WT1 Trio Peptide-Based Cancer Vaccine for Rare Cancers Expressing Shared Target WT1.

No standard treatment has been established for most rare cancers. Here, we report a clinical trial of a biweekly WT1 tri-peptide-based vaccine for recurrent or advanced rare cancers. Due to the insufficient number of patients available for a traditional clinical trial, the trial was designed for rare cancers expressing shared target molecule WT1. The recruitment criteria included WT1-expressing tumors as well as HLA-A*24:02 or 02:01. The primary endpoints were immunoglobulin G (IgG) antibody (Ab) production against the WT1-235 cytotoxic T lymphocyte (CTL) epitope and delayed-type hypersensitivity (DTH) skin reactions to targeted WT1 CTL epitopes. The secondary endpoints were safety and clinical efficacy. Forty-five patients received WT1 Trio, and 25 (55.6%) completed the 3-month protocol treatment. WT1-235 IgG Ab was positive in 88.0% of patients treated with WT1 Trio at 3 months, significantly higher than 62.5% of the weekly WT1-235 CTL peptide vaccine. The DTH positivity rate in WT1 Trio was 62.9%, which was not significantly different from 60.7% in the WT1-235 CTL peptide vaccine. The WT1 Trio safety was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer. Fifteen (33.3%) patients achieved stable disease after 3 months of treatment. In conclusion, the biweekly WT1 Trio vaccine containing the WT1-332 helper T lymphocyte peptide induced more robust immune responses targeting WT1 than the weekly WT1-235 CTL peptide vaccine. Therefore, WT1-targeted immunotherapy may be a potential therapeutic strategy for rare cancers.

Complication rate, functional outcomes, and risk factors associated with carbon ion radiotherapy for patients with unresectable pelvic bone sarcoma.

BACKGROUND: To the authors' knowledge, carbon ion radiotherapy (CIRT) is one of the few curative treatments for unresectable pelvic bone sarcoma. The current study investigated the complications, functional outcomes, and risk factors of CIRT. METHODS: Of 112 patients who were treated with CIRT for unresectable pelvic bone sarcoma, the authors enrolled 29 patients who were without local disease recurrence or distant metastasis. The mean follow-up was 93 months. Complications, functional outcomes, and quality of life scores were assessed. Risk factors were analyzed, including the dose-volume histogram of the femoral head. RESULTS: Femoral head necrosis occurred in approximately 37% of patients, pelvic fractures were reported in 48% of patients, and neurological deficits were noted in 52% of patients. Femoral head necrosis was found to be significantly more prevalent among patients with periacetabular tumors (P = .018). The dose-volume histogram of the femoral head indicated tolerable volume percentages of the femoral head to be <33% for 40 grays (relative biological effectiveness) and 16% for 60 grays ( relative biological effectiveness). The mean Musculoskeletal Tumor Society score and Toronto Extremity Salvage Score were 53% and 64%, respectively, and the mean EuroQol 5 dimensions questionnaire index was 0.587. Patients aged >50 years and those with periacetabular tumors were found to have significantly lower Toronto Extremity Salvage Scores. CONCLUSIONS: Femoral head necrosis, pelvic fracture, and nerve damage are common complications with the use of CIRT for pelvic bone sarcoma. To prevent femoral head necrosis, the radiation dose to the femoral head should be kept below the estimated tolerance curve presented in the current study. The functional outcome is nearly equivalent to that of surgery. CIRT may be a promising alternative to surgery for patients with unresectable pelvic bone sarcoma.

Clinical Outcomes of Osteoarticular Extracorporeal Irradiated Autograft for Malignant Bone Tumor.

METHODS: We retrospectively reviewed 33 patients who underwent osteoarticular ECIA after bone tumor resection from 1988 to 2014. We investigated complications, radiographic changes by the International Society of Limb Salvage graft evaluation criteria, and functional outcomes according to the Musculoskeletal Tumor Society scoring system. RESULTS: Fifteen patients were reoperated upon due to infection (n = 9), protruding fixation implant (n = 4), or fracture of the grafted bone (n = 2). The average radiographic evaluation score was 66.4%, and the median functional score was 23 (77%). The radiographic score for the proximal humerus or proximal tibia was lower than that for the other locations. The functional score was not different among the autograft sites but was related to the radiographic score. CONCLUSION: Although osteoarticular ECIA is one of the reasonable surgical options for patients with tumors for which reliable prostheses are not available, we do not recommend osteoarticular ECIA as a routine procedure because of high complication rate.

Silent subperiosteal iliac hematoma with bone radiolucency in adolescent male athletes.

BACKGROUND: Subperiosteal hematoma of the iliac bone is reported as a rare disorder that specifically occurs in young patients after obvious trauma. This report presents seven cases of male adolescent athletes with subperiosteal iliac hematoma without blunt trauma that was identified as an incidental radiolucent lesion on the iliac bone mimicking neoplasm. The purpose of this report is to describe clinical features and radiological findings of silent subperiosteal hematoma of the iliac bone. SUBJECTS AND METHODS: We retrospectively reviewed the clinical data and radiological appearance of 7 patients who presented with subperiosteal hematoma of iliac bone. RESULTS: All seven patients had no obvious trauma and no serious symptoms. All patients were middle school or high school male students who regularly participated in vigorous sports activity; five soccer players, one baseball catcher, and one basketball player. The X-ray showed radiolucent lesion on the iliac bone. Lens-shaped mass without involvement of psoas muscle with ghost native cortex sign and overlying periosteal calcification on CT scan was characteristic radiological finding on subperiosteal iliac hematoma. CONCLUSION: Subperiosteal iliac hematomas can be caused by vigorous sports activity in adolescence. This condition is not always associated with serious symptom. It can be identified as a radiolucent lesion mimicking a neoplasm.

Trabectedin is a promising antitumour agent for synovial sarcoma.

Synovial sarcoma (SS) is an aggressive soft tissue tumour with poor prognosis. Using five human SS cell lines, we examined the cytotoxic effects of trabectedin (ET-743; Yondelis(®)), a novel marine natural product, which was approved in Europe for the treatment of soft tissue sarcomas (STS). The significant growth inhibitory effects were observed in all SS cell lines below nanomolar concentration of trabectedin. Furthermore, trabectedin significantly suppressed the tumour growth in xenograft models. Flow cytometer analysis in vitro and immunohistochemical analysis in vivo revealed its effect of cell cycle inhibition and apoptosis induction. We also examined the expression of ERCC1, 5 and BRCA1 in SS cell lines and clinical samples, and majority of them showed highly trabectedin-sensitive pattern as previously reported in other cancers. Our preclinical data indicated that trabectedin could be a promising therapeutic option for patients with SS.

Treatment outcomes of Japanese patients with Ewing sarcoma: differences between skeletal and extraskeletal Ewing sarcoma.

OBJECTIVE: The incidence of Ewing sarcoma is lower in non-Caucasian populations, compared with Caucasian populations, for unknown reasons. Most studies from western countries have reported improvement in outcomes following multi-agent chemotherapy, with no difference in outcome between skeletal and extraskeletal Ewing sarcoma. However, there are few studies of Ewing sarcoma in non-Caucasian populations, with especially few comparing outcomes between skeletal and extraskeletal Ewing sarcoma. Thus, the purpose of this study is to determine whether the outcomes and prognostic factors of Ewing sarcoma in the Japanese population are similar to those in Caucasian populations and to determine whether skeletal and extraskeletal Ewing sarcoma have similar outcomes in Japanese patients. METHODS: We retrospectively evaluated the outcomes of 74 Japanese patients with Ewing sarcoma treated between 1981 and 2011 from the Osaka University Orthopaedic Oncology Group. RESULTS: Extraskeletal Ewing sarcoma, tumors in the extremities, localized disease at presentation and diagnosis after 2000 were significantly associated with a favorable outcome. Among patients with localized disease at presentation, a significantly better outcome was observed for those with extraskeletal Ewing sarcoma, those who underwent a VDC/IE based or VAIA chemotherapy protocol, and those who were diagnosed after 2000. In the multivariable analyses, extraskeletal Ewing sarcoma was an independent predictor of increased overall survival among all patients and the subset of patients with localized disease. CONCLUSIONS: The outcome of patients with Ewing sarcoma in Japan has improved in the last decade. The outcomes and prognostic factors are similar for Japanese and Caucasian patients, though in this series of Japanese patients, a better prognosis was observed for patients with extraskeletal rather than skeletal Ewing sarcoma.

Tailored therapeutic strategies for synovial sarcoma: receptor tyrosine kinase pathway analyses predict sensitivity to the mTOR inhibitor RAD001.

We examined efficacy of the mTOR inhibitor RAD001 to seek novel therapies for synovial sarcoma (SS). Although RAD001 had significant anti-tumor effects, its sensitivity differed among cell lines. Phospho-receptor tyrosine kinase (RTK) array analyses revealed c-MET phosphorylation in highly mTOR inhibitor-sensitive cells and PDGFRα (which induces intrinsic resistance to mTOR inhibitor) activation in less sensitive cells. Combined treatment with RAD001 and the PDGFR inhibitor pazopanib showed anti-tumor effects in xenograft models with less sensitive cells. Thus, evaluating activated RTKs in clinical samples may predict sensitivity to mTOR inhibitors, raising the possibility of a tailored therapy for SS.

A novel angiomatoid epithelioid sarcoma cell line, Asra-EPS, forming tumors with large cysts containing hemorrhagic fluid in vivo.

BACKGROUND: Whereas we can use several human epithelioid sarcoma (ES) cell lines for basic and preclinical research, an angiomatoid ES cell line has not been reported to date. We have treated a case of an angiomatoid ES developing in the right upper extremity of a 67-year-old man. METHODS: An angiomatoid ES cell line, Asra-EPS was newly established and characterized for its morphology, growth rate and chromosomal analysis. Tumorigenicity of Asra-EPS cells was also analyzed in athymic nude mice. RESULTS: Asra-EPS cells were round, polygonal or spindle-shaped with an abundant cytoplasm and have been maintained continuously in vitro for over 150 passages during more than 15 months. These cells secreted cancer antigen 125 (CA 125), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) into the culture medium. Asra-EPS cells were tumorigenic when implanted in nude mice with tumors reaching a volume of 1000 mm3 at around 50 days. Histological features of tumors formed in mice were essentially the same as those of the original tumor, exhibiting a multinodular proliferation of eosinophilic epithelioid and spindle-shaped cells with prominent areas of hemorrhage and blood-filled cystic spaces strikingly corresponding to the potential of hemorrhagic cyst formation in the original tumor. They showed immunopositive staining for cytokeratins (AE1/AE3 and CAM5.2), epithelial membrane antigen (EMA), vimentin, CD31, CD34 and CA 125, but negative for integrase interactor 1 (INI-1) and factor VIII-related antigen. CONCLUSIONS: The established cell line represents a biologically relevant new tool to investigate the molecular pathology of human angiomatoid ES and to evaluate the efficacy of novel therapeutics both in vitro and in vivo.

SYT-SSX breakpoint peptide vaccines in patients with synovial sarcoma: a study from the Japanese Musculoskeletal Oncology Group.

In the present study, we evaluated the safety and effectiveness of SYT-SSX-derived peptide vaccines in patients with advanced synovial sarcoma. A 9-mer peptide spanning the SYT-SSX fusion region (B peptide) and its HLA-A*2402 anchor substitute (K9I) were synthesized. In Protocols A1 and A2, vaccines with peptide alone were administered subcutaneously six times at 14-day intervals. The B peptide was used in Protocol A1, whereas the K9I peptide was used in Protocol A2. In Protocols B1 and B2, the peptide was mixed with incomplete Freund's adjuvant and then administered subcutaneously six times at 14-day intervals. In addition, interferon-α was injected subcutaneously on the same day and again 3 days after the vaccination. The B peptide and K9I peptide were used in Protocols B1 and B2, respectively. In total, 21 patients (12 men, nine women; mean age 43.6 years) were enrolled in the present study. Each patient had multiple metastatic lesions of the lung. Thirteen patients completed the six-injection vaccination schedule. One patient developed intracerebral hemorrhage after the second vaccination. Delayed-type hypersensitivity skin tests were negative in all patients. Nine patients showed a greater than twofold increase in the frequency of CTLs in tetramer analysis. Recognized disease progression occurred in all but one of the nine patients in Protocols A1 and A2. In contrast, half the 12 patients had stable disease during the vaccination period in Protocols B1 and B2. Of note, one patient showed transient shrinkage of a metastatic lesion. The response of the patients to the B protocols is encouraging and warrants further investigation.

Case of an unusual clinical and radiological presentation of pulmonary metastasis from a costal chondrosarcoma after wide surgical resection: a transbronchial biopsy is recommended.

Chondrosarcomas are the most frequently occurring primary malignant chest wall tumors. Furthermore, the lungs serve as the most frequent sites for metastases. Pulmonary metastases from sarcomas usually appear as round nodules of varying sizes on roentgenograms. Here, we report an unusual clinical and radiographic presentation of pulmonary metastasis from a costal chondrosarcoma. Bilateral pulmonary metastases developed soon after wide surgical resection. Thoracic computed tomography revealed unusual radiological findings: consolidation accompanied with ground-glass opacity. To confirm the metastasis, we recommend a transbronchial biopsy in cases where unusual pulmonary findings are detected.

Extracorporeally irradiated autograft-prosthetic composite arthroplasty with vascular reconstruction for primary bone tumor of the proximal tibia.

The proximal tibia is a common site for primary bone tumors. Proximal tibial tumors may invade the adjacent soft-tissue by destroying the cortex and may further invade neurovascular bundles. We treated a patient with primary bone tumor of the proximal tibia with neurovascular invasion by extracorporeally irradiated autograft-prosthetic composite arthroplasty with vascular reconstruction. In cases of concomitant allograft arthroplasty and vascular reconstruction, we recommend that vascular reconstruction be performed before arthroplasty to minimize ischemia time. Good oncological and functional outcomes were achieved 75 months after surgery. Therefore, this reconstruction technique can be considered as a good treatment option.

Synovial sarcoma is a stem cell malignancy.

Synovial sarcoma (SS) is a malignant soft tissue tumor characterized by its unique t(X;18)(p11;q11) chromosomal translocation leading to the formation of the SS18-SSX fusion gene. The resulting fusion protein product is considered to play as an aberrant transcription factor and transform target cells by perturbing their gene expression program. However, the cellular origin of SS is highly debated. We herein established two novel human SS cell lines, named Yamato-SS and Aska-SS, and investigated their biological properties. We found the self-renewal ability of these cells to generate sarcospheres, to form tumors in serial xenotransplantation and reconstitute the tumor phenotypes without fractionation by any surface markers. Both SS cells as well as clinical tissue specimens from 15 patients expressed the marker genes-associated stem cell identity, Oct3/4, Nanog, and Sox2. We also found that both SS cells displayed limited differentiation potentials for mesenchymal lineages into osteocytes and chondrocytes albeit with the expression of early mesenchymal and hematopoietic lineage genes. Upon SS18-SSX silencing with sequence-specific siRNAs, these SS cells exhibited morphological transition from spherical growth in suspension to adherent growth in monolayer, additional expression of later mesenchymal and hematopoietic lineage genes, and broader differentiation potentials into osteocytes, chondrocytes, adipocytes, and macrophages in appropriate differentiation cocktails. Collectively, these data suggest that a human multipotent mesenchymal stem cell can serve as a cell of origin for SS and SS is a stem cell malignancy resulting from dysregulation of self-renewal and differentiation capacities driven by SS18-SSX fusion protein.

Soft-tissue sarcomas in the inguinal region may present as deep vein thrombosis.

Swelling, pain, and discoloration in the affected extremities are classic symptoms of deep vein thrombosis (DVT), although these symptoms are often unreliable for diagnosis, and wide differential diagnoses exist. Soft-tissue sarcomas arising in the inguinal region may encompass or be adjacent to femoral vessels and result in venous obstruction, mimicking symptoms of DVT. We present two cases of soft-tissue sarcomas in the inguinal region that were initially misdiagnosed as spontaneous DVT and administered unnecessary long-term anticoagulation therapy. In patients diagnosed with unprovoked DVT, especially young patients aged <45 years, we recommend a careful physical examination and ultrasonography in the inguinal region to prevent overlooking possible underlying malignancy, as delays in diagnosis may affect prognosis.

Prognostic significance of phosphorylated FOXO1 expression in soft tissue sarcoma.

BACKGROUND: Forkhead box O1 (FOXO1; forkhead in rhabdomyosarcoma, FKHR) is a key transcription factor that regulates the cell cycle and apoptosis, and therefore is considered to be involved in cell transformation and tumorigenesis. Expression of FOXO1 in soft tissue sarcoma (STS) and its correlation with clinicopathological factors and prognostic significance were evaluated. METHODS: Expression of phosphorylated FOXO1 (p-FOXO1) in localized STS from 84 adult patients, 50 male and 34 female, aged 15-89 (median 54) years, was evaluated by immunohistochemistry. Staining intensity of p-FOXO1 in the tumors was judged separately for the nucleus and cytoplasm and categorized as follows: level 0, absent or faint staining; level 1, weaker than that of endothelial cells in the same specimen; and level 2, equal to or stronger than that of endothelial cells. RESULTS: Twenty-three (27.3%), 26 (31.0%), and 35 (41.7%), and 32 (38.1%), 30 (35.7%), and 22 (26.2%) of the tumors showed level 0, 1, and 2 expression of p-FOXO1 for the nucleus and cytoplasm, respectively. Nuclear p-FOXO1 expression correlated with mitotic count, and cytoplasmic p-FOXO1 expression with histological subtype, mitotic count, cellularity, myxoid change, Ki-67 labeling index, histological grade, American Joint Committee on Cancer stage, and patient age. Multivariate analysis revealed nuclear and cytoplasmic p-FOXO1 expression, mitotic count, and tumor size to be independent prognostic indicators for overall survival, and cytoplasmic p-FOXO1 expression for disease-free survival, respectively. CONCLUSIONS: The prognostic significance of p-FOXO1 expression level in STS was demonstrated.

Prognostic significance of CD100 expression in soft tissue sarcoma.

BACKGROUND: CD100, a class IV semaphorin, promotes angiogenesis, invasive growth, proliferation, and antiapoptosis of cancer cells in vitro. The expression of CD100 in soft tissue sarcoma (STS) and its correlation with clinicopathologic factors and prognostic significance were evaluated. METHODS: Expression levels of CD100 in patients with localized STS were evaluated immunohistochemically on paraffin-embedded sections from 81 patients, including 47 men and 34 women with a median age of 54 years. Staining intensity was categorized into weaker than (level 1) or equal to that of lymphocytes with a rate of <10% stained tumor cells (level 2) or >10% stained tumor cells (level 3). Ki-67 staining was performed in parallel. RESULTS: Forty-two tumors (52%) had level 1 CD100 expression, 18 tumors (22%) had level 2 CD100 expression, and 21 tumors (26%) had level 3 CD100 expression. Tumors that had level 2 and 3 CD100 expression were correlated significantly with higher mitotic count, cellularity, ratio of necrosis, and Ki-67 labeling index (LI) compared with tumors that had level 1 CD100 expression. There was no correlation between CD100 expression and other characteristics. Among the 3 levels of CD100 expression, higher expression levels were correlated with poorer overall and disease-free survival. Multivariate analysis revealed that CD100 expression (levels 1 and 2 vs level 3) and tumor size (5 cm) were independent prognosticators for overall survival (P < .05 for both), and CD100 expression (levels 1 and 2 vs level 3) was an independent prognosticator for disease-free survival (P < .05). CONCLUSIONS: The results from this study indicated the demonstrated prognostic significance of CD100 expression in STS.

Prognostic significance of activated AKT expression in soft-tissue sarcoma.

PURPOSE: AKT is a serine/threonine kinase which is important in tumorigenesis. Several molecules involved in AKT pathway are dysregulated in various kinds of human cancers. PATIENTS AND METHODS: Ninety-three patients (53 males and 40 females), ages ranging from 19 to 77 years (median, 57 years), with localized soft-tissue sarcomas arising in the trunk and extremities, were analyzed. Immunoperoxidase procedure (avidin-biotin complex method) was done on paraffin-embedded sections with anti-phosphorylated AKT (Thr308), anti-phosphorylated p44/42 extracellular signal-regulated kinase 1 and 2 (ERK1/2) (Thr202/Tyr204), anti-phosphorylated forkhead in rhabdomyosarcoma (FKHR) (Ser256), and anti-Ki 67 antibodies. Expression levels of phosphorylated AKT (p-AKT), phosphorylated ERK1/2 (p-ERK1/2), and phosphorylated FKHR (p-FKHR) were categorized as either weaker (level 1) or equal to or stronger (level 2) compared with those in the endothelial cells of the same specimens. Percentage of cells showing intranuclear staining with Ki-67 was shown as the Ki-67 labeling index (LI). Cases were divided into two groups: level 1, Ki-67 LI < 20%; level 2, Ki-67 LI > or = 20%. RESULTS: Twenty-six (28.0%), 6 (6.5%), and 46 (44.1%) of the tumors showed level 2 expression for p-AKT, p-ERK1/2, and Ki-67 LI, respectively. Tumors with level 2 p-AKT expression showed a higher ratio of level 2 p-FKHR expression (P < 0.01). Multivariate analysis revealed p-AKT expression and Ki-67 LI to be independent prognosticators for overall survival, and p-AKT expression for disease-free survival. CONCLUSION: p-AKT expression level is a significant prognosticator in soft-tissue sarcoma.

Estimation of particulate organic carbon flux in relation to photosynthetic production in a shallow coastal area in the Seto Inland Sea.

Sediment trap experiments were carried out three times from 1999 to 2000, in the western part of the Seto Inland Sea (Suo-Sound), Japan. We investigated both the particulate flux and the composition of chemical substances in the sediment trap samples. Based on the results, we discuss the origin of particulate organic carbon (POC) collected by the sediment traps in a coastal area. Moreover, we purposed to estimate the flux of the portion of the POC that is derived from phytoplankton photosynthesis. The fluxes of POC varied between 677 and 3424 mgC m(-2) d(-1). Significant positive correlations between POC and aluminum (Al) fluxes suggested that these components show almost the same behaviour. The mean value of the Al flux was about eight times higher than that of Al burial rates on the sediment surface. Therefore, it seems that the POC flux observed with the sediment traps was considerably overestimated. Moreover, judging from the fact that Al is a typical terriginous element, it seems that most of the POC collected in the sediment traps derived from the re-suspended surface sediment or sediment transported laterally from shallow flanks such as intertidal mudflats. The fluxes of chlorophyll a (Chl a) were independent of the POC fluxes, and a relatively consistent correlation was found between Chl a abundance in the water column and the Chl a flux. Moreover, surface sediment Chl a content was approximately 100 times lower than that of suspended matter. Therefore, resuspension and terriginous contributions to Chl a collected in sediment traps are likely to be negligible. The POC content in the trap samples varied between 22.4 and 70.7 mg g(-1) dry weight. The variations of POC contents were positively correlated with the Chl a contents: POC(mg g(-1))=76.5 x Chl a(mg g(-1)) + 26.0 (r=0.95, p<0.01, n=9). This result shows that POC contents strongly corresponded with phytoplankton and their debris. It was also considered that the fraction of POC derived from phytoplankton primary production could be estimated as Chl a content times a certain factor. In this study, we estimated the flux of the portion of the POC originating from phytoplankton production by multiplying the Chl a fluxes by 76.5 (the mean POC:Chl a ratio in the trap samples). These values varied between 308 and 758 mgC m(-2) d(-1), and accounted for 35.1+/-21.2% of total POC flux. Although the amount of POC that originates from phytoplankton photosynthesis was a small portion of total POC flux, it seems to be a large portion of potential primary production in the water column.