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Chordoma is a rare bone tumor that frequently recurs after surgery, and the prognosis is poor with current treatments. This study aimed to identify potential novel immunotherapeutic targets for chordomas by identifying target proteins in clinical samples as well as tumor microenvironmental factors to enhance efficacy. Fourteen chordoma samples were analyzed by single-cell RNA sequencing, and B7-H3 and IL-7 were identified as potential targets and potentiators, respectively. B7-H3-targeted chimeric antigen receptor T (CAR-T) cells and B7-H3 CAR-T cells expressing IL-7 were synthesized and their anti-tumor activity evaluated in vitro, including in primary chordoma organoid models. The B7-H3 CAR-T/IL-7 therapy showed enhanced cytotoxicity and prolonged duration of action against tumor cells. Additionally, IL-7 modulated favorable subpopulations of cultured CAR-T cells, diminished immune checkpoint expression on T-cell surfaces, and enhanced T-cell functionality. The incorporation of IL-7 molecules into the B7-H3 CAR structure augmented CAR-T-cell function and improved CAR-T-cell efficacy, thus providing a novel dual therapeutic strategy for chordoma treatment., (© 2024. The Author(s).)

Background: Chordoma, a rare bone tumor, presents limited treatment options and patients typically exhibit poor survival outcomes. While immunotherapy has shown promising results in treating various tumors, research on the immune microenvironment of chordomas is still in its early stages. Therefore, understanding how the immune microenvironment of chordomas influences the outcomes of immunotherapy is crucial., Methods: We employed single-cell RNA sequencing (scRNA-seq), bulk RNA-seq, CellChat, gene set variation analysis, as well as calculation of immune features to further dissect the complex immune microenvironment of chordoma., Results: Previous research by van Oost et al argued that compared with other sarcomas, chordomas typically exhibit an immunologically "hot" microenvironment, a conclusion with which we concur based on their research findings. Additionally, the authors suggest that T cell-mediated immunotherapy is feasible for the majority of chordomas. However, we are inclined to categorize them as an immune-excluded phenotype according to the latest classification methods, rather than persisting with the concepts of "cold" and "hot". Unlike them, we explored immune infiltration scores (IS), T lymphocyte scoring (TLS), and human leucocyte antigen class I (HLA-I) using Bulk RNA-seq data from 126 chordoma patients and found that higher IS, TLS, and higher HLA-I expression were associated with poorer patient prognosis. Additionally, CellChat analysis of scRNA-seq results from six chordoma patients revealed no direct interaction between T cells and tumor cells., Conclusions: These findings suggested that the efficacy of T cell-based immunotherapy may be limited or even ineffective for patients with chordoma., Competing Interests: Competing interests: The authors declare that they have no competing interests, (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Chordomas are rare osseous neoplasms with a dismal prognosis when they recur. Here we identified cell surface proteins that could potentially serve as novel immunotherapeutic targets in patients with chordoma., Methods: Fourteen chordoma samples from patients attending Xuanwu Hospital Capital Medical University were subjected to single-cell RNA sequencing. Target molecules were identified on chordoma cells and cancer metastasis-related signalling pathways characterised. VEGFR-targeting CAR-T cells and VEGFR CAR-T cells with an additional TGF-β scFv were synthesised and their in vitro antitumor activities were evaluated, including in a primary chordoma organoid model., Results: Single-cell transcriptome sequencing identified the chordoma-specific antigen VEGFR and TGF-β as therapeutic targets. VRGFR CAR-T cells and VEGFR/TGF-β scFv CAR-T cells recognised antigen-positive cells and exhibited significant antitumor effects through CAR-T cell activation and cytokine secretion. Furthermore, VEGFR/TGF-β scFv CAR-T cells showed enhanced and sustained cytotoxicity of chordoma cell lines in vitro compared with VRGFR CAR-T cells., Conclusions: This study provides a comprehensive single-cell landscape of human chordoma and highlights its heterogeneity and the role played by TGF-β in chordoma progression. Our findings substantiate the potential of VEGFR as a target for CAR-T cell therapies in chordoma which, together with modulated TGF-β signalling, may augment the efficacy of CAR-T cells., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1) loss is associated with a poor prognosis in chordoma, while the mechanism remains largely unclear. Here, we aim to explore the function and regulatory mechanisms of SMARCB1 in chordoma., Materials and Methods: The effect of SMARCB1 on chordoma cells was investigated in vitro and in vivo. Chromatin immunoprecipitation (ChIP) sequencing was used to investigate the mechanisms of SMARCB1 in chordoma. The association between SMARCB1 and autophagy was validated by Western blot, immunofluorescence and transmission electron microscopy. In addition, the ATG5 expression in chordoma tissue was assessed using immunohistochemistry and correlated with patient survival., Results: SMARCB1 inhibited the malignant phenotype of chordoma cells in vitro and in vivo, supporting a tumour suppressor role of SMARCB1 in chordoma. ATG5-mediated autophagy was identified as a potential downstream pathway of SMARCB1. Mechanistically, SMARCB1 bound directly to the ATG5 promoter and epigenetically inhibited its transcription, which decreased ATG5 expression and impaired autophagy. Additionally, autophagy inhibitor chloroquine had a potential anti-cancer effect on chordoma cells in vitro. Moreover, high ATG5 expression was observed in recurrent chordoma patients, which independently correlated with adverse outcomes., Conclusions: Taken together, our results revealed that the SMARCB1/ATG5 axis is a promising therapeutic target for chordoma and autophagy inhibitors may be effective agents for chordoma treatment., (© 2021 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Background: There are no effective systemic therapies for chordoma. The recent successes of immunotherapeutic strategies in other cancers have resulted in a resurgence of interest in using immunotherapy in chordoma. These approaches rely on a functional interaction between the host's immune system and the expression of tumor peptides via the human leukocyte antigen (HLA) Class I antigen. It is not known whether chordoma cells express the HLA Class I antigen., Questions/purposes: (1) Do chordoma tumors exhibit defects in HLA Class I antigen expression? (2) What is the pattern of lymphocyte infiltration in chordoma tumors?, Methods: Patients with chordoma treated at Massachusetts General Hospital between 1989 and 2009 were identified with permission from the institutional review board. Of the 75 patients who were identified, 24 human chordoma tumors were selected from 24 distinct patients based on tissue availability. Histology slides from these 24 formalin-fixed paraffin-embedded chordoma tissue samples were deparaffinized using xylene and ethanol and underwent heat-induced antigen retrieval in a citrate buffer. Samples were incubated with monoclonal antibodies directed against HLA Class I antigen processing machinery components. Antibody binding was detected via immunohistochemical staining. Staining intensity (negative, weakly positive, strongly positive) was assessed semiquantitatively and the percentage of chordoma cells stained for HLA Class I antigen subunits was assessed quantitatively. Hematoxylin and eosin-stained histology slides from the same 24 chordoma samples were assessed qualitatively for the presence of tumor-infiltrating lymphocytes and histologic location of these lymphocytes. Immunohistochemical staining with monoclonal antibodies directed against CD4 and CD8 was performed in a quantitative manner to identify the lymphocyte subtype present in chordoma tumors. All results were scored independently by two investigators and were confirmed by a senior bone and soft tissue pathologist., Results: Seven of 24 chordoma samples exhibited no staining by the anti-HLA-A heavy chain monoclonal antibody HC-A2, two had weak staining intensity, and eight had a heterogeneous staining pattern, with fewer than 60% of chordoma cells exhibiting positive staining results. Four of 24 samples tested were not stained by the anti-HLA-B/C heavy chain monoclonal antibody HC-10, five had weak staining intensity, and 11 displayed a heterogeneous staining pattern. For the anti-β-2-microglobulin monoclonal antibody NAMB-1, staining was detected in all samples, but 11 had weak staining intensity and four displayed a heterogeneous staining pattern. Twenty-one of 24 samples tested had decreased expression in at least one subunit of HLA Class I antigens. No tumors were negative for all three subunits. Lymphocytic infiltration was found in 21 of 24 samples. Lymphocytes were primarily found in the fibrous septae between chordoma lobules but also within the tumor lobules and within the fibrous septae and tumor lobules. Twenty-one of 24 tumors had CD4+ T cells and 11 had CD8+ T cells., Conclusion: In chordoma tissue samples, HLA Class I antigen defects commonly were present, suggesting a mechanism for escape from host immunosurveillance. Additionally, nearly half of the tested samples had cytotoxic CD8+ T cells present in chordoma tumors, suggesting that the host may be capable of mounting an immune response against chordoma tumors. The resulting selective pressure imposed on chordoma tumors may lead to the outgrowth of chordoma cell subpopulations that can evade the host's immune system., Clinical Relevance: These findings have implications in the design of immunotherapeutic strategies for chordoma treatment. T cell recognition of tumor cells requires HLA Class I antigen expression on the targeted tumor cells. Defects in HLA Class I expression may play a role in the clinical course of chordoma and may account for the limited or lack of efficacy of T cell-based immunity triggered by vaccines and/or checkpoint inhibitors., Competing Interests: All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request., (Copyright © 2020 by the Association of Bone and Joint Surgeons.)

Purpose: Chordoma is a rare tumor of the skeletal system that is characterized by a high recurrence rate and treatment resistance. Given the common finding of immune dysregulation in chordoma, immunotherapy has emerged as potential treatment option. As an important immune checkpoint regulator, we evaluated cytotoxic T-lymphocyte antigen-4 (CTLA-4) expression and its prognostic significance for patients with chordoma of the spine., Methods: CTLA-4 expression was analyzed immunohistochemically in 32 chordoma tissues and 14 nucleus pulposus tissues to examine the specificity of CTLA-4 expression in chordoma. Univariate log-rank analysis was used to evaluate the association of CTLA-4 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) with survival. Cox multivariate analysis was used to identify independent factors of survival., Results: Positive CTLA-4 expression was observed in all of the TILs and tumor cell cytoplasm, and partial in the membrane or in both the membrane and nucleus, with a markedly higher positivity rate than that observed in normal nucleus tissues. Higher CTLA-4 expression in the tumor but not in TILs was significantly associated with shorter continuous disease-free survival (CDFS) and overall survival (OS). CTLA-4 expression in tumor cells and TILs were independent predictors for CDFS, whereas only tumor cell expression was a significant predictor of OS. Furthermore, the combination of CTLA-4 expression in the tumor and TILs had higher prognostic value., Conclusions: Targeting CTLA-4 may be a potential novel therapeutic strategy for chordoma patients.

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.

Chordomas are rare malignant tumours, which typically occur in the axial skeleton and skull base. They arise from embryonic remnants of the notochord. They constitute less than 5 % of primary bone tumours. They are characterised by their locally aggressive potential with high frequency of recurrences and a median overall survival of 6 years. The initial therapeutic strategy must be discussed in an expert centre and may involve surgery, preoperative radiotherapy, exclusive radiotherapy or therapeutic abstention. Despite this, more than 50 % of patients will be facing recurrences with few therapeutic options available at this advanced stage. This review aims to outline current treatment options available in chordomas, as well as discussing potentiality of new therapeutic approaches through their molecular characterization and the comprehension of their immunological environment., (Copyright © 2019 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Background: Currently, little is known about the clinical relevance of tumor-stroma ratio (TSR) in chordoma and data discussing the relationship between TSR and immune status of chordoma are lacking., Objective: To characterize TSR distribution in spinal chordoma, and investigated its correlation with clinicopathologic or immunological features of patients and outcome., Methods: TSR was assessed visually on hematoxylin and eosin-stained sections from 54 tumor specimens by 2 independent pathologists. Multiplex immunofluorescence was used to quantify the expression levels of microvessel density, Ki-67, Brachyury, and tumor as well as stromal PD-L1. Tumor immunity status including the Immunoscore and densities of tumor-infiltrating lymphocytes (TILs) subtypes were obtained from our published data and reanalyzed., Results: Bland-Altman plot showed no difference between mean TSR derived from the two observers. TSR was positively associated with stromal PD-L1 expression, the Immunoscore and CD3+ as well as CD4+ TILs density, but negatively correlated with tumor microvessel density, Ki-67 index, surrounding muscle invasion by tumor and number of Foxp3+ and PD-1+ TILs. Low TSR independently predicted poor local recurrence-free survival and overall survival. Moreover, patients with low TSR and low Immunoscore chordoma phenotype were associated with the worst survival. More importantly, combined TSR and Immunoscore accurately reflected prognosis and enhanced the ability of TSR or Immunoscore alone for outcome prediction., Conclusion: These data reveal the significant impact of TSR on tumor progression and immunological response of patients. Subsequent use of agents targeting the stroma compartment may be an effective strategy to treat chordoma especially in combination with immune-based drugs., (Copyright © 2019 by the Congress of Neurological Surgeons.)

Chordoma is difficult to eradicate due to high local recurrence rates. The immune microenvironment is closely associated with tumor prognosis; however, its role in skull base chordoma is unknown. The expression of Galectin-9 (Gal9) and tumor-infiltrating lymphocyte (TIL) markers was assessed by immunohistochemistry. Kaplan-Meier and multivariate Cox analyses were used to assessing local recurrence-free survival (LRFS) and overall survival (OS) of patients. MiR-455-5p was identified as a regulator of Gal9 expression. Immunopositivity for Gal9 was associated with tumor invasion (p = 0.019), Karnofsky performance status (KPS) score (p = 0.017), and total TIL count (p < 0.001); downregulation of miR-455-5p was correlated with tumor invasion (p = 0.017) and poor prognosis; and the T-cell immunoglobulin and mucin-domain 3 (TIM3) + TIL count was associated with chordoma invasion (p = 0.010) and KPS score (p = 0.037). Furthermore, multivariate analysis indicated that only TIM3 + TIL density was an independent prognostic factor for LRFS (p = 0.010) and OS (p = 0.016). These results can be used to predict clinical outcome and provide a basis for immune therapy in skull base chordoma patients.

Background: Currently, clinical implications of immune system cells in chordoma remain to be elucidated., Objective: To characterize in situ immune cell infiltrates, the Immunoscore, and investigate their correlation with clinicopathologic data of spinal chordoma patients and outcome., Methods: Tumor-infiltrating lymphocytes (TILs) subtypes were assessed in 54 tumor specimens using immunohistochemistry for CD3, CD4, CD8, CD20, Foxp3, PD-1, and PD-L1., Results: Overall, immune cell infiltrates were present in all samples and there was low or moderate correlation among several TILs subsets. PD-1+ TILs density, CD3+, and CD8+ TILs densities in the tumor interior (TI) subarea were associated with surrounding muscle invasion by tumor, whereas PD-L1+ TILs showed inverse association with tumor pathological grade and stage. The density of PD-1+ TILs, PD-L1+ TILs, CD4+ TILs, and CD3+ TILs both in the TI and combined tumor regions (TI and invasion margin) were significantly associated with local recurrence-free survival and overall survival (OS). However, Foxp3+ TILs (P = .024) and CD8+ TILs evaluated in the TI (P < .001) only correlated with OS. The Immunoscore predicted less aggressive clinical features and favorable outcomes. Patients with an Immunoscore of 4 had a median OS of 128 mo, while I0 (Immunoscore of 0) patients survived only 27 mo. Multivariate analysis demonstrated that the Immunoscore was an independent favorable prognostic factor of both local recurrence-free survival (P = .026) and OS (P = .046)., Conclusion: Our data suggest a clinically relevant role of the immune microenvironment in spinal chordoma and identify the Immunoscore as promising prognostic marker., (Copyright © 2018 by the Congress of Neurological Surgeons.)

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for chordoma.

Currently, little is known about the interactions between microRNAs (miRNAs) and the PD-1/PD-L1 signaling pathway in chordoma, and data discussing the role of the immune milieu in chordoma prognosis are limited. We aimed to analyze the relationship between PD-L1, miR-574-3p, microenvironmental tumor-infiltrating lymphocytes (TILs) and clinicopathological features of spinal chordoma patients. PD-L1 expression and TILs (including Foxp3 + , CD8 + , PD-1 + and PD-L1 + ) were assessed by immunohistochemistry in tumor specimens of 54 spinal chordoma patients. MiRNAs microarray and bioinformatical analysis were used to identify miRNAs potentially regulating PD-L1 expression, which were further validated by quantitative RT-PCR. miR-574-3p was identified to potentially regulate PD-L1 expression in chordoma, which inversely correlated with PD-L1. Positive PD-L1 expression on tumor cells was associated with advanced stages (P = 0.041) and TILs infiltration (P = 0.005), whereas decreased miR-574-3p level correlated with higher muscle invasion (P = 0.012), more severe tumor necrosis (P = 0.022) and poor patient survival. Importantly, a patient subgroup with PD-L1 + /miR-574-3p low chordoma phenotype was significantly associated with worse local recurrence-free survival (LRFS) (P = 0.026). PD-1 + TILs density was associated with surrounding muscle invasion (P = 0.014), and independently portended poor LRFS (P = 0.040), while PD-L1 + TILs showed tendencies of less aggressive clinical outcomes. Multivariate analysis of OS only found CD8 + /Foxp3 + ratio to be independent prognostic factor (P = 0.022). These findings may be useful to stratify patients into prognostic groups and provide a rationale for the use of checkpoint blockade therapy, possibly by administering miR-574-3p mimics, in spinal chordoma.

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.

Chordoma is a locally aggressive primary malignancy of the axial skeleton. The gold standard for treatment is en bloc resection, with some centers now advocating for the use of radiation to help mitigate the risk of recurrence. Local recurrence is common, and salvaging local failures is quite difficult. Chemotherapy has been ineffective and small molecule targeted therapy has had only marginal benefits in small subsets of patients with rare tumor phenotypes or refractory disease. Recent successes utilizing immunotherapy in a variety of cancers has led to a resurgence of interest in modifying the host immune system to develop new ways to treat tumors. This review will discuss these studies and will highlight the early studies employing immune strategies for the treatment of chordoma.

Background: The Response Evaluation Criteria in Solid Tumors (RECIST) are the current standard for evaluating disease progression or therapy response in patients with solid tumors. RECIST 1.1 calls for axial, longest-diameter (or perpendicular short axis of lymph nodes) measurements of a maximum of five tumors, which limits clinicians' ability to adequately measure disease burden, especially in patients with irregularly shaped tumors. This is especially problematic in chordoma, a disease for which RECIST does not always adequately capture disease burden because chordoma tumors are typically irregularly shaped and slow-growing. Furthermore, primary chordoma tumors tend to be adjacent to vital structures in the skull or sacrum that, when compressed, lead to significant clinical consequences., Methods: Volumetric segmentation is a newer technology that allows tumor burden to be measured in three dimensions on either MR or CT. Here, we compared the ability of RECIST measurements and tumor volumes to predict clinical outcomes in a cohort of 21 chordoma patients receiving immunotherapy., Results: There was a significant difference in radiologic time to progression Kaplan-Meier curves between clinical outcome groups using volumetric segmentation (P = 0.012) but not RECIST (P = 0.38). In several cases, changes in volume were earlier and more sensitive reflections of clinical status., Conclusion: RECIST is a useful evaluation method when obvious changes are occurring in patients with chordoma. However, in many cases, RECIST does not detect small changes, and volumetric assessment was capable of detecting changes and predicting clinical outcome earlier than RECIST. Although this study was small and retrospective, we believe our results warrant further research in this area.

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC., Competing Interests: The authors declare no conflicts of interest.

Purpose: To provide the foundation for combining immunotherapy to induce tumor antigen-specific T cells with proton radiation therapy to exploit the activity of those T cells., Methods and Materials: Using cell lines of tumors frequently treated with proton radiation, such as prostate, breast, lung, and chordoma, we examined the effect of proton radiation on the viability and induction of immunogenic modulation in tumor cells by flow cytometric and immunofluorescent analysis of surface phenotype and the functional immune consequences., Results: These studies show for the first time that (1) proton and photon radiation induced comparable up-regulation of surface molecules involved in immune recognition (histocompatibility leukocyte antigen, intercellular adhesion molecule 1, and the tumor-associated antigens carcinoembryonic antigen and mucin 1); (2) proton radiation mediated calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells; and (3) cancer stem cells, which are resistant to the direct cytolytic activity of proton radiation, nonetheless up-regulated calreticulin after radiation in a manner similar to non-cancer stem cells., Conclusions: These findings offer a rationale for the use of proton radiation in combination with immunotherapy, including for patients who have failed radiation therapy alone or have limited treatment options., (Published by Elsevier Inc.)

The nuclear transcription factor brachyury has previously been shown to be a strong mediator of the epithelial-to-mesenchymal transition (EMT) in human carcinoma cells and a strong negative prognostic factor in several tumor types. Brachyury is overexpressed in a range of human carcinomas as well as in chordoma, a rare tumor for which there is no standard systemic therapy. Preclinical studies have shown that a recombinant Saccharomyces cerevisiae (yeast) vaccine encoding brachyury (GI-6301) can activate human T cells in vitro. A phase I dose-escalation (3+3 design) trial enrolled 34 patients at 4 dose levels [3, 3, 16, and 11 patients, respectively, at 4, 16, 40, and 80 yeast units (YU)]. Expansion cohorts were enrolled at 40- and 80-YU dose levels for analysis of immune response and clinical activity. We observed brachyury-specific T-cell immune responses in the majority of evaluable patients despite most having been heavily pretreated. No evidence of autoimmunity or other serious adverse events was observed. Two chordoma patients showed evidence of disease control (one mixed response and one partial response). A patient with colorectal carcinoma, who enrolled on study with a large progressing pelvic mass and rising carcinoembryonic antigen (CEA), remains on study for greater than 1 year with stable disease, evidence of decreased tumor density, and decreased serum CEA. This is the first-in-human study to demonstrate the safety and immunogenicity of this therapeutic cancer vaccine and provides the rationale for exploration in phase II studies. A randomized phase II chordoma study is now enrolling patients., (©2015 American Association for Cancer Research.)

Chordomas are primary malignant tumors of the notochord that are resistant to conventional chemotherapy. Expression of programmed cell death ligand 1 (PD-L1), prevalence of tumor-infiltrating lymphocytes (TILs), and their clinical relevance in chordoma remain unknown. We evaluated PD-L1 expression in three chordoma cell lines and nine chordoma tissue samples by western blot. Immunohistochemical staining was performed on a chordoma tissue microarray (TMA) that contained 78 tissue specimens. We also correlated the expression of PD-L1 and TILs with clinical outcomes. PD-L1 protein expression was demonstrated to be induced by IFN-γ in both UCH1 and UCH2 cell lines. Across nine human chordoma tissue samples, PD-L1 protein was differentially expressed. 94.9% of chordoma samples showed positive PD-L1 expression in the TMA. The expression score of PD-L1 for metastatic chordoma tumors was significant higher as compared with non-metastatic chordoma tumors. Expression of PD-L1 protein significantly correlates with the presence of elevated TILs, which correlates with metastasis. In summary, our study showed high levels of PD-L1 are expressed in chordoma, which is correlated with the prevalence of TILs. The current study suggests targeting PD-L1 may be a novel immunotherapeutic strategy for chordoma clinical trials.

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.

A 67-year-old white man with a remote history of a chordoma of the clivus presented with myasthenia gravis. We investigated the possibility that these conditions were related immunologically. Tissue sections of various chordoma specimens were reacted with dilutions of patient serum and control serum by an indirect immunoperoxidase method. In addition, sections were reacted with antibodies to muscle antigens. Of six chordomas, five reacted positively to patient serum. None reacted to control serum. One chordoma reacted positively to desmin, and all six reacted positively to myoglobin. We propose that the patient may have produced antibody to muscle-like antigens of the chordoma that subsequently cross-reacted with acetylcholine receptor and led to clinical myasthenia gravis.

The expression of the embryonal and of the differentiational proteins in 12 cases of chordoma and in the notochord of a 4-month-old and a 5-month-old human embryo have been examined immunohistologically by the ABC method using polyclonal antibodies to CEA, AFP, or S-100, and the monoclonal antibody to cytokeratin. It was found that S-100 was expressed in all cases of chordoma and in the notochords examined. CEA and cytokeratin also were found expressed in some cases of chordoma but not in the notochords. These proteins were found expressed more strongly in chordomas without a metastasis than in those with a metastasis. In the metastatic lesions, these proteins were expressed more strongly than in the primary lesions. The antibody to AFP reacted with neither the chordomas nor the notochords tested. These results suggest a possible link between the gene-expression of the tumor cells and the microenvironment in which they are harbored.

The application of immunohistochemical staining with anti-epithelial monoclonal antibodies to the differential diagnosis of chordomas is described. Cytokeratins and an epithelial membrane-specific oligosaccharide sequence are found in chordomas but not in chondrosarcomas or normal cartilage. The same cytokeratins and oligosaccharide sequence are demonstrated in human fetal notochord. Immunohistochemical staining with antiepithelial antibodies is therefore of value in distinguishing chordomas from cartilaginous tumours. The staining of notochord with the same monoclonal antibodies adds weight to the proposition that chordomas arise from embryonic rests of notochordal cells.

Fourteen chordomas, five myxoid chondrosarcomas, and 15 chondroid tumors (four mesenchymal and 11 conventional chondrosarcomas) were stained for cytokeratin, S-100 protein, carcinoembryonic antigen (CEA), and vimentin. The epithelial markers stained 93 per cent of the chordomas, whereas none of the tumors of other types showed any staining. Sixty-four per cent of the chordomas, 20 per cent of the myxoid chondrosarcomas, and 87 per cent of the other chondroid tumors were positive for S-100 protein. All of the tumors were positive for vimentin. Three of the 14 chordomas were positive for CEA. The present study confirms the utility of these markers in the differential diagnosis of chordoma and tumors with similar histologic characteristics.

Seven chordomas, chondrosarcomas, and mucinous colorectal carcinomas, five liposarcomas, and five ependymomas were evaluated for the presence of epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA) by using an immunoperoxidase technique. All of the chordomas and mucinous carcinomas were cytoplasmic EMA positive, while the chondrosarcomas, liposarcomas, and ependymomas were negative. Among the tumors that were studied, only the mucinous carcinomas were positive for CEA. The EMA positivity of a chordoma reflects its epithelial nature and is a valuable aid in making the differential diagnosis between a chordoma and nonepithelial tumors, while the absence of CEA in a chordoma aids in making the distinction between a chordoma and a mucinous carcinoma.

Chondroid chordomas are shown to possess cytokeratins and to stain with HMFG-2 as do ordinary chordomas. These findings support the concepts that these neoplasms are a variant of chordoma and that they are unrelated to myxoid chondrosarcoma.

Tumor cells in primary cultures derived from 11 untreated nonhemopoietic cancer patients were reacted with specifically coated sheep erythrocytes. Rosette formation between tumor and indicator cells was assessed, Eight of the primary cultures reacted positively with both IgG-coated (EAIgG) and with IgM-human complement coated (EAIgMC or EAIgMC 4,3) sheep erythrocytes. EAIgG rosette formation in positive cultures ranged from 25 to 85%, and for EAIgMC/EAIgMC 4,3 reactivity ranged between 22--95%. Rosette formation with E (uncoated) and EAIgM was negligible. These findings suggest that human nonhemopoietic tumor cells may carry on their surface receptor sites for an IgG component of immune complexes and for human complement. These receptor sites may be important in the host-tumor relationship.

A cartilaginous tumor of the cervical spine in a 31-year-old man is described, with discussion of the differential diagnostic problems both in aspiration biopsy cytology and in histopathology. The tumor mass caused nasopharyngeal swelling and infiltrated the base of the skull and the spinal canal. Aspiration biopsy cytology of the tumor demonstrated mostly papillary epithelial groupings, causing differential diagnostic problems with thyroid and salivary gland carcinomas. Most of the tumor mass was resected, with postoperative radiation performed. Immunohistochemical staining for type II procollagen on the histologic sections confirmed the presence of cartilage. The epithelial components of the tumor appeared to be cytokeratin-positive. Immunohistochemical staining for laminin and type IV collagen revealed positivity only in the blood vessel walls. The diagnosis of cervical chordoma was established by the histologic examination. The patient is without symptoms two years after removal of the tumor.

An indirect immunoperoxidase method was used in an attempt to detect carcinoembryonic antigen (CEA) in 17 well-differentiated colonic adenocarcinomas, five osteosarcomas, and four chordomas. Fifteen of the adenocarcinomas were positive, and five of them had been fixed and embedded in 1952 and one in 1942. Two other adenocarcinomas from 1942 were not positive. CEA was not demonstrated in any of the osteosarcomas or chordomas.

Lymphocyte blastogenesis induced by autologous tumor biopsy cells, by allogeneic lymphocytes and by PHA, was performed in the presence of autologous or allogeneic serum collected from normal blood donors. In 13 of the 15 cases lymphocyte stimulation by autologous tumor cells was inhibited in the presence of autologous serum. In contrast, autologous serum somewhat enhanced the blastogenic effect of PHA and allogeneic lymphocytes.