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The characteristics of the tumor immune microenvironment remains unclear in liposarcomas, and here we aimed to determine the prognostic impact of the tumor immune microenvironment across separate liposarcomas subtypes. A total of 70 liposarcoma patients with three subtypes: myxoid liposarcoma (n = 45), dedifferentiated liposarcoma (n = 17), and pleomorphic liposarcoma (n = 8) were enrolled. The presence of tumor infiltrating lymphocytes (CD4+ , CD8+ , FOXP3+ lymphocytes) and CD163+ macrophages and expression of HLA class I and PD-L1 were assessed by immunohistochemistry in the diagnostic samples; overall survival and progression-free survival were estimated from outcome data. For infiltrating lymphocytes and macrophages, dedifferentiated liposarcoma and pleomorphic liposarcoma patients had a significantly higher number than myxoid liposarcoma patients. While myxoid liposarcoma patients with a high number of macrophages were associated with worse overall and progression-free survival, dedifferentiated liposarcoma patients with high macrophage numbers showed a trend toward favorable prognosis. Expression of HLA class I was negative in 35 of 45 (77.8%) myxoid liposarcoma tumors, whereas all dedifferentiated liposarcoma and pleomorphic liposarcoma tumors expressed HLA class I. The subset of myxoid liposarcoma patients with high HLA class I expression had significantly poor overall and progression-free survival, while dedifferentiated liposarcoma patients with high HLA class I expression tended to have favorable outcomes. Only four of 17 (23.5%) dedifferentiated liposarcomas, two of eight (25%) pleomorphic liposarcomas, and no myxoid liposarcoma tumors expressed PD-L1. Our results demonstrate the unique immune microenvironment of myxoid liposarcomas compared to other subtypes of liposarcomas, suggesting that the approach for immunotherapy in liposarcomas should be based on subtype.

Objectives There are few reports of aortic leiomyosarcoma arising from the mural region with extraluminal expansion. We experienced 3 cases of mural leiomyosarcoma, 2 originating from the thoracic aorta and 1 from the abdominal aorta, causing back pain with spinal involvement. The objectives of this study were to describe the clinical presentation and course of these 3 cases. Methods We retrospectively reviewed 3 consecutive patients with primary tumors of the aorta who underwent tumor resection and were anatomopathologically diagnosed with mural leiomyosarcoma at Niigata University Medical and Dental Hospital between July 2012 and June 2015. Results Three male patients were aged from 47 to 61 years. For all patients, the chief complaint was back pain attributed to spinal involvement with direct invasion of the tumor. The extraluminal expansion was shown around the aorta, and pathological vertebral fractures and spinal canal stenosis were observed in all cases. For one patient, tumor resection and vertebral reconstruction were performed after aortic replacement through endovascular stenting. Another patient presented with progressive paraplegia, and cytoreductive surgery was performed. The remaining patient was diagnosed with a ruptured abdominal aortic aneurysm resulting in open surgery and the implantation of an aortic prosthesis. All patients were resistant to any adjuvant therapy and died between 4 and 19 months after surgery. Conclusions In all cases, spinal involvement was shown. Back pain is a considerable initial presentation of aortic mural leiomyosarcoma with extraluminal expansion. To our knowledge, this is the first report to show in minute detail the clinical course of mural leiomyosarcoma of the aorta with spinal involvement.

Background: Musculoskeletal sarcomas (MSSs) are rare cancers and often aggressive tumors that originate from mesenchymal tissue. Patients with advanced MSS often report difficulties with symptom burden, which can reduce their health-related quality-of-life. Objective: The aim of this study was to describe the patterns of the physical symptoms of MSS patients in the palliative setting and to detail the palliative treatment used in the last two weeks of life. Design: Retrospective study using the electronic patient records from a single institution. Setting/Subjects: A retrospective study was carried out in a sample of 46 consecutive MSS patients with locally advanced/metastatic disease, who were hospitalized and died in our department. The median age of these patients was 56 years at death. Measurements: Symptom burden and medical intervention during the last two weeks of life were collected. Results: The most frequent physical symptoms were pain and dyspnea in 93% and 78% of patients, respectively, while only 17% of patients suffered from nausea. A total of 98% of patients required opioids, and most patients were treated with morphine through either subcutaneous or intravenous continuous injection. Nonsteroidal anti-inflammatory drugs and acetaminophen were administered to 79% of patients. Corticosteroids were administered for the relief of dyspnea to 83% of patients. Of the patients, 46% received palliative chemotherapy within the last two weeks of life, and the oral treatment was continued until a median of 5.6 days before death. In addition, 39% of patients received a sedative treatment during the last two weeks of life for uncontrolled refractory symptoms. Conclusions: The symptom burden experienced by advanced MSS patients is profound at the end of life for all palliative approaches. Therefore, palliative medicine is an important and even crucial component of the continuum of care, allowing for aggressive symptom management with a variety of medical interventions, including palliative sedation.

Bone and soft tissue tumors are derived from mesenchymal cells, and they are hard to treat. Receptor-activator of nuclear factor-kappa B ligand (RANKL) is an essential cytokine for osteoclast differentiation and activation and is expressed on the surface of osteoblasts or stromal cells. In this study, to explore the potential of denosumab treatment for soft tissue tumors, we analyzed the expression profiles of RANKL mRNA in 425 tumor specimens of 33 histological types by real-time RT-PCR. Denosumab is a monoclonal antibody that prevents the binding of RANKL to receptor-activator of nuclear factor-kappa B (RANK). For comparison, the relative expression levels of RANK and osteoprotegerin (OPG) mRNAs were also measured. OPG functions as a soluble decoy receptor for RANKL. Higher expression levels of RANKL mRNA were detected in calcifying aponeurotic fibroma, fibrosarcoma, calcifying epithelioma, myositis ossificans, heterotopic calcification, giant cell tumor of the tendon sheath (GCTTS), and pigmented villonodular synovitis (PVNS), compared with the levels of other tumor types. Moreover, the expression levels of RANK mRNA were highest in GCTTS, followed by myositis ossificans and PVNS, whereas the expression levels of OPG mRNA were greatly varied among these histological types. We then analyzed RANKL protein expression by immunohistochemistry in 57 tumor specimens with higher expression levels of RANKL mRNA. RANKL-positive cells were detected in GCTTS, PVNS, myositis ossificans, heterotopic calcification, and calcifying aponeurotic fibroma. In conclusion, RANKL is expressed in subsets of soft tissue tumors with calcification, and denosumab is a potential therapeutic option for soft tissue tumors expressing RANKL.

Fine needle aspiration cytology (FNAC) and core needle biopsy (CNB) can provide tissue samples for the diagnoses of bone and soft tissue tumors. We evaluated the diagnostic accuracy of FNAC and CNB, the usefulness of the image-guided needle procedures, and assessed whether a discordance can influence the prognosis.We retrospectively examined the accuracy rates of FNAC and CNB procedures by analyzing results of 405 specimens of 389 patients. We evaluated the diagnostic accuracy of FNAC and CNB, compared the clinical effectiveness between the image-guided procedures and the blind procedures, and also compared survival rates between the true positive and the false negative cases for patients with high-grade malignant tumors.The accuracy rates of FNAC were 86.6% and 93.8% for CNB. In cases with non-palpable masses, there were significantly low sampling error rates in the image-guided procedure. There were no significant differences in progression-free-survival and overall survival rates in patients between the false negative and true positive cases.Both FNAC and CNB procedures had high accuracy rates. Limited to cases with no palpable masses, the image-guided procedure had a low sampling error rate and was an effective method for obtaining tissue samples.

Stanniocalcin-1 (STC1) is a glycoprotein that was originally identified as a calcium-regulating hormone in bony fish, and that has been shown to also critically mediate cell growth, proliferation and differentiation, etc. in humans. Increased STC1 expression levels have been previously detected in different human cancer samples, such as those isolated from lung, breast, ovary, colon, pancreas, and liver tumors; thus, the present study evaluated STC1 expression in various soft-tissue tumors. STC1 mRNA isolated from 16 cell lines and 186 clinical soft-tissue tumor specimens were analyzed via quantitative real-time PCR, and the calculated expression levels were normalized to those exhibited by STC1-expressing MDA-MB-231 cells. The results of these analyses did not reveal any specific histological tumor types that displayed significantly increased STC1 expression; however, they did not indicate that STC1 expression was significantly higher in malignant compared to benign soft-tissue tumors. Furthermore, in adipocytic tumors, STC1 expression in dedifferentiated liposarcomas was found to be highest and lowest in lipoma tissues, respectively, suggesting that adipocytic tumors may express increasely high levels of STC1 mRNA as they become histologically more advanced. STC1 expression correlates with the malignancy grade in soft-tissue tumors.

The association between the immune status within the tumor microenvironment and prognosis in synovial sarcoma is not well understood. We aimed to investigate the tumor immune microenvironment and analyze its prognostic impact for patients with synovial sarcoma. A total of 36 primary patients who were treated in our institution were retrospectively evaluated. Infiltration of lymphocytes (CD4+, CD8+, and FOXP3+), CD163+ macrophages, and expression of human leukocyte antigen (HLA) class I and programmed death ligand 1 (PD‐L1) were evaluated by immunohistochemistry. Moreover, we investigated PD‐L1 and programmed death ligand 2 (PD‐L2) mRNA expression in 19 of the 36 cases, using real‐time PCR. The Kaplan‐Meier method was used to estimate overall survival and progression‐free survival. Infiltration of lymphocytes and macrophages varied among the patients. Furthermore, the expression of HLA class I was negative or downregulated in 11 specimens. No PD‐L1 expression was observed using immunohistochemistry. Moreover, although PD‐L1 mRNA expression was observed in 18 of 19 specimens, the expression level was low. A higher infiltration of CD8+ or FOXP3+ lymphocytes in patients was associated with a favorable overall survival. In addition, a higher infiltration of CD163+ macrophages indicated a significantly worse overall and progression‐free survival. Infiltration of CD4+ lymphocytes, HLA class I, PD‐L1, and PD‐L2 expression were not associated with patient prognosis. This represents the first report investigating the tumor immune microenvironment as a prognostic factor in synovial sarcoma, indicating that CD163+ macrophages are associated with tumor progression. Our results underscore the clinical significance of the tumor immune microenvironment in synovial sarcoma.

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.

Background Reconstruction of the extensor mechanism after resection of the proximal tibia is challenging, and several surgical procedures are available. The purpose of this study was to determine the outcome of the fibular transposition technique for reconstruction of the extensor mechanism of the knee after proximal tibial resection. Methods We retrospectively reviewed five consecutive patients who underwent resection of the proximal tibia with prosthetic reconstruction and reconstruction of the extensor using fibular transposition between 1997 and 2011. There were two female and three male patients with a mean age of 50years (range, 27 to 76years). A follow-up evaluation included both passive and active range of motion, extensor lag, the MSTS score and complications. Results Patients were followed up for 93months (range, 44 to 160months). The mean extensor lag and active flexion were four degrees (range, 0 to 10°) and 103° (range, 85 to 110°), respectively. The mean MSTS score was 80% (range, 73 to 90%). All patients were able to ambulate without crutches at the latest follow-up. Conclusions The utilization of the fibular transposition technique is a simple, reliable, and successful procedure for extensor reconstruction after proximal tibial resection.

The prognosis of alveolar soft part sarcoma is poor, despite the slow growth of the tumor. A number of cases with spontaneous regression of this rare tumor have been reported. Although the mechanisms underlying spontaneous regression remain uncertain, local immune reaction may be a possible contributing factor. Immunohistochemical expression of human leukocyte antigen (HLA) class I, cluster of differentiation (CD) 3, CD4, CD8, CD20, CD45, CD56, CD68, CD138 and CD163 were assessed in a series of 10 alveolar soft part sarcomas, and the expression profiles were associated with patients' clinicopathological parameters. Expression of HLA class I was observed in almost all the tumor cells of all cases. CD8(+) cells were identified in all tumors with varying densities. Moderate infiltration of CD8(+) cells was detected in three patients; one of these patients survived with long-term tumor remission. Infiltration of CD10(+), CD20(+), CD56(+) or CD138(+) cells was not revealed in all tumors. Moderate-diffuse infiltration of CD163(+) cells was observed in all tumors. To the best of our knowledge, the present study represents the first report of intratumoral immune cells in alveolar soft part sarcoma. Frequent expression of HLA class I in tumor cells was observed. CD8(+) cells were identified at various densities and CD163(+) cells were observed in alveolar soft part sarcoma. Moderate infiltration of CD8(+) cells in patients with a good prognosis may indicate the antitumor effects of immune cells in alveolar soft part sarcoma.

AimsWe analyzed the long-term outcomes of patients observed over ten years after resection en bloc and reconstruction with extracorporeal irradiated autograftsPatients and MethodsThis retrospective study included 27 patients who underwent resection en bloc and reimplantation of an extracorporeal irradiated autograft. The mean patient age and follow-up period were 31.7 years (9 to 59) and 16.6 years (10.3 to 24.3), respectively. The most common diagnosis was osteosarcoma (n = 10), followed by chondrosarcoma (n = 6). The femur (n = 13) was the most frequently involved site, followed by the tibia (n = 7). There were inlay grafts in five patients, intercalary grafts in 15 patients, and osteoarticular grafts in seven patients. Functional outcome was evaluated with the Musculoskeletal Tumor Society (MSTS) scoring system.ResultsThere were no recurrences in the irradiated autograft and the autograft survived in 24 patients (88.9%). Major complications included nonunion (n = 9), subchondral bone collapse (n = 4), and deep infection (n = 4). Although 34 revision procedures were performed, 25 (73.5%) and four (11.8%) of these were performed less than five years and ten years after the initial surgery, respectively. The mean MSTS score at the last follow-up was 84.3% (33% to 100%).ConclusionConsidering long-term outcomes, extracorporeal irradiated autograft is an effective method of reconstruction for malignant musculoskeletal tumours Cite this article: Bone Joint J 2019;101-B:1151–1159

Background Patellar tendon autograft after intraoperative extracorporeal radiotherapy has been used for reconstruction of the extensor mechanism following limb-sparing wide tumor resection around the knee. The purpose of this study was to determine the clinical outcome of this reconstruction technique. Methods We retrospectively reviewed six consecutive patients with peripatellar tendon and proximal tibial sarcoma who underwent reconstruction of the knee extensor mechanism. The resection area was planned to be contained with the patellar tendon in order to obtain a wide margin. First, the patella was osteotomized at the midline, and the inferior half of patella, patellar tendon, and tibial tuberosity were excised en bloc. The resected segments were devitalized with intraoperative extracorporeal radiotherapy and reimplanted into the original site. A follow-up evaluation included an assessment of the range of motion, extensor lag, the International Society of Limb Salvage score, and complications. Results Six patients were followed up for 121–270 months. One patient underwent an additional reconstruction with total knee arthroplasty due to a collapse of the tibial subchondral bone. A supracondylar fracture of the femur occurred in two patients, and a delayed union of the osteosynthesis site of the tibial shaft was observed in one patient. At the latest follow up, extensor lag had a median of five degrees, and International Society of Limb Salvage scores had a median of 83%. No local recurrence or rupture of the patellar tendon was observed. Conclusions Reconstruction of the knee extensor mechanism using a patellar tendon treated with intraoperative radiotherapy is a reliable and successful method.

Myoepithelial carcinoma of the breast is an extremely rare tumor composed entirely of malignant spindle cells with myoepithelial differentiation. The majority of previously reported cases have mainly described the clinicopathological features of the disease, and few have presented cytogenetic data. We herein present the case of a 48-year-old woman who was admitted with a left-sided breast lump in the inner upper quadrant that was initially diagnosed as a myoepithelioma with potentially malignant disorder. At 12 months after resection, she complained about a newly developed solid mass in the subareolar region of the ipsilateral breast that was diagnosed as an invasive ductal carcinoma. In addition, 16 months after the initial admission, a re-growing remnant lesion recurred in the inner upper quadrant and was ultimately diagnosed as a myoepithelial carcinoma. Lymph node metastasis of the myoepithelial carcinoma was also observed in her left axillary region 11 months after local recurrence. A cytogenetic analysis showed recurring specific chromosomal alterations both in the locally recurrent and in the lymph-node metastatic lesion: 48, XX, t(5;18)(q13;q23),del(6)(q?),+14. + mar1. To our knowledge, this is the first published report of clonal chromosomal rearrangements in myoepithelial carcinoma of the breast that presented as metachronic double cancer with invasive ductal carcinoma in the ipsilateral breast.

Undifferentiated round cell sarcomas, which carry the BCOR-CCNB3 fusion gene, have traditionally been called “Ewing-like sarcomas” and have been treated with Ewing sarcoma treatment regimens. While there has been emerging evidence that these BCOR-CCNB3 sarcomas (BCS) are molecularly distinct from Ewing sarcomas, the biology of these tumors is poorly understood, and patients have few options when they do not respond to Ewing sarcoma-based therapies. Here, we report the establishment of a novel BCS cell line, which we utilized to identify a unique BCS-specific therapeutic vulnerability using a hybrid screening approach consisting of a high-throughput drug screen combined with a CRISPR/Cas9-based knock out genetic screen. The BCOR-CCNB3-positive sarcoma cell line NBC-1 was established from the tumor tissue obtained from an 18-year-old male with a soft tissue tumor of the buttock with lymph node metastasis, and the expression of the fusion BCOR-CCNB3 transcript was confirmed by RT-PCR. An in vitro high-throughput screening was performed using the L1300 library of 2570 drugs, where the cells were incubated for 48 hours with drug before cell viability was measured. This drug screen identified 148 drugs, which included 84 FDA-approved drugs. While these drugs predictably included a significant number of conventional cytotoxic chemotherapy drugs currently used to treat BCS, such as vincristine and doxorubicin, several other drug classes emerged, identifying twenty tyrosine kinase inhibitors, proteasome inhibitors such as Carfizomib, Ixazomib, and Bortezomib, and a histone deacetylase inhibitor, Panobinostat, all of which had distinctly higher cytotoxicity compared to the Ewing sarcoma cell controls. Next, we utilized a custom-made CRISPR panel consisting of 9771 sgRNAs targeting 1221 genes where we were able to identify a specific pharmacologic inhibitor in the literature. Using this library, we carried out a genetic loss-of-function screen to identify genes that are required for the proliferation of BCS. Based on these initial findings, we were able to demonstrate that BCS tumors have distinct vulnerabilities from Ewing sarcoma, suggesting that a BCS-specific therapeutic approach is necessary. Citation Format: Naoki Oike, Hiroyuki Kawashima, Akira Ogose, Takashi Ariizumi, Yudai Murayama, Hajime Umezu, Molishree Joshi, Colin Sempeck, Deandra Warker, Avery Bodlak, Naoto Endo, Masanori Hayashi. High-throughput drug screening combined with a druggable target CRISPR drop-out screen identifies therapeutic vulnerabilities of BCOR-CCNB3 fusion positive sarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3411.

The value of routine blood tests in malignant soft tissue tumors remains uncertain. To determine if these tests can be used for screening, the routine pretreatment blood test findings were retrospectively investigated in 359 patients with benign and malignant soft tissue tumors. Additionally, the prognostic potential of pretreatment blood abnormalities was evaluated in patients with soft tissue sarcomas. We compared clinical factors and blood tests findings between patients with benign and malignant soft tissue tumors using univariate and multivariate analysis. Subsequently, patients with malignant tumors were divided into two groups based on blood test reference values, and the prognostic significance of each parameter was evaluated. In the univariate analysis, age, tumor size, and tumor depth were significant clinical diagnostic factors. Significant increases in the granulocyte count, C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), and γ-glutamyl transpeptidase (γ-GTP) levels were found in patients with malignant soft tissue tumors. Multiple logistic regression showed that tumor size and ESR were independent factors that predicted malignant soft tissue tumors. The Kaplan-Meier survival analysis revealed that granulocyte counts, γ-GTP levels, and CRP levels correlated significantly with overall survival. Thus, pretreatment routine blood tests are useful diagnostic and prognostic markers for diagnosing soft tissue sarcoma.

The animal cap-the presumptive ectoderm of the blastula embryo-can differentiate into a variety of tissues belonging to the three germ layers following exposure to specific inducers. The "animal cap assay" was devised based on the pluripotency of presumptive ectodermal cells and enabled many important discoveries in the field of embryonic induction and cell differentiation. Using this system, investigators can test multiple factors in solution simultaneously to determine their inducing activities qualitatively, quantitatively, and synergistically. Furthermore, after dissociation and induction, reaggregated animal cap cells can be induced to form higher-order organs. This protocol details preoperative preparations, followed by the basic animal cap assay. Advanced protocols for the induction of kidney, pancreas, and heart are also described.

Background A solitary fibrous tumour (SFT) is an unusual neoplasm typically found in soft tissues. Although SFTs can arise in the bones, they very rarely arise in the vertebral arch. Here, we describe a case of a SFT that arose in the vertebral arch of the first lumbar (L1) spinal vertebrae and mimicked osteosarcoma. Case presentation A 49-year-old woman presented with a 2-month history of lower back pain and a lumbar region mass. Magnetic resonance imaging demonstrated a heterogeneously enhanced mass in the L1 vertebral arch. The patient received neoadjuvant chemotherapy, followed by a surgical procedure comprising an anterior spinal fusion and en bloc resection. Histologically, our initial diagnosis was osteosarcoma. The postoperative course was uneventful, and the patient received adjuvant chemotherapy. However, the tumour metastasised to the lung 5 years after the first surgery, and a second surgery was performed for lung tumour resection. The histology of the metastatic lung tumour appeared similar to that of the malignant SFT, and the specimen from the first surgery was re-examined. Immunohistochemically, the tumour was positive for STAT6. Reverse transcription-polymerase chain reaction revealed a NAB2-STAT6 fusion gene, thus confirming our final diagnosis of malignant SFT. The patient died of disease progression 8 years after the first surgery; however, there was no evidence of local recurrence. Conclusions Malignant SFT in the vertebral arch is extremely rare and very difficult to distinguish histologically an osteoid from lace-like collagen. STAT6 immunostaining is useful for distinguishing malignant SFTs from other neoplasms. Although it is difficult to completely resect a SFT arising from the spine, we demonstrated the feasibility of an en bloc resection of spinal tumours arising from posterior elements, without local recurrence.

Bone leiomyosarcoma is a rare primary osseous malignant tumor with a high metastatic potential. Similar to other bone sarcomas, high histological grade and tumor stage are predictive of a poor outcome. We herein present our experience with treating a 64-year-old woman with bone leiomyosarcoma accompanied by multiple bone metastases. A biopsy revealed occasional osteoclast-like giant cells. In addition to radiation therapy, the osteoclastogenesis inhibitor denosumab was administered but the patient did not undergo adjuvant chemotherapy or surgery. Good clinical and short-term radiological responses to denosumab have been observed for 2 years. Therefore, denosumab may represent a viable treatment option without the need for adjuvant chemotherapy.

Background: The prognosis of patients with metastatic soft tissue sarcoma (STS) remain poor. Although immunotherapies have been attempted in patients with STS, the prognostic significance of the immune status within the tumor microenvironment in STS is not well understood. The purpose of this study is to investigate the tumor immune microenvironment and evaluate its prognostic impact for patients with STS. Material and Methods: We retrospectively evaluated the tumor immune microenvironment in 116 primary untreated STS patients with five subtypes: synovial sarcoma (SS: n=36), myxoid liposarcoma (MLS: n=32), undifferentiated pleomorphic sarcoma (UPS: n=27), leiomyosarcoma (LMS: n=11), and dedifferentiated liposarcoma (DDLS: n=10). The infiltration of CD8+ and FOXP3+ lymphocytes, CD163+ macrophages, and the expression of HLA class I and PD-L1 in the tumor were evaluated by immunohistochemistry. Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). Results: UPS had a higher infiltration of CD8+ and FOXP3+ lymphocytes than SS and MLS. Infiltration of CD163 + macrophages were higher in UPS, LMS, and DDLS than in SS and MLS. HLA class I expression was detected in all of UPS, LMS, and DDLS. However, HLA class I was negative in 17% of SS and 69% of MLS. Although no PD-L1 expression was observed in SS and MLS, PD-L1 expression was detected in 36% of UPS, 28% of LMS, and 25% of DDLS. Moreover, PD-L1 expression was associated with poor outcome in these subtypes. Increasing number of CD8+ lymphocytes was associated with a better OS in patients with SS. A higher infiltration of FOXP3+ lymphocytes was associated with an unfavorable OS in patients with MLS but favorable outcome in SS. Moreover, a higher infiltration of CD163+ macrophages showed a significantly worse OS and PFS in patients with SS and MLS. Conclusion: Lymphocytes and macrophages were variably infiltrated across STS subtype. UPS, LMS and DDLS, which have numerous genetic mutations, demonstrated high infiltration of lymphocytes, HLA class I and PD-L1 expression. These suggest that T-cell based immunotherapy such as immune checkpoint inhibitors may be effective in these tumor subtypes. Although the SS and MLS are less mutated and do express cancer-testis antigen NY-ESO-1 in common, expression level of HLA class I is significantly different between SS and MLS. In addition, infiltration of FOXP3+ lymphocytes have opposite effect in these tumors. These suggests different immunotherapeutic strategy might be required in these subtypes. Our results might be useful in the clinical strategy of immunotherapy in STS. Citation Format: Naoki Oike, Hiroyuki Kawashima, Akira Ogose, Hiroshi Hatano, Takashi Ariizumi, Taro Sasaki, Tetsuro Yamagishi, Hajime Umezu, Naoto Endo. Clinical significance of tumor immune microenvironment in soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1501.

We herein report a case of a huge suprascapular arteriovenous malformation (AVM) that was infected after endovascular embolization and then resected to good clinical outcome. The infection occurred after the fourth embolization with N-butyl-cyanoacrylate . The contrast-enhanced CT scan revealed abscesses surrounding the embolized material. The infection was not completely healed by antibiotic treatment, so we performed a resection of the AVM by the circumferential parallel ligation method after preoperative embolization. Fifteen months after the surgery, the patient is healthy without sign of infection. Infection is a rare complication of endovascular embolization. But, once it occurs, it is a very intractable disease.

This study investigated the pathway underlying the antitumor activity of telomelysin, a telomerase‐dependent, replication‐selective oncolytic adenovirus, in soft tissue sarcoma cells. Treatment with telomelysin alone resulted in simultaneous induction of apoptosis and autophagy, whereas cotreatment with telomelysin and 3‐methyladenine significantly reduced cell viability and increased apoptosis and the cellular ATP level compared to treatment with telomelysin alone, indicating that telomelysin‐mediated autophagy is a death‐protective but not death‐promoting process. Cotreatment with Z‐Val‐Ala‐Asp‐CH2F significantly increased cellular ATP depletion compared to telomelysin‐alone treatment while inhibiting telomelysin‐induced apoptosis and having no significant effect on cell viability, indicating that it promotes transition from apoptotic to necrotic cell death.

Porous β-tricalcium phosphate (TCP) displays osteoinductivity in certain animals in the absence of osteoinductive agents. We evaluated whether the microstructure may be an important determinant of osteoinduction, and also investigated how bone formation was promoted using β-TCP combined with bone marrow aspirates. We prepared two types of β-TCP, namely, β-TCP A, which possessed interconnected macropores and micropores, and β-TCP B, which possessed macropores but had less detectable micropores. These were implanted with or without marrow in canine muscles. Bone formation and the resorption of each β-TCP implant were evaluated histologically. Newly formed bone began to appear at day 42 in the implants of β-TCP A alone, but the implants of β-TCP B alone did not show any bone formation by day 42. Meanwhile, bone formation was already evident on day 14 by loading with bone marrow aspirates with or without micropores. By immunohistochemistry, the number of cathepsin K-positive cells (osteoclasts) increased as time passed in the implants of β-TCP A alone, while the number of the osteoclasts did not change obviously in the implants of β-TCP B alone from day 14 to 56. Reticular fibrils were evident within the β-TCP A, and were barely observed in the β-TCP B in the silver impregnation. The present result would bring about the possible role to enhance the importance of the surface microstructure for the better osteoinductivity. Our findings suggest that the combination of porous β-TCP and bone marrow facilitates bone formation.

Intravertebral cleft (IVC) is a common finding in osteoporotic compression fracture. However, since the vertebral collapse attributable to cancer metastasis is rarely associated with IVC, the phenomenon is generally considered as a sign of a benign lesion. In this study, we retrospectively reviewed the radiographs, computed tomography scans, and magnetic resonance images of 111 patients with spinal metastasis. Three cases (2.7 %) had IVC in the collapsed thoracic vertebral bodies (T7, T8, and T11) attributable to cancer metastasis. IVC alone is not necessarily an indicator of a benign vertebral collapse.

We established a dedifferentiated liposarcoma cell line (NDDLS-1) that produces interleukin-6 (IL-6) and granulocyte-colony stimulating factor (G-CSF). The parental tumor showed high leukemoid reactions. The NDDLS-1 cell line was established from a pleural effusion associated with a lung metastasis. Pleomorphic tumor cells arranged in a haphazard growth pattern were seen in xenograft tumors. Numerous inflammatory cells including neutrophils or eosinophils were present throughout the tumor cells. This finding resembled the dedifferentiated area of the parental tumor. The mice bearing NDDLS-1 showed marked leukocytosis. In addition, the NDDLS-1 cells expressed IL-6 and G-CSF at both the mRNA and protein levels, while the NDDLS-1 cells produced near normal levels of tumor necrosis factor alpha (TNF-α). In the cytogenetic analysis, both the parental tumor and the NDDLS-1 cells showed a ring or giant marker chromosomes. The NDDLS-1 cell line demonstrated the amplification and expression of both MDM2 and CDK4 by fluorescence in situ hybridization and immunohistochemical analysis. The NDDLS-1 cell line is consistent with the parental dedifferentiated liposarcoma, and it should therefore be useful for further investigations of human dedifferentiated liposarcomas.

This study investigated the development of Ca2+ signaling mechanisms and their role in initiating morphogenetic cell movement in the presumptive ectoderm of Japanese newt (Cynops pyrrhogaster) during gastrulation. Histochemical staining using fluorescently labeled ryanodine and dihydropyridine probes revealed that dihydropyridine receptor (L-type Ca2+ channels) appeared in stage 12b embryos, while ryanodine receptors were expressed in both stage 11 and 12b embryos. Transmission electron microscopy of stage 12b embryos showed abundant peripheral couplings, which are couplings of the endoplasmic reticulum and cell membrane with an approximate 12 nm gap. Caffeine increased the intracellular free Ca2+ concentration ([Ca2+]i) in presumptive ectodermal cells isolated from both stage 11 and 12b embryos, while (±)-Bay K 8644 ((±)-BayK) increased [Ca2+]i in cells isolated from stage 12b embryos, but not in cells isolated from stage 11 embryos. Dantrolene and nifedipine completely inhibited increases in [Ca2+]i after treatment with caffeine and (±)-BayK, respectively. Caffeine activated the motility of cells isolated from both stage 11 and 12b embryos, but (±)-BayK only activated the motility of cells isolated from stage 12b embryos. These findings suggested that formation of the Ca2+-induced Ca2+ release system in presumptive ectodermal cells during gastrulation plays an important role in the initiation and execution of epibolic extension.

A telomerase-specific oncolytic adenovirus, Telomelysin, can selectively kill cancer cells, and be attenuated in normal cells. We herein describe the oncolytic effect of Telomelysin on human osteosarcoma both in vitro and in vivo. The anti-tumor effects of Telomelysin were evaluated on human osteosarcoma cell lines in vitro and in a mouse xenograft model of human osteosarcoma in vivo. The replication efficiencies of Telomelysin in human osteosarcoma cell lines and normal cell lines and in osteosarcoma xenografts were determined by the expression levels of E1 mRNA and E1A protein using real-time quantitative PCR, Western blot analysis and immunohistochemistry. The in vitro telomerase-specific replication and the viral infection rate were also confirmed by TelomeScan (Telomelysin-GFP), using fluorescent microscopy and flow cytometry, respectively. The cell viabilities were examined by XTT assay, and the tumor volumes were measured every 2 days. The induction of apoptosis was assessed by Western blot analysis, as well as by TUNEL assay. TelomeScan and Telomelysin were efficiently replicated in human osteosarcoma cell lines and led to a dose- and time-dependent expression of GFP, E1 mRNA and E1A protein. Telomelysin infection induced marked cytolysis and apoptosis in osteosarcoma cell lines in vitro. Neither cytotoxicity nor apoptosis were induced in normal human cell lines. In the human osteosarcoma cell xenograft model, intratumoral injection of Telomelysin resulted in increased viral replication, significant tumor growth suppression and distinct apoptotic cell death. This study indicated that virotherapy with Telomelysin may provide a promising strategy for the treatment of human osteosarcoma.

Claudin proteins are the major components of tight junctions connecting adjacent cells, where they regulate a variety of cellular activities. In the present paper we identified two Xenopus claudin5 genes (cldn5a and 5b), which are expressed early in the developing cardiac region. Precocious cldn5 expression was observed in explants of non-heart-forming mesoderm under inhibition of the canonical Wnt pathway. Cardiogenesis was severely perturbed by antisense oligonucleotides against cldn5 or by Cldn5 proteins lacking the cytoplasmic domain. Results of light- and electron-microscopic observations suggested that cldn5a and 5b are required for Xenopus heart tube formation through epithelialization of the precardiac mesoderm.

We report here, our experience of seven patients with epithelioid sarcomas and their serum CA 125 levels, as well as the results of an in vitro and in vivo study of CA 125 expression in epithelioid sarcoma cells and xenografts using three epithelioid sarcoma cell lines.In the clinical study, the serum CA 125 levels of seven epithelioid sarcoma patients were examined at multiple time points. Expression of the MUC16 gene that encodes the CA 125 sequence was examined using RT-PCR methods in three epithelioid sarcoma cell lines, FU-EPS-1, SFT-8606 and NEPS, and the CA 125 protein in each cell lysate was examined by Western blot using anti-CA 125 clone OC125 antibody. The concentration of CA 125 in the conditioned medium of each cell line was also measured.In five of the seven epithelioid sarcoma patients, CA 125 levels reflected regression and progression of their disease. The CA 125 concentrations in the conditioned medium of FU-EPS-1, SFT-8606 and NEPS cells were 259, 252, and 6 U/ml, respectively. Strong expression of MUC16 mRNA was shown in FU-EPS-1 and SFT-8606 cells: correspondingly, a thick band was observed by Western blot analysis in only FU-EPS-1 and SFT-8606 cells.We concluded that epithelioid sarcoma cells produce and secrete CA 125 into the blood serum and that the elevation of serum CA 125 correlates with disease progression. Therefore, measuring the serum CA 125 level should provide an useful index for diagnosing and monitoring the course of epithelioid sarcoma.

Pleomorphic rhabdomyosarcoma (PRMS) is a rare variant of rhabdomyosarcoma that occurs mostly in adults. A few cytogenetic studies of PRMS have been reported, but no consistent specific chromosome aberrations were detected. We herein report a cytogenetic study of three cases of pleomorphic rhabdomyosarcoma using a conventional G-banded karyotyping analysis. The three cases appeared to exhibit an extremely complex karyotype with numeric and structural rearrangements. Although the three cases displayed several common aberrations, including -2, -4, -9, -13, -14, -15, -19, -21, add(X)(p11), add(1)(q11), add(7)(p11), and add(13)(p11), no recurrent characteristic chromosomal aberrations could be detected. In addition, among these cases and seven other cases of previously reported PRMS, the most frequent chromosomal alterations were -2, -13, -14, -15, -16, and -19. No obviously consistent structural alterations can be found in these 10 PRMS cases, however, thereby suggesting that it is difficult to confirm whether these complex karyotypes correlated with the diagnosis or clinical outcome in PRMS. In this study, we detected MyoD1 and myogenin gene transcripts at the mRNA level in four cases of PRMS together with other soft-tissue sarcomas, including seven cases of malignant fibrous hitiocytoma, five cases of liposacroma, and three cases of leiomyosacroma using a real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. High-level expressions of MyoD1 and myogenin gene transcripts were determined in all cases of PRMS. In contrast, the other non-PRMS sarcomas showed either no expression or extremely weak expressions for both genes. Our findings suggest that the detections of MyoD1 and myogenin transcripts using real-time quantitative RT-PCR, combined with immunohistochemical stains, are extremely sensitive and useful for the diagnosis of PRMS.

Directional migration of neuronal cells over long distances is critical for the developing and regenerating nervous system. A scaffold, which includes radial glia, provides a route between the germinal zone and the destination, with the direction of migration being attributed mainly to chemotaxis. However, the decrease in chemokine concentration with distance complicates cell guidance control over long distances. Our working hypothesis is that neuronal cells migrate directionally on an anisotropic and periodic scaffold. We tested this in a model involving neuronal PC12 cells cultured in a ratchet wheel-shaped microchamber. The microchamber was constructed by printing a patterned, thin film of polydimethylsiloxane (PDMS), to which the cells adhere weakly, onto a collagen-coated dish, to which the cells adhere strongly, using a microcontact printing technique. The cells can attach, extend neurites, and migrate on the anisotropic and periodic collagen-coated area between the ratchet wheel-shaped outer frame and round inner frame of the PDMS. We found that the microchamber geometry affected the direction of migration, even though the mean length of the longest neurite was independent of microchamber geometry. The time-course trace of cell body migration and neurite tips showed that the neurite tips remained around the tips of the ratchet teeth. These results suggested that neuronal cells migrate directionally on a scaffold, even in the absence of chemokine, and reveal a new conceptual framework for neuronal migration.

Amphibians have been used for over a century as experimental animals. In the field of developmental biology in particular, much knowledge has been accumulated from studies on amphibians, mainly because they are easy to observe and handle. Xenopus laevis is one of the most intensely investigated amphibians in developmental biology at the molecular level. Thus, Xenopus is highly suitable for studies on the mechanisms of organ differentiation from not only a single fertilized egg, as in normal development, but also from undifferentiated cells, as in the case of in vitro organogenesis. Based on the established in vitro organogenesis methods, we have identified many genes that are indispensable for normal development in various organs. These experimental systems are useful for investigations of embryonic development and for advancing regenerative medicine. Developmental Dynamics 238:1309-1320, 2009. (c) 2009 Wiley-Liss, Inc.

Musculoskeletal sarcomas (MSS) are a heterogeneous group of malignancies with relatively high mortality rates. The prognosis for patients with MSS is poor, with few drugs inducing measurable activity. Alkylating agents, namely ifosfamide and dacarbazine, which act nonspecifically on proliferating cells, are the typical therapy prescribed for advanced MSS. A novel alkylating agent, temozolomide (TMZ), has several advantages over existing alkylating agents. TMZ induces the formation of O6-methylguanine in DNA, thereby inducing mismatches during DNA replication and the subsequent activation of apoptotic pathways. However, due to conflicting data in the literature, the mechanism of TMZ action has remained elusive. Therefore, the present study aimed to evaluate apoptosis in MSS cells treated with TMZ, and to evaluate the correlation between TMZ action and survival pathways, including the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen activated protein kinase (MAPK) pathways. Cell proliferation was evaluated by performing an XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay. Apoptotic morphological changes, for example chromatin condensation, were evaluated by fluorescence confocal microscopy. The expression of the apoptosis-associated proteins caspase-3, poly adenosine diphosphate ribose polymerase (PARP), Akt and ERK1/2, was determined by western blotting. The results of the present study indicated that, in certain MSS cells, the IC50 value was lower than that in TMZ-sensitive U-87 MG cells. Furthermore, TMZ treatment was associated with apoptotic morphological changes and the expression levels of pro-apoptotic cleaved caspase-3 and PARP were also increased in TMZ-treated MSS cells. In addition, the results indicated that PI3K/Akt and ERK1/2 MAPK were constitutively phosphorylated in MSS cells, and phosphorylation of PI3K/Akt was suppressed in certain cells, and maintained in other cells, by TMZ. These observations emphasized the plasticity of MSS cells, and suggested that this plasticity may contribute to the variance in cell sensitivity to TMZ and TMZ-resistance in MSS.

Na+-HCO3– cotransporter (NBC1) plays a major role in bicarbonate reabsorption from proximal tubules. In a previous immunohistochemical study on human kidney, we showed that the kidney-type transporter (kNBC1) was abundantly expressed in the basolateral membranes of proximal tubules while the expression of pancreatic-type transporter (pNBC1) was undetectable. In the present study we tried to determine the localization of NBC1 variants in rat kidney using the antibodies against the unique N-terminal regions of kNBC1 and pNBC1. In Western blot analysis on the membrane-enriched fraction from rat kidney both anti-kNBC1 and anti-pNBC1 antibodies yielded a ∼130 kDa band. In immunohistochemical analysis with confocal microscopy the anti-kNBC1 antibody produced a strong and exclusively basolateral labeling in proximal tubules. On the other hand, the occasional pNBC1 labeling was detected in the apical membranes of proximal tubules. The electron microscopic observation further supported the basolateral localization of kNBC1 as well as the localization of pNBC1 on the basis of the brush border. Acute metabolic acidosis did not change the protein expression levels as well as the intracellular distribution of both NBC1 variants in rat kidney. These results are consistent with a view that kNBC1 is the dominant variant that mediates bicarbonate reabsorption from rat renal proximal tubules. They also indicate that species difference may exist regarding the distribution of NBC1 variants in kidney.

The animal cap—the presumptive ectoderm of the blastula embryo—can differentiate into a variety of tissues belonging to the three germ layers following exposure to specific inducers. The “animal cap assay” was devised based on the pluripotency of presumptive ectodermal cells and enabled many important discoveries in the field of embryonic induction and cell differentiation. Using this system, investigators can test multiple factors in solution simultaneously to determine their inducing activities qualitatively, quantitatively, and synergistically. Furthermore, after dissociation and induction, reaggregated animal cap cells can be induced to form higher-order organs. This protocol details preoperative preparations, followed by the basic animal cap assay. Advanced protocols for the induction of kidney, pancreas, and heart are also described.

Various historical eras in the distant as well as the recent past of amphibian embryology are briefly reviewed. The concepts which emerged from the early years matured, then were laid to rest for several decades. A resurgence, driven by key discoveries with peptide growth factors, and fueled by modern molecular biology methods, is underway. The future for several amphibian research projects should be promising since interest in basic concepts remains strong, and application of frontier methodologies is yielding novel findings.

The discovery that peptide growth factors are capable of embryonic induction has led to rapid growth in our understanding of this process. One of these factors, activin, can control mesoderm formation inXenopus animal caps in a dose-dependent manner. Activin may also participate in kidney and heart organogenesis in combination with other factors. Further, animal caps treated with a high concentration of activin clearly show organizer actions, which are closely related to the fundamental body plan along the anteroposterior axis. These in vitro experiments will serve as an excellent model system for analysis of cell differentiation, organogenesis, and morphogenesis at the molecular level.

Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin-treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin-treated exp ants. Co-treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin-treated explants.

The inductive interactions between activin-induced and non-induced cells were investigated in newt animal cap explants. A wide range of concentrations of activin A (0.1–100 ng/ml) induced mesodermal tissues in the animal caps, but at generally low frequencies. Animal caps treated with 100 ng/ml of activin A, on the other hand, differentiated solely into nonspecific endoderm. At this concentration, various mesodermal tissues were induced in addition to endoderm as the animal caps increased in size. They were more frequently induced in explants in which the activin-treated animal caps were combined with untreated animal caps. Central nervous systems were frequently induced in sandwich explants with larger amounts of untreated animal caps. Differentiation of endodermal organs such as the liver, the pancreas and the intestine in the long-term cultured sandwich explants was confirmed by electron microscopy. Lineage tracing of the combination and sandwich explants revealed that the activin-treated anim...

The differentiation patterns of animal cap explants from the Japanese salamanders Hynobius lichenatus and Hynobius nigrescens were examined after exposure to various concentrations of activin A. A wide range of concentrations of activin A (0.5-100 ng/ml) induced various mesodermal tissues such as ventral mesoderm, somitic muscle, and notochord. At concentrations higher than 50 ng/ml, yolk-rich endodermal tissue was induced in many of the explants. Activin A is also known to have mesoderm-and/or endoderm-inducing activity on the animal caps of Xenopus laevis and Cynops pyrrhogaster, but their response patterns are slightly different. The mode of differentiation of activin-treated Hynobius animal caps was compared with that of Xenopus and Cynops in relation to the structure of the animal caps.

We have demonstrated that the tissue differentiation patterns along the dorsoventral and anteroposterior axes can be controlled by a combination of activin A, concanavalin A (Con A), and retinoic acid. Xenopus blastula animal caps, normally fated to form epidermal tissues, differentiated into ventral mesoderm tissues such as coelomic epithelium and blood-like cells following treatment with activin A (0.5 ng/ml). Dorsal mesoderm tissues like muscle and notochord, were induced by graded addition of Con A. Conversely, Con A (1 mg/ml) induced anterior neural tissues, forebrain accompanied by eyes and cement glands, in the animal caps. Posterior neural tissues, hindbrain with ear vesicles and spinal cord, were induced by graded addition of activin A. Retinoic acid was also capable of shifting the Con A-induced anterior neural tissues to more posterior tissue phenotypes, however, its caudalizing activity was slightly different from that of activin A. These results suggest that the concentration gradien...