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Immune checkpoint inhibitors (ICIs) such as PD1/PD-L1 blockers are an established treatment for many solid cancers. There are currently no approved ICIs for sarcomas, but satisfactory results have been seen in some patients with disseminated disease in certain histological types. Most studies on PD-L1 in sarcoma have used small specimens and there are no clear cutoff values for scoring. We investigated PD-L1 immunoreactivity in high-grade chondrosarcomas (CS), abdominal liposarcoma (LS) and undifferentiated pleomorphic sarcomas (UPS). In total, 230 tumors were stained with SP142 and SP263 assays and evaluated by two clinical pathologists. Immunoreactivity in tumor and immune cells was correlated with clinical outcome. Overall, ≥1% PD-L1 immunoreactivity in tumor cells was found in 11 CS, 26 LS and 59 UPS (SP142 assay) and in 10 CS, 26 LS and 77 UPS (SP263 assay). Most tumors exhibited ≤10% PD-L1 immunoreactivity, but a subset across all three subtypes had >50%. Kaplan-Meier survival analysis showed no significant difference in metastasis-free or overall survival in relation to PD-L1 immunoreactivity in tumor or immune cells for any subtype. As there is a lack of clinical data regarding PD-L1/PD-1 status and therapy response, it is not currently possible to establish clear cutoff values. Patients with high (>50%) PD-L1 immunoreactivity in tumor cells (TC) with the SP263 assay would be a logical group to investigate for potentially beneficial PD1/PD-L1-targeted treatment.

The levels of sPD-1 and sPD-L1 were analyzed in blood serum of 132 patients (age 14-70 years) with primary bone tumors: osteosarcoma (N=39), chondrosarcoma (N=42), Ewing sarcoma (N=9), chordoma (N=12), giant-cell bone tumor (GCBT) (N=16), benign neoplasms (N=14) and in and practically healthy subjects (age 19-58 years; N=27). sPD-L1 levels in all studied bone neoplasms were significantly higher than in the control. Serum sPD-1 level in GCBT patients was significantly higher than in the control, benign neoplasms, chondrosarcoma, and chordoma patients, but did not differ from osteosarcoma group. sPD-1 concentration in Ewing sarcoma was significantly higher than in chordoma and chondrosarcoma, but did not differ from the control. sPD-1 level in chondrosarcoma patients was also lower than in osteosarcoma, Ewing sarcoma, and in the control. Both sPD-1 and sPD-L1 concentrations were not significantly associated with the type of affected bone, process localization, disease stage, tumor histological grade, patients' age and sex. These results suggest the possibility of using these biological markers for preliminary assessment of the character of the process in the bone.

Matrix metalloproteinase-3 (MMP-3) plays a pivotal role in the destruction of articular cartilage in osteoarthritis (OA). The regulation of gene expression of MMP-3 is complicated. Interferon regulatory factor 5 (IRF5) is a member of the interferon regulatory factor family of transcription factors. Little information regarding the biological function of IRF5 on chondrocytes and the pathogenesis of OA has been reported. In the current study, for the first time, we report that IRF5 is expressed in human primary chondrocytes and human chondrosarcoma cell line SW1353 cells. In addition, IRF5 is upregulated in response to TNF-α treatment in a dose dependent manner. Interestingly, IRF5 is significantly higher in chondrocytes from OA patients compared to those from normal subjects. Notably, IRF5 mediates TNF-α- induced expression of MMP-3 in chondrocytes. Overexpression of IRF5 promotes the expression of MMP-3, however, knockdown of IRF5 reduces the expression of MMP-3. Mechanistically, IRF5 is able to enhance the transcription of MMP-3 by binding to its promoter. Also, we found that NF-κB was involved in the effects of IRF-5 on MMP-3 expression. These findings suggest that IRF5 might be a novel pharmacological target for the treatment of OA and RA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chordomas and chondrosarcomas are two major malignant bone neoplasms located at the skull base. These tumors are rarely metastatic, but can be locally invasive and resistant to conventional chemotherapies and radiotherapies. Accordingly, therapeutic approaches for the treatment of these tumors can be difficult. Additionally, their location at the skull base makes them problematic. Although accurate diagnosis of these tumors is important because of their distinct prognoses, distinguishing between these tumor types is difficult due to overlapping radiological and histopathological findings. However, recent accumulation of molecular and genetic studies, including extracranial location analysis, has provided us clues for accurate diagnosis. In this report, we review the genetic aberrations and molecular biology of these two tumor types. Among the abundant genetic features of these tumors, brachyury immunohistochemistry and direct sequencing of IDH1/2 are simple and useful techniques that can be used to distinguish between these tumors. Although it is still unclear why these tumors, which have such distinct genetic backgrounds, show similar histopathological findings, comparison of their genetic backgrounds could provide essential information.

Therapies targeting the programmed cell death 1 (PD-1) or its ligand (PD-L1) promote antitumor T-cell activity, leading to unprecedented long-lasting tumor responses in some advanced cancers. Because of radiotherapy and chemotherapy resistance, no effective treatments have been defined for advanced chondrosarcomas. We here report an immunohistochemical analysis of PD-L1 expression in a large series of conventional, mesenchymal, clear cell and dedifferentiated chondrosarcomas using tissue microarrays. In the PD-L1-positive tumors, we analyzed the immune microenvironment (T-cell and macrophage infiltration as well as HLA class I expression) using whole sections. PD-L1 expression was absent in conventional (n=119), mesenchymal (n=19) and clear cell (n=20) chondrosarcomas. Forty-one percent (9 of the 22) of dedifferentiated chondrosarcomas displayed PD-L1 positivity. These results were confirmed in an independent cohort using whole tissue sections of dedifferentiated chondrosarcomas in which PD-L1 expression was detected in 52% (11 of the 21) of cases. PD-L1 expression was exclusively found in the dedifferentiated component and expression positively correlated with other immune parameters such as high number of tumor-infiltrating lymphocytes (P=0.014) and positive HLA class I expression (P=0.024) but not with patient overall survival (P=0.22). The presence of PD-L1 expression in association with immune-infiltrating cells and HLA class I expression in nearly 50% of the dedifferentiated chondrosarcomas provides rationale for including these patients in clinical trials with PD-1/PD-L1-targeted therapies.

Chondrosarcoma (CS) is a cartilaginous malignant neoplasm characterized by resistance to conventional adjuvant therapy. The prognosis of unresectable or metastatic CS is poor. Therefore, it is imperative to explore novel therapeutic approaches to improve the treatment efficacy for those CS patients. Emerging data has implicated the synergistic antitumor activity of zoledronate (ZOL) and Vγ9Vδ2 T cells. However, whether ZOL-stimulated Vγ9Vδ2 T cells could infiltrate bone sarcoma and inhibit tumor growth has not been thoroughly answered yet. In this study, Vγ9Vδ2 T cells from healthy donors and CS patients were expanded in the presence of ZOL (1 μM) and IL-2 (400 IU/ml). The antitumor activity of Vγ9Vδ2 T cells to ZOL-pretreated human CS was examined both in vitro and in vivo. ZOL pretreatment substantially enhanced the cytotoxicity of Vγ9Vδ2 T cells to SW1353 and primary CS cells. ZOL potentiated the migration and cytotoxicity of Vγ9Vδ2 T cells to SW1353 in dose- and time-dependent manner. Moreover, weekly intravenous ZOL followed by Vγ9Vδ2 T cells inhibited subcutaneous xenograft growth. Thus, Vγ9Vδ2 T cells were able to infiltrate bone tumor and significantly suppressed the development of orthotopic SW1353 xenografts. Altogether, the study raises the possibility of combining ZOL with Vγ9Vδ2 T cells for CS treatment.

Isocitrate dehydrogenase 2 (IDH2) mutations have been reported in gliomas, osteosarcomas, cartilaginous tumors, giant cell tumors of bone, and acute myeloid leukemias. Although IDH2 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate (α-KG) in mitochondria, mutated IDH2 proteins possess the ability to change α-KG into the oncometabolite R(-)-2-hydroxyglutarate (2-HG). To date, several monoclonal antibodies (mAbs) specific for IDH2 mutations have been established, such as KMab-1 against IDH2-R172K, MMab-1 against IDH2-R172M, and WMab-1 against IDH2-R172W. Although a multi-specific mAb MsMab-1 reacted with IDH2-R172G and IDH2-R172S, a mono-specific mAb against IDH2-R172S has not been established. In this study, we established a novel mAb SMab-2, which recognizes IDH2-R172S but not with wild type IDH2 in ELISA. Although SMab-2 reacted with both IDH1-R132S and IDH2-R172S expressed in Escherichia coli, it reacted with only IDH2-R172S expressed in U-2 OS osteosarcoma cells. Furthermore, SMab-2 recognized endogenous IDH2-R172S protein expressed in SW1353 chondrosarcoma cells in Western blot and immunocytochemical analyses. SMab-2 is expected to be useful for diagnosis of IDH2-R172S-bearing tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cartilage degradation is the most predominant pathological change during osteoarthritis (OA). Furthermore, accumulating evidence suggests that an excess of matrix metalloproteinase-13 (MMP-13) plays a critical role in the breakdown of cartilage. Here, the effects of Icariin on the expression of MMP-13 in IL-1β-induced SW 1353 chondrosarcoma cells were investigated. In addition, the in vivo effects of Icariin on an experimental rat model of OA induced by anterior cruciate ligament transection (ACLT) was examined. SW1353 chondrosarcoma cells were pretreated with or without Icariin and MAPK and Wnt/β-catenin signaling pathway inhibitors, then were stimulated with IL-1β. In rats, experimental OA was induced by ACLT. These rats then received intra-articular injections of vehicle, signaling pathway inhibitors, and/or Icariin. Expression of MMP-13, phosphorylated p38, phosphorylated JNK, and β-catenin were verified by western blotting. In addition, levels of MMP-13 mRNA were detected using quantitative real-time PCR. In histological analyses, treatment with Icariin reduced the number of cartilage lesions present. In addition, treatment with Icariin was associated with lower levels of phosphorylated p38, phosphorylated JNK, and β-catenin in both IL-1β-induced SW1353 chondrosarcoma cells and in the rat OA model. Furthermore, the suppressive effect of Icariin on MMP-13 was greater than that exhibited by other signaling pathway inhibitors. Overall, these data suggest that Icariin has therapeutic potential for the treatment of OA., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Primary malignant bone tumors are heterogeneous groups of neoplasms, which affect mainly children and adolescents. The most common types are Osteosarcoma, Ewing sarcoma and chondrosarcoma. Elevation of sCD30 and sCD40L has been observed in lymphoma, leukemia and autoimmune disorders., Objective: To evaluate serum concentrations of sCD30 and sCD40L in patients with primary malignant bone tumors., Method: Fifty-four cases (31 Osteosarcomas, 14 Ewing sarcomas, and 9 Chondrosarcomas) and 54 healthy controls enrolled in this study. Cases with the history of prior treatment (surgery, chemotherapy and radiotherapy) were excluded from the study. Serum levels of sCD30 and sCD40L were detected by an enzyme linked immunosorbent assay (ELISA)., Results: Mean serum concentration of sCD30 in Ewing sarcoma was significantly higher than that of the control groups (p=0.007), but mean serum concentrations of sCD30 in osteosarcoma and chondrosarcoma groups were not significantly different, compared to the controls (p=0.41 and p=0.11, respectively). Mean serum concentrations of sCD40L in osteosarcoma, Ewing sarcoma and chondrosarcoma groups were significantly higher than that of the control group (p<0.0001). In addition, the mean serum level of sCD40L in chondrosarcoma patients was higher than that of both Ewing sarcoma and osteosarcoma groups (p<0.001)., Conclusion: sCD30 and sCD40L increase in primary bone tumors; however the significant of these findings for diagnosis or prognosis of these tumors needs further investigation.

The carbohydrate polymer, hyaluronan, is a major component of the extracellular matrix in animal tissues. Exogenous hyaluronan has been used to treat osteoarthritis (OA), a degenerative joint disease involving inflammatory changes. The underlying mechanisms of hyaluronan in OA are not fully understood. Pro-inflammatory interleukin (IL)-1β downregulates peroxisome proliferator-activated receptor gamma (PPARγ), and increases expression of matrix metalloproteinases (MMPs) which are responsible for the degeneration of articular cartilage. The effects of low- and high-molecular-weight hyaluronan (oligo-HA and HMW-HA) on the inflammatory genes were determined in human SW-1353 chondrosarcoma cells. HMW-HA antagonized the effects of IL-1β by increasing PPARγ and decreasing cyclooxygenase (COX)-2, MMP-1, and MMP-13 levels. It promoted Akt, but suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB) signaling, indicating anti-inflammatory effects. In contrast, the cells had overall opposite responses to oligo-HA. In conclusion, HMW-HA and oligo-HA exerted differential inflammatory responses via PPARγ in IL-1β-treated chondrosarcoma cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA) family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157-165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTA's have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC) in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression., Methods: We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTA's with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment., Results: A minority (36%) of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment., Conclusion: These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic chondrosarcoma patients where no proven systemic therapies exist.

The aim of the present study was to investigate whether the expression of ezrin, a membrane-cytoskeleton linker and regulator of cellular signaling, is associated with clinical features of chondrosarcoma. For this purpose, we studied the expression of ezrin in 54 chondrosarcomas by immunohistochemistry and correlated the expression with other tumor characteristics, markers of proliferation, apoptosis and with clinical parameters. The intensity of ezrin staining increased with the histologic grade, and a significant positive association existed between the tumor grade and ezrin expression (p = 0.0475). In addition, there was a positive correlation between the expression of ezrin and Bcl-2, an anti-apoptotic protein (r = 0.83, p < 0.0001), as well as between ezrin expression and increased proliferation as measured by Ki-67 index (r = 0.70, p < 0.0001). The positive correlation of ezrin expression with Bcl-2 and Ki-67 as well as with tumor grade suggests that an aggressive behavior of chondrosarcoma may be related to activation of ezrin and that ezrin inhibitors could provide a much needed adjuvant therapy in chondrosarcomas. In conclusion, our results indicate that high ezrin expression correlates with aggressive features of chondrosarcomas. Further analyses on the pathways downstream of ezrin are warranted., (© 2010 The Authors. Journal Compilation © 2010 APMIS.)

Extraskeletal myxoid chondrosarcomas usually arise deep in the proximal extremities and limb girdles. Patients with this type of sarcoma have high rates of local recurrence and metastases, but do not typically have paraneoplastic syndromes. We report an unusual case of a 49-year-old man with anti-Hu syndrome in the setting of an extraskeletal myxoid chondrosarcoma. This case shows the importance of searching for antineural antibodies in oncologic patients with new neurologic deficits, and of having a judicious workup for occult malignancies in patients with known antineural antibodies.

Objective: Proinflammatory cytokine-induced expression of matrix metalloproteinases (MMPs) is a major cause of arthritic cartilage destruction. The neuropeptide, alpha-melanocyte-stimulating hormone (alpha-MSH), has been detected in the synovial fluid of arthritis patients, where it is thought to play an anti-inflammatory role. Here, we examined whether alpha-MSH acts via downregulation of MMP expression, and sought to elucidate the intracellular signal pathways underlying this effect., Design: Human chondrosarcoma cell line, HTB-94 (SW1353) was pretreated with or without alpha-MSH and then treated with tumor necrosis factor-alpha (TNF-alpha). The effect of alpha-MSH on TNF-alpha-induced MMP-13 expression and mitogen-activated protein kinases' (MAPKs) activation were determined by reverse transcriptase-polymerase chain reaction and Western blot analysis. Additionally, the intracellular signaling of alpha-MSH was investigated using the inhibitors of MAPK and nuclear factor kappaB (NF-kappaB) and plasmids encoding dominant negative (dn) forms of inhibitor kappaB kinase-alpha (IKKalpha) and inhibitor kappaB kinase-beta (IKKbeta)., Results: We found that alpha-MSH pretreatment inhibited TNF-alpha-induced MMP-13 expression and p38 kinase phosphorylation in HTB-94 human chondrosarcoma cells. TNF-alpha-induced MMP-13 expression was not suppressed by extracellular signal-regulated kinase (ERK) inhibitors (PD98059 and U0126) or a c-jun terminal kinase (JNK) inhibitor (SP600125), but was inhibited by inhibitors of p38 kinase (SB203580) and NF-kappaB (SN-50 peptide) and dnIKKalpha and dnIKKbeta., Conclusions: Our results suggest that alpha-MSH regulates TNF-alpha-induced MMP-13 expression by decreasing p38 kinase phosphorylation and subsequent NF-kappaB activation in human chondrocytes and may be an effective inhibitor of MMP-13-mediated collagen degradation, providing new potential opportunities for the development of anti-arthritis therapeutics.

We present a case report of patient with intracranial chondrosarcoma and attempt to use vaccination of dendritic cells as the salvage therapy. To our knowledge, this is the first case report of DCs vaccination in the head and neck chondrosarcoma. Immunotherapy with allogeneic DCs stimulated with tumor cell lysates in this case was demonstrated to be feasible, safe and well tolerated. Unfortunately we did not observe any clinical or immune response during vaccination. CD4+ and CD8+ regulatory cells could be responsible for ineffectiveness of immunotherapy.

Treatment of chondrosarcomas is limited to resection because these tumors are unresponsive to standard adjuvant treatments, such as chemotherapy and radiation. We have previously shown that high-grade chondrosarcomas express unspecified members of the Melanoma Antigen (MAGE) gene family. We show here that FS human chondrosarcoma (FS) cells express MAGE-A3 gene and HLA-A1 molecules. In vitro assays show that a cytolytic T-lymphocyte clone (CTL) specific for a MAGE-A3 peptide presented by HLA-A1 specifically lysed FS chondrosarcoma cells. Addition of antigenic peptide did not increase the susceptibility of FS cells to CTL mediated lysis, suggesting that HLA-A1 expression by the chondrosarcoma cells limited their susceptibility to lysis by the anti-MAGE-A3 CTL clone. Incubation of FS cells with 50 U/mL interferon-gamma increased surface expression of HLA class-I molecules, increased their susceptibility to lysis, and had no effect on MAGE-A3 gene expression. These results suggest that immunotherapy targeted against chondrosarcoma cells is possible., ((c) 2007 Orthopaedic Research Society.)

Objective: To investigate mitogen activated protein (MAP) kinase pathways for their ability to differentially regulate the expression of matrix metalloprotease (MMP)-1 and -13 in human chondrosarcoma cells using pathway-selective inhibitors., Design: Human chondrosarcoma cell lines (SW1353 and JJ012) and human articular chondrocytes (HACs) were treated with cytokines (IL-1beta and TNFalpha) and the expression of MMP-1 and -13 was analyzed. The effects of MAP kinase inhibitors on cytokine-induced expression of MMP-1 and -13 were evaluated using ELISA and Western blot analyses. The possible involvement of the Runx2 pathway in mediating p38 effects on MMP-13 expression was analyzed using promoter-reporter assays, ELISA and immunoprecipitation analyses., Results: IL-1beta and TNFalpha strongly induced the expression of MMP-1 and -13 in SW1353 cells and HACs, whereas only TNFalpha was found to induce the expression of these two MMPs in JJ012 cells. Cytokine treatment did not result in a significant increase in the activity of MMPs because of the excess production of endogenous tissue inhibitors of metalloproteases (TIMPs). Treatment with p38 kinase inhibitors (SB203580 and SB242235) strongly inhibited cytokine-induced MMP-13 expression in a dose-dependent fashion while having a somewhat weaker inhibitory effect on MMP-1 expression. In contrast, inhibitors of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways did not inhibit the expression of either MMP. Overexpression of Runx2 robustly stimulated the transcriptional activation of MMP-13 but had no effect on MMP-1 expression. Furthermore, IL-1beta induced the phosphorylation of Runx2, and this effect was blocked by a p38 kinase inhibitor. Our data suggest that Runx2 is likely to be a key downstream mediator of p38 effects in the differential regulation of IL-1beta induced MMP-13 expression., Conclusions: These studies demonstrate the differential inhibition of cytokine-induced MMP-1 and -13 expression by p38 kinase inhibitors in human chondrosarcoma cells. Our studies also suggest the involvement of Runx2, at least in part, in mediating the effects of p38 on MMP-13 expression.

Chondrosarcoma is a malignant cartilage-forming tumour of bone, of which distinct clinicopathological subtypes are known. Conventional chondrosarcoma is notorious for its locally aggressive behaviour as well as for its resistance to chemotherapy and radiotherapy; so far surgery is the only effective therapeutic option. During the past 10 years, substantial new insights have been gained about molecular cell biology, molecular cytogenetics, and immunopathology, leading to better understanding of chondrosarcoma development at the molecular level, which will ultimately lead to better clinical understanding and possibly to the development of targeted treatment.

A mixed population of lymphocytes from a healthy donor co-existed with an established culture of allogeneic chondrosarcoma cells, during which time the tumor cells changed from malignantly transformed to benign fibroblast-like morphology; from multilayered to a monolayered growth pattern; lost their potency to grow in colonies in soft agar; and showed signs of senescence. A discussion of possible molecular mechanisms for this event is offered. If there are as yet undiscovered lymphokines that can induce reversal of the malignant geno/phenotype, the cognate gene(s) should be cloned for genetic engineering and for the mass production of the corresponding molecular mediators for clinical trials.

We investigated the effects of treatment with anti-parathyroid hormone-related protein (1-34) monoclonal murine antibody (anti-PTHrP MoAb) on apoptosis and the differentiation of chondrosarcoma HTB-94 cells. Treatment with anti-PTHrP MoAb accelerated apoptosis of HTB-94 cells in a dose-dependent manner, and anti-PTHrP MoAb also promoted the chondrogenic differentiation of HTB-94 cells. The induction of apoptosis by anti-PTHrP MoAb via imbalance of Bcl-2/Bax ratio and activation of caspase-3 may provide a mechanistic explanation for its potential antitumor effects. Our results suggest the possibility that anti-PTHrP MoAb may be beneficial as a new treatment for chondrosarcoma.

In this work, we describe a technique for the in-situ observation of cells adhered on opaque biomaterial surfaces. The visualisation of the morphology of cells adhered onto a surface allows to derive nuclear apoptotic signs or even the existence of organisation between groups of these cells. The technique is based on the use of an auto-immune reaction combined with a fluorescent agent that allows a direct inspection of the cell behaviour. The versatility of the technique is demonstrated by presenting several examples with different cultured cells (human chondrosarcome cells (CSRCs) and pluripotential mesenchymal stem cells (MSCs) from bone marrow) seeded over two different Ti-based surfaces (TiO(2) and TiN, respectively). These in-vitro observations are compared with the behaviour of the same cells on bare TiAlV alloy. From our results it is concluded that both TiO(2) and TiN surfaces show enhanced biological responses.

As an adjuvant to chemotherapy hyperthermia has proven to be successful as a treatment for osteo- and chondrosarcoma patients. The aim of this study was to investigate whether hyperthermia could increase cellular expression of heat-shock-protein 72 in human osteo- and chondrosarcoma cells and how heat treatment would affect their susceptibility to natural killer cell (NK-cell)-mediated lysis. About 5-10% of the peripheral mononuclear blood cells (PBMC) in the human peripheral blood are natural killer cells (NK-cells). Natural cytotoxicity, mediated by NK-cells, is believed to play an important role in host defense against cancer. The exact mechanisms of recognition of target cells and subsequent NK-cell activation are not yet known. NK-cells, isolated from the peripheral blood of healthy donors, were enriched by magnetic cell-separation to a purity of 85-97%, assessed by FACS-analysis. The susceptibility of heat-treated (42.5 degrees C, 90 minutes) and untreated osteosarcoma (MG63) and chondrosarcoma (HTB94) cell lines to NK-killing was determined by a release assay of lactate dehydrogenase (LDH). Lysis by NK-cells was increased by heat treatment of the target cells from 16.6% + 4.5% to 33% + 15%, p=0.035, for osteosarcoma cells, (E/T ratio of 5:1) and from 13.7% + 3.1% to 27.9% + 16.9%, p=0.021, (E/T ratio of 20:1) for chondrosarcoma cells. An increased expression of HSP72 of chondro- and osteosarcoma cells after heat treatment was detected by the Western blot technique. The results of this study show that hyperthermia increases HSP72 expression in osteo- and chondrosarcoma cells and their susceptibility to NK-cell-mediated lysis. These findings may lead to new therapeutic strategies, using hyperthermia to improve immunological defense against chondro- and osteosarcoma cells.

Although loss of HLA expression by malignant cells has also been demonstrated, it has not been clarified how the loss of HLA expression observed in vitro actually results in immune escape. We demonstrated two major findings: (i) a part of chromosome 6 coding for HLA haplotypes was deleted from the genome of chondrosarcoma cell line, OUMS-27; furthermore, immunohistostaining for HLA-A11 showed that the original chondrosarcoma tissue lost the expression of HLA-A11, implicating that HLA haplotype loss was already present in the original tumor tissue and (2) HLA class I-restricted and autologous tumor-specific cytotoxic T cells (CTL) were generated from peripheral blood lymphocytes of the patient with chondrosarcoma, from whom OUMS-27 originated. This CTL line was maintained by weekly stimulation with OUMS-27, and lysed OUMS-27 in an HLA-A24 dependent manner but did not either K562 or autologous (EBV)-transformed B cells. These observations indicated that OUMS-27 and its original tumor are still immunogenic and can present antigen peptides with the remaining HLA-A24, even if HLA expression is partially lost. Tumor specific immunotherapy can be applied to the treatment of malignancies, even if HLA expression is partially lost., (Copyright 2001 Academic Press.)

Osteosarcoma is the most frequent malignant bone tumor, mainly occurring in the second and third decade of life. Diagnosis is limited to clinical symptoms, radiology and histology, but so far no diagnostic laboratory tests are available. Heat shock proteins (hsp), highly conserved proteins performing vital intracellular chaperoning functions and preventing cells from death, have been shown to be involved in tumor immunity. We analyzed 75 sera from 23 patients with high-grade osteosarcoma, 8 patients with chondrosarcoma, 10 patients with Ewing's sarcoma, 5 patients with soft tissue sarcoma, 11 patients with benign bone tumors at the time of diagnosis and from 18 healthy controls with an indirect one-site enzyme linked immunosorbent assay (ELISA) for the presence of anti-hsp60 and 70 antibodies. In these assays 10/23 osteosarcoma patients (43%) had anti-hsp60 antibodies with a mean +/- S.D. titer of 0.382 +/- 0.243 U/ml. Only one of the 18 healthy controls (1/18, 5.6%; titer 0.22 U/ml), two of the Ewing's sarcoma patients (2/10, 20%; titer 0.2 +/- 0.09 U/ml), two of the patients with a benign bone tumor (2/11, 18%; titer 0.22 +/- 0.16 U/ml) and one of the chondrosarcoma patients (1/8, 12.5%; titer 0.14 U/ml) were positive, whereas all others, including all soft tissue sarcomas were negative throughout. Anti-hsp60 antibodies in patients with osteosarcoma are therefore significantly increased (p < 0.05). 19/23 (83%) of osteosarcoma biopsy specimens expressed hsp60 immunohistochemically and all specimens from patients with a positive anti-hsp60 serum titer expressed hsp60. The level of the anti-hsp60 antibodies did not correlate with clinical parameters such as response to preoperative chemotherapy, duration of symptoms, age, gender, tumor size, serum alkaline-phosphatase levels and metastases. Although no difference in anti-hsp70 antibodies could be observed between sera from patients and healthy controls, a positive correlation was found for the presence of anti-hsp70 serum antibodies and lung metastases at the time of diagnosis in osteosarcoma patients. These data suggest an increase of anti-hsp60 antibodies at the time of first diagnosis of osteosarcoma. These findings should therefore give rise to further investigations on a group of new markers for the diagnosis of osteosarcoma.

We investigated whether antibodies specific to autologous cancer cells are produced in the peripheral blood of patients with chondrosarcoma. There have been few reports on the investigation of the immune responses, such as autologous antibody production, to chondrosarcoma. Here, tumor-associated antigens were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and detected by immunoenzymatic amplification. A 56 kDa molecule (M56) was detected in the serum from patients' peripheral blood. M56 is ubiquitously expressed in various kinds of tissue-derived cells. However, the molecule seemed to be retained mostly in the cytosolic compartment of lymphoid cells, while it was expressed on the cell surface of nonlymphoid cancer cells. Furthermore, the antibodies reactive to the 56 kDa molecule were frequently observed in sera derived from patients with other cancers and autoimmune diseases as compared to the sera from healthy control donors, suggesting that M56 is a common target molecule of immune responses in patients with various cancers and autoimmune diseases.

To find specific humoral antibodies in sera from patients with temporomandibular joint (TMJ) osteoarthritis (OA), an immortal human chondrocyte (HCS-2/8) and osteoblast (Saos-2) cell line derived from a chondrosarcoma and an osteosarcoma, respectively, were used as source proteins of human antigens. Patients with chronically painful TMJ OA (n = 18) but no other joints symptoms were selected from a consecutive series of patients with temperomandibular disorders and sex-matched asymptomatic controls (n = 8) were also recruited. Cellular proteins of the HCS-2/8 and Saos-2 cells were subjected to Western blotting with the OA and control sera as probes. Band-recognition frequency and the peak optical density of the band were compared between groups by chi2 and t-tests. OA sera recognized various bands for the chondrocytes, and one of these (47-kDa) was specific for the OA sera. In two OA patients whose treatment outcome was less favorable, the reactivity against the 47-kDa protein was relatively high. In addition, the OA sera clearly cross-reacted with recombinant HSP47. Based on these findings, an autoimmune reaction against chondrocytes could be one of the exaggerating and/or perpetuating mechanisms in the pathophysiology of osteoarthritic TMJs, and the humoral antibody titre against the HSP47-like protein derived from the chondrocytes could be one of the possible markers for the prognosis of the joint pathology.

Aim: Clear cell chondrosarcoma (CCC) is a rare malignant cartilaginous neoplasm of bone. CCC is characterized by clear cells (CCC cells), osteoclasts and osteoblasts. Many important questions concerning the varied histological features of CCC, and the interactions between CCC cells and coexisting osteoclasts and osteoblasts have not been fully investigated and remain controversial. The aim of this study is to clarify and explain the varied histological features and the possible interaction between tumour cells (CCC cells) and stromal cells such as osteoclasts and osteoblasts., Methods and Results: Four cases of CCC were histologically and immunohistochemically studied in order to elucidate the biological nature and histological characteristics. A comparative study with chondroblastoma and grade I conventional chondrosarcoma (CC) was also performed. S100 protein and type II collagen were expressed in CCC cells, chondroblastoma cells and CC cells. CD68 and matrix metalloproteinase-9 were expressed in coexisting histiocytes and osteoclasts. Parathyroid hormone-like protein (PTH-LP) was expressed in histiocytes, osteoclasts, osteoblasts, chondroblastoma cells and CCC cells. Platelet-derived growth factor (PDGF) and its receptor (PDGF-R) were observed in osteoblasts, chondroblastoma cells and CCC cells. However, PTH-LP, PDGF and PDGF-R were not expressed in CC cells. PCNA (proliferating-cell nuclear antigen) was expressed more intensely in CCC than in chondroblastoma., Conclusion: These observations suggest that CCC cells trigger the varied histological changes in association with several cytokines. The difference of PCNA expression between CCC and chondroblastoma seemed to be related to the biological difference between the two tumours.

Thirty-five chordomas and more than 100 other tumors that have to be considered in the differential diagnosis, were immunohistochemically analyzed using a panel of antibodies including those to subsets of keratins (K), HBME-1, a monoclonal antibody recognizing an unknown antigen on mesothelial cells, and neuroendocrine markers. The patterns of immunoreactivities in chordoma were compared with those in renal cell carcinoma, colorectal mucinous adenocarcinoma, pituitary adenoma, skeletal chondrosarcoma, and extraskeletal myxoid chondrosarcoma (ESMC). Chordomas were consistently positive for keratin cocktail AE1/AE3, and for the individual keratins K8 and K19, and nearly always positive for K5, but they showed negative or only sporadic reactivity for K7 and K20. The keratin K8 and K19 reactivity was retained in those chordomas showing solid sheets of epithelioid, spindle cells, or cartilaginous metaplasia, and in one of two cases showing overtly sarcomatous transformation. In comparison, keratins were never present in skeletal chondrosarcoma, although K8 and to a lesser extent K19 were seen in occasional cases of ESMC with chordoid features. HBME-1 reacted strongly with chordoma and skeletal chondrosarcoma but was almost never positive in renal or colorectal carcinoma. These carcinomas lacked K5-reactivity, in contrast to chordoma. Chordomas were also consistently positive for neuron-specific enolase and occasionally focally for synaptophysin, but never for chromogranin. In contrast, pituitary adenomas regularly expressed the full spectrum of neuroendocrine markers and differed from chordoma by having a narrower repertoire of keratins, often showing negative or focal keratin 8- or AE1/AE3 reactivity and being almost always K19-negative. These findings indicate that chordoma can be immunohistochemically separated from tumors that can resemble it. Immunohistochemistry is especially useful in the diagnosis of small biopsy specimens that offer limited material for morphological observation.

Unlabelled: In order to investigate the clinicopathological and immunohistochemical features of chordomas, 34 chordomas, with 5 chondrosarcomas for comparison, were studied by clinicopathological and immunohistochemical methods., Results: Based on the presence or absence of cartilaginous areas, chordomas are classified into two subtypes: chondroid chordoma (14 cases) and classic chordoma (20 cases). Chondroid chordoma occurred in a younger age group (mean age 40.9 years) than classic chordoma (mean age 51.1 years). 7/14 (50%) of chondroid chordomas occurred in the sacrococcygeal region, 4/ 14 (28.6%) occurred in the spheno-occipital region. Immunohistochemical staining showed that all chordomas were positive for cytokeratin, and 16 (47.1%) chordomas were also positive for EMA. In contrast, 5 chondrosarcomas were immunonegative for both cytokeratin and EMA. Vimentin and S-100 protein were positive in the majority of chordomas (29 & 24 respectively) and in the 5 chondrosarcomas. The present study confirms the dual features of chordoma-epithelial and mesenchymal, and also the utility of immunohistochemical staining in the differential diagnosis of chordoma and chondrosarcoma. The pathologic diagnosis of chondroid chordoma and other issues were also discussed in the study.

To investigate the prognostic significance of the Ki67 (MIBI)-proliferation index and p53 over-expression in chondrosarcomas, we retrospectively analyzed a cohort of 29 patients with chondrosarcomas using immunohistochemical assays with MIBI and p53 monoclonal antibodies on formalin-fixed, paraffinembedded tissue samples with microwave preparation. We also assessed 19 patients with benign cartilaginous tumors as a control group. There was a significant positive correlation between MIBI index and tumor grade in chondrosarcomas, while there was no significant difference in the MIBI index between the grade-1 chondrosarcomas and the benign cartilaginous tumors. Patients categorized in the high-MIBI-index group had a significantly lower survival rate than those in the low-index group. Moreover, in analyzing the sub-set of the patients with grade-II chondrosarcomas, it was found that they could be prognostically sub-divided according to MIBI index. The p53 index also significantly correlated with patient survival, and there was significant correlation between the MIBI index and the p53 index. However, in multivariate analysis, only the MIBI index and tumor grade proved to be independent prognostic indicators of chondrosarcomas. These results demonstrate that the MIBI index can be a useful procedure for assessing tumor grade in chondrosarcomas, especially for determining the prognosis of patients with grade-II chondrosarcoma.

Eight syngeneic rat monoclonal antibodies that recognize structurally overlapping epitopes on the chondroitin proteoglycan NG2, a tumour-specific antigen on the chemically induced rat chondrosarcoma HSN, have been analysed for the sequence of their immunoglobulin heavy (H) and light (L) chain variable (V) regions. This analysis defined five groups of antibodies which are very similar for both the H and L chains and revealed that a wide range of different V regions are capable of binding to the same antigenic determinant. However, three mAbs, 11/160, ALN/12/17 and ALN/9/94, which recognize a sequential epitope, were found to use almost identical heavy (V-D-J) and light (V-J) chains in regions demonstrating an exclusivity in specific protein-protein interaction for this particular epitope. Two other mAbs, ALN/11/53 and AL/3/12, used similar V and J segments but totally different D regions. With the exception of the pair ALN/11/53 and AL/3/12, this grouping of antibodies matches that derived from the idiotypic specificity study we have reported previously. The reactivity pattern of Ab1 11/160, ALN/12/17 and ALN/9/94 with six anti-idiotopic mAbs raised against 11/160 demonstrated that the idiotope recognized by Ab2 HIM/3/41 was defined by a single amino acid, Asn, at position 52 within the CDR2 loop of the VH region; whereas the D region of Ab1 ALN/11/53 was implicated as the structural correlate of idiotypy. The substitution of AsnH52 influenced the Id recognition but Ag binding was not affected suggesting that Ab2 HIM/3/41 did not mimic the NG2 Ag.

Background: The availability of Ki-67 monoclonal antibody has opened new possibilities for an extensive analysis of cell kinetics in human neoplasms. Ki-67 antibody reveals a nuclear antigen that is expressed in proliferating but not in quiescent cells. Although the reliability of Ki-67 immunostaining has been evaluated in different tumor types, little information has been reported on bone neoplasms., Methods: Cell proliferation, as determined by Ki-67 expression, was measured by immunofluorescence on representative cytospins obtained from 205 patients with bone tumors. In each sample, the percentage of Ki-67-positive cells was quantified on at least 500 cells and expressed as Ki-67 labeling index (LI)., Results: Ki-67 LI was lower in benign and low grade lesions as compared with high grade malignant lesions. A correlation between Ki-67 LI and histologic grade was observed in osteosarcoma and chondrosarcoma. In osteosarcoma, among the 43 primary lesions included in this study, 30 patients, all treated with the same regimen of chemotherapy and limb-salvage surgery, were selected to establish the prognostic significance of cell proliferation. The Ki-67 labeling was higher in patients with a good histologic response to chemotherapy. However, at a 24-month follow-up, a worse prognosis was associated with a higher proliferative activity, whereas no correspondence was found between the histologic response to preoperative chemotherapy and the disease free survival, suggesting that in high grade osteosarcoma the biologic aggressiveness expressed by high levels of Ki-67 LI may be clinically more relevant than the responsiveness to antineoplastic agents., Conclusions: In bone tumors, the level of Ki-67 expression correlates with the level of malignancy and is diagnostically and prognostically useful.

S-100 protein (S-100) appears to be a marker for bone tumors of cartilaginous origin. Any analyses of proliferative activity in S-100-positive tumor cells, however, has not yet been presented. This study assessed the proliferative activity of those cells by means of a double-immunohistochemical staining method using proliferating cell nuclear antigen (PCNA) and S-100. The most intense reactivity for S-100 was found in the well-differentiated chondrocytes of enchondromas, osteochondromas, and osteosarcomas. On the contrary, the more immature the tumor cells were, the more intensely positive they were for PCNA. In parosteal chondrosarcoma, exceptionally, PCNA-positive as well as S-100-positive cells were abundant, suggesting that these proliferating cells produced S-100. In periosteal osteosarcoma, however, the proliferating cells labeled by PCNA revealed little reactivity for S-100. This immunohistochemical method is potentially useful to know the identity and origin of proliferating cells and may sometimes be diagnostic for bone tumors containing cartilaginous elements.

N-terminal sequences of 19 and 11 amino acids obtained from 2 different tryptic fragments of the tumour-specific antigen on the chemically induced rat chondrosarcoma HSN show a 100% homology with the rat chondroitin sulfate proteoglycan NG2. Using a scheme of overlapping oligonucleotide primers we have cloned by PCR amplification the cDNA for the specific antigen of the HSN tumour that is immunogenic in immunocompetent CBH/Cbi rats and now report that its cDNA sequence is identical to that of NG2. The cDNA codes for a transmembrane protein of 2,325 amino acids with a large extracellular domain of 2,224 amino acids containing 2 cysteine-rich regions, a transmembrane domain (25 amino acids) and a short cytoplasmic tail (76 amino acids). The tumour-specific determinant was found to lie between amino acid residues 556 and 992 on the core glycoprotein.

Cytogenetic analysis of four specimens (biopsy, definitive surgical, and two separately occurring lung metastases) of a dedifferentiated chondrosarcoma with a rhabdomyosarcomatous component revealed clonal karyotypic abnormalities in each. Anomalies seen in all specimens included a structurally aberrant chromosome 17 and extra copies of chromosomes 5, 7, 12, and 20. The derivation of the chromosomally abnormal cells was determined by a combined immunocytochemical/cytogenetic approach that allowed simultaneous assessment of cytogenetic aberrations and immunophenotypic features of individual cells. S-100 protein and desmin antibodies were used to evaluate the chondrosarcomatous and rhabdomyosarcomatous components, respectively. A chromosome 7-specific centromeric probe was used for determination of aneuploidy. In both specimens obtained from the primary lesion, S-100 protein and desmin-positive and -negative aneuploid cells were observed. These findings: 1) suggest that both the chondrocytic and rhabdomyoblastic cells arose from the same abnormal clone, 2) support the theory of a common primitive mesenchymal cell progenitor with the ability to differentiate or express features of more than one line of mesenchymal differentiation, and 3) indicate that the term dedifferentiated may be an inaccurate designation for this neoplasm.

A murine monoclonal antibody (E10) was made against cultured cartilage cells. The E10 antibody binding is localized to the surface of cultured cartilage cells in suspension and is present in the cytoplasm in paraffin embedded sections. There is no reactivity with cartilage matrix, or with the matrix of cartilaginous tumors. Reactivity is removed by treatment with trypsin and hyaluronidase, but not by treatment with heparinase, neuraminidase, and chondroitinase. Regeneration of E10 antigen after trypsinization takes 48 hours in chondrocytes in tissue culture. SDS-polyacrylamide gel electrophoresis of an E10 immune precipitate of cultured chondrocytes results in two peaks: one at a very high molecular weight and a small fragment at approximately 250 kd. Specificity has been demonstrated by cytofluorometry, immunofluorescence, and immunohistochemistry, in both frozen and paraffin-embedded tissues. Positive reactivity was seen in cultured cartilage cells, chondrocytes in fetal and adult cartilage, chondrosarcomas, and chordomas. Minimal reactivity was found in a chondromyxoid liposarcoma. Acinar cells of salivary and sweat glands and mast cells in various tissues and tumors were also positive. There was no reactivity with other tissues and tumors, including myxoid and mucinous tumors and epithelial tissues.

Non-neoplastic mesenchymal cells, along with 33 benign and 87 malignant soft-tissue tumors (STT) were examined for expression of HLA-A,B,C, beta 2-microglobulin (beta 2m), HLA-DR, -DP, and -DQ molecules and the HLA-D associated invariant chain (Ii). Serial frozen sections were immunostained using monoclonal antibodies (MAbs) to monomorphic framework determinants of HLA sublocus products, beta 2m and Ii, and to CD53, a recently defined broadly distributed pan-leucocyte molecule. Compared with the normal state, an induction/neo-expression of HLA-A,B,C/beta 2m was found in a considerable number of tumors of muscle, peripheral nerve, cartilage-forming, adipose, and vascular tissues. Conversely, some tumors of fibrous origin and of autonomic ganglia showed an abnormal abrogation/loss of HLA-A,B,C/beta 2m with respect to their cells of origin. Small, round tumor cells present in various types of STT exhibited a heterogenous pattern of expression of these molecules with a preponderance of HLA-A, B,C/beta 2m-negativity. HLA-D/Ii determinants were rarely detectable in STT. Besides their expression in some fibrohistiocytic tumors, they were only occasionally found in tumors of smooth-muscle, peripheral-nerve and vascular origin as well as in one clear-cell sarcoma. In all tumors but one, there was no microtopographic association between HLA-D/Ii-positive tumor cells and inflammatory cells. CD53 allowed discrimination between dendritic interstitial cells (DIC) and neoplastic cells and additionally revealed that, in contrast to other solid tumors, STT are generally characterized by an extreme scarcity of lymphohistiocytic infiltrates. Our data indicate that, aside from very rare exceptions, aberrant induction or abrogation of MHC molecules in STT occurs in the absence of lymphohistiocytic stromal infiltrates, suggesting that these alterations might not be a consequence of local cytokine effects.

High molecular weight DNA, which was isolated from a chondrosarcoma cell line, was transfected into human neonatal foreskin fibroblasts and NIH/3T3 cells. Both types of transfected cells expressed anchorage-independent growth in soft agar and produced tumors in nude mice. The tumors which developed in nude mice following injection of transfected human fibroblasts grew to approximately 0.8 cm in diameter in four weeks. The tumors which developed from transfected NIH/3T3 cells grew to greater than 2.0 cm in diameter in 6 weeks. After growth in soft agar the transfected human fibroblasts expressed a cell surface sarcoma-associated epitope recognized by the monoclonal antibody 345.134S. In addition to the transfected human fibroblasts, the original human chondrosarcoma tumor, the chondrosarcoma cell line derived from the tumor, and the nude mouse tumor which developed from transfected human fibroblasts all exhibited positive reactivity with the monoclonal antibody 345.134S. Transfected NIH/3T3 cells that exhibited anchorage-independent growth and tumorigenicity did not exhibit detectable reactivity with the monoclonal antibody. These results suggest that the expression of the tumor-associated cell surface antigen appears to be an early event correlated with transformation of the transfected human cells but not directly related to the tumorigenic potential of the DNA. The transfected cells which expressed anchorage independent growth exhibited the sarcoma cell surface antigen prior to attaining the potential for tumorigenicity.

Mesenchymal chondrosarcoma is an uncommon small-cell neoplasm of bone and soft tissue, the chondrogenic nature of which has been generally accepted. However, the phenotypic attributes of the small-cell population in this neoplasm have not been well characterized, and its relationship to "precartilage mesenchyme" remains unclear. In an attempt to address this issue, we performed an immunohistochemical analysis of nine cases, using antibodies to vimentin, S100 protein, Leu-7 antigen, neuron-specific enolase, synaptophysin, desmin, muscle-specific actin, cytokeratin, and epithelial membrane antigen, and the avidin-biotin-peroxidase complex (ABC) method. The small cells of mesenchymal chondrosarcoma failed to express S100 protein, whereas all components of the tumors (small cells, lacunar chondroblasts, and chondroid matrix) stained for Leu-7 antigen in six cases. Neuron-specific enolase was identified in the small cells of four cases and in the lacunar cells of seven. None contained desmin, actin, cytokeratin, epithelial membrane antigen, or synaptophysin. The immunophenotype of mesenchymal chondrosarcoma resembled that of embryonic cartilage and thus did not contradict the premise that this tumor was the neoplastic counterpart of fetal chondroid tissues. However, immunohistologic studies are not overly helpful in the differential diagnosis between mesenchymal chondrosarcoma and other small round cell lesions.

Levels of serum tumor markers including tissue polypeptide antigen (TPA), CA 15-3, CA 19-9, squamous cell carcinoma antigen, carcinoembryonic antigen, alpha-fetoprotein, and PAP were measured in 26 patients with bone metastasis and in 9 patients with primary bone tumors. More than one markers was elevated in 19 of the 26 patients with bone metastasis, although there was no elevation of the markers in 3 patients with renal cell carcinoma. TPA was the most sensitive marker in the diagnosis of metastasis. CA 15-3 was also a sensitive marker in this study, since metastasis from breast carcinoma may be the most common of all metastases in the skeleton. On the other hand, alpha-fetoprotein was uniformly unresponsive except in one case of gastric cancer. Combinations of markers are valuable for metastasis screening tests. No definite correlations were found between the markers in this study. On the other hand, there was a slight elevation of the markers observed in two of the nine patients with primary bone lesions. Serum tumor markers are useful in the diagnosis of bone metastasis to differentiate it from primary bone lesions. Especially in solitary bone lesions, serum markers may be the only way to make a differential diagnosis between the two.

A new "nude" mouse model was developed by successive crossings and backcrossings between athymic nu/nu mice, on an N:NIH(S) background, and female CBA/N mice that have an X-linked immune defect in B-lymphocyte function. The resulting doubly congenic N:NIH(S(II-nu/nu mice maintained the marked thymic hypoplasia and poor development of hair of the nu/nu mice. In contrast to nu/nu and CBA/N mice, in this new mouse model both T-cell zones of lymph nodes and the spleen were depleted of lymphocytes. Lymphocytic follicles were rare and diminutive; not germinal centers were noted. The nodal cortical and paracortical areas were represented principally by connective tissue, endothelial cells, and macrophages, including giant multinucleated cells. No medullary cords were recognized. The mice with combined immunodeficiency supported the growth of human tumor xenografts and were susceptible to murine viral hepatitis.

Blood small lymphocyte (not exceeding 7.5 micron in diameter) counts obtained from patients with malignant bone tumors in the course of primary examination were 40-75% those in healthy subjects. The said changes were registered only in some patients with osteoblastoclastoma; they were not observed in cases of trauma, osteomyelitis, sepsis, spontaneous osteolysis and chronic synovitis. The study failed to establish a correlation between blood small lymphocyte count, on the one hand, and concentrations of total, stable and active T-lymphocytes as well as autologous E-rosettes, on the other. In the course of separation of lymphocytes in percoll density gradients, small lymphocytes concentrated in high density fractions. Purified small lymphocytes of healthy subjects appeared to be mainly T-lymphocytes, particularly, "activated" ones. Proliferative response to PHA and production of interleukin-2 in cell cultures showing high levels of small lymphocytes were higher than in those with moderate or low concentration of the said cells. Small lymphocytes are considered to be a special subset of T-cells which exhibit high functional activity and may be identified only morphologically. Lowered counts of these cells are attributed to neoplastic growth.

2,6,10,14-Tetramethylpentadecane (pristane), s.c. injected simultaneously with plasmacytoma inocula, enhances the transplantability of tumor cells. The effect is dose and time dependent. The enhancement is shown only by plasmacytoma (MOPC-315 and MPC-11) and not by the murine lymphosarcoma and chondrosarcoma tested. Various mechanisms, such as stress and depression of humoral and cellular immunity, have been considered.

We have studied 32 patients with different bone neoplasias. 18 had osteogenic sarcoma. Clinical and immunological observations led us to consider our results as a general approach to the problem. No specific conclusions can be made. We describe 3 patients treated with levamisole therapy and two with transfer factor. In single patients some useful observations are connected about relationship between treated and non-treated patients.

By means of a micromethod of lymphocytotoxic test the content of eleven HL-A antigens (1, 2, 3, 5, 7, 8, 9, 10, 11, 12 and 13) was studied in 200 healthy human subjects, 100 patients with rheumatoid polyarthritis and 82 patients with bone tumors. The latter included 50 patients with benign tumors (osteoblastoclastomas, chondroblastomas, chondromas, non-osteogenic fibromas) and 32 patients with malignant tumors (chondro- and osteosarcomas, Ewing sarcoma, malignant osteoblastoclastomas). It was found that in patients with different bone tumors antigen HL-7 was encountered reliably more frequently than in control groups, in patients with malignant tumor also antigen HL-A10 was more often detected. The most frequently observed haplotyes in oncological patients were HL-A3/7 and HL-A2/7. In patients with rheumatoid polyarthritis antigen HL-A3 was found more rarely and antigen HL-A10 more frequently than in healthy subjects. The most frequent haplotypes in this group were as follows: HL-A2/7, HL-A2/8 and HL-A11/12. The possible mechanisms of the relationship between HL-A antigens and the development of pathology are discussed. Some considerations are offered concerning the possibility to utilize HL-A typing for an accessory differential diagnosis.