ALCAM-mediated cDC1 CD8 T cells interactions are suppressed in advanced lung tumors.

Conventional type 1 dendritic cells (cDC1) are critical regulators of anti-tumoral T-cell responses. The structure and abundance of intercellular contacts between cDC1 and CD8 T cells in cancer tissues is important to determine the outcome of the T-cell response. However, the molecular determinants controlling the stability of cDC1-CD8 interactions during cancer progression remain poorly investigated. Here, we generated a genetic model of non-small cell lung cancer crossed to a fluorescent cDC1 reporter (KP-XCR1venus) to allow the detection of cDC1-CD8T cell clusters in tumor tissues across tumor stages. We found that cDC1-CD8 clusters are abundant and productive at the early stages of tumor development but progressively diminish in advanced tumors. Transcriptional profiling and flow cytometry identified the adhesion molecule ALCAM/CD166 (Activated Leukocyte Cell Adhesion Molecule, ligand of CD6) as highly expressed by lung cDC1 and significantly downregulated in advanced tumors. Analysis of human datasets indicated that ALCAM is downregulated in non-small cell lung cancer and its expression correlates to better prognosis. Mechanistically, triggering ALCAM on lung cDC1 induces cytoskeletal remodeling and contact formation whereas its blockade prevents T-cell activation. Together, our results indicate that ALCAM is important to stabilize cDC1-CD8 interactions at early tumor stages, while its loss in advanced tumors contributes to immune evasion.

FHOD1 is upregulated in glioma cells and attenuates ferroptosis of glioma cells by targeting HSPB1 signaling.

BACKGROUND: As a new type of regulatory cell death, ferroptosis has been proven to be involved in cancer pathogenesis and therapeutic response. However, the detailed roles of ferroptosis or ferroptosis-associated genes in glioma remain to be clarified. METHODS: Here, we performed the TMT/iTRAQ-Based Quantitative Proteomic Approach to identify the differentially expressed proteins between glioma specimens and adjacent tissues. Kaplan-Meier survival was used to estimate the survival values. We also explored the regulatory roles of abnormally expressed formin homology 2 domain-containing protein 1 (FHOD1) in glioma ferroptosis sensitivity. RESULTS: In our study, FHOD1 was identified to be the most significantly upregulated protein in glioma tissues. Multiple glioma datasets revealed that the glioma patients with low FHOD1 expression displayed favorable survival time. Functional analysis proved that the knockdown of FHOD1 inhibited cell growth and improved the cellular sensitivity to ferroptosis in glioma cells T98G and U251. Mechanically, we found the up-regulation and hypomethylation of HSPB1, a negative regulator of ferroptosis, in glioma tissues. FHOD1 knockdown could enhance the ferroptosis sensitivity of glioma cells via up-regulating the methylated heat-shock protein B (HSPB1). Overexpression of HSPB1 significantly reversed FHOD1 knockdown-mediated ferroptosis. CONCLUSIONS: In summary, this study demonstrated that the FHOD1-HSPB1 axis exerts marked regulatory effects on ferroptosis, and might affect the prognosis and therapeutic response in glioma.

Uterine Sarcoma With FGFR1-TACC1 Gene Fusion: A Case Report and Review of the Literature.

With the growing availability of RNA sequencing technology in the pathology laboratory, new gene fusion-associated malignancies are increasingly being characterized. In this article, we describe the second ever reported case of a uterine sarcoma harboring a FGFR1-TACC1 gene fusion. The patient, a 53-yr-old perimenopausal woman, was found to have a 6 cm mass spanning the lower uterine segment and endocervix. Histologically, this was a spindle cell neoplasm with coagulative necrosis, moderate cytologic atypia, and increased mitotic activity. By immunohistochemistry, the neoplastic cells coexpressed CD34 and S100, and lacked smooth muscle marker expression. RNA sequencing revealed the presence of a FGFR1-TACC1 gene fusion. This report provides further evidence to suggest that FGFR1-TACC1 may be a recurrent fusion in a subset of uterine sarcomas. RNA sequencing using a panel that includes FGFR-TACC family fusions should be considered for uterine sarcomas that do not fit conventional diagnostic criteria, particularly as tumors with these fusions may be amenable to targeted therapy.

FGFR1/MAPK-directed brachyury activation drives PD-L1-mediated immune evasion to promote lung cancer progression.

Immune checkpoint inhibitors provide promising benefits for patients with cancer. However, efficacy has been encumbered by high resistance rates. It is critical to understand the basic mechanisms of tumor-mediated resistance to this treatment modality. Previous studies have found that the transcription factor brachyury is highly expressed in lung cancer. Here, we show that brachyury activation induces the upregulation of PD-L1 leading to inactivation of T cell proliferation in vitro and inhibited infiltration of CD8+ and CD3+ T cells into tumor in an immunocompetent mouse model. We further demonstrate that FGFR1/MAPK activation regulates brachyury and PD-L1 expressions and promotes immunosuppression. Blocking FGFR1/MAPK suppresses brachyury and PD-L1 expressions, revives immune activity, and reverses the resistance to anti-PD-1 treatment to produce a durable therapeutic response. We also find that lung cancer patients with high activation of the FGFR1-MAPK-brachyury-PD-L1 signature and low expression of CD8A, CD3D, or PDCD1 have worse survival outcomes. These findings elucidate a novel mechanism of immune escape from immune checkpoint therapy and provide an opportunity to enhance its therapeutic efficacy in the treatment of a subset of FGFR1/MAPK/brachyury/PD-L1-driven lung cancer.

Proteome profiling of enzalutamide-resistant cell lines and serum analysis identified ALCAM as marker of resistance in castration-resistant prostate cancer.

Enzalutamide (ENZA) is a frequently used therapy in metastatic castration-resistant prostate cancer (mCRPC). Baseline or acquired resistance to ENZA have been observed, but the molecular mechanisms of resistance are poorly understood. We aimed to identify proteins involved in ENZA resistance and to find therapy-predictive serum markers. We performed comparative proteome analyses on ENZA-sensitive parental (LAPC4, DuCaP) and -resistant prostate cancer cell lines (LAPC4-ENZA, DuCaP-ENZA) using liquid chromatography tandem mass spectrometry (LC-MS/MS). The top four most promising candidate markers were selected using bioinformatic approaches. Serum concentrations of selected markers (ALCAM, AGR2, NDRG1, IDH1) were measured in pretreatment samples of 72 ENZA-treated mCRPC patients using ELISA. In addition, ALCAM serum levels were measured in 101 Abiraterone (ABI) and 100 Docetaxel (DOC)-treated mCRPC patients' baseline samples. Results were correlated with clinical and follow-up data. The functional role of ALCAM in ENZA resistance was assessed in vitro using siRNA. Our proteome analyses revealed 731 significantly differentially abundant proteins between ENZA-sensitive and -resistant cells and our filtering methods identified four biomarker candidates. Serum analyses of these proteins revealed only ALCAM to be associated with poor patient survival. Furthermore, higher baseline ALCAM levels were associated with poor survival in ABI- but not in DOC-treated patients. In LAPC4-ENZA resistant cells, ALCAM silencing by siRNA knockdown resulted in significantly enhanced ENZA sensitivity. Our analyses revealed that ALCAM serum levels may help to identify ENZA- and ABI-resistant patients and may thereby help to optimize future clinical decision-making. Our functional analyses suggest the possible involvement of ALCAM in ENZA resistance.

LINC01140 inhibits nonsmall cell lung cancer progression and cisplatin resistance through the miR-4742-5p/TACC1 axis.

Recent studies show that lncRNAs participate in drug resistance and nonsmall cell lung cancer (NSCLC) progression. This study aimed to study the roles and mechanisms of long intergenic nonprotein coding RNA 01140 (LINC01140) in regulating NSCLC progression and drug resistance. Real-time quantitative polymerase chain reaction and western blot analysis were used to detect LINC01140, miR-4742-5p, and transforming acidic coiled-coil 1 (TACC1) expression in NSCLC cells. The interaction between two molecules was examined by luciferase reporter and/or RNA immunoprecipitation assays. Cell invasion, apoptosis, and cisplatin cytotoxicity were assessed by transwell invasion assay, flow cytometry analysis, and CCK-8 assay, respectively. LINC01140 was downregulated and miR-4742-5p was upregulated in NSCLC. LINC01140 inhibited miR-4742-5p expression by competitively binding to miR-4742-5p, while miR-4742-5p targeted TACC1 to inhibit TACC1 expression in NSCLC cells. LINC01140 enrichment repressed the invasive potential and cisplatin resistance and triggered apoptosis, which was reversed by miR-4742-5p overexpression. miR-4742-5p inhibition suppressed cell invasion and cisplatin resistance and accelerated apoptosis in NSCLC cells, while TACC1 silencing abolished these effects. Mechanistically, LINC01140 positively regulated TACC1 expression by sponging miR-4742-5p. In conclusion, LINC01140 inhibited NSCLC progression and cisplatin resistance via functioning as a ceRNA for miR-4742-5p to modulate TACC1.

Significant co-expression of putative cancer stem cell markers, EpCAM and CD166, correlates with tumor stage and invasive behavior in colorectal cancer.

BACKGROUND: The crucial oncogenic role of cancer stem cells (CSCs) in tumor maintenance, progression, drug resistance, and relapse has been clarified in different cancers, particularly in colorectal cancer (CRC). The current study was conducted to evaluate the co-expression pattern and clinical significance of epithelial cell adhesion molecules (EpCAM) and activated leukocyte cell adhesion (CD166 or ALCAM) in CRC patients. METHODS: This study was carried out on 458 paraffin-embedded CRC specimens by immunohistochemistry on tissue microarray (TMA) slides. RESULTS: Elevated expression of EpCAM and CD166 was observed in 61.5% (246/427) and 40.5% (164/405) of CRC cases. Our analysis showed a significant positive association of EpCAM expression with tumor size (P = 0.02), tumor stage (P = 0.007), tumor differentiate (P = 0.005), vascular (P = 0.01), neural (P = 0.01), and lymph node (P = 0.001) invasion. There were no significant differences between CD166 expression and clinicopathological parameters. Moreover, the combined analysis demonstrated a reciprocal significant correlation between EpCAM and CD166 expression (P = 0.02). Interestingly, there was a significant positive correlation between EpCAM/CD166 phenotypes expression and tumor stage (P = 0.03), tumor differentiation (P = 0.05), neural, and lymph node invasion (P =0.01). CONCLUSIONS: The significant correlation of EpCAM and CD166 expression and their association with tumor progression and aggressive behavior is the reason for the suggestion of these two CSC markers as promising targets to promote novel effective targeted-therapy strategies for cancer treatment in the present study.

The novel finding of an FGFR1::TACC1 fusion in an undifferentiated spindle cell sarcoma of soft tissue with aggressive clinical course.

We report an aggressive soft tissue sarcoma with FGFR1-TACC1 fusion occurring in the thigh of an 83-year-old man. Microscopically, the tumor was composed of monomorphic spindle cells arranged in intersecting fascicles. The tumor cells showed ovoid nuclei, fine chromatin, indistinct nucleoli, and elongated eosinophilic cytoplasm. Focal increase in nuclear atypia was noted. Immunohistochemistry showed only focal rare positivity to S100, but negative to SOX10, CD34, STAT6, TLE-1, SMA, and other myogenic markers. An extensive panel of immunostains did not reveal a definite lineage of cellular differentiation. The fusion junction of the chimeric transcript involved FGFR1 exon 16 and TACC1 exon 7, which was similar to those reported in other types of neoplasms such as glioblastomas and epithelial cancers. The transcript was predicted to be in-frame and confirmed by Sanger sequencing after reverse transcriptase-polymerase chain reaction. The patient was treated by marginal excision of tumor without receiving adjuvant therapy. He experienced rapid tumor recurrence with distant metastases and succumbed at 3.5 months after presentation. The finding of FGFR1-TACC1 fusion in a high-grade, undifferentiated spindle cell sarcoma of soft tissue is novel and may offer a potential therapeutic target in the near future.

Association between TBXT rs2305089 polymorphism and chordoma in Iranian patients identified by a developed T-ARMS-PCR assay.

BACKGROUND: Chordoma is a locally aggressive bone tumor with a high capability of recurrence. Because chordoma often occurs at critical locations next to neurovascular structures, there is an urgent need to introduce validated biomarkers. T-box transcription factor T (TBXT; OMIM: 601397) plays an important role in the pathogenesis and survival of chordoma cells. METHODS: Herein, we aimed to show whether rs2305089 polymorphism is correlated with chordoma in the Iranian population. In order to detect rs2305089, tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) was used. In total, 19 chordoma patients and 108 normal healthy individuals were recruited and screened using T-ARMS-PCR. The results were subsequently validated by Sanger sequencing. RESULTS: The genotype distributions and allele frequencies were significantly different among the patient and healthy groups (p-value <0.05). The A allele of rs2305089 showed a significant positive association with chordoma risk (p-value <0.05). DNA sequencing verified the T-ARMS-PCR results as well. This study demonstrated the association between TBXT rs2305089 and chordoma in an Iranian population using a simple, accurate, and cost-effective T-ARMS-PCR assay. CONCLUSIONS: Our results were in line with those of previous studies showing that TBXT rs2305089 is associated with chordoma development. We also developed an efficient T-ARMS-PCR assay to determine the genotype of rs2305089.

TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth.

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.

Recurrent loss of chromosome 22 and SMARCB1 deletion in extra-axial chordoma: A clinicopathological and molecular analysis.

Extra-axial chordoma is a rare neoplasm of extra-axial skeleton and soft tissue that shares identical histomorphologic and immunophenotypic features with midline chordoma. While genetic changes in conventional chordoma have been well-studied, the genomic alterations of extra-axial chordoma have not been reported. It is well known that conventional chordoma is a tumor with predominantly non-random copy number alterations and low mutational burden. Herein we describe the clinicopathologic and genomic characteristics of six cases of extra-axial chordoma, with genome-wide high-resolution single nucleotide polymorphism array, fluorescence in situ hybridization and targeted next-generation sequencing (NGS) analysis. The patients presented at a mean age of 33 years (range: 21-54) with a female to male ratio of 5:1. Four cases were histologically conventional type, presented with bone lesions and three of them had local recurrence. Two cases were poorly differentiated chordomas, presented with intra-articular soft tissue masses and both developed distant metastases. All cases showed brachyury positivity and the two poorly differentiated chordomas showed in addition loss of INI-1 expression by immunohistochemical analysis. Three of four extra-axial conventional chordomas showed simple genome with loss of chromosome 22 or a heterozygous deletion of SMARCB1. Both poorly differentiated chordomas demonstrated a complex hyperdiploid genomic profile with gain of multiple chromosomes and homozygous deletion of SMARCB1. Our findings show that heterozygous deletion of SMARCB1 or the loss of chromosome 22 is a consistent abnormality in extra-axial chordoma and transformation to poorly differentiated chordoma is characterized by homozygous loss of SMARCB1 associated with genomic complexity and instability such as hyperdiploidy.

Aurora kinase A is essential for meiosis in mouse oocytes.

The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile, and their oocytes fail to complete meiosis I. In determining AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at acentriolar microtubule organizing centers (aMTOCs; meiotic spindle poles). This activation induces fragmentation of the aMTOCs, a step essential for building a bipolar spindle. We also show that AURKA is required for regulating localization of TACC3, another protein required for spindle building. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.

Machine learning based tissue analysis reveals Brachyury has a diagnosis value in breast cancer.

BACKGROUND: The aim of the present study was to confirm the role of Brachyury in breast cancer and to verify whether four types of machine learning models can use Brachyury expression to predict the survival of patients. METHODS: We conducted a retrospective review of the medical records to obtain patient information, and made the patient's paraffin tissue into tissue chips for staining analysis. We selected 303 patients for research and implemented four machine learning algorithms, including multivariate logistic regression model, decision tree, artificial neural network and random forest, and compared the results of these models with each other. Area under the receiver operating characteristic (ROC) curve (AUC) was used to compare the results. RESULTS: The chi-square test results of relevant data suggested that the expression of Brachyury protein in cancer tissues was significantly higher than that in paracancerous tissues (P=0.0335); patients with breast cancer with high Brachyury expression had a worse overall survival (OS) compared with patients with low Brachyury expression. We also found that Brachyury expression was associated with ER expression (P=0.0489). Subsequently, we used four machine learning models to verify the relationship between Brachyury expression and the survival of patients with breast cancer. The results showed that the decision tree model had the best performance (AUC = 0.781). CONCLUSIONS: Brachyury is highly expressed in breast cancer and indicates that patients had a poor prognosis. Compared with conventional statistical methods, decision tree model shows superior performance in predicting the survival status of patients with breast cancer.

Phase I study of a multitargeted recombinant Ad5 PSA/MUC-1/brachyury-based immunotherapy vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: Antitumor vaccines targeting tumor-associated antigens (TAAs) can generate antitumor immune response. A novel vaccine platform using adenovirus 5 (Ad5) vectors [E1-, E2b-] targeting three TAAs-prostate-specific antigen (PSA), brachyury, and MUC-1-has been developed. Both brachyury and the C-terminus of MUC-1 are overexpressed in metastatic castration-resistant prostate cancer (mCRPC) and have been shown to play an important role in resistance to chemotherapy, epithelial-mesenchymal transition, and metastasis. The transgenes for PSA, brachyury, and MUC-1 all contain epitope modifications for the expression of CD8+ T-cell enhancer agonist epitopes. We report here the first-in-human trial of this vaccine platform. METHODS: Patients with mCRPC were given concurrently three vaccines targeting PSA, brachyury, and MUC-1 at 5×1011 viral particles (VP) each, subcutaneously every 3 weeks for a maximum of three doses (dose de-escalation cohort), followed by a booster vaccine every 8 weeks for 1 year (dose-expansion cohort only). The primary objective was to determine the safety and the recommended phase II dose. Immune assays and clinical responses were evaluated. RESULTS: Eighteen patients with mCRPC were enrolled between July 2018 and September 2019 and received at least one vaccination. Median PSA was 25.58 ng/mL (range, 0.65-1006 ng/mL). The vaccine was tolerable and safe, and no grade >3 treatment-related adverse events or dose-limiting toxicities (DLTs) were observed. One patient had a partial response, while five patients had confirmed PSA decline and five had stable disease for >6 months. Median progression-free survival was 22 weeks (95% CI: 19.1 to 34). Seventeen (100%) of 17 patients mounted T-cell responses to at least one TAA, whereras 8 (47%) of 17 patients mounted immune responses to all three TAAs. Multifunctional T-cell responses to PSA, MUC-1, and brachyury were also detected after vaccination in the majority of the patients. CONCLUSIONS: Ad5 PSA/MUC-1/brachyury vaccine is well tolerated. The primary end points were met and there were no DLTs. The recommended phase II dose is 5×1011 VP. The vaccine demonstrated clinical activity, including one partial response and confirmed PSA responses in five patients. Three patients with prolonged PSA responses received palliative radiation therapy. Further research is needed to evaluate the clinical benefit and immunogenicity of this vaccine in combination with other immuno-oncology agents and/or palliative radiation therapy. TRIAL REGISTRATION NUMBER: NCT03481816.

The Co-mutational Spectrum Determines the Therapeutic Response in Murine FGFR2 Fusion-Driven Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and a highly lethal malignancy. Chemotherapeutic options are limited, but a considerable subset of patients harbors genetic lesions for which targeted agents exist. Fibroblast growth factor receptor 2 (FGFR2) fusions belong to the most frequent and therapeutically relevant alterations in ICC, and the first FGFR inhibitor was recently approved for the treatment of patients with progressed, fusion-positive ICC. Response rates of up to 35% indicate that FGFR-targeted therapies are beneficial in many but not all patients. Thus far, no established biomarkers exist that predict resistance or response to FGFR-targeted therapies in patients with ICC. APPROACH AND RESULTS: In this study, we use an autochthonous murine model of ICC to demonstrate that FGFR2 fusions are potent drivers of malignant transformation. Furthermore, we provide preclinical evidence that the co-mutational spectrum acts not only as an accelerator of tumor development, but also modifies the response to targeted FGFR inhibitors. Using pharmacologic approaches and RNA-interference technology, we delineate that Kirsten rat sarcoma oncogene (KRAS)-activated mitogen-activated protein kinase signaling causes primary resistance to FGFR inhibitors in FGFR2 fusion-positive ICC. The translational relevance is supported by the observation that a subset of human FGFR2 fusion patients exhibits transcriptome profiles reminiscent of KRAS mutant ICC. Moreover, we demonstrate that combination therapy has the potential to overcome primary resistance and to sensitize tumors to FGFR inhibition. CONCLUSIONS: Our work highlights the importance of the co-mutational spectrum as a significant modifier of response in tumors that harbor potent oncogenic drivers. A better understanding of the genetic underpinnings of resistance will be pivotal to improve biomarker-guided patient selection and to design clinically relevant combination strategies.

Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity.

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop.

Randomized, Double-Blind, Placebo-Controlled Phase II Study of Yeast-Brachyury Vaccine (GI-6301) in Combination with Standard-of-Care Radiotherapy in Locally Advanced, Unresectable Chordoma.

BACKGROUND: Brachyury is a transcription factor overexpressed in chordoma and is associated with chemotherapy resistance and epithelial-to-mesenchymal transition. GI-6301 is a recombinant, heat-killed Saccharomyces cerevisiae yeast-based vaccine targeting brachyury. A previous phase I trial of GI-6301 demonstrated a signal of clinical activity in chordomas. This trial evaluated synergistic effects of GI-6301 vaccine plus radiation. MATERIALS AND METHODS: Adults with locally advanced, unresectable chordoma were treated on a randomized, placebo-controlled trial. Patients received three doses of GI-6301 (80 × 107 yeast cells) or placebo followed by radiation, followed by continued vaccine or placebo until progression. Primary endpoint was overall response rate, defined as a complete response (CR) or partial response (PR) in the irradiated tumor site at 24 months. Immune assays were conducted to evaluate immunogenicity. RESULTS: Between May 2015 and September 2019, 24 patients enrolled on the first randomized phase II study in chordoma. There was one PR in each arm; no CRs were observed. Median progressive-free survival for vaccine and placebo arms was 20.6 months (95% confidence interval [CI], 5.7-37.5 months) and 25.9 months (95% CI, 9.2-30.8 months), respectively. Hazard ratio was 1.02 (95% CI, 0.38-2.71). Vaccine was well tolerated with no vaccine-related serious adverse events. Preexisting brachyury-specific T cells were detected in most patients in both arms. Most patients developed T-cell responses during therapy, with no difference between arms in frequency or magnitude of response. CONCLUSION: No difference in overall response rate was observed, leading to early discontinuation of this trial due to low conditional power to detect statistical difference at the planned end of accrual. IMPLICATIONS FOR PRACTICE: Chordoma is a rare neoplasm lacking effective systemic therapies for advanced, unresectable disease. Lack of clinically actionable somatic mutations in chordoma makes development of targeted therapy quite challenging. While the combination of yeast-brachyury vaccine (GI-6301) and standard radiation therapy did not demonstrate synergistic antitumor effects, brachyury still remains a good target for developmental therapeutics in chordoma. Patients and their oncologists should consider early referral to centers with expertise in chordoma (or sarcoma) and encourage participation in clinical trials.

Neurofibrosarcoma Revisited: An Institutional Case Series of Uterine Sarcomas Harboring Kinase-related Fusions With Report of a Novel FGFR1-TACC1 Fusion.

Uterine sarcomas with variable CD34 and S100 expression represent an emerging class of tumor in the female genital tract which commonly presents in the endocervix of premenopausal women. Initial molecular characterization identified NTRK1 and NTRK3 gene fusions as oncogenic drivers in these tumors; however, the repertoire of genetic alterations is likely more diverse given the recent discovery of PDGFB and RET gene fusions in similarly described tumors. Importantly, these fusion events lead to the aberrant activation of kinases that are potentially therapeutically targetable; therefore, recognizing this class of tumor becomes critical for initiating the molecular testing required for an accurate diagnosis and identification of clinically actionable fusions. Here, we report our institutional experience with 12 cases of uterine spindle cell sarcomas harboring kinase-related fusions. Patients ranged from 21 to 80 years old (median, 38 y) and presented either asymptomatically or with pelvic pain and/or uterine bleeding. Eleven (92%; 11/12) tumors were localized to the cervix and 1 (8%; 1/12) tumor was localized in the anterior fundus of the uterine corpus. Tumors ranged in size from 1.5 to 15.0 cm (median, 6.0 cm) and were histologically characterized by a moderately cellular, infiltrative proliferation of spindle cells with features of benign gland entrapment, stromal collagen deposition, perivascular hyalinization, occasionally myxoid stroma, a lymphocytic infiltrate, occasional nuclear pseudoinclusions, and a pseudophyllodes architecture. RNA-sequencing identified NTRK1 (8/12), NTRK3 (1/12), and PDGFB (2/12) gene fusions, which have been previously implicated in this tumor class, as well as a novel FGFR1-TACC1 (1/12) fusion. All tumors in this cohort showed coexpression of CD34 and S100 by immunohistochemistry except for those tumors with PDGFB fusions which showed solely CD34 expression. Of the 10 surgically resected tumors with follow-up, outcomes best correlated with the stage of disease. One of 4 patients with stage IA tumors (1/4) had recurrences, half of the stage IB (2/4) tumors had recurrences and all of the stage IIB tumors (2/2) had recurrences and died of disease. Future studies are still required to better understand the spectrum of genetic alterations as well as evaluate the efficacy of targeted kinase inhibitors in this class of tumor.

Brachyury engineers cardiac repair competent stem cells.

To optimize the regenerative proficiency of stem cells, a cardiopoietic protein-based cocktail consisting of multiple growth factors has been developed and advanced into clinical trials for treatment of ischemic heart failure. Streamlining the inductors of cardiopoiesis would address the resource intensive nature of the current stem cell enhancement protocol. To this end, the microencapsulated-modified-mRNA (M3 RNA) technique was here applied to introduce early cardiogenic genes into human adipose-derived mesenchymal stem cells (AMSCs). A single mesodermal transcription factor, Brachyury, was sufficient to trigger high expression of cardiopoietic markers, Nkx2.5 and Mef2c. Engineered cardiopoietic stem cells (eCP) featured a transcriptome profile distinct from pre-engineered AMSCs. In vitro, eCP demonstrated protective antioxidant capacity with enhanced superoxide dismutase expression and activity; a vasculogenic secretome driving angiogenic tube formation; and macrophage polarizing immunomodulatory properties. In vivo, in a murine model of myocardial infarction, intramyocardial delivery of eCP (600 000 cells per heart) improved cardiac performance and protected against decompensated heart failure. Thus, heart repair competent stem cells, armed with antioxidant, vasculogenic, and immunomodulatory traits, are here engineered through a protein-independent single gene manipulation, expanding the available regenerative toolkit.

Poorly differentiated chordoma with whole-genome doubling evolving from a SMARCB1-deficient conventional chordoma: A case report.

Evolution of poorly differentiated chordoma from conventional chordoma has not been previously reported. We encountered a case of a poorly differentiated chordoma with evidence of whole-genome doubling arising from a SMARCB1-deficient conventional chordoma. The tumor presented as a destructive sacral mass in a 43-year-old man and was comprised of a highly cellular poorly differentiated chordoma with small, morphologically distinct nodules of conventional chordoma accounting for <5% of the total tumor volume. Immunohistochemistry (IHC) revealed both components were strongly reactive for brachyury and lacked normal staining for INI1. Single nucleotide polymorphism (SNP) array analysis identified multiple genomic imbalances in the conventional component, including deletions of 1p, 3p, and 22q (involving SMARCB1) and loss of chromosomes 5 and 15, while the poorly differentiated component exhibited the same aberrations at a more profound level with additional loss of chromosome 4, low level focal deletion of 17p (involving TP53), and tetraploidy. Homozygous deletion of SMARCB1 was present in both components. Fluorescence in situ hybridization (FISH) analysis confirmed the relevant deletions in both components as well as genome doubling in the poorly differentiated tumor. This case suggests that SMARCB1 loss is an early event in rare conventional chordomas that could potentially evolve into poorly differentiated chordoma through additional genomic aberrations such as genome doubling. Further studies with additional patients will be needed to determine if genome doubling is a consistent pathway for evolution of poorly differentiated chordoma.