[Surgically treated pleomorphic carcinoma of the lung].

We experienced 3 resected cases of pleomorphic carcinoma of the lung. Each cases were 74-year-old man (case 1), 74-year-old woman (case 2) and 69-year-old man (case 3). Two patients (case 1 and 2) were histologically diagnosed as pleomorphic carcinoma composed of spindle cell carcinoma with giant cell carcinoma. One patient (case 3) was similarly diagnosed as pleomorphic carcinoma composed of spindle cell carcinoma with adenocarcinoma and squamous cell carcinoma. Although lymph nodes metastasis were not recognized in all patients, invasion to vessels were recognized in 2 patients (case 1 and 3). In one patient (case 1), recurrence was recognized at contralateral side 1 month after surgery and he died of other disease 2 months after surgery. The other 2 patients were alive without recurrence 24 and 5 months after surgery. Recently it is reported that recurrence is recognized at early phase after surgery and prognosis is poor in a case with vessel invasions in spite of pathological NO state. Since one patient (case 3) had nonmetastatic lymph nodes with vessel invasions, careful observation is considered to be necessary.

[Clinical analysis of tracheoplasty and bronchoplasty in primary lung cancer].

In our department, there were 482 thoracic surgeries for primary lung cancer between 1994 and 2007. We clinically reviewed cases that underwent tracheoplasty or bronchoplasty (n = 22, 4.6%). The patients consisted of 21 males and 1 female (66.5 +/- 12.0 years-old). All patients were smokers. The tissue forms were 19 squamous cell carcinomas, 2 adenocarcinomas, 1 large cell carcinoma, 1 adenoid cystic carcinoma and 1 carcinoid, including 2 multiple carcinomas. Sleeve resections involved the trachea in 1, upper lobes in 13, lower lobes in 3, upper-middle lobes in 2 and intermediate bronchus in 1. Wedge resections were performed in the upper lobes in 2. Fourteen reconstructions were performed. We ordinarily sutured the trachea and bronchus in any case, using a single outside knot. There was no leakage at the anastomosis. There were 2 hospital deaths. There were 4 cancer deaths, including 2 local recurrences. There were 4 patients demonstrating stenosis post operatively. There were 3 stenoses among 4 preoperative radiation therapies. We considered that radiation therapy disturbed the repair of the anastomosis. There were 8 pneumonia patients who developed post operatively. There were 2 operative hospital deaths among 3 angio-bronchoplasties without coverage. Recently, we have routinely covered the anastomosis at the reconstruction site and have not experienced any major complications.

[Approach by clamshell incision for bilateral pulmonary metastasis].

In recent years thoracotomy by "clamshell incision" has frequently been chosen for heart-lung transplantation, bilateral lung tumors and mediastinal tumor merging into lung tumor because this approach provides very good visibility to access the whole bilateral lung including the lower lobe and mediastinal organs. In our hospital, 4 patients underwent bilateral thoracotomy by clamshell incision for pulmonary metastasectomy between 2001 and 2005. All cases had bilateral pulmonary metastases, and multiple wedge resection was performed. All lesions that were planned for resection on preoperative computed tomography (CT) could be resected. Regarding the surgical approach to bilateral pulmonary metastases that did not need lobectomy, clamshell incision is one of the useful approaches that can allow wedge resection anywhere in the whole lung. In cases that are expected multiple procedures, bilateral thoracotomy by clamshell incision is recommended because it allows another route for thoracotomy at reoperation.

[Clinical experience with 16 cases of mediastinal lymphoma].

Sixteen cases of mediastinal lymphoma in our hospital were reviewed clinically. There were 7 men and 9 women, whose mean age is 35 years old. The histological types were non-Hodgkins' disease in 12 (B-cell type in 6 and T-cell type in 6), Hodgkins' disease in 2, mucosa-associated lymphoid tissue (MALT) lymphoma in 2. All cases except 1 in which percutaneous needle biopsy was performed were diagnosed histologically. Even small specimens by percutaneous needle biopsy can be helpful in diagnosing histological type and subtype with immunohistlogy, recommending percutaneous needle biopsy as an mitial step for diagnosis. When histological diagnosis can not be made by needle biopsy, open biopsy should be done.

The balance of VEGF-C and VEGFR-3 mRNA is a predictor of lymph node metastasis in non-small cell lung cancer.

A positive association between vascular endothelial growth factor-C (VEGF-C) expression and lymph node metastasis has been reported in several cancers. However, the relationship of VEGF-C and lymph node metastasis in some cancers, including non-small cell lung cancer (NSCLC), is controversial. We evaluated the VEGF-C and vascular endothelial growth factor receptor-3 (VEGFR-3) expression in NSCLC samples from patients who had undergone surgery between 1998 and 2002 using real-time quantitative RT-PCR and immunohistochemical staining. We failed to find a positive association between VEGF-C and VEGFR-3 mRNA expression and lymph node metastasis in NSCLC. An immunohistological study demonstrated that VEGF-C was expressed not only in cancer cells, but also in macrophages in NSCLC, and that VEGFR-3 was expressed in cancer cells, macrophages, type II pneumocytes and lymph vessels. The VEGF-C/VEGFR-3 ratio of the node-positive group was significantly higher than that of the node-negative group. Immunohistochemical staining showed that VEGFR-3 was mainly expressed in cancer cells. The immunoreactivity of VEGF-C and VEGFR-3 was roughly correlated to the mRNA levels of VEGF-C and VEGFR-3 in real-time PCR. VEGF-C mRNA alone has no positive association with lymph node metastasis in NSCLC. The VEGF-C/VEGFR-3 ratio was positively associated with lymph node metastasis in NSCLC. This suggests that VEGF-C promotes lymph node metastasis while being influenced by the strength of the VEGF-C autocrine loop, and the VEGF-C/VEGFR-3 ratio can be a useful predictor of lymph node metastasis in NSCLC.

[Clinical analysis of operative and hospital deaths in primary lung cancer].

In our department, there were 313 thoracic surgeries for primary lung cancer from January 1994 to December 2003. We clinically reviewed for the operative and hospital death (n=18, 5.8%). The patients were 16 males and 2 females (70.6 +/- 5.6 years old). The surgical procedures were 4 pneumonectomies, 13 lobectomies (3 bronchoplasties) and 1 partial resection. The mean interval until postoperative death was 122.5 +/- 156.1 days. There were 5 direct operative deaths within 30 days (1.6%). There were 4 cancer deaths, 2 hemoptyses, 2 operative bleeding, 2 thromboses, 2 cerebral hemorrhages, 1 pyothorax, 1 pneumonia, 1 respiratory failure, 1 multiple organ failure after chemotherapy and 2 unexplained deaths. The patients with pneumonectomy or aged significantly had high mortality. For postoperative complications such as hemoptysis or bleeding, perioperative management that takes these issues into consideration is needed. Furthermore, we must carefully review the preoperative evaluation and combined treatment, because there were many cancer deaths among cases showing early recurrence and metastasis.

A multidisciplinary treatment strategy that includes high-dose chemotherapy for metastatic retinoblastoma without CNS involvement.

The prognosis of patients with metastatic retinoblastoma is poor with conventional chemotherapy and radiation. Since retinoblastoma is highly chemosensitive, dose-escalation of chemotherapeutic agents with stem cell support should be promising. We report our experience with high-dose chemotherapy (HDC) and autologous stem cell transplantation (SCT) in patients with metastatic retinoblastoma. Five patients with metastatic retinoblastoma underwent HDC with autologous SCT following conventional chemotherapy and local radiation therapy. Stem cells (bone marrow in four and peripheral blood stem cells in one) were collected after marrow involvement was cleared. Melphalan was a key drug in all patients, and was administered in combination with other agents such as cisplatin, cyclophosphamide, carboplatin or thiotepa. Three patients are currently alive disease-free at 113, 107 and 38 months, respectively, from the time of SCT. They had no central nervous system (CNS) involvement. The two patients who died of disease had CNS involvement. No long-term sequelae of HDC have been noted. Our treatment strategy using HDC appears to be effective for treating metastatic retinoblastoma without CNS involvement.

Transbronchial administration of adenoviral-mediated interleukin-10 gene to the donor improves function in a pig lung transplant model.

Interleukin-10 (IL-10) gene transfection of donor lungs prior to transplantation is an attractive strategy to reduce ischemia-reperfusion induced lung injury. However, experimental data with gene therapy in large animal models of lung transplantation are generally lacking. We have developed a simple clinically applicable technique for adenoviral-mediated gene delivery of human IL-10 to the lung of large animals that provides homogenous gene expression after 12-24 h of transfection. Using this technique of gene delivery, we have studied the dynamics of adenoviral gene delivery to the lung in the setting of lung transplantation. Although there is a persistent inflammatory response to the adenoviral vector, we achieved significant expression of human IL-10 in lung tissue before lung retrieval to obviate the deleterious impact of the adenoviral vector on the donor lung. The administration of adenoviral-mediated human IL-10 to the donor lung reduced ischemia-reperfusion injury and improved graft function after lung transplantation in this pig lung transplantation model. Transfection of adenoviral-mediated human IL-10 to the donor lung prevented the release of inflammatory cytokines such as IL-6 in lung tissue and plasma. We have demonstrated that IL-10 gene therapy has significant potential to prevent or treat the inflammatory response associated with ischemia-reperfusion injury in lung transplantation. In the future, IL-10 gene therapy could also be used for immunomodulation or tolerance induction.

Reduced inflammatory pain in mice deficient in the differential screening-selected gene aberrative in neuroblastoma.

Differential screening-selected gene aberrative in neuroblastoma (Dan) protein is produced in small neurons of dorsal root ganglia. Thermal and mechanical allodynia and Fos expression in the spinal dorsal horn evoked by inflammation and neuropathic pain were investigated using Dan-deficient mice. Mice showed pain reactions induced by the introduction of complete Freund's adjuvant (CFA) into their hind paw (inflammatory pain model) and after sciatic nerve ligation (neuropathic pain model). In the inflammatory pain model, thermal and mechanical pain thresholds in Dan-deficient mice were significantly higher than those of wild-type mice. The number of Fos-immunoreactive cells in the dorsal horn during the inflammatory period was significantly less in Dan-deficient mice. However, in the neuropathic pain model, no differences in thermal hypersensitivity, mechanical allodynia, or the number of Fos-immunoreactive cells in the dorsal horn were observed between the mice. These data suggest that Dan may be a neuromodulator in inflammatory pain.

A promoter region of the midkine gene that is frequently expressed in human hepatocellular carcinoma can activate a suicide gene as effectively as the alpha-fetoprotein promoter.

We examined the expression of the midkine (MK) and alpha-fetoprotein (AFP) genes in 15 paired human specimens obtained from hepatocellular carcinoma (HCC) and the corresponding noncancerous regions of the same patients. A total of 14 HCC but none of the noncancerous specimens were positive for the MK mRNA. In contrast, three HCC specimens and one corresponding noncancerous sample out of the three AFP-positive HCC cases expressed the AFP gene. A 2.3-kb genomic fragment in the regulatory region of the MK gene could activate a fused reporter gene in both AFP-producing and -nonproducing HCC lines, and the MK fragment-mediated transcriptional activity was comparable to the AFP enhancer-linked AFP promoter in AFP-producing cell lines. The AFP-producing but not AFP-nonproducing HCC cell lines that were transfected with the MK promoter-linked herpes simplex virus-thymidine kinase (HSV-TK) gene became susceptible to a prodrug ganciclovir to a similar degree of the HCC transfected with the enhancer-linked AFP promoter-fused HSV-TK gene. These data suggest that the MK promoter can activate a therapeutic gene preferentially in HCC and is as useful as the AFP promoter in clinical settings.

Differential screening-selected gene aberrative in neuroblastoma protein modulates inflammatory pain in the spinal dorsal horn.

Differential screening-selected gene aberrative in neuroblastoma (DAN) belongs to a novel gene family that includes the Xenopus head-inducing factor, Cerberus and the dorsalizing factor, Gremlin. It has been suggested that members of this family control diverse processes in growth, development and the cell cycle.Here, we demonstrate that the DAN protein is produced in the small neurons of the dorsal root ganglion and is transported to the nerve terminals in the spinal dorsal horn in adult rats. Furthermore, intrathecal injection of an antibody to the DAN protein suppressed inflammatory pain caused by the introduction of complete Freund's adjuvant or carrageenan into the rat hindpaw. The amount of mRNA for DAN in dorsal root ganglion neurons and of its expressed protein in the spinal dorsal horn were both increased in inflammatory models.Together, these data suggest that the DAN protein may be a novel neuromodulator in primary nociceptive nerve fibers.

Expression of the Dan gene during chicken embryonic development.

The Dan gene was first identified as the putative rat tumor suppressor gene and encodes a protein structurally related to Cerberus and Gremlin in vertebrates. Xenopus DAN, as with Cerberus and Gremlin, was demonstrated to block bone morphogenetic protein (BMP) signaling by binding BMPs, and to be capable of inducing additional anterior structures by ectopic overexpression in Xenopus embryos. DAN, thus, is suggested to play pivotal roles in early patterning and subsequent organ development, as in the case of other BMP antagonists. In this report, we isolated the chicken counterpart of Dan. Chicken Dan is mainly expressed in the cephalic and somitic mesoderm and several placodes during organ development.

Induction of immunity in peripheral tissues combined with intracerebral transplantation of interleukin-2-producing cells eliminates established brain tumors.

Cytokine gene therapy for the induction of potent immune responses against central nervous system tumors has proven to have significant potential. However, this strategy needs improvement in the process of antigen presentation and/or insufficient recruitment of immunocompetent cells to achieve successful eradication of established brain tumors. We investigated the therapeutic potential of induced systemic immunity in peripheral tissues combined with interleukin-2 (IL-2) production in the vicinity of brain tumors to treat established brain tumors. Sequential magnetic resonance image monitoring showed that the combinatory therapy consisting of intracerebral (i.c.) transplantation of IL-2-producing rat gliosarcoma 9L (9L/IL-2) cells and s.c. vaccination using irradiated 9L or 9L/IL-2 cells could cure 9L-bearing rats, whereas either the i.c. injection of 9L/IL-2 cells or the s.c. vaccination produced little or marginal antitumor effects, respectively. Xenogeneic murine neuroblastoma cells secreting IL-2 could substitute for 9L/IL-2 cells, producing significant antitumor effects in the vaccinated rats. Tumor-specific cytotoxic activity was induced in the vaccinated rats but not fully in the rats treated only with i.c. injection of 9L/IL-2 cells. Immunohistochemical analysis revealed that a number of CD4(+) and CD8(+) T cells infiltrated into the brain tumors which were treated with the combinatory therapy. The level of cell infiltration was similar to that found in s.c. 9L/IL-2 tumors which were subsequently rejected. In contrast, the brain tumors treated with either i.c. transplantation of 9L/IL-2 cells or the s.c. vaccination showed only moderate infiltration of T cells. The combinatory strategy, i.c. grafting of IL-2-producing cells, and s.c. immunization of irradiated whole tumor cell vaccine, is, thus, effective for recruiting activated T cells into the brain tumor site and could be a potential therapy for brain tumors.

Electroporation-mediated transfer of cytokine genes into human esophageal tumors produces anti-tumor effects in mice.

Electroporation facilitates transfer of chemicals or plasmid DNA from extracellular milieu into cells by increasing the permeability of the cell membrane. Delivery of electric pulses to established tumors thereby can improve the susceptibility of tumors to an anti-cancer agent administered. We examined whether electroporation-mediated transfer of cytokine genes into solid tumors could produce anti-tumor effects in the tumor-bearing mice. Plasmid DNA containing cytokine genes were injected into human esophageal T.Tn tumors developed in nude mice and electric pulses were then delivered. Administration of murine GM-CSF or human IL-2 gene followed by electroporation significantly suppressed the subsequent growth of T.Tn tumors and prolonged the survival of the inoculated mice. In contrast, electroporation-mediated introduction of a control gene, human GM-CSF gene, whose products do not bind to murine GM-CSF receptors, did not achieve any anti-tumor effects. In vivo transfection of cytokine genes with electroporation could be a possible therapeutic strategy for established solid tumors.

Irradiation with ultrasound of low output intensity increased chemosensitivity of subcutaneous solid tumors to an anti-cancer agent.

Ultrasound is a possible mechanical method to deliver small molecules into target cells. In order to evaluate the therapeutic potentials provided by ultrasound irradiation, we compared anti-tumor effects of electroporation- and ultrasound-mediated chemotherapy and efficacy of gene transfer by the two methods. Electric pulses (5 Hz, 100 V/cm, 8 square-wave/100 microsec) or ultrasound (1 MHz, 2 W/cm(2), 5 min) was delivered to subcutaneous solid tumors of murine lymphoma after the tumor-bearing mice received an intraperitoneal injection of bleomycin (BLM) (2.5 mg) or intratumoral injection of plasmid DNA containing the luciferase reporter gene. Administration of BLM alone did not affect the subsequent tumor growth but additive treatment with ultrasound irradiation suppressed the growth to the same level as electroporation. The luciferase activity of the DNA-injected tumors showed that ultrasound irradiation achieved better transfection efficiency than plasmid DNA injection alone but the efficacy was not as great as that by electroporation. The low energy level of ultrasound that is currently used for a diagnostic purpose and physical therapy in clinical fields can thereby increase the in vivo chemosensitivity of treated tumors but further modifications are necessary to achieve better efficacy of the ultrasound-mediated gene transfer.

Multilocular thymic cyst associated with Sjögren's syndrome.

A 61-year-old woman, who had been diagnosed as having Sjögren's syndrome, developed an anterior mediastinal mass. She was diagnosed with Sjögren's syndrome with thymoma, preoperatively. Extended thymectomy was performed. Macroscopically, the mediastinal mass showed thick-walled multiloculated cavities filled with turbid yellow fluid. Microscopically, the cyst lining was continuous with thymic lobules in the wall with inflammatory process, cholesterol granuloma formation, and prominent lymph follicular hyperplasia. She was diagnosed with multilocular thymic cysts associated with Sjögren's syndrome.

[A novel neurotransmitter, DAN, mediates pain sensation in the spinal dorsal horn].

Differential screening-selected gene aberrative in neuroblastoma(DAN) belongs to a novel gene family(DAN family) that includes the head-inducing factor, Cerberus, and dorsaling factor, Gremlin. It has been suggested that DAN family members control diverse processes in growth, development and the cell cycle. Here, we demonstrate that DAN is produced in the small neurons of the dorsal root ganglion(DRG) and transported to the nerve terminals in the spinal dorsal horn in adult rats. Furthermore, intrathecal injection of an antibody to DAN suppressed pain sensations induced by the application of complete Freund's adjuvant and carageenan into the rat hindpaw, and the amount of DAN mRNA in the DRG neurons and of DAN in the spinal dorsal horn were increased in the inflammatory models. These data suggest that DAN in a novel neurotransmitter and/or modulator in the primary sensory nerve fibers for pain sensation.

Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells.

Tumoricidal "bystander effect" observed in the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy was studied between different rat glioma cell lines (9L and C6 cells) under both in vitro and in vivo conditions. For that purpose, mixed populations of wild-type cells (9Lwt and C6wt) and respective HSVtk gene-transduced cells (9Ltk and C6tk) were examined for their sensitivity to GCV. A potent in vitro bystander effect was observed in 9Lwt/9Ltk and 9Lwt/C6tk combinations but not in C6wt/9Ltk and C6wt/C6tk combinations. In vivo bystander effect studied in a subcutaneous tumor model in athymic nude mice was also potent in 9Lwt/9Ltk and 9Lwt/C6tk combinations. Because the expression of connexin43, a major protein in the connexin family gene products, in 9L cells is much higher than that in C6 cells, the results suggest that the amount of connexin in target (wild-type) cells but not in effector (HSVtk gene-bearing) cells is important for the generation of the bystander effect. This hypothesis was further confirmed by the observation that in vitro bystander effect in C6wt/C6tk combination was potentiated by transduction of the connexin43 gene to the target cells.

Preclinical study of adenoviral p53 gene therapy for esophageal cancer.

An alteration of the p53 gene function is a major factor in the development of esophageal cancer. Recently, p53 gene therapy has been applied for clinical studies in lung cancer and head and neck cancer. However, no preclinical studies have yet demonstrated an anticancer effect of adenoviral-mediated wild-type p53 gene therapy on esophageal cancer. We herein evaluated the effect of p53 adenoviral gene therapy on human esophageal squamous cell carcinoma to test the ability of clinical application. A normal esophageal epithelial cell line (EN53F) and two human esophageal cancer cell lines (ECGI-10 and T.Tn) with a p53 alteration were used. The transduction efficiency, p53 protein expression, p21 protein expression, the induction of apoptosis, and growth suppression were assessed by using the recombinant adenoviral vector Ad5CMV-p53. The transduction efficiency was 60%-80% at 100 plaque-forming units (PFU)/cell and 80%-100% at 300PFU/cell. A significant growth suppression following an Ad5CMV-p53 infection was observed in both cancer cell lines. A Western blot analysis confirmed the presence of both exogenous p53 protein expression and p21 protein induction. Apoptotic cell death was observed with TUNEL staining. T.Tn xenografts in nude mice transduced with Ad5CMV-p53 demonstrated significant growth suppression. These data suggest that Ad5CMV-p53 may thus be a potentially effective therapeutic agent for locally advanced esophageal cancer.

DNA polymerase kappa, implicated in spontaneous and DNA damage-induced mutagenesis, is overexpressed in lung cancer.

DNA polymerase kappa (Pol kappa) is a newly identified low-fidelity polymerase implicated in spontaneous and DNA damage-induced mutagenesis. As an initial study to investigate its possible involvement in tumorigenesis, we compared the expression level of Pol kappa in tumors and adjacent nontumorous tissues by Northern blot, semiquantitative RT-PCR, and Western blot analyses. In this study, paired tumor and normal specimens from 29 patients with stages I to IIIb non-small cell lung cancer (NSCLC), including 13 adenocarcinomas, 15 squamous cell cancers, and 1 adenosquamous carcinoma, were analyzed, among which different levels of tumor-associated Pol kappa overexpression were observed in 21 of 29 matched specimens. In addition, five matched specimens exhibited elevated Pol kappa expression in both tumor and control tissues, whereas only one nontumorous tissue expressed a higher level of Pol kappa than its tumor counterpart. The preferential up-regulation of Pol kappa expression in tumors was highly significant (P < 0.001). There was no apparent correlation of Pol kappa expression levels with tumor histology, grade, and stage or with smoking history. Southern blot analysis did not show amplification of the Pol kappa gene, indicating that the elevated Pol kappa expression is likely attributable to dysregulated transcription. Our data suggest that Pol kappa may contribute to lung tumor development by accelerating the accumulation of mutations.