Predicted Rate of Secondary Malignancies Following Adjuvant Proton Versus Photon Radiation Therapy for Thymoma.

PURPOSE: Thymic malignancies are the most common tumors of the anterior mediastinum. The benefit of adjuvant radiation therapy for stage II disease remains controversial, and patients treated with adjuvant radiation therapy are at risk of late complications, including radiation-induced secondary malignant neoplasms (SMNs), that may reduce the overall benefit of treatment. We assess the risk of predicted SMNs following adjuvant proton radiation therapy compared with photon radiation therapy after resection of stage II thymic malignancies to determine whether proton therapy improves the risk-benefit ratio. METHODS AND MATERIALS: Ten consecutive patients treated with double-scattered proton beam radiation therapy (DS-PBT) were prospectively enrolled in an institutional review board-approved proton registry study. All patients were treated with DS-PBT. Intensity modulated radiation therapy (IMRT) plans for comparison were generated. SMN risk was calculated based on organ equivalent dose. RESULTS: Patients had a median age of 65 years (range, 25-77 years), and 60% were men. All patients had stage II disease, and many had close or positive margins (60%). The median dose was 50.4 Gy (range, 50.4-54.0 Gy) in 1.8-Gy relative biological effectiveness daily fractions. No differences in target coverage were seen with DS-PBT compared with IMRT plans. Significant reductions were seen in mean and volumetric lung, heart, and esophageal doses with DS-PBT compared with IMRT plans (all P≤.01). Significant reductions in SMNs in the lung, breast, esophagus, skin, and stomach were seen with DS-PBT compared with IMRT. For patients with thymoma diagnosed at the median national age, 5 excess secondary malignancies per 100 patients would be avoided by treating them with protons instead of photons. CONCLUSIONS: Treatment with proton therapy can achieve comparable target coverage but significantly reduced doses to critical normal structures, which can lead to fewer predicted SMNs compared with IMRT. By decreasing expected late complications, proton therapy may improve the therapeutic ratio of adjuvant radiation therapy for patients with stage II thymic malignancies.

Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas.

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.

Use of a "replication-restricted" herpes virus to treat experimental human malignant mesothelioma.

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.

Successful liver, kidney, and pancreas transplantation from a donor with cerebral emboli from a left atrial myxoma.

Although transmission and engraftment of donor-derived malignancies is rare in recipients of solid organ transplants, it is associated with unfavorable allograft and patient survival. Therefore, a recent history of malignancy is considered a contraindication to organ donation. Although atrial myxomas are benign cardiac tumors of stromal origin, they can lead to systemic embolization with ectopic myxoma formation. We report successful liver, kidney, and pancreas transplantation into 3 recipients from a donor with cerebral emboli from a left atrial myxoma. Eighteen months after transplantation, all 3 patients enjoy good allograft function and are free of donor-derived atrial myxoma. Although the duration of follow-up in this report is limited, we suggest that the presence of atrial myxoma should not be viewed as an absolute contraindication to organ recovery, particularly in view of the shortage of organ donors and the attendant morbidity and mortality for patients on waiting lists.

Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a.

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.

Transfer of the CFTR gene to the lung of nonhuman primates with E1-deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study.

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.

Treatment of pleural mesothelioma in an immunocompetent rat model utilizing adenoviral transfer of the herpes simplex virus thymidine kinase gene.

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.

Safety of intrapleurally administered recombinant adenovirus carrying herpes simplex thymidine kinase DNA followed by ganciclovir therapy in nonhuman primates.

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.

Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma.

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.

A pilot study of systemic corticosteroid administration in conjunction with intrapleural adenoviral vector administration in patients with malignant pleural mesothelioma.

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.

Bilateral sequential lung transplantation for pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is characterized by deposition of calcium phosphate within the alveolar airspaces. There is currently no effective medical therapy and affected individuals may progress to end-stage lung disease requiring transplantation. Two patients with PAM underwent bilateral sequential lung transplantation. This study reviews the clinical manifestations of PAM and discusses the particular difficulties that may be encountered in the use of lung transplantation as treatment for this uncommon disease. Also addressed is the question of recurrence in the allograft.

Primary graft failure following lung transplantation.

STUDY OBJECTIVES: To determine the incidence of primary graft failure (PGF) following lung transplantation, assess possible risk factors, and characterize its effect on outcomes. METHODS: Retrospective review of 100 consecutive patients undergoing lung transplantation at the University of Pennsylvania Medical Center. Fifteen patients meeting diagnostic criteria for PGF (PGF+ group) were compared with 85 patients without this complication (PGF- group). RESULTS: The incidence of PGF was 15%. There was no significant difference in age, sex, underlying pulmonary disease, preoperative pulmonary artery systolic pressure, type of transplant, allograft ischemic times, use of cardiopulmonary bypass, or use of postoperative prostaglandin E1 infusion between the PGF+ and PGF- groups. Induction therapy with antilymphocyte globulin was used less frequently in the PGF+ group (p<0.005). Duration of mechanical ventilatory support was 36+/-43 days vs 4+/-6 days for the PGF+ and PGF- groups, respectively (p<0.0001). Hospital stay was significantly longer in the PGF+ group, averaging 75+/-105 days, compared with 27+/-38 days in the PGF group (p<0.005). One-year actuarial survival for the PGF+ group was only 40% compared with 69% for the PGF- group (p<0.005). Five of the six PGF+ survivors were ambulatory by 1 year; three were completely independent while two continued to require assistance with activities of daily living. Six-minute walk test distance among the ambulatory patients averaged 883+/-463 feet (range, 200 to 1,223 feet) compared with 1513+/-424 feet for the PGF- group (p<0.005). Among the subset of survivors who underwent single lung transplantation for COPD, the mean percent predicted FEV1 at 1 year was 43% for the PGF+ group and 55% for the PGF- groups, but this difference was not statistically significant. CONCLUSIONS: PGF is a devastating postoperative complication, occurring in 15% of patients in the current series, and it is associated with a high mortality rate, lengthy hospitalization, and protracted and often compromised recovery among survivors.

Rapid method for detection of Aspergillus 5S ribosomal RNA using a genus-specific oligonucleotide probe.

Ribosomal RNA (rRNA) is present in all prokaryotic and eukaryotic cells. Although sequences are conserved, variations in nucleic acid composition are known to be species specific. Formalin or Bouin's fixed, paraffin-embedded tissue specimens from 17 cases of culture proven Aspergillus sp infections were studied by a rapid (< 30 minutes) in situ hybridization procedure using a biotinylated oligonucleotide DNA probe complementary to nucleic acids 1-22 of Aspergillus sp 5S rRNA sequence. Positivity was noted within fungal organisms in all 17 cases and was identified in both hyphal forms and within fruiting bodies. Signal was weak or absent within the center of Aspergillus abscess cavities with increasing signal located toward the periphery of the cavity, suggesting that rRNA in situ hybridization may detect viable fungal forms. In situ hybridization with the oligonucleotide probe on tissues containing several different organisms including Candida sp, Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis carinii, Pseudallescheria boydii, Fusarium sp, and Mucor were negative. Use of site specific oligonucleotide probes specific for a variety of rRNA sequences may aid in the diagnosis of several medically important bacterial, fungal, and protozoal pathogens.

Analysis of Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder after lung transplantation.

BACKGROUND: Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disorder (PTLD) is a serious complication of lung transplantation. Besides immunosuppression the risk factors for PTLD development are largely unknown. METHODS: The incidence of PTLD was ascertained in a lung transplant population consisting of 45 patients. Nine patients (20%) experienced PTLD. The clinical, histologic, and human leukocyte antigen (HLA) data were collected on all patients. The incidence of EBV infection in lymphoid tissue taken at the time of engraftment was studied by using EBV in situ hybridization. RESULTS: All patients with PTLD had polymorphous lymphoproliferations, seven of which were polymorphous B-cell hyperplasias and two of which were polymorphous B-cell lymphomas. EBV was identified in all lesions. All patients with polymorphous B-cell hyperplasias had clinically unsuspected disease, five of which were identified at autopsy. The two polymorphous B-cell lymphoma lesions were monoclonal and regressed with immunosuppression reduction. EBV in situ hybridization on donor or recipient lymph nodes obtained at engraftment from the 45 transplant recipients showed no difference in the number of EBV positive cells in patients with and without PTLD. Cyclosporine and PTLD and azathioprine dosages and cyclosporine levels were similar between patients with and without PTLD. PTLD was seen in patients with high cumulative doses of antilymphocyte globulin. Analysis of HLA status showed a predominance of HLA A2 and DR7 in the donors of the patients with PTLD, whereas donor HLA B7 was more common in patients without PTLD> CONCLUSIONS: Detailed studies are necessary to further elucidate the risk factors for PTLD development in the lung transplant population.

Pleural-based mesothelioma in immune competent rats: a model to study adenoviral gene transfer.

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.

The pathology of fungal disease in the lung.

The gross and histological appearance of pulmonary mycotic disease is rarely pathognomonic for a particular entity. Tissue obtained through an invasive procedure is usually necessary for a specific diagnosis. Pathological diagnosis is directed by the pattern of inflammation and based on the morphological identification of fungi. The interventional radiologist plays a critical role in the diagnosis of pulmonary mycoses by obtaining additional material for culture. An open and inquisitive collaboration between the radiologist and pathologist can improve diagnostic accuracy for both specialists.

Evaluation of EGFR mutation status in cytology specimens: an institutional experience.

Epidermal growth factor receptor (EGFR) mutation status has been shown to predict response to anti-EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). In patients with advanced-stage NSCLC, evaluation of mutational status is increasingly requested on biopsy or fine-needle aspiration specimens, which often have limited material. There are limited data on the suitability of cytology cell blocks (CB) for EGFR mutation testing. In this study, we report our institutional experience with cytology cell block material for EGFR mutation testing. We retrospectively reviewed EGFR mutation analyses performed on 234 surgical (SP) and cytology (CB) from October 2007 to May 2010. One hundred ninety-two SP specimens and 42 CB specimens were evaluated for EGFR mutation. CB specimens were evaluated for overall specimen size based on aggregate cellularity in comparison to small biopsy specimens, and percent tumor. Of the 192 SP and 42 CB specimens, 31 (16.1%) and 11 (26.2%) were positive for EGFR mutation, respectively; there does not appear to be an association between mutation detection rate and the source of the specimen (P = 0.124). Limited DNA was obtained from 70.0% (29/42), including 81.8% (9/11) of those which were mutation positive. Additionally, 45.4% (5/11) of mutation positive specimens had extremely low DNA yields. Although 16.6% (7/42) of CB specimens had <10% tumor, all 11 mutation positive CB cases had >10% tumor. These data indicate that CB specimens provide an alternative source for molecular evaluation of NSCLC, and that tumor percentage may be more important than specimen size and/or DNA yield in determining the suitability of these specimens for testing.