Clinical Significance of Tumor-Infiltrating T Cells and Programed Death Ligand-1 in Patients with Pancreatic Cancer.

The associations of the immunological status of the pancreatic ductal adenocarcinoma (PDA) microenvironment with prognosis were assessed. A high tumor-infiltrating lymphocyte (TIL) density was associated with a better prognosis. Importantly, even with a high density of TILs, the PDA cells with programed cell death-ligand 1 (PD-L1) expression showed a worse prognosis than the patients with negative PD-L1 expression. A significant association between a better prognosis and a tumor microenvironment with a high TIL density/negative PD-L1 expression was observed. Assessments of a combined immunological status in the tumor microenvironment may predict the prognosis of PDA patients following surgical resection.

Predicted Markers of Overall Survival in Pancreatic Cancer Patients Receiving Dendritic Cell Vaccinations Targeting WT1.

BACKGROUND: We have developed a Wilms' tumor 1 (WT1)-targeting dendritic cell (DC)-based cancer vaccine combined with standard chemotherapy for patients with advanced pancreatic ductal adenocarcinoma (PDA). METHODS: We evaluated predictive markers of overall survival (OS) in PDA patients treated with multiple major histocompatibility complex class I/II-restricted, WT1 peptide-pulsed DC vaccinations (DC/WT1-I/II) in combination with chemotherapy. Throughout the entire period of immunochemotherapy, the plasma levels of soluble factors derived from granulocytes of 7 eligible PDA patients were examined. Moreover, systemic inflammatory response markers (neutrophil-to-lymphocyte ratio [NLR], monocyte-to-lymphocyte ratio [MLR], and granulocyte-to-lymphocyte ratio [GLR]) were assessed. In addition, cytoplasmic WT1 expression in PDA cells was examined. RESULTS: Compared to the 4 non-super-responders (OS <1 year), the remaining 3 super-responders (OS ≥1 year) showed significantly decreased low plasma matrix metalloproteinase-9 levels throughout long-term therapy. The NLR, MLR, and GLR after 5 DC/WT1-I/II vaccinations and 3 cycles of gemcitabine were significantly lower in the super-responders than in the non-super-responders. Furthermore, the cytoplasmic WT1 expression in the PDA cells of super-responders was relatively weak compared to that in the PDA cells of non-super-responders. CONCLUSIONS: Prolonged low levels of a granulocyte-related systemic inflammatory response after the early period of therapy and low cytoplasmic WT1 expression in PDA cells may be markers predictive of OS in PDA patients receiving WT1-targeting immunochemotherapy.

Lung adenocarcinoma may be a more susceptive subtype to a dendritic cell-based cancer vaccine than other subtypes of non-small cell lung cancers: a multicenter retrospective analysis.

OBJECTIVE: The J-SICT DC Vaccine Study Group provides dendritic cell (DC) vaccines for compassionate use under unified cell production and patient treatment regimens. We previously reported beneficial effects of DC vaccines on the overall survival of 62 patients with advanced non-small cell lung cancer (NSCLC) in a single-center analysis. Here, we extended analysis to 260 patients with NSCLC who were treated at six centers. METHODS: Of the 337 patients who met the inclusion criteria, we analyzed 260 patients who received ≥5 peptide-pulsed DC vaccinations once every 2 weeks. RESULTS: The mean survival time (MST) from diagnosis was 33.0 months (95 % confidence interval [CI]: 27.9-39.2), and that from time of first vaccination was 13.8 months (95 % CI 11.4-16.8). An erythema reaction at the injection site that was ≥30 mm in diameter was correlated most strongly with overall survival from the first vaccine (≥30 vs. < 30 mm: MST 20.4 vs. 8.8 months, P < 0.001). We reported a similar finding in our previous analysis of patients with advanced pancreatic cancer. Interestingly, although such findings were common between patients with adenocarcinoma and those with other subtypes, the former group experienced significantly prolonged overall survival and a higher response rate for erythema (56.3 vs. 37.3 %, respectively, P = 0.014). CONCLUSIONS: This is the first multicenter study that suggests a possible clinical benefit of DC vaccines for patients with advanced NSCLC, especially those with adenocarcinoma. These findings suggest a specific potential responder population for DC vaccines and warrant further investigation in well-controlled prospective randomized trials.

Comprehensive assessment of the prognosis of pancreatic cancer: peripheral blood neutrophil-lymphocyte ratio and immunohistochemical analyses of the tumour site.

OBJECTIVE: Several studies have suggested that an elevated neutrophil-lymphocyte ratio (NLR) is associated with a poorer prognosis in patients with pancreatic cancer (PC). The correlations between the NLR and immunohistochemical (IHC) analysis with regard to the prognosis of patients with PC remain to be elucidated. By using IHC findings, we determined the value of the NLR as a prognostic factor in patients with PC. MATERIAL AND METHODS: We collected the clinico-pathological data of 28 consecutive patients who underwent surgical resection for PC between January 2008 and December 2012 at The Jikei University Kashiwa Hospital. We investigated whether the NLR and IHC results were related and ensured the consistency of the prognosis of patients with PC. RESULTS: The Kaplan-Meier curves for the disease-free survival (DFS) and the overall survival (OS) revealed that an NLR ≥ 5 is an implicit factor for decreased DFS and OS in patients with PC (p = 0.003, p < 0.001, log-rank test). The density of CD163(+) macrophages and CD66b(+) neutrophils was significantly higher in the high NLR group; on the contrary, the density of CD20(+) lymphocytes was significantly higher in the low NLR group. Moreover, a Mann-Whitney U test showed that the NLR was significantly correlated with a high density of CD20(+) lymphocytes (p = 0.031) and CD163(+) macrophages (p = 0.023), while the NLR was not significantly correlated with CD66b(+) neutrophils (p = 0.397). CONCLUSIONS: Our results demonstrated the validity of the NLR by IHC analyses and we determined that a higher value of NLR is a trustworthy prognostic factor for patients with PC.

Phase I pilot study of Wilms tumor gene 1 peptide-pulsed dendritic cell vaccination combined with gemcitabine in pancreatic cancer.

This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide-pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first-line therapy among patients with advanced pancreatic cancer. Ten HLA-A*2402 patients were treated with WT1 peptide-pulsed DC vaccination (1 × 10(7) cells) on days 8 and 22 and gemcitabine (1000 mg/m(2) ) on days 1, 8 and 15. Induction of a WT1-specific immune response was evaluated using the delayed-type hypersensitivity (DTH) skin test, interferon-γ enzyme-linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well-tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C-reactive protein and interleukin-8 (IL-8) showed poor survival even though a WT1-specific immune response was induced in them. WT1 peptide-pulsed DCGEM is feasible and effective for inducing anti-tumor T-cell responses. Our results support future investigations for pancreatic cancer patients with non-liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry (http://www.umin.ac.jp/ctr/ number: UMIN-000004855).

Safety and tolerability of allogeneic dendritic cell vaccination with induction of Wilms tumor 1-specific T cells in a pediatric donor and pediatric patient with relapsed leukemia: a case report and review of the literature.

A 15-year-old girl with acute lymphoblastic leukemia received allogeneic dendritic cell vaccination, pulsed with Wilms tumor 1 (WT1) peptide, after her third hematopoietic stem cell transplantation (HSCT). The vaccines were generated from the third HSCT donor, who was her younger sister, age 12 years. The patient received 14 vaccines and had no graft-versus-host disease or systemic adverse effect, aside from grade 2 skin reaction at the injection site. WT1-specific immune responses were detected after vaccination by both WT1-tetramer analysis and enzyme-linked immunosorbent spot assay. This strategy may be safe, tolerable and even feasible for patients with a relapse after HSCT.

Up-regulation of HER2 by gemcitabine enhances the antitumor effect of combined gemcitabine and trastuzumab emtansine treatment on pancreatic ductal adenocarcinoma cells.

BACKGROUND: Although pancreatic ductal adenocarcinomas (PDAs) widely express HER2, the expression level is generally low. If HER2 expression in PDA cells could be enhanced by treatment with a given agent, then combination therapy with that agent and trastuzumab emtansine (T-DM1), a chemotherapeutic agent that is a conjugate of trastuzumab, might lead to significant antitumor effects against PDA. METHODS: Cell proliferation was examined by spectrophotometry. HER2 expression was examined by flow cytometry, immunoblot and quantitative reverse transcription polymerase chain reaction. T-DM1 binding to cells was examined by flow cytometry and enzyme-linked immunosorbent assay. RESULTS: Out of 5 tested human PDA cell lines, including MIA PaCa-2, three showed increases in HER2 expression after gemcitabine (GEM) treatment. The binding of T-DM1 to GEM-treated MIA PaCa-2 cells was higher than to untreated MIA PaCa-2 cells. Treatment with GEM and T-DM1 showed synergic cytotoxic effects on MIA PaCa-2 cells in vitro. Cells in the G2M phase of the cell cycle were retained after GEM treatment and showed higher levels of HER2 expression, possibly contributing to the synergic effect of GEM and T-DM1. CONCLUSIONS: Combined treatment with GEM and T-DM1 might confer a potent therapeutic modality against PDA as a result of GEM-mediated HER2 up-regulation.

[A Large Number of Circulating Tumor Cells(CTCs)Can Be Isolated from Samples Obtained by Using Leukapheresis Procedures].

We hypothesized that a large number of circulating tumor cells(CTCs)may be isolated from samples obtained by using the leukapheresis procedures that are utilized to collect peripheral blood mononuclear cells for dendritic cell vaccine therapy. We utilized the CellSearch System to determine the number of CTCs in samples obtained by using leukapheresis in 7 patients with colorectal cancer, 5 patients with breast cancer, and 3 patients with gastric cancer. In all patients, a large number of CTCs were isolated. The mean number of CTCs per tumor was 17.1(range 10-34)in colorectal cancer, 10.0(range 2-27)in breast cancer, and 24.0(range 2-42)in gastric cancer. We succeeded in culturing the isolated CTCs from 7 patients with colorectal cancer, 5 patients with breast cancer, and 3 patients with gastric cancer. In conclusion, compared to conventional methods, a large number of CTCs can be obtained by using leukapheresis procedures. The molecular analyses of the CTCs isolated by using this method should be promising in the development of personalized cancer treatments.

Vaccination with vascular progenitor cells derived from induced pluripotent stem cells elicits antitumor immunity targeting vascular and tumor cells.

Vaccination of BALB/c mice with dendritic cells (DCs) loaded with the lysate of induced vascular progenitor (iVP) cells derived from murine-induced pluripotent stem (iPS) cells significantly suppressed the tumor of CMS-4 fibrosarcomas and prolonged the survival of CMS-4-inoculated mice. This prophylactic antitumor activity was more potent than that of immunization with DCs loaded with iPS cells or CMS-4 tumor cells. Tumors developed slowly in mice vaccinated with DCs loaded with iVP cells (DC/iVP) and exhibited a limited vascular bed. Immunohistochemistry and a tomato-lectin perfusion study demonstrated that the tumors that developed in the iVP-immunized mice showed a marked decrease in tumor vasculature. Immunization with DC/iVP induced a potent suppressive effect on vascular-rich CMS-4 tumors, a weaker effect on BNL tumors with moderate vasculature, and nearly no effect on C26 tumors with poor vasculature. Treatment of DC/iVP-immunized mice with a monoclonal antibody against CD4 or CD8, but not anti-asialo GM1, inhibited the antitumor activity. CD8(+) T cells from DC/iVP-vaccinated mice showed significant cytotoxic activity against murine endothelial cells and CMS-4 cells, whereas CD8(+) T cells from DC/iPS-vaccinated mice did not. DNA microarray analysis showed that the products of 29 vasculature-associated genes shared between genes upregulated by differentiation from iPS cells into iVP cells and genes shared by iVP cells and isolated Flk-1(+) vascular cells in CMS-4 tumor tissue might be possible targets in the immune response. These results suggest that iVP cells from iPS cells could be used as a cancer vaccine targeting tumor vascular cells and tumor cells.

Prognostic factors related to add-on dendritic cell vaccines on patients with inoperable pancreatic cancer receiving chemotherapy: a multicenter analysis.

OBJECTIVE: Dendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced pancreatic cancer. However, variations among clinical studies make it difficult to compare clinical outcomes. Here, we identified factors that determined the clinical benefits by analyzing data obtained at seven Japanese institutions that employed the same DC preparation and treatment regimens. METHODS: Of 354 patients who met the inclusion criteria, 255 patients who received standard chemotherapy combined with peptide-pulsed DC vaccines were analyzed. RESULTS: The mean survival time from diagnosis was 16.5 months (95 % CI 14.4-18.5) and that from the first vaccination was 9.9 months (95 % CI 8.0-12.9). Known prognostic baseline factors related to advanced pancreatic cancer, namely ECOG-PS, peritoneal metastasis, liver metastasis, and the prognostic nutrition index, were also representative. Importantly, we found that erythema reaction after vaccination was an independent and treatment-related prognostic factor for better survival and that OK-432 might be a good adjuvant enhancing the antitumor immunity during DC vaccination. CONCLUSIONS: This is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of an add-on DC vaccine in patients with advanced pancreatic cancer who are undergoing chemotherapy. These findings need to be addressed in well-controlled prospective randomized trials.

Therapeutic effect of intratumoral injections of dendritic cells for locally recurrent gastric cancer: a case report.

An 80-year-old man with a history of gastric cancer and pulmonary emphysema underwent a distal gastrectomy for gastric cancer in 1997. In 2010, an endoscopic examination revealed a depressed-type lesion at the oral side of the anastomosis, which was diagnosed as signet-ring adenocarcinoma. Surgical management was considered, but was rejected because of obstructive and restrictive respiratory events. Chemotherapy was terminated because of adverse events. Endoscopy was used to administer intratumoral injections of dendritic cells (DCs) targeting synthesized peptides of Wilms tumor 1 (WT1) and mucin 1, cell-surface associated (MUC1). An immunohistochemical analysis of the tumor samples indicated positivity for WT1 and MUC1. One month after seven cycles of DC had been administered (between November 2010 and April 2011), no suspicious lesions were evident, and his biopsy results were normal. The patient has been in remission for 30 months. Intratumoral injections of DCs showed therapeutic effects in this patient, who could not undergo endoscopic submucosal dissection or surgery.

Selective inhibition of NF-κB suppresses bone invasion by oral squamous cell carcinoma in vivo.

Nuclear factor-κB (NF-κB) is constitutively activated in many cancers, including oral squamous cell carcinoma (OSCC), and is involved in the invasive characteristics of OSCC, such as growth, antiapoptotic activity and protease production. However, the cellular mechanism underlying NF-κB's promotion of bone invasion by OSCC is unclear. Therefore, we investigated the role of NF-κB in bone invasion by OSCC in vivo. Immunohistochemical staining of OSCC invading bone in 10 patients indicated that the expression and nuclear translocation of p65, a main subunit of NF-κB, was increased in OSCC compared with normal squamous epithelial cells. An active form of p65 phosphorylated at serine 536 was present mainly in the nucleus in not only differentiated tumor cells but also tumor-associated stromal cells and bone-resorbing osteoclasts. We next injected mouse OSCC SCCVII cells into the masseter region of C(3) H/HeN mice. Mice were treated for 3 weeks with a selective NF-κB inhibitor, NBD peptide, which disrupts the association of NF-κB essential modulator (NEMO) with IκB kinases. NBD peptide treatment inhibited TNFα-induced and constitutive NF-κB activation in SCCVII cells in vitro and in vivo, respectively. Treatment with NBD peptide decreased zygoma and mandible destruction by SCCVII cells, reduced number of osteoclasts by inhibiting RANKL expression in osteoblastic cells and SCCVII cells, increased apoptosis and suppressed the proliferation of SCCVII cells. Taken together, our data clearly indicate that inhibition of NF-κB is useful for inhibiting bone invasion by OSCC.

The inhibition of RANKL/RANK signaling by osteoprotegerin suppresses bone invasion by oral squamous cell carcinoma cells.

Oral squamous cell carcinomas (OSCCs) are malignant tumors that frequently invade the maxilla and mandibular bone. However, the molecular mechanisms underlying bone invasion by OSCC are unclear. Recent studies showed that receptor activator of nuclear factor κB (RANK) was expressed not only in osteoclast precursors but also in tumor cells. Therefore, we examined whether RANK ligand (RANKL)/RANK signaling regulates bone invasion by OSCC cells in vivo and in vitro. We first injected human OSCC B88 cells into the masseter region of nude mice. Mice were treated for 3 weeks with osteoprotegerin (OPG), the decoy receptor for RANKL. Treatment with OPG decreased bone invasion by B88 cells, reduced the number of osteoclasts and increased B88 cell apoptosis. However, OPG did not affect apoptosis and proliferation in B88 cells in vitro, suggesting that the effects of OPG on apoptosis in B88 cells are restricted in a bone environment. RANK was expressed in the B88 cells and in OSCC cells from patients. RANKL induced NF-κB activation and extracellular signal-regulated kinase phosphorylation in B88 cells and enhanced B88 cell migration in a modified chemotaxis chamber equipped with a gelatin-coated filter. OPG inhibited RANKL-induced NF-κB activation, extracellular signal-regulated kinase phosphorylation and cell migration. Our data clearly indicate that RANKL/RANK inhibition suppresses bone invasion by inhibiting osteoclastogenesis and cancer cell migration and by inducing apoptosis of cancer cells via indirect anticancer action in vivo.

Long-term survival of pancreatic cancer patients treated with multimodal therapy combined with WT1-targeted dendritic cell vaccines.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive tumors with a dismally poor prognosis. Although surgical resection remains the only potentially curative treatment, most PDAs are not surgically resectable at diagnosis. Therefore, multimodal therapy is urgently needed to improve the long-term survival of PDA patients. METHODS: Six eligible PDA patients underwent multimodal therapy comprising dendritic cells (DCs) pulsed with Wilms' tumor 1 (WT1) peptide (DC/WT1-I) restricted by the human leukocyte antigen (HLA) class I (A*24:02 or A*02:06) allele, chemotherapy, radiation, and/or surgery. Patient laboratory data, DC/WT1-I-specific delayed-type hypersensitivity (DTH) reactions, and WT1-specific immune responses were analyzed to assess the prognostic markers of multimodal therapy. RESULTS: Compared to 2-treatment type combinations, multimodal therapy involving 3 to 4 treatment types was significantly associated with longer overall survival (p = 0.0177). Moreover, after 7 DC/WT1-I vaccinations, the progression-free survival (PFS) of PDA patients with a neutrophil to lymphocyte ratio (NLR) or C-reactive protein (CRP) level less than the median was superior to that of PDA patients with values above the median (p = 0.0246). PDA patients with an overall survival (OS)>1000 days had significantly more lymphocytes after one DC/WT1-I vaccination course than did those with an OS<1000 days. CONCLUSION: Multimodal therapy involving the DC/WT1-I vaccination may benefit patients with advanced PDA. However, comparing the limited number of PDA patients in terms of survival is difficult because the patients were at different disease stages and received different treatments. Further studies are needed to evaluate the clinical benefits of this multimodal therapy.

Pilot study of WT1 peptide-pulsed dendritic cell vaccination with docetaxel in esophageal cancer.

In the present study, the immune response to Wilms tumor gene 1 (WT1) peptide-pulsed dendritic cell (DC) vaccination combined with docetaxel (DCDOC) in advanced esophageal cancer patients who had already received first-line chemotherapy was investigated. Ten HLA-A*2402 patients were treated with docetaxel (50 mg/m2) on day 1 and WT1 peptide-pulsed DC vaccination (1×107 cells) on days 15 and 22 (repeated every 4 weeks for 3 cycles). The delayed-type hypersensitivity skin test, HLA tetramer assay and interferon-γ enzyme-linked immunospot (ELISPOT) assay were used to evaluate the induction of a WT1-specific immune response. Median overall survival was 5 months (range, 1.1-11.6). The clinical effect of DCDOC therapy was not observed and only 1 patient could complete the protocol therapy. Disease progression was observed in 9 patients and 1 patient succumbed to fatality during the second cycle of therapy. As a pilot study, it was not possible to evaluate the safety of WT1 peptide-pulsed DCDOC therapy for esophageal squamous cell cancer. However, a WT1-specific response in 6 patients, as indicated by the ELISPOT or HLA/WT1-tetramer assay, was demonstrated. The results suggested that the positive immune response had significant relevance on the low percentage of CD11b+ and CD66b+ granulocytic myeloid-derived suppressor cells in CD15+ cells. Furthermore, DCDOC elicited a WT1-specific immune response regardless of the myelosuppression associated with docetaxel. The present findings support future studies and further work to assess DCDOC as an adjuvant therapy for esophageal cancer will be performed. The present clinical trial was registered in the University Hospital Medical Information Network (UMIN) Clinical Trials Registry on November 11th, 2011, no. UMIN000006704.

WT1-pulsed Dendritic Cell Vaccine Combined with Chemotherapy for Resected Pancreatic Cancer in a Phase I Study.

BACKGROUND/AIM: Wilms' tumor 1 (WT1) is a tumor-associated antigen highly expressed in cancer. We examined the safety of WT1-peptide pulsed dendritic cell (WT1-DC) vaccine in combination with chemotherapy in patients with surgically resected pancreatic cancer. PATIENTS AND METHODS: Eight patients with resectable pancreatic cancer undergoing surgery either combined with S-1 or S-1 plus gemcitabine therapy were enrolled. Immunohistochemical analysis of WT1 was performed in 34 cases of pancreatic cancer. RESULTS: No serious side-effects were observed, except grade I fever in five and grade I reactions at the injection site in all patients. WT1-specific cytotoxic T-lymphocytes were detected in seven patients, and WT1 and human leukocyte antigen class I antigens were positive in all 34 cases. CONCLUSION: Our study clarified the safety and potential acquisition of immunity after vaccination targeting WT1. Further efficacy of WT1-DC vaccine to improve prognosis would be determined by a prospective clinical trial for resectable pancreatic cancer.

Feasibility and Immune Response of WT1 Peptide Vaccination in Combination with OK-432 for Paediatric Solid Tumors.

BACKGROUND/AIM: Wilms' tumor 1 (WT1) peptide-based vaccination has been reported for its potential usefulness in targeting several cancers. The adjuvant drug OK-432 is known to have potent immunomodulation and therapeutic properties when applied in cancer treatment and may, thus, be important to trigger the appropriate immunological response in paediatric patients with a solid tumor that are vaccinated with a WT1 peptide. PATIENTS AND METHODS: Paediatric patients with a solid tumor were vaccinated with a WT1 peptide and OK-432 once every 2 weeks, for a total of seven times. RESULTS: Of the 24 patients, 18 completed the scheduled vaccinations. Sixteen patients had local skin symptoms and/or fever. In 1 patient, anaphylactic symptoms emerged at the time of the final injection, but these quickly subsided after the treatment. WT1-specific immunological responses were observed in 4 patients (22.2%). WT1 and HLA class I expression were confirmed in 100% and 85% of primary tumors, respectively. CONCLUSION: WT1 peptide vaccine therapy combined with OK-432 appears to be relatively safe for children. However further studies in a larger number of patients are necessary to confirm its safety and efficacy.

Prognostic significance of Wilms' tumor 1 expression in patients with pancreatic ductal adenocarcinoma.

The only current curative treatment for patients with pancreatic ductal adenocarcinoma (PDA) is surgical resection, and certain patients still succumb to disease shortly after complete surgical resection. Wilms' tumor 1 (WT1) serves an oncogenic role in various types of tumors; therefore, in the present study, WT1 protein expression in patients with PDA was analyzed and the association with overall survival (OS) and disease-free survival (DFS) time in patients with PDA was assessed following surgical resection. A total of 50 consecutive patients with PDA who received surgical resection between January 2005 and December 2015 at the Jikei University Kashiwa Hospital (Kashiwa, Chiba, Japan) were enrolled. WT1 protein expression in PDA tissue was measured using immunohistochemical staining. Furthermore, laboratory parameters were measured within 2 weeks of surgery, and systemic inflammatory response markers were evaluated. WT1 protein expression was detected in the nucleus and cytoplasm of all PDA cells and in tumor vessels. WT1 exhibited weak staining in the nuclei of all PDA cells; however, the cytoplasmic expression of WT1 levels was classified into four groups: Negative (n=0), weak (n=19), moderate (n=23) and strong (n=8). In patients with PDA, it was demonstrated that the OS and DFS times of patients with weak cytoplasmic WT1 expression were significantly prolonged compared with those of patients with moderate-to-strong cytoplasmic WT1 expression, as determined by log-rank test (P=0.0005 and P=0.0001, respectively). Furthermore, an association between the density of WT1-expressing tumor vessels and worse OS/DFS times was detected. Multivariate analysis also indicated a significant association between the overexpression of WT1 in PDA tissue and worse OS/DFS times. To the best of our knowledge, the present study is the first to demonstrate that moderate-to-strong overexpression of WT1 in the cytoplasm of PDA cells is significantly associated with worse OS/DFS times. Therefore, overexpression of WT1 in the cytoplasm of PDA cells may impact the recurrence and prognosis of patients with PDA following surgical resection. The results further support the development of WT1-targeted therapies to prolong survival in all patients with PDA.

Significance of Wilms' tumor 1 antigen as a cancer vaccine for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by a very poor prognosis, despite novel chemotherapeutic treatments. Moreover, the majority of PDA patients with complete surgical resection show recurrence within 5 years of resection. Therefore, new targeted cancer vaccines are urgently needed to extend PDA patient survival. The Wilms' tumor 1 (WT1) antigen was identified as an excellent antigen in a list of 75 tumor-associated antigens by a National Cancer Institute prioritization project based on several factors, such as therapeutic function. WT1-targeted cancer vaccines are not only efficient in the regression of PDA cells but also angiogenesis and immune suppressive cells, such as myeloid-derived suppressor cells (MDSCs), in the PDA microenvironment. Moreover, WT1 is increased in PDA cells; thus, WT1 may represent an effective therapeutic target for PDA, resulting in a survival benefit for PDA patients. A novel WT1 peptide with increased immunogenicity was developed and used in clinical trials to induce more successful clinical results. This review summarizes the clinical trials of PDA patients receiving WT1-targeted cancer vaccines.

Prognostic impact of preoperative serum interleukin-6 levels in patients with early-stage oral squamous cell carcinoma, defined by sentinel node biopsy.

Failure to detect recurrence and lymph node metastasis early represents a fundamental barrier to the improvement of survival rate in early stage oral squamous cell carcinoma (OSCC). The present study evaluated the association between serum interleukin-6 (IL-6) level and clinical outcomes in patients with early stage OSCC patients defined by sentinel node biopsy (SNB). A total of 53 patients with clinical stage I/II OSCC who underwent SNB were enrolled. SNB was determined by a radioisotope method, and was evaluated by histopathological examination and genetic analysis. Preoperative sera were measured for IL-6 by ELISA. In the clinical stage I/II patients, disease-free survival (DFS) was demonstrated to be higher in patients with negative SNB compared with patients with positive SNB. In total, 13 patients were demonstrated to exhibit lymph node metastasis by SNB or were reclassified to pathological stage T4 subsequent to analysis of the surgically resected specimens. Thus, 40 patients were diagnosed with early stage OSCC. Of these 40 patients, DFS of the patients with low serum IL-6 was significantly higher compared with the patients with high serum IL-6 (P=0.012). In 19 patients with negative SNB and low serum IL-6, the disease-free rate was 100%. These findings suggested that SNB staging and serum IL-6 level have a high prognostic value in patients with early stage OSCC. Additional investigation and longer follow-up times are warranted to improve understanding of the group of patients that may benefit from this procedure.