Targeting of Tumor-Associated Glycoforms of MUC1 with CAR T Cells.

We read with interest the manuscript by June and colleagues published recently in Immunity in which they describe targeting of aberrantly glycosylated tumor-associated cell membrane mucin MUC1 using chimeric antigen receptor-engineered human T cells (Posey et al., 2016). In that study, the authors used a second generation 4-1BB costimulatory-molecule-based chimeric antigen receptor (CAR) (Imai et al., 2004) in which targeting was achieved using a single-chain variable fragment (scFv) derived from the 5E5 antibody. This CAR selectively binds MUC1 that carries the Tn or sialyl (S)Tn glycan. Both of these truncated glycans are aberrantly expressed on the MUC1 glycoprotein in a spectrum of malignancies and consequently represent attractive targets for immunotherapeutic exploitation.

Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma.

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.

Tumor-associated O-glycans of MUC1: Carriers of the glyco-code and targets for cancer vaccine design.

The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. It has frequently been reported that MUC1, the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern, in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play a key role in tumor initiation, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with tumor escape from immune defenses. In this report, we will give an overview of the oncogenic functions of MUC1 that are exerted through TACA interactions with endogenous carbohydrate-binding proteins (lectins). These interactions often lead to creation of a pro-tumor microenvironment, favoring tumor progression and metastasis, and tumor evasion. In addition, we will describe current efforts in the design of cancer vaccines with special emphasis on synthetic MUC1 glycopeptide vaccines. Analysis of the key factors that govern structure-based design of immunogenic MUC1 glycopeptide epitopes are described. The role of TACA type, position, and density on observed humoral and cellular immune responses is evaluated.

Recent advances in tumor associated carbohydrate antigen based chimeric antigen receptor T cells and bispecific antibodies for anti-cancer immunotherapy.

Tumor associated carbohydrate antigens (TACAs) are a class of attractive antigens for the development of anti-cancer immunotherapy. Besides monoclonal antibodies and vaccines, chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) targeting TACA are exciting directions to harness the power of the immune system to fight cancer. In this review, we focus on two TACAs, i.e., the GD2 ganglioside and the mucin-1 (MUC1) protein. The latest advances in CAR T cells and bispecific antibodies targeting these two antigens are presented. The roles of co-stimulatory molecules, structures of the sequences for antigen binding, methods for CAR and antibody construction, as well as strategies to enhance solid tumor penetration and reduce T cell exhaustion and death are discussed. Furthermore, approaches to reduce "on target, off tumor" side effects are introduced. With further development, CAR T cells and BsAbs targeting GD2 and MUC1 can become powerful agents to effectively treat solid tumor.

Tiliroside Combined with Anti-MUC1 Monoclonal Antibody as Promising Anti-Cancer Strategy in AGS Cancer Cells.

Specific changes in mucin-type O-glycosylation are common for many cancers, including gastric ones. The most typical alterations include incomplete synthesis of glycan structures, enhanced expression of truncated O-glycans (Tn, T antigens and their sialylated forms), and overexpression of fucosylation. Such altered glycans influence many cellular activities promoting cancer development. Tiliroside is a glycosidic dietary flavonoid with pharmacological properties, including anti-cancer. In this study, we aim to assess the effect of the combined action of anti-MUC1 and tiliroside on some cancer-related factors in AGS gastric cancer cells. Cancer cells were treated with 40, 80, and 160 µM tiliroside, 5 µg/mL anti-MUC1, and flavonoid together with mAb. Real-Time PCR, ELISA, and Western blotting were applied to examine MUC1 expression, specific, tumor-associated antigens, enzymes taking part in their formation, Gal-3, Akt, and NF-κB. MUC1 expression was significantly reduced by mAb action. The combined action of anti-MUC1 and tiliroside was more effective in comparison with monotherapy in the case of C1GalT1, ST3GalT1, FUT4, Gal-3, NF-κB, Akt mRNAs, and Tn antigen, as well as sialyl T antigen expression. The results of our study indicate that applied combined therapy may be a promising anti-gastric cancer strategy.

Modulating MUC1 Function on T Cells.

Immunotherapy is a promising approach to treating cancer. Mucin1 (MUC1), an epithelial glycoprotein, is hypo-glycosylated and overexpressed on epithelial cancers. This renders it a promising target for potential immunotherapeutic approaches. However, MUC1 has also been identified on T cells, which might complicate its potential as a target for immunotherapies.

Synthesis and immunological evaluation of the unnatural ß-linked mucin-1 Thomsen-Friedenreich conjugate.

MUC1 glycopeptides are attractive antigens for anti-cancer vaccine development. One potential drawback in using the native MUC1 glycopeptide for vaccine design is the instability of the O-glycosyl linkage between the glycan and the peptide backbone to glycosidase. To overcome this challenge, a MUC1 glycopeptide mimic has been synthesized with the galactose-galactosamine disaccharide linked with threonine (Thomsen-Friedenreich or Tf antigen) through an unnatural ß-glycosyl bond. The resulting MUC1-ß-Tf had a much-enhanced stability toward a glycosidase capable of cleaving the glycan from the corresponding MUC1 glycopeptide with the natural α-Tf linkage. The MUC1-ß-Tf was subsequently conjugated with a powerful carrier bacteriophage Qß. The conjugate induced high levels of IgG antibodies in clinically relevant human MUC1 transgenic mice, which cross-recognized not only the natural MUC1-α-Tf glycopeptide but also MUC1 expressing tumor cells, supporting the notion that a simple switch of the stereochemistry of the glycan/peptide linkage can be a strategy for anti-cancer vaccine epitope design for glycopeptides.

An altered HLA-A0201-restricted MUC1 epitope that could induce more efficient anti-tumor effects against gastric cancer.

MUC1 is a tumor-associated antigen (TAA) overexpressed in many tumor types, which makes it an attractive target for cancer immunotherapy. However, this marker is a non-mutated antigen without high immunogenicity. In this study, we designed several new altered peptides by replacing amino acids in their sequences, which were derived from a low-affinity MUC1 peptide, thus bypassing immune tolerance. Compared to the wild-type (WT) peptide, the altered MUC1 peptides (MUC11081-1089L2, MUC11156-1164L2, MUC11068-1076Y1) showed higher affinity to the HLA-A0201 molecule and stronger immunogenicity. Furthermore, these altered peptides resulted in the generation of more cytotoxic T lymphocytes (CTLs) that could cross-recognize gastric cancer cells expressing WT MUC1 peptides, in an HLA-A0201-restricted manner. In addition, M1.1 (MUC1950-958), a promising antitumor peptide that has been tested in multiple tumors, was not able to induce stronger antitumor responses. Collectively, our results demonstrated that altered peptides from MUC1, as potential HLA-A0201-restricted CTL epitopes, could serve as peptide vaccines or constitute components of peptide-loaded dendritic cell vaccines for gastric cancer treatment.

The combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb elicited antitumor immunity by T-cell adoptive immunotherapy in lung cancer.

Lung cancer remains a global challenge due to high morbidity and mortality rates and poor response to treatment, and there are still no effective strategies to solve it. The bispecific antibody (BsAb) is a novel antibody, which can target two different antigens and mediate specific killing effects by selectively redirecting effector cells to the target cells. In this study, we combined two BsAbs to achieve a dual-target therapy strategy of EpCAM+ and MUC-1+ with high affinity and specificity. The results showed that the combination of two BsAbs against EpCAM and MUC-1 could inhibit the growth of lung cancer more effectively in cell lines and primary tumors. The superior antitumor effect of two BsAbs could be attributable to enhanced CTL and increased production of type I IFNs. At the same time, the combination of EpCAM/CD3 BsAb and MUC-1/CD3 BsAb significantly regulated T population in the TDLNs. Therefore, we have found a potential immunotherapeutic strategy, which was the combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb for the treatment of non-small cell lung cancer.

A novel humanized MUC1 antibody-drug conjugate for the treatment of trastuzumab-resistant breast cancer.

Mucin 1 (MUC1) has been regarded as an ideal target for cancer treatment, since it is overexpressed in a variety of different cancers including the majority of breast cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1. In this study, we generated a humanized MUC1 (HzMUC1) antibody from our previously developed MUC1 mouse monoclonal antibody that only recognizes MUC1 on the surface of tumor cells. Furthermore, an antibody-drug conjugate (ADC) was generated by conjugating HzMUC1 with monomethyl auristatin (MMAE), and the efficacy of HzMUC1-MMAE on the MUC1-positive HER2+ breast cancer in vitro and in 'Xenograft' model was tested. Results from western blot analysis and immunoprecipitation revealed that the HzMUC1 antibody did not recognize cell-free MUC1-N in sera from breast cancer patients. Confocal microscopy analysis showed that HzMUC1 antibody bound to MUC1 on the surface of breast cancer cells. Results from mapping experiments suggested that HzMUC1 may recognize an epitope present in the interaction region between MUC1-N and MUC1-C. Results from colony formation assay and flow cytometry demonstrated that HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in trastuzumab-resistant HER2-positive breast cancer cells. Meanwhile, HzMUC1-MMAE significantly reduced the growth of HCC1954 xenograft tumors by inhibiting cell proliferation and enhancing cell death. In conclusion, our results indicate that HzMUC1-ADC is a novel therapeutic drug that can overcome trastuzumab resistance of breast cancer. HzMUC1-ADC should also be an effective therapeutic drug for the treatment of different MUC1-positive cancers in clinic.

A sandwich-type electrochemical immunosensor based on Au-rGO composite for CA15-3 tumor marker detection.

A sensitive detection of carbohydrate antigen 15-3 (CA15-3) levels may allow for early diagnosis and monitoring the treatment of breast cancer, but this can only be made in routine clinical practice if low-cost immunosensors are available. In this work, we developed a sandwich-type electrochemical immunosensor capable of rapid detection of CA15-3 with an ultra-low limit of detection (LOD) of 0.08 fg mL-1 within a wide linear concentration range from 0.1 fg mL-1 to 1 µg mL-1. The immunosensor had a matrix of a layer-by-layer film of Au nanoparticles and reduced graphene oxide (Au-rGO) co-electrodeposited on screen-printed carbon electrodes (SPCE). The high sensitivity was achieved by using secondary antibodies (Ab2) labeled with horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) as signal amplifiers, and hydroquinone (HQ) was used as an electron mediator. The immunosensor was selective for CA15-3 in human serum and artificial saliva samples, robust, and stable to permit storage at 4 °C for more than 30 days. With its high performance, the immunosensor may be incorporated into future point-of-care (POC) devices to determine CA15-3 in distinct biological fluids, including in blood and saliva samples.

Preparation, quality control, and biodistribution assessment of [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors.

In this study, [111 In]In-DOTA-PR81 was developed, and its preliminary preclinical qualifications were assessed for single photon emission computed tomography (SPECT) imaging of breast cancer. DOTA-NHS-ester was practiced and successively purified by molecular filtration. The chelate:mAb ratio was determined by spectrophotometry. DOTA-PR81 was radiolabeled with In-111 and its radiochemical yield, in vitro stability, in vitro internalization, and immunoreactivity tests were performed. SPECT imaging and tissue counting were applied to evaluate the tissue distribution of [111 In]In-DOTA-hIgG and [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors. The radiochemical yield of [111 In]In-DOTA-PR81 complex was >95.0 ± 0.5% (ITLC), and the specific activity was 170 ± 44 MBq/mg. Conjugation reaction resulted in the average number of chelators attached to a mAb (c/a) of 3.4 ± 0.3:1. The radioimmunoconjugate showed immunoreactivity towards MCF7 cell line and MUC1 antigen while its significant in vitro and in vivo stability were investigated over 48 h, respectively (93.0 ± 1.2% in phosphate-buffered saline (PBS) and 84.0 ± 1.3% in human serum). The peak concentration of internalized activity of [111 In]In-DOTA-PR81 was between 4 to 6 h. In comparison with control probes, the complex was accumulated with high specificity and sensitivity at the tumor site. Achieved results indicated that [111 In]In-DOTA-PR81 could be contemplated as an appropriate radiotracer for prognostic imaging of antigens in oncology.

A Possible Inhibitory Role of Sialic Acid on MUC1 in Peritoneal Dissemination of Clear Cell-Type Ovarian Cancer Cells.

The role of sialic acids on MUC1 in peritoneal dissemination of ovarian cancer cells was investigated. A human ovarian carcinoma cell line, ES-2, was transfected with full-length MUC1 containing 22 or 42 tandem repeats. These transfectants were less adherent to monolayers of patient-derived mesothelial cells than ES-2/mock transfectants. When these cells were inoculated into the abdominal cavity of female nude mice, mice that had received the transfectants showed better survival. When the transfectants were mixed with sialidase and injected, the survival was poorer, whereas when they were mixed with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid, a sialidase inhibitor, the survival was significantly prolonged. These behaviors, concerned with peritoneal implantation and dissemination observed in vitro and in vivo, were dependent on the expression of MUC1. Therefore, sialic acid linked to MUC1 in the form, at least in part, of sialyl-T, as shown to be recognized by monoclonal antibody MY.1E12, is responsible for the suppression of adhesion of these cells to mesothelial cells and the suppression of peritoneal implantation and dissemination.

In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies.

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

TF-containing MUC1 glycopeptides fail to entice Galectin-1 recognition of tumor-associated Thomsen-Freidenreich (TF) antigen (CD176) in solution.

Aberrant Mucin-1 (MUC1) glycosylation with the Thomsen-Friedenreich (TF) tumor-associated antigen (CD176) is a hallmark of epithelial carcinoma progression and poor patient prognosis. Recognition of TF by glycan-binding proteins, such as galectins, enables the pathological repercussions of this glycan presentation, yet the underlying binding specificities of different members of the galectin family is a matter of continual investigation. While Galectin-3 (Gal-3) recognition of TF has been well-documented at both the cellular and molecular level, Galectin-1 (Gal-1) recognition of TF has only truly been alluded to in cell-based platforms. Immunohistochemical analyses have purported Gal-1 binding to TF on MUC1 at the cell surface, however binding at the molecular level was inconclusive. We hypothesize that glycan scaffold (MUC1's tandem repeat peptide sequence) and/or multivalency play a role in the binding recognition of TF antigen by Gal-1. In this study we have developed a method for large-scale expression of Gal-1 and its histidine-tagged analog for use in binding studies by isothermal titration calorimetry (ITC) and development of an analytical method based on AlphaScreen technology to screen for Gal-1 inhibitors. Surprisingly, neither glycan scaffold or multivalent presentation of TF antigen on the scaffold was able to entice Gal-1 recognition to the level of affinity expected for functional significance. Future evaluations of the Gal-1/TF binding interaction in order to draw connections between immunohistochemical data and analytical measurements are warranted.

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope.

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding-competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution-state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre-selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.

The Effect of Different Immunization Cycles of a Recombinant Mucin1-Maltose-Binding Protein Vaccine on T Cell Responses to B16-MUC1 Melanoma in Mice.

We explored the effect of a recombinant mucin1-maltose-binding protein vaccine, including immunization cycles of recombinant mucin1-maltose-binding protein (MUC1-MBP) and CpG 2006 on T cell responses to human MUC1-overexpressing mouse melanoma B16 cells (B16-MUC1) melanoma in mice. We found that the vaccine had a significant antitumor effect, with the most obvious tumor-suppressive effect being observed in mice immunized five times. After more than five immunizations, the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations). To study the possible mechanism, Mucin-1(MUC1)-specific antibodies, IFN-γ secretion by lymphocytes, and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and a real-time cell analyzer (RTCA). T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. These results showed that five immunizations activated MUC1-specific Th1 and CTL and reduced the ratio of myeloid-derived suppressor cells (MDSCs) and Th17 in mice more significantly than eight immunizations, indicating that excessive frequency of the immune cycle leads to the increased numbers of immunosuppressive cells and decreased numbers of immunostimulatory cells, thereby inhibiting antitumor immune activity. This data provide an experimental foundation for the clinical application of a recombinant MUC1-MBP vaccine.

Synthesis and Immunological Evaluation of a Single Molecular Construct MUC1 Vaccine Containing l-Rhamnose Repeating Units.

A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.

Combination Immunotherapy of MUC1 mRNA Nano-vaccine and CTLA-4 Blockade Effectively Inhibits Growth of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC), which constitutes 10%-20% of all breast cancers, is associated with aggressive progression, a high rate of metastasis, and poor prognosis. The treatment of patients with TNBC remains a great clinical challenge. Preclinical reports support the combination immunotherapy of cancer vaccines and immune checkpoint blockades in non-immunogenic tumors. In this study, we constructed nanoparticles (NPs) to deliver an mRNA vaccine encoding tumor antigen MUC1 to dendritic cells (DCs) in lymph nodes to activate and expand tumor-specific T cells. An anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) monoclonal antibody was combined with the mRNA vaccine to enhance the anti-tumor benefits. In vivo studies demonstrated that the NP-based mRNA vaccine, targeted to mannose receptors on DCs, could successfully express tumor antigen in the DCs of the lymph node; that the NP vaccine could induce a strong, antigen-specific, in vivo cytotoxic T lymphocyte response against TNBC 4T1 cells; and that combination immunotherapy of the vaccine and anti-CTLA-4 monoclonal antibody could significantly enhance anti-tumor immune response compared to the vaccine or monoclonal antibody alone. These data support both the NP as a carrier for delivery of mRNA vaccine and a potential combination immunotherapy of the NP-based mRNA vaccine and the CTLA-4 inhibitor for TNBC.

Myoepithelioma-like tumor of the vulvar region showing infiltrative growth and harboring only a few estrogen receptor-positive cells: A case report.

Recently, a new entity "myoepithelioma-like tumor of the vulvar region (MELTVR)" was proposed as a rare mesenchymal neoplasm arising in vulvar regions of adult women. While MELTVRs morphologically resemble soft tissue myoepitheliomas and extraskeletal myxoid chondrosarcomas, they have a unique immunohistochemical profile (positive for epithelial membrane antigen and estrogen receptor, negative for S100 protein and glial fibrillary acidic protein, and loss of INI1/SMARCB1 expression), and lack EWSR1 and NR4A3 gene rearrangement, as seen by fluorescence in situ hybridization. MELTVRs are usually well-demarcated tumors, with no reports of extensive infiltrative growth. In the current report, we present an unusual case of MELTVR showing infiltrative growth and harboring only a few estrogen receptor-positive cells, which might indicate a variation in this rare tumor.