Efficacy, safety, and optimal timing of single-trocar video-assisted flexible thoracoscopic debridement under local anesthesia for complicated parapneumonic empyema.

OBJECTIVE: Thoracoscopic debridement under local anesthesia is a useful approach for complicated parapneumonic effusion or empyema (CPE) and is a less invasive procedure than video-assisted thoracoscopic surgery under general anesthesia. There are various methods of thoracoscopic debridement under local anesthesia, although the optimal timing of treatment is unknown. The objective of this study was to verify the efficacy and safety of our video-assisted flexible thoracoscopic debridement (VAFTS-D) procedure under local anesthesia, and to investigate the clinical features associated with the success of VAFTS-D. METHODS: The study included 71 consecutive patients with CPE who underwent VAFTS-D. The primary outcome was success of VAFTS-D. We retrospectively analyzed the efficacy and safety of VAFTS-D from the clinical data obtained from hospital medical records, and used univariate logistic analyses to identify potential predictors of the outcome. RESULTS: VAFTS-D was considered successful in 62 of 71 patients (87.3%). Two of the remaining nine patients died and the other seven patients required subsequent operation under general anesthesia. Complications due to VAFTS-D occurred in six patients (8.5%). Duration of empyema < 10 days (P = 0.024) and negative bacterial culture in pleural effusion (P = 0.029) were independently associated with the success of VAFTS-D by univariate logistic regression analysis. CONCLUSION: VAFTS-D might be an acceptable first-line procedure in patients with suspected CPE. VAFTS-D should be performed as early as possible for a successful outcome, and to obtain useful information on the pleural cavity.

[Costochondritis and Osteomyelitis of the Ribs after Intercostal Thoracotomy].

A 53-year-old man underwent surgical repair and drainage of a spontaneous esophageal rupture through a left anterolateral intercostal thoracotomy. Thereafter, wound infection persisted for 3 years with formation of cutaneous fistulae and granulation tissue. Chest computed tomography revealed osteolysis, swollen ribs and costal cartilage, and cutaneous fistulous tracts. Bone scintigraphy with 99mTechnetium revealed abnormal accumulation in the ribs and costal cartilage, indicating costochondritis and osteomyelitis of ribs with cutaneous fistulae. Surgical resection of the skin including the cutaneous fistulae, infected ribs and costal cartilage were performed successfully.

Single-trocar thoracoscopic pericardio-pleural fenestration under local anesthesia for malignant pleural effusion: a case report.

BACKGROUND: Pericardio-pleural fenestration by video-assisted thoracoscopic surgery is an efficient procedure for malignant pericardial effusion, but requires general anesthesia with single-lung ventilation. CASE PRESENTATION: A 43-year-old woman was referred with complaints of deteriorating dyspnea and orthopnea. Chest computed tomography revealed right massive pleural effusion and pericardial effusion. Echocardiography demonstrated collapse of both the right atrium and right ventricle due to cardiac tamponade. Semi-rigid thoracoscopic pleural biopsy and pericardio-pleural fenestration were successfully performed under local anesthesia via a single trocar, because surgical procedures under general anesthesia with single-lung ventilation might have been intolerable for the patient. Adequate biopsy specimens of pleura and pericardium and immediate relief of serious symptoms were obtained without perioperative complications. No recurrence of pleural or pericardial effusion was observed for 3 months postoperatively. CONCLUSION: Thoracoscopic pericardio-pleural fenestration under local anesthesia via a single trocar is feasible as an alternative approach in critically ill patients, allowing effective pericardial drainage, evaluation of the pleural cavity, and accurate biopsies of the pericardium and parietal pleura simultaneously.

Predictive biomarkers and effectiveness of MUC1-targeted dendritic-cell-based vaccine in patients with refractory non-small cell lung cancer.

BACKGROUND: The dendritic cell (DC)-based vaccine targeting the highly immunogenic tumor antigen, MUC1, has been promising for a cancer immunotherapy; however, predictive biomarkers for beneficial clinical responses of the vaccine remain to be determined. METHODS: DCs loaded with MUC1-derived peptide were subcutaneously administered to patients with MUC1-positive non-small cell lung cancer (NSCLC) that was refractory to standard anticancer therapies, every 2 weeks. The effectiveness and tolerability of the vaccine were evaluated, and predictive biomarkers of clinical responses were explored. RESULTS: Between August 2005 and May 2015, 40 patients received the vaccines. The median survival time (MST) after the initial vaccination was 7.4 months, and the 1-year survival rate was 25.0%. The MST for patients who received more than six vaccinations was 9.5 months, and the 1-year survival rate was 39.3%. In this cohort, patients who experienced immune-related adverse events, including skin reactions at the vaccination site and fever, had significantly longer survival times compared with patients without those immune-related adverse events (12.6 versus 6.7 months, p = 0.042). Longer survival times were also observed in patients whose peripheral white blood cells contained >20.0% lymphocytes (12.6 versus 4.5 months; p = 0.014). MUC1-specific cytotoxic immune responses were achieved in all of seven patients analyzed who received six vaccinations. CONCLUSION: The MUC1-targeted DC-based vaccine induced an antitumor immune response that promoted prolonged survival of patients with refractory NSCLC. The occurrence of immune-related adverse events and having a higher percentage of peripheral lymphocytes were predictive biomarkers of a beneficial clinical response during cancer immunotherapy for NSCLC.

Systemic air embolism during pleural lavage for empyema.

Pleural lavage has been considered a convenient and safe method that is often performed for empyema. We report a case of systemic air embolism that developed during pleural lavage. A 53-year-old man with empyema in the organizing phase suddenly developed paralysis of the left side of the body and altered level of consciousness during pleural lavage, which was performed in a sitting position without negative pressure suction. Systemic air embolism was diagnosed based on computed tomography. In this case, use of fibrinolytic agents, positioning during pleural lavage, and pressure in an empyema cavity may have predisposed to development of systemic air embolism. Conversion from thoracoscopic therapy to open decortication or fenestration should be considered to prevent this type of complication.

Thoracolithiasis diagnosed by thoracoscopy under local anesthesia.

A 68-year-old woman was referred to our hospital for a lung nodule identified on chest radiography. Computed tomography (CT) showed a 10-mm calcified nodule in the left thoracic cavity. On follow-up CT, the nodule was found in a different location within the left thoracic cavity. Thoracoscopy was performed under local anesthesia, removing a pearl-like pleural stone. Thoracolithiasis was therefore diagnosed without any complications. To the best of our knowledge, this is the first report on thoracoscopy under local anesthesia for the diagnosis of thoracolithiasis. When thoracolithiasis is suspected, thoracoscopy under local anesthesia is minimally invasive and useful, and could be considered as an option for definite diagnosis.

Single-trocar thoracoscopy under local anesthesia for pleural space infection.

OBJECTIVE: The role of single-trocar thoracoscopy for complicated parapneumonic effusion (CPE) and pleural empyema is not established as yet. The aim of this study was to report our experience and analyze the efficacy and safety of debridement by single-trocar thoracoscopy for the patients with CPE and multiloculated empyema. METHODS: We performed a retrospective study reviewing the medical records of the patients treated parapneumonic effusion and multiloculated empyema by single-trocar thoracoscopy under local anesthesia at our department from January 2000 to December 2012. RESULTS: A total 29 patients with CPE and multiloculated empyema were treated by single-trocar thoracoscopy. As the staging of pleural infection, class 5 and class 7 by Light classification were 21 and 8 patients, respectively. The onset of the symptom was on average 13.9 ± 11.7 days before the procedure. This procedure was successful in 23 of 29 patients (79.3%) without further operation under general anesthesia. Complication occurred in 1 case of 29 patients (3.4%). Six patients required subsequently the operation under general anesthesia, and one of the 6 patients died to multiple organ failure caused by sepsis. A microbiological diagnosis could be made in fifteen patients (51.7%). CONCLUSIONS: Debridement by single-trocar thoracoscopy can be an acceptable approach as the first-line procedure in patients with CPE and empyema. This procedure can provide not only appropriate and expeditious treatment but also information of pleural cavity to decide indication for thoracotomy under general anesthesia.

A phase II study of docetaxel plus nedaplatin in patients with metastatic non-small-cell lung cancer.

PURPOSE: Nedaplatin is a cisplatin derivative, which has similar activity to cisplatin in non-small-cell lung cancer (NSCLC) when combined with vindesine, and causes less nausea/vomiting and nephrotoxicity compared with cisplatin. The aim of this study was to evaluate the efficacy and safety of combination chemotherapy with docetaxel plus nedaplatin in patients with metastatic NSCLC. METHODS: Patients with metastatic stage IIIB excluding locally advanced diseases or stage IV NSCLC were enrolled between March 2004 and March 2006. They were treated with docetaxel (60 mg/m(2)) and nedaplatin (80 mg/m(2)) on day 1 every 3-4 weeks until progression or intolerable toxicity for up to 4 cycles. RESULTS: Forty-four patients (mean age, 65 years; range, 40-79 years) received a total of 140 treatment cycles. Responses could be assessed in all patients (complete response, 0; partial response, 22; stable disease, 11; and progressive disease, 11). Response rate was 50.0 % (95 % confidence interval [CI], 35.2-64.8 %) with a disease control rate of 75.0 % (95 % CI, 62.2-87.8 %). A high response rate was achieved in patients with squamous cell carcinoma (66.7 %) compared with that in patients with adenocarcinoma (41.4 %). Median survival time from the start of the combination chemotherapy was 13.0 months, and the progression-free survival time was 7.4 months. Grade 3 or 4 hematologic toxicities included leukopenia (28.6 %) and neutropenia (61.4 %). Nonhematologic toxicities were mild. CONCLUSION: The combination of docetaxel plus nedaplatin was well tolerated and demonstrated potent activity in patients with metastatic NSCLC, particularly squamous cell carcinoma of the lung.

[A case of anesthetic management using levobupivacaine in epidural anesthesia combined with general anesthesia for thymectomy with thoracoscopy for generalized type myasthenia gravis].

Combined epidural and general anesthesia has become a standard anesthetic method for thymectomy. We employed levobupivacaine for epidural anesthesia combined with general anesthesia using remifentanil and sevoflurane for thymectomy with thoracoscopy for generalized type of myasthenia gravis. After decreasing the high level of antibodies to acethylcholine receptor by plasmapheresis, we could perform a stable and successful anesthetic management without the use of any muscle relaxants for the thymectomy. And a myasthenic crisis did not occur after the procedure. We concluded that levobupivacaine would be one of the appropriate options in epidural anesthesia for thymectomy for myasthenia gravis.

[Case of miliary tuberculosis with esophageal perforation and a tracheal inflammatory polyp secondary to mediastinal lymphadenitis causing massive hematemesis and hemosputum].

A 62-year-old man with a history of left nephrectomy due to tuberculosis was referred to our hospital, because chest radiography showed diffuse miliary shadows in the bilateral lung fields, and acid-fast bacilli were detected from his hemosputum after steroid therapy for fever of unknown origin. Chest computed tomography showed mediastinal lymph node enlargement with partial calcification of these lymph nodes together with the presence of air. He was diagnosed with miliary tuberculosis and tuberculous mediastinal lymphadenitis and anti-tuberculosis drug therapy was started. Massive hematemesis occurred 11 days after the start of the treatment. Although gastroendoscopy was performed, the bleeding point could not be identified. The patient's symptoms improved after conservative therapy. Repeat gastroendoscopy showed a submucosal nodule with laceration of the esophageal mucosa, 30 days after admission for the examination of melena and progression of anemia. The episodes occurred because of esophageal perforation secondary to tuberculous mediastinal lymphadenitis. Bronchoscopic examination for hemosputum showed an inflammatory polypoid lesion in the left tracheal wall. These symptoms improved with anti-tuberculosis drug therapy. In our case, mediastinal lymphadenitis progressed to miliary tuberculosis because of endogenous reactivation. We report a rare case of esophageal perforation with a tracheal inflammatory polyp secondary to tuberculous mediastinal lymphadenitis. In cases of tuberculous mediastinal lymphadenitis, if hematemesis or hemosputum is observed, an endoscopic examination should be performed.

Podoplanin expression in cancerous stroma induces lymphangiogenesis and predicts lymphatic spread and patient survival.

CONTEXT: Podoplanin is a mucin-type glycoprotein and a lymphatic endothelial marker. Immunohistochemical staining for podoplanin is currently used as a routine pathologic diagnosis tool in Japan to identify lymphatic invasion of cancer cells. Recent reports suggest that podoplanin and other proangiogenic molecules are expressed in stromal fibroblasts and myofibroblasts. OBJECTIVE: To analyze the distribution of podoplanin expression in tumor stroma and its clinical and biologic significance. DESIGN: We performed immunohistochemistry for podoplanin on tissue microarrays from 1350 cases of 14 common cancer types. RESULTS: Two hundred eighty-seven of 662 cases (43%) showed podoplanin expression in the stromal cells within cancer nests. Stromal podoplanin expression in 14 common cancer types was significantly associated with tumor stage (P < .001), lymph node metastases (P < .001), lymphatic invasion (P  =  .02), and venous invasion (P < .001). The stromal cells positive for podoplanin were also positive for α-smooth muscle actin but negative for desmin, confirming a myofibroblasts phenotype. In contrast, myofibroblasts in inflammatory fibrotic lung diseases were podoplanin negative. Lymphatic vessel density was greater in the stromas with podoplanin expression than in the stroma lacking podoplanin-expressing stromal cells (P  =  .01). Survival data were available for non-small cell lung cancer. Stromal podoplanin expression was associated with poorer prognosis in adenocarcinoma (P < .001) and remains statistically significant after adjustment for sex, age, and stage (P  =  .01). CONCLUSION: Our data indicate that podoplanin expression in stromal myofibroblasts may function as a proangiogenic biomarker and may serve as a predictive marker of lymphatic/vascular spread of cancer cells and a prognostic marker of patient survival.

Inhibition of transforming growth factor-beta-mediated immunosuppression in tumor-draining lymph nodes augments antitumor responses by various immunologic cell types.

Tumor-draining lymph nodes (DLN) are the most important priming sites for generation of antitumor immune responses. They are also the location where an immunosuppressive cytokine, transforming growth factor-beta (TGF-beta), plays a critical role in suppressing these antitumor immune responses. We focused on TGF-beta-mediated immunosuppression in DLNs and examined whether local inhibition of TGF-beta augmented antitumor immune responses systemically in tumor-bearing mice models. For inhibition of TGF-beta-mediated immunosuppression in DLNs, C57BL/6 mice subcutaneously bearing E.G7 tumors were administered plasmid DNA encoding the extracellular domain of TGF-beta type II receptor fused to the human IgG heavy chain (TGFR DNA) i.m. near the established tumor. In DLNs, inhibition of TGF-beta suppressed the proliferation of regulatory T cells and increased the number of tumor antigen-specific CD4(+) or CD8(+) cells producing IFN-gamma. Enhancement of antitumor immune responses in DLNs were associated with augmented tumor antigen-specific cytotoxic and natural killer activity in spleen as well as elevated levels of tumor-specific antibody in sera. The growth of the established metastatic as well as primary tumors was effectively suppressed via augmented antitumor immune responses. Inhibition of TGF-beta-mediated immunosuppression in DLNs is significantly associated with augmented antitumor responses by various immunocompetent cell types. This animal model provides a novel rationale for molecular cancer therapeutics targeting TGF-beta.

Good syndrome coexisting with leukopenia.

A 61-year-old man was admitted to our hospital for further examinations of a mediastinal mass. He had underwent an extended thymothymectomy, and had a tumor that was diagnosed as a type B1 thymoma, according to the World Health Organization. One year after surgery he was admitted again for recurrent diarrhea and pneumonia. Laboratory data revealed severe hypogammaglobulinemia with leukopenia. He was diagnosed with Good syndrome with leukopenia. Regular gamma globulin and figrastim injections were successful in keeping the patient symptom free. The prognosis of patients with Good syndrome and leukopenia is very poor; therefore, immediate diagnosis is important. The development of infectious diseases in a patient with thymoma or after the resection of thymoma mandates early and comprehensive immunologic investigation.

Preparation of fully activated dendritic cells capable of priming tumor-specific cytotoxic T lymphocytes in patients with metastatic cancer using penicillin-killed streptococcus pyogenes (OK432) and anti-CD40 antibody.

In order to achieve sufficient therapeutic potency, it has been proposed that vaccine therapy with dendritic cells needs to be combined with manipulation of immunological checkpoints, such as inhibition of regulatory T cells and blockade of negative signals, and enhancement of T cell trafficking to tumor sites. In the combinatorial cancer immunotherapy, use of matured/activated dendritic cells (DCs) with more potent antigen presenting capacity seems to be essential for eliciting anti-tumor immune responses. We herein established an ex vivo induction strategy for activated DCs capable of eliciting efficient tumor antigen-specific cytotoxic T lymphocytes (CTLs) from patients with metastatic cancer as well as healthy donors. Immature DCs were matured by 48-h culture in the presence of anti-CD40 antibody and penicillin-killed streptococcus pyogenes (OK432). Supplementation with both anti-CD40 and OK432 resulted in induction of activated DCs with higher surface expression of CD80, CD83, CD86 and major histocompatibility complex class II antigens, compared with other mature DCs that were induced by the combination of anti-CD40 with tumor necrosis factor-alpha or lipopolysaccharide. In analysis of the produced cytokine profiles, the activated DCs produced the highest T-helper 1-type cytokines for at least 72 h. Furthermore, the activated DCs, pulsed with tumor-associated antigen peptide, elicited in vitro tumor-specific CTLs, but DCs activated with other combinations did not in cancer patients. Therefore, we suggest that the activated DCs studied here might be used as a basic element for the combinatorial cancer immunotherapy.

Successful tumor eradication was achieved by collaboration of augmented cytotoxic activity and anti-angiogenic effects following therapeutic vaccines containing helper-activating analog-loaded dendritic cells and tumor antigen DNA.

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4(+) and CD8(+) T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes in an HLA-A24 restricted manner.

We previously reported that 90K/Mac-2 binding protein (M2BP) is highly expressed in lung cancer and that M2BP-specific immunity was observed in many patients with lung cancer. These findings suggested the possibility of using M2BP as a target antigen in cancer immunotherapy. In this study, we selected 11 peptides derived from M2BP with an HLA-A24 binding motif and analyzed their ability to induce M2BP-specific cytotoxic T lymphocytes (CTL). CTLs were generated with the M2BP-derived peptides from peripheral blood CD8-positive T lymphocytes of HLA-A24-positive healthy donors in multiple in vitro stimulations. Two CTLs, one induced with M2BP(241-250) (GYCASLFAIL) and the other with M2BP(568-576) (GFRTVIRPF), produced interferon-gamma in response to HLA-A24-positive TISI cells pulsed with the same peptide used for the in vitro stimulation. Although the CTLs induced with M2BP(241-250) reacted with both peptide-pulsed TISI cells and BT20 cells expressing both M2BP and HLA-A24, the CTLs induced with M2BP(568-576) did not react with BT20 cells. The cytokine production was blocked by antibodies against HLA class I in CTLs induced using M2BP(241-250), but not in CTLs induced using M2BP(568-576). These findings suggest that M2BP(241-250) is naturally processed from the native M2BP molecule in cancer cells and recognized by M2BP-specific CTLs in an HLA-A24 restriction. An M2BP-derived CTL epitope with an HLA-A24 binding motif was identified for the first time in this study, and it is expected to be useful as a target antigenic epitope in clinical immunotherapy for lung cancer.

Involvement of 90K/Mac-2 binding protein in cancer metastases by increased cellular adhesiveness in lung cancer.

90K/Mac-2 Binding Protein (M2BP) plays a role in regulation of immune responses and cell adhesive ability in patients with cancer and infectious diseases. We previously reported that M2BP was highly expressed in lung cancer and that immune responses to M2BP were increased in many patients with lung cancer. To determine the involvement of M2BP in metastatic processes of cancer progression, we examined the ability of M2BP DNA-transduced lung carcinoma cell lines to adhere to extracellular matrices. Although expressions of cell-surface integrins were not modulated in the M2BP transfectants, they showed increased adhesiveness to fibronectin and collagen IV. We next analyzed the serum levels of M2BP in patients with lung cancer and normal donors and the relationships between M2BP expression and clinicopathological factors in the patients. The M2BP level was markedly elevated in the patients and was strongly correlated with nodal involvement and clinical staging. To determine whether expression of M2BP by cancer cells is modulated in the environment of tumor-bearing hosts, M2BP expression in M2BP-positive QG56 cells following exposure of the cells to pro-inflammatory cytokines was examined. The M2BP expression in QG56 cells was up-regulated by many of the cytokines that activate host protective immunity. The findings in this study suggest that M2BP plays a role in cancer metastasis by increased adhesiveness of cancer cells and that M2BP is increasingly produced even in a state of exposure to the host immune system. This molecule may be useful as a predictive factor of disease progression in lung cancer.

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy.

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.

Deoxyribonucleic acid (DNA) encoding a pan-major histocompatibility complex class II peptide analogue augmented antigen-specific cellular immunity and suppressive effects on tumor growth elicited by DNA vaccine immunotherapy.

Vaccine immunotherapy must induce helper and cytotoxic cell-mediated immunity to generate the powerful antitumor immune responses needed to suppress cancer progression. We reported previously that a 16-amino acid peptide analogue derived from pigeon cytochrome c can bind broad ranges of MHC class II types and activate helper T cells in mice. To determine whether DNA encoding the Pan-MHC class II IA peptide (Pan-IA) can increase the efficacy of tumor suppression by DNA vaccine immunotherapy targeting tumor antigens, Pan-IA DNA was administered with ovalbumin (OVA) DNA to C57BL/6 mice bearing the OVA-expressing tumor cell line E.G7. Specific proliferative responses to and cytotoxic activities against OVA-expressing targets were induced in mice vaccinated with both OVA and Pan-IA DNA but not in those vaccinated with OVA DNA alone or control DNA plus Pan-IA DNA. Growth of E.G7 cells was suppressed only by combined vaccination with OVA and Pan-IA DNA, and tumors in five of the nine mice that received this combined vaccination were eradicated completely. In those mice, the frequency of CD8-positive T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased in the tumor site. Furthermore, immunofluorescent study of the inoculated tumors revealed increased accumulation of both CD4- and CD8-positive T cells producing IFN-gamma in the tumor only by this vaccine protocol. The data suggest that Pan-IA DNA can augment suppressive effects of DNA vaccines on tumor growth by increasing numbers of antigen-specific CTLs and helper T cells. This is the first study in which established tumors have been eradicated successfully by vaccination with DNA corresponding to CTL epitopes and helper T cell epitopes. Our animal model may contribute to the development of therapeutic DNA vaccines against cancer.