5'-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-beta protein and no IFN- or VSV-induced changes in PKR, MxA, and 2'5'-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-beta by immunostaining for the presence of p48 protein.

One of the primary limitations of adenoviral (Ad) -mediated gene therapy is the generation of anti-Ad inflammatory responses that can induce clinical toxicity and impair gene transfer efficacy. The effects of immunosuppression on these inflammatory responses, transgene expression, and toxicity have not yet been systematically examined in humans undergoing Ad-based gene therapy trials. We therefore conducted a pilot study investigating the use of systemic corticosteroids to mitigate antivector immune responses. In a previous phase I clinical trial, we demonstrated that Ad-mediated intrapleural delivery of the herpes simplex virus thymidine kinase gene (HSVtk) to patients with mesothelioma resulted in significant, but relatively superficial, HSVtk gene transfer and marked anti-Ad humoral and cellular immune responses. When a similar group of patients was treated with Ad.HSVtk and a brief course of corticosteroids, decreased clinical inflammatory responses were seen, but there was no demonstrable inhibition of anti -Ad antibody production or Ad-induced peripheral blood mononuclear cell activation. Corticosteroid administration also had no apparent effect on the presence of intratumoral gene transfer. Although limited by the small numbers of patients studied, our data suggest that systemic administration of steroids in the context of Ad-based gene delivery may limit acute clinical toxicity, but may not inhibit cellular and humoral responses to Ad vectors.

Malignant pleural mesothelioma is a fatal neoplasm that is unresponsive to standard modalities of cancer therapy. We conducted a phase I dose-escalation clinical trial of adenoviral (Ad)-mediated intrapleural herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy in patients with mesothelioma as a model for treatment of a localized malignancy. The goals of this phase I trial were to assess the safety, toxicity, and maximally tolerated dose of intrapleural Ad.HSVtk, to examine patient inflammatory response to the viral vector, and to evaluate the efficiency of intratumoral gene transfer. Twenty-one previously untreated patients were enrolled in this single-arm, dose-escalation study with viral doses ranging from 1 x 10(9) plaque-forming units (pfu) to 1 x 10(12) pfu. A replication-incompetent recombinant adenoviral vector containing the HSVtk gene under control of the Rous sarcoma virus (RSV) promoter-enhancer was introduced into the pleural cavity of patients with malignant mesothelioma followed by 2 weeks of systemic therapy with GCV at a dose of 5 mg/kg twice a day. The initial 15 patients underwent thoracoscopic pleural biopsy prior to, and 3 days after, vector delivery. The last six patients underwent only the post-vector instillation biopsy. Dose-limiting toxicity was not reached. Side effects were minimal and included fever, anemia, transient liver enzyme elevations, and bullous skin eruptions, as well as a temporary systemic inflammatory response in those receiving the highest dose. Strong intrapleural and intratumoral immune responses were generated. Using RNA PCR, in situ hybridization, immunohistochemistry, and immunoblotting, HSVtk gene transfer was documented in 11 of 20 evaluable patients in a dose-related fashion. This study demonstrates that intrapleural administration of an adenoviral vector containing the HSVtk gene is well tolerated and results in detectable gene transfer when delivered at high doses. Further development of therapeutic trials for treatment of localized malignancy using this vector is thus warranted.

To investigate the role of telomerase in the multistage pathogenesis of lung cancer, we examined 205 fresh and archival tissue samples obtained from 40 patients, 34 of whom had invasive lung carcinoma, 5 with carcinoma in situ (CIS) without invasion, and 1 without lung carcinoma. We analyzed samples for telomerase enzyme activity using the semiquantitative PCR-based telomeric repeat amplification protocol assay (131 samples) or by a radioactive in situ hybridization method for expression of the RNA component of human telomerase (hTR; 74 samples). A subset of samples was assayed by both methods, and the correlation was excellent (30 of 36; 83%). With the exception of a carcinoid tumor and a necrotic squamous cell carcinoma, all tumor cells were moderate to strongly positive for both hTR and telomerase activity, except for foci of keratinization in squamous cell carcinomas. Telomerase positivity, with weak enzyme activity and/or low hTR expression, was present in basal epithelial cells of large bronchi, both histologically normal (26%) and hyperplastic (71%), and in 23% of peripheral lung samples (in epithelium of small bronchi and bronchioles or lymphoid aggregates). More advanced epithelial changes (metaplasia, dysplasia, and CIS) were associated with telomerase dysregulation. Dysregulation in preneoplasia was manifested in three ways: almost all such lesions expressed hTR, although enzyme activity levels were several-fold lower than in the corresponding invasive tumors; cells throughout these multilayered processes expressed hTR; and intense, focal up-regulation of hTR occurred in CIS foci in the vicinity of invasive cancers. Alveolar cells and areas of atypical adenomatous hyperplasia (possible precursor lesions for peripheral adenocarcinomas) were negative. Our studies demonstrate that dysregulation of telomerase occurs early in the multistage pathogenesis of bronchogenic lung carcinomas and that intense focal localized hTR expression in CIS may indicate imminent invasion.

Modified, nonneurovirulent herpes simplex viruses (HSVs) have shown promise in the treatment of brain tumors. However, HSV-1 can infect and lyse a wide range of cell types. In this report, we show that HSV-1716, a mutant lacking both copies of the gene coding ICP-34.5, can effectively treat a localized i.p. malignancy. Human malignant mesothelioma cells supported the growth of HSV-1716 and were efficiently lysed in vitro. i.p. injection of HSV-1716 into animals with established tumor nodules reduced tumor burden and significantly prolonged survival in an animal model of non-central nervous system-localized human malignancy without dissemination or persistence after i.p. injection into SCID mice bearing human tumors. These findings suggest that this virus may be efficacious and safe for use in localized human malignancies of nonneuronal origin such as malignant mesothelioma.

Preclinical safety and toxicity studies of intrapleural administration of recombinant adenovirus carrying the herpes simplex thymidine kinase gene (H5.010RSVtk) were performed. Previously reported experimental evidence has demonstrated the efficacy of this approach in animal models of a localized thoracic cancer, malignant mesothelioma. H5.010RSVtk was delivered at high dose (10(12) pfu) into the pleural cavity of three non-human primates followed by systemic administration of ganciclovir. No clinical toxicity was noted. Although an inflammatory reaction observable by microscopy was noted in the serosal spaces of the chest cavity, these changes were reversible and were not associated with radiographic sequelae. Extrathoracic viral dissemination was minimal and detectable only by sensitive polymerase chain reaction techniques. This low level of viral dissemination was not associated with detectable clinical, biochemical, or pathologic abnormalities.

HSV-1716 is a replication-restricted, neuroattenuated ICP 34.5 gene mutant of herpes simplex virus type 1 (HSV-1). Because of the attenuated phenotype of ICP 34.5 mutants in rodent models of HSV disease, they have been promoted as potential vaccine strains and gene therapy vectors and have been used by us and others as therapeutic agents for the treatment of experimental malignant tumors. However, all data on the phenotype of HSV-1716 and other ICP 34.5 mutants are from animal model systems, while humans are the natural hosts of HSV-1. To achieve an initial characterization of the phenotype of 1716 in human tissue, we have studied its replication in mature human skin xenografts on SCID mice. We find that replication of 1716 is severely restricted in such human skin grafts relative to both parental wild-type HSV-1 strain 17+ and the HSV-1716 revertant virus 1716R, in which the 759-bp ICP 34.5 gene deletions have been repaired. Moreover, the replication of both 1716 and 17+ is significantly better in the human skin grafts than it is in mouse skin. The implications of these findings are discussed.

Previously, we have treated malignant mesothelioma (MM) growing in the peritoneal cavity of immunodeficient mice utilizing a recombinant adenovirus vector carrying the herpes simplex virus-thymidine kinase gene (Ad.RSVtk) followed by administration of the anti-viral drug ganciclovir (GCV). To mimic more closely the clinical situation in human MM, a syngeneic model of pleural MM was developed in immunocompetent Fischer rats. Administration of Ad.RSVtk into the pleural space of animals with established multifocal tumor followed by systemic GCV therapy resulted in significant tumor regression at 20 days in HSVtk/GCV-treated animals (average tumor weight 0.6 +/- 0.2 gram; n = 12) versus control animals (average weight 5.4 +/- 0.2 grams; n = 21; p < 0.001). In additional studies, Ad.RSVtk/GCV-treated animals had a mean survival of 34 days (average tumor weight 1.0 +/- 0.3 gram at death) versus 26 days in control animals (average tumor weight 6.2 +/- 0.6 grams at death). A significant reduction in tumor burden was also seen when more advanced, bulkier disease was treated. These studies demonstrate the Ad.RSVtk/GCV system is effective in the treatment of pleural-based tumors in an immunocompetent host. However, there are limitations to this treatment approach that result in only small increments in survival.

Background: Despite multimodality approaches, pleural-based malignant mesothelioma remains a disease with a very poor prognosis. Novel therapeutic strategies such as gene therapy clearly are needed to improve the survival of patients with this neoplasm. To aid in the evaluation of new treatment strategies, animal models that closely mimic human disease are required. This article describes the establishment of a pleural-based model of malignant mesothelioma in immune-competent Fischer rats., Methods: Via a modified left anterior lateral thorocotomy, a syngeneic malignant mesothelioma cell line, called II-45, was placed into the pleural cavity of Fischer rats., Results: Placement of II-45 cells into the pleural cavity of Fischer rats results in a model of pleural mesothelioma that closely resembles the disease seen in patients and is highly reproducible, with animals dying within 1 month. We also demonstrate the feasibility of adenoviral-mediated gene transfer to normal mesothelial cells lining the pleural cavity, as well as to malignant cells deep within the substance of pleural-based malignant mesothelioma., Conclusions: The model described here offers the opportunity to study a variety of new treatment modalities, especially somatic gene transfer, against pleural-based malignant mesothelioma in an immune competent setting.

Objective: To determine whether the immune system limits or improves the therapeutic efficacy of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene in a subcutaneous tumor model., Background Data: Enhanced immune reactions against tumors may be therapeutically useful. However, recent studies with adenoviral vectors show that immune responses limit the efficacy and persistence of gene expression. The effect of the immune response on cancer gene therapy with HSVtk gene delivery by an adenovirus vector followed by treatment with ganciclovir is unclear., Methods: After adenoviral transduction of a Fischer rat syngeneic mesothelioma cell line with the HSVtk gene in vitro, subcutaneous flank tumors were established. The ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Fischer rats, immunodeficient nude rats, and Fischer rats immunosuppressed with cyclosporin., Results: HSVtk/ganciclovir therapy was more effective in nude rats and immunosuppressed Fischer rats than in immunocompetent Fischer rats., Conclusion: These results indicate that the immune response against adenovirally transduced cells limits the efficacy of the HSVtk/ganciclovir system and that immunosuppression appears to be a useful adjunct. These findings have important implications for clinical trials using currently available adenovirus vectors as well as for future vector design.

This paper describes a preclinical toxicology study designed to investigate the biological efficacy and safety profile of second-generation adenovirus for CFTR gene transfer into the baboon lung. This second-generation virus is deleted of E1 and contains a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein. Two distinct projects were undertaken. Group A animals received a first-generation adenovirus (i.e., deleted of E1) in an upper lobe at the time a second-generation virus was instilled into the contralateral upper lobe. The goal of study A was to compare the biology of each construct directly and to determine if an immune response to the first-generation virus affected the performance of the second-generation virus. Group B animals received a lacZ second-generation virus in an upper lobe at the same time the CFTR second-generation virus was instilled in the other upper lobe. Necropsies were performed 4 or 21 days after gene transfer and tissues were evaluated for recombinant gene expression and histopathology. Using a second-generation adenovirus, recombinant gene stability was prolonged and associated with a diminished level of perivascular inflammation as compared to first-generation vectors. Markedly diminished levels of hexon protein were present in tissues infected with second-generation as compared to first-generation virus. No evidence of viral shedding was evident. Furthermore, coadministration of first- and second-generation adenovirus did not affect the stability of transgene expression from the second-generation virus. These data suggest that second-generation adenoviral vectors provide an improved gene delivery vehicle, and thus may be useful in gene therapy for cystic fibrosis.

The distinction between malignant mesothelioma and other neoplastic processes involving the pleura is difficult, partly due to the lack of specific markers expressed on mesothelioma. Because of evidence suggesting that the Wilms' tumor susceptibility gene (WT1), unlike other tumor suppressor genes, is restricted mostly to mesenchymally derived tissues, we hypothesized that the WT1 gene products could serve as a potential marker for mesothelioma. The expression of WT1 mRNA was analyzed in 19 malignant mesothelioma cell lines and 9 tumors and compared with the expression of WT1 in 10 non-small cell lung cancer lines and 9 lung cancer specimens. WT1 mRNA was detectable by Northern analysis in 16 of 19 mesothelioma cell lines and in 5 of 8 malignant mesothelioma tumors. In contrast, WT1 mRNA was not detected by Northern analysis in non-small cell lung cancer lines or carcinomas. Immunoprecipitation with an anti-WT1 monoclonal antibody showed that a 52- to 54-kd protein was present in 4 mesothelioma cell lines. Immunostaining with this antibody localized the WT1 protein to the nucleus in two mesothelioma lines and in 20 of 21 mesothelioma tumors examined. This distinctive pattern of nuclear immunoreactivity was absent in 26 non-mesothelioma tumors involving the lung, including 20 non-small cell lung carcinomas. The detection of WT1 mRNA or protein may thus provide a specific molecular or immunohistochemical marker for differentiation of mesothelioma from other pleural tumors, in particular, adenocarcinoma.

Recombinant adenoviruses have tremendous potential for gene therapy of cystic fibrosis (CF) lung disease. First-generation recombinant viruses, rendered replication defective by deleting E1, have been associated with high-level recombinant gene expression in airway epithelial cells when administered directly to the lung. Experience in mice and non-human primates indicates that transgene expression is transient (i.e., lasting less than 21 days) and associated with the development of inflammation. We suggest an hypothesis to explain these findings that is based on expression of viral proteins in genetically modified cells that leads to destructive cellular immune responses and repopulation of lung epithelia with non-transgene-containing cells. This hypothesis has been evaluated in the current study using the cotton rat model. Instillation of the first-generation lacZ virus, H5.010CBlacZ, into cotton rat airway led to high-level gene expression in conducting and respiratory airway that was transient and associated with a substantial mononuclear, CD8-dominated, infiltrates. Treatment of the animals with cyclosporine blunted the inflammatory response and prolonged recombinant gene expression in both conducting and respiratory airways. Expression of viral early and late genes was detected in a subpopulation of lacZ-expressing epithelial cells of conducting airway and alveoli. Instillation of virus into cotton rat tracheal xenografts grown in athymic nu/nu mice led to efficient and stable transgene expression in the absence of pathology, underscoring the importance of T cell-mediated immunity. A recombinant adenovirus was constructed that is disabled in its capacity to replicate by the introduction of a temperature-sensitive mutation in the E2a gene as well deletion of E1 sequences. Instillation of this virus into cotton rat airway led to high-level transgene expression that was more stable than that achieved with the first-generation virus and was associated with less early and late gene expression as well as a diminished infiltration of CD8+ T cells in conducting airway epithelium. Interestingly, the introduction of the E2a mutation had no effect on the persistence of transgene expression, the pattern of late viral gene expression, nor the CD8+ T cell response within alveolar cells. These data suggest that cell-specific variation in the cell biology of recombinant adenoviruses exists in the lung. The present studies in cotton rats confirm the role of cellular immunity in the biology of adenovirus-mediated gene therapy to the lung and suggest that modifications in the design of recombinant adenoviruses to minimize or ablate transgene expression will be useful in improving the potential of this technology for gene therapy of CF.

We have used in situ hybridization for RNA to localize cells containing mRNA for the 92 kd gelatinase in carcinomas of the lung. We used archival material to analyze sections from 12 cases of squamous cell carcinomas of the lung including six stage I and three stage II and from three cases of adenocarcinoma of the lung. Presence of mRNA in the tissue was verified by in situ hybridization for gamma actin. The 92 kd gelatinase mRNA was found in all 12 squamous cell carcinomas tumors and was highly expressed in the tumor cells themselves. In addition, it was found in host stromal cells surrounding the tumor, but not in normal lung fibroblasts. In contrast it was not found in the adenocarcinomas of the lung or in the stroma surrounding these tumors. The mRNA for the 92 kd gelatinase was present in normal pulmonary tissue, bronchial epithelium, basal cell hyperplasia of bronchial epithelium, alveolar macrophages, and focally in bronchial mucous glands. It was not present in normal alveoli, vascular cells, cartilage, or most lymphocytes. We corroborated the presence of the mRNA for the 92 kd gelatinase by ribonuclease protection assay. The levels of mRNA for the 92 kd gelatinase in two specimens of squamous cell carcinoma were 6- to 10-fold greater than in the nonneoplastic tissue and two adenocarcinoma specimens.