Malignant and atypical meningiomas are resistant to standard therapies and associated with poor prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach has not been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinically meaningful but underutilized model for preclinical testing of novel strategies for aggressive human meningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and only one dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-γ–elaborating T cells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responses developed in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surface antigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, showing common antigens across breed and species. Histologic analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatment compared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility and immunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrant further development toward human trials. Cancer Res; 73(10); 2987–97. ©2013 AACR.

Meningioma is the most common primary brain neoplasm, with more than 100,000 patients diagnosed in the United States between 2004 and 2008 (1). Newly diagnosed tumors are managed by surgical resection alone. Roughly 6,000 patients will need additional treatment in the United States every year due to recurrence (2), which often occurs with invasive or malignant disease (3, 4). Current salvage approaches include reoperation, radiation, radiosurgery, and chemotherapy; there is controversy regarding the perceived clinical benefit of these interventions (5–8). The 3-year recurrence rate in reoperated World Health Organization (WHO) grade 1 meningioma is 50% (9), with risk of recurrence even greater in grade 2 and 3 tumors (10–12). Radiation, although modestly effective in benign disease, has been associated with cognitive deficits, secondary malignancies, and the transformation of the tumor to a higher-grade neoplasm (2).

One challenge in treating aggressive meningiomas is the paucity of animal models with which to test combinations of surgery and systemic therapy in a meaningful manner. Spontaneous meningiomas in dogs make up 45% of primary canine brain tumors (13) and are an underutilized resource for preclinical studies. Canine and human meningiomas share many features, including histologic resemblance, overexpression of growth factor receptors, deletion of chromosomal segments, and losses of function in tumor suppressor genes (14–16). More than 40% of canine meningiomas were atypical or malignant in one study (17). Dogs develop many other cancers that are prevalent in humans, and tumors progress 5- to 7-fold faster in dogs relative to humans (18). We hypothesized that preclinical studies in canine meningioma would enable accurate and rapid testing of immunotherapy for aggressive meningioma.

Cancer vaccines have been tested in multiple malignancies, including gliomas, with evidence of clinical activity (19, 20). A meta-analysis covering more than 100 clinical trials revealed that response rates to vaccination with peptides containing defined T-cell epitopes were less than half of that achieved with whole-cell–based vaccines (21). The higher response rate achieved with whole-cell vaccines (as used in this study) could be due to greater antigenic coverage or the potential to induce tumor-binding antibodies. Meningiomas are not subject to the same principles of immune privilege as cells within the brain parenchyma such as gliomas (22). Relative to gliomas, meningiomas may be better candidates for immunotherapy because they: (1) are not insulated by the endothelial tight junctions that limit large-molecule (e.g., antibody) diffusion; (2) are in direct contact with cerebrospinal fluid that drains to the venous circulation and cervical lymph nodes for antigen presentation to T and B lymphocytes; (3) lack the T-cell trafficking checkpoints present in the Virchow–Robin space (e.g., glia limitans); and (4) are relatively slow growing tumors that may be more susceptible to adaptive immune responses. However, invasive, atypical, and malignant meningiomas can infiltrate the brain parenchyma, requiring penetration of antibodies and/or lymphocytes for control of postsurgical, microscopic disease.

Tumor cell lysates mixed with synthetic toll-like receptor (TLR) ligands function as effective vaccines to induce antitumor immune responses in glioma-bearing animals (23, 24). The mechanisms of lysate/TLR ligand vaccines have been thoroughly characterized and tested in many patients with cancer; however, the activity of this type of vaccine against meningioma is unknown. TLR activation on antigen-presenting cells facilitates the induction of adaptive immune responses by enhancing antigen presentation, expression of costimulatory molecules, cytokine production, and homing to secondary lymphoid organs. The TLR9 ligand CpG olidgodeoxynucleotide induced clinical responses in patients with select melanoma and renal cell carcinoma (25, 26). The U.S. Food and Drug Administration-approved TLR7 ligand imiquimod exhibited adjuvant activity in cancer clinical trials (20, 27), with excellent efficacy as a single topical agent against skin cancers (28).

We conducted a vaccine immunotherapy trial for pet dogs with symptomatic, spontaneous meningiomas. Dogs underwent surgery followed by vaccination with autologous tumor cell lysate that was combined with imiquimod or CpG. Herein, we report safety, robust extension of survival, homology among antibody epitopes between dogs and humans, and vaccine-induced, local antibody production in the brain. Vaccination for canine meningioma reveals promising avenues for further development toward human trials.

Surgery, vaccination production, and administration

Dogs were enrolled after obtaining owner consent according to an approved protocol from the University of Minnesota (Minneapolis, MN) Institutional Animal Care and Use Committee. A presumptive diagnosis of meningioma was based on the MRI characteristics of a solitary extra-axial mass in the brain, with heterogeneous T1W signal, usually isointense, homogenous T2W signal, sharply defined borders, homogenous contrast enhancement, evidence of a dural tail, that may have associated cysts, peritumoral edema, and falx-shift. Surgical resection was conducted under general anesthesia using the appropriate approach based on tumor location. Dogs were hospitalized with supportive care for 1 to 2 days after surgery. Corticosteroids used to minimize peritumoral edema were discontinued by 48 hours before vaccination. Part of the tumor specimen was used for histologic diagnosis, and the remainder of the tumor was cultured for vaccine production.

Cultures were established by mincing specimens with scalpels and digestion at 37°C for 15 minutes in suspension with TrypLE Express (Invitrogen/Life Technologies). Cell suspensions were filtered through a 100 μm filter, washed with PBS, and placed in culture in 10 cm culture plates precoated with 1:10 Matrigel (BD Biosciences), in serum-free neural stem cell media consisting of DMEM:F12 (1:1), with l-glutamine, sodium bicarbonate, penicillin/streptomycin (100 U/mL), B27 and N2 supplements (Gibco), and 0.1 mg/mL Normocin (InVivoGen). Semi-weekly, cells were given 20 ng/mL recombinant human EGF and recombinant human FGF (R&D Systems) and were cultured at 5% O2 and 5% CO2. Harvest for vaccination involved scraping cells from one 10 cm dish. Cells were washed thrice in PBS, and underwent 5 freeze-thaw alternations by transfer from warm water bath to liquid N2 followed by radiation (200 Gy). Protein was measured by Bradford assay with standard Coomassie reagent (Pierce), and lysates were stored at −80°C. GMP-grade CpG 685 ODN was provided by SBI Biotech Co., Ltd (Tokyo, Japan), and imiquimod cream was acquired through the University of Minnesota Boynton Health Services Pharmacy. In 5 dogs, CpG (2.0 mg) was mixed with thawed lysates immediately before intradermal injection at 2 sites in the back of the neck. Six other dogs received imiquimod cream (5%; 0.5 g) at 2 intradermal injection sites in the back of the neck 15 minutes before lysate injection. The maximally achievable lysate protein concentration was given to each dog, which varied by tumor volume and the ability of the tumor to proliferate in culture. Lysate doses ranged from 200 to 1,500 μg protein (average of 595 μg), and average dose did not vary significantly between CpG and imiquimod-treated dogs.

Tumor volume measurements

Twenty consecutive surgical human cases (of M.A. Hunt; Department of Neurosurgery, University of Minnesota, Minneapolis, MN) were analyzed for volume by MRI, as calculated by (length × width × height)/2 for tumor and intracranial volume. The same procedure was carried out for the 11 dogs in this study, and the results were compared as described in Supplementary Materials and Methods.

Western immunoblot analysis

Cultured tumor cells or homogenized flash-frozen tumors were used for blotting. Cells were lysed, protease and phosphatase inhibitors (Calbiochem) were added, and for SDS-PAGE, lysates were diluted in Laemmli-reducing sample buffer, heated, and centrifuged. Protein standards (Bio-Rad) were loaded next to each 40 μg lysate and resolved on NuPAGE 4% to 12% Bis/Tris gels (Invitrogen). Proteins were transferred to nitrocellulose (Amersham) at 5 V constant voltage using semidry transfer (BioRad). The membranes were blocked in 5% nonfat dry milk (NFDM)/Tris-buffered saline with 0.1% Tween-20 (TTBS) at room temperature for 1 hour and cut appropriately into identical blots, each with a molecular weight standard (BioRad) run adjacent to lysate. Each membrane was incubated at room temperature for 1 hour in normal, pre or postvaccination sera diluted 1:1000 in 5% NFDM/TTBS, washed 6 times for 10 minutes each in TTBS, followed by room temperature for 1 hour in rabbit anti-canine immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch) at 1:50,000 in 5% NFDM/TTBS. Bands were detected using ECL Western Blotting Detection System (Amersham) and HyBlot CL autoradiography film (Denville Scientific). Densitometry was conducted by the Gel Analysis tool in ImageJ 1.45s software (NIH), and values were normalized by dividing by heavy- and light-chain densities (areas under the curve) from prevaccination lanes.

Detection of tumor cell surface-reactive antibodies

A Becton Dickinson Custom Canto three-laser flow cytometer was used for data acquisition. Tumor cells were removed from Matrigel-coated 10 cm culture plates by scraping or BD Dispase (BD Biosciences), washed with PBS thrice, and incubated with heat-inactivated normal dog, prevaccination, or 3-month postvaccination serum at 1:100 dilutions at 4°C for 30 minutes. Cells were then washed thrice in PBS and incubated with 1 μg anti-canine IgG (H+L)—fluorescein isothiocyanate (American Qualex) at 4°C for an additional 30 minutes before washing and analyzing.

Immunohistochemistry staining and quantification of lymphocyte infiltration

For lymphocyte analysis, 5 μm tissue sections were cut, prepared with standard procedures, and the following antibodies were used: CD3 (AbD Serotec) at 1:2,000, CD20 (Thermo Scientific) at 1:2,000, and IgG (H+L; Jackson ImmunoResearch) at 1:2,000. Tissue sections were incubated with the primary antibodies for 1 hour, rinsed, and a biotinylated secondary antibody was applied for 30 minutes. CD20 and IgG antibodies were followed with undiluted Rabbit Link (Covance), and a biotin-conjugated donkey anti-rat IgG (H+L; Jackson ImmunoResearch) at 1:500 was used with CD3. Sections were rinsed, incubated in hydrogen peroxidase, and a tertiary streptavidin HRP link (Covance) for 30 minutes. The immune complex was visualized using 3,3′-diaminobenzidine as the chromogen. Sections were lightly counterstained with hematoxylin, dehydrated, coverslipped, and scanned using the Aperio Scanscope XT.

All surgical resection specimens and all necropsy blocks containing tumor or inflammation as seen by hematoxylin and eosin stain (H&E) staining were included in counting of CD3+ and CD20+ cells. Samples were blinded, and all 10× fields in slides were captured and saved as image files. Automated cell quantification was conducted with a customized macro and the particle analysis tool in Fiji software (ImageJ 1.46j, NIH). Counts underwent statistical analysis as described in Supplementary Materials and Methods before unblinding.

Antibody-dependent cell-mediated cytotoxicity

Dog blood from healthy donors was collected in anticoagulant tubes, lysed osmotically, and peripheral blood leukocytes (PBL) were washed thrice with PBS. PBLs were resuspended in complete RPMI-1640 (supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.1 mg/mL Normocin from InVivoGen) in a 96-well plate at 2.5 × 106 cells/mL, and stimulated 14 hours with 30 μmol/L of the TLR7/8 ligand resiquimod before being washed and cocultured with antibody-coated tumor cells. Primary meningioma cultures were coated with antibody as described in the cell surface-binding procedure. Two washes were conducted before addition to PBLs at an effector:target ratio of 25:1. Tumor cell lysis was determined by measurement of lactate dehydrogenase (LDH) activity as indicated by the manufacturer's protocol after 7 hours of coculture (Roche Applied Science). Percentage-specific lysis of tumor cells was calculated by: [Sample−(tumor only + PBL only)]/(lysed tumor + PBL only) × 100.

Statistical analysis

Samples were analyzed by unpaired t test (tumor/brain volume, flow cytometry, LDH activity). Histologic cell counts were analyzed by an unpaired t test with Welch's correction for unequal variances with 95% confidence intervals. Survival was analyzed using a log-rank test with 95% confidence intervals. All statistics were conducted using GraphPad Prism version 4.0c for OS X (GraphPad Software www.graphpad.com).

Canine meningiomas model aggressive human disease

Meningiomas from dogs treated in our study share histologic features with human tumors of the same subtype (Fig. 1A), in addition to features that signify poor survival in humans. Brain invasion is an independent negative prognostic factor that prompted the reclassification of otherwise WHO grade I meningiomas as grade II (29, 30). Two canine cases exhibited gross tumor invasion into the brain at resection, and 3 additional cases showed brain invasion upon postmortem analysis (Fig. 1B, left and Supplementary Table S1). Tumor invasion into the skull was also present in 2 cases (Fig. 1B, right). In addition, canine meningiomas occupied more than twice the volume of the brain relative to human tumors (Fig. 1C). All dogs were symptomatic at the time of diagnosis, and in 10 dogs, the presenting clinical sign was seizures. The rapid recurrence and progression of this disease in canines is consistent with the common appearance of invasion into brain, which, in humans, predicts poor outcomes.

Figure 1.

Canine meningiomas model aggressive human disease. A, meningioma surgery specimens stained with H&E, left to right, from cases 3, 4, and 1. B, necropsy specimens from 2 dogs with tumors that, in humans, are categorized as WHO grade II on biopsy. C, tumor volume and brain volume of the 11 dogs treated with surgery plus immunotherapy and 20 consecutive intrainstitutional patients with human meningioma. *, P < 0.05.

Figure 1.

Canine meningiomas model aggressive human disease. A, meningioma surgery specimens stained with H&E, left to right, from cases 3, 4, and 1. B, necropsy specimens from 2 dogs with tumors that, in humans, are categorized as WHO grade II on biopsy. C, tumor volume and brain volume of the 11 dogs treated with surgery plus immunotherapy and 20 consecutive intrainstitutional patients with human meningioma. *, P < 0.05.

Close modal

Extension of survival following vaccine immunotherapy

Eleven dogs underwent craniotomy for tumor removal after radiographic diagnosis of an intracranial neoplasm consistent with meningioma. Following histologic confirmation, dogs were administered vaccinations biweekly with lysate derived from their tumors in combination with CpG or imiquimod (Fig. 2A). Mean follow-up time was 585 days, with 36% (4/11) of dogs alive (Supplementary Table S1; Fig. 2B and C). Relative to historic controls, median survival is extended in the immunotherapy cohort (645 vs. 222 days, P < 0.05), with no censoring of dogs that died from other causes (Fig. 2B and Supplementary Table S2). Neither vaccination cohort contained a case of frank tumor progression, although more deaths occurred in the CpG cohort (Fig. 2C). One CpG-treated dog developed 2 meningiomas, of which the second was not identified before vaccination due to small size. The second tumor was subsequently removed after its growth caused breakthrough seizures, and 4 other vaccines were given using tumor lysate of the second tumor.

Figure 2.

Biweekly tumor lysate/adjuvant vaccination schedule results in prolonged survival of spontaneous canine meningioma. A, study design and immune monitoring scheme. B, surgery controls were conducted at 4 institutions, and all cases were confirmed to be meningioma by histopathology. C, comparison of CpG with imiquimod treatment groups.

Figure 2.

Biweekly tumor lysate/adjuvant vaccination schedule results in prolonged survival of spontaneous canine meningioma. A, study design and immune monitoring scheme. B, surgery controls were conducted at 4 institutions, and all cases were confirmed to be meningioma by histopathology. C, comparison of CpG with imiquimod treatment groups.

Close modal

Tumor-reactive antibodies bind cell surface antigens and cross-react with human meningiomas

Immune responses before and after vaccination were measured in peripheral blood mononuclear cells (PBMC) and sera. IFN-γ–elaborating tumor-reactive T cells were significantly increased in postvaccination PBMCs in 2 of 9 dogs tested, suggesting a low frequency of circulating T cells 3 months after surgery (Supplementary Table S3). In contrast, polyclonal tumor-reactive antibody responses to whole-tumor lysate (Fig. 3A) and cell surface antigens (Fig. 3B and C) were detected in all 11 dogs (Supplementary Table S3). Probing of allogeneic tumor cells and lysates indicated recognition of common tumor antigens among dogs, some of which were present on the cell surface (Fig. 4A–C). Postvaccination antibodies recognized recombinant human HSP60 and HSP60 from autologous and allogeneic tumor (Fig. 4A), showing the common expression and recognition of this antigen. Remarkably, postvaccination canine serum recognized human meningioma tissue, indicating antigen conservation between species (Fig. 4D).

Figure 3.

Vaccination induces antibody responses to meningioma surface antigens. A, Western immunoblot analysis of autologous tumor cells from culture (cases 10 and 3) or snap-frozen tumor specimen (case 5). Normal dog serum (ND) was used as a control. B, live autologous tumor cells were stained with serum from prevaccination or postvaccination (at 82 days). C, aggregate data of 3 dogs from B. *, P < 0.05; **, P < 0.005.

Figure 3.

Vaccination induces antibody responses to meningioma surface antigens. A, Western immunoblot analysis of autologous tumor cells from culture (cases 10 and 3) or snap-frozen tumor specimen (case 5). Normal dog serum (ND) was used as a control. B, live autologous tumor cells were stained with serum from prevaccination or postvaccination (at 82 days). C, aggregate data of 3 dogs from B. *, P < 0.05; **, P < 0.005.

Close modal
Figure 4.

Antibodies recognize allogeneic dog and human meningiomas. A, Western immunoblot analysis of tumor cells from 3 cases and recombinant human HSP60. B, representative histograms of cell surface binding of primary tumor cells from case 7. C, quantification of serum antibody surface binding to an allogeneic papillary (nonstudy dog—recognized by case 3) and a meningothelial meningioma (case 7—recognized by cases 4, 6, 7, and 9). D, Western immunoblot analysis of a human meningioma probed with postvaccination serum from case 3. *, P < 0.05; **, P < 0.005.

Figure 4.

Antibodies recognize allogeneic dog and human meningiomas. A, Western immunoblot analysis of tumor cells from 3 cases and recombinant human HSP60. B, representative histograms of cell surface binding of primary tumor cells from case 7. C, quantification of serum antibody surface binding to an allogeneic papillary (nonstudy dog—recognized by case 3) and a meningothelial meningioma (case 7—recognized by cases 4, 6, 7, and 9). D, Western immunoblot analysis of a human meningioma probed with postvaccination serum from case 3. *, P < 0.05; **, P < 0.005.

Close modal

Vaccination induces T-, B-, and plasma cell infiltration into peritumoral brain

We conducted analyses of B- and T-cell infiltration in surgical and necropsy tissue in the 4 cases in which they were available. Three CpG-treated dogs died within 3 weeks of the final vaccination and exhibited robust, focal increases in B- (with relatively less T)-cell infiltration into peritumoral brain (Fig. 5A and B; cases 1, 3, and 5). One imiquimod-treated dog developed acute lymphoblastic lymphoma and was euthanized 25 weeks after the final vaccination. This dog showed mild B-cell infiltration (and no T-cell infiltration) into the brain (Fig. 5B, case 8). As mentioned above, case 3 presented with a second meningioma in the contralateral hemisphere between the fifth and sixth vaccinations, which was subsequently resected. Western blot analysis revealed a profound increase in IgG penetration into the second tumor relative to the first, prevaccination tumor (Fig. 5C and D). Moreover, staining for canine IgG exposed plasma cells in the brains of 3 CpG-treated dogs (Fig. 5E). Although previous studies have reported plasma cell entry into the brain in antibody-mediated autoimmune (31) and antipathogen (32) responses, this is the first account of induced plasma cell homing into brain tumors. In situ antibody production was further suggested by focal areas of extracellular IgG staining seen in plasma cell-containing areas of brain tissue but not others (data not shown).

Figure 5.

Vaccination induces B- and plasma cell infiltrates in peritumoral brain. A, representative images from CD3 and CD20 immunohistochemistry of biopsies and necropsies from case 1. B, quantification of CD3 and CD20 stains from cases 1, 3, 5, and 8. C, IgG blotted on case 3 primary tumor (prevaccination) and contralateral tumor (postvaccination). D, densitometry of heavy and light chain bands from C. E, immunohistochemical stain of canine IgG (H+L), indicating plasma cell infiltrates in normal brain areas of cases 3 and 5. *, P < 0.005.

Figure 5.

Vaccination induces B- and plasma cell infiltrates in peritumoral brain. A, representative images from CD3 and CD20 immunohistochemistry of biopsies and necropsies from case 1. B, quantification of CD3 and CD20 stains from cases 1, 3, 5, and 8. C, IgG blotted on case 3 primary tumor (prevaccination) and contralateral tumor (postvaccination). D, densitometry of heavy and light chain bands from C. E, immunohistochemical stain of canine IgG (H+L), indicating plasma cell infiltrates in normal brain areas of cases 3 and 5. *, P < 0.005.

Close modal

Recognition of nonneoplastic brain and meningeal antigens by postvaccination sera

Two dogs (cases 3 and 5) were euthanized 7 and 20 days after the most recent vaccination. Both animals presented with uncontrollable seizures and tumor recurrence was assumed. At necropsy, case 3 had a microscopic focus of residual tumor and case 5 had no evidence of tumor. To evaluate vaccine-induced autoantibody production, sera from these and 3 other dogs was probed against normal dog brain, arachnoid/pia mater, and dura mater. Secondary antibody revealed heavy and light chain IgG and IgM deposited in meninges, but not brain parenchyma (Fig. 6A, left). These results are consistent with the physiologic permeability of immunoglobulin into these tissues. Serum from cases 3 and 5 reacted to arachnoid/pia and brain parenchyma, respectively. Consistent with the autoreactivity of case 5 sera, analysis of necropsy brain tissue from this dog revealed IgG accumulation on or in neurons distal to the site of resection (Fig. 6B). The binding of the sera to normal brain antigens in these dogs sets them apart from 2 CpG-treated dogs and one imiquimod-treated dog that remained healthy and had nonreactive sera (Fig. 6A).

Figure 6.

Reactivity with normal brain correlates with neurologic symptoms in CpG-treated dogs. A, brain, arachnoid/pia mater, and dura mater were probed with secondary anti-canine IgG (H+L) alone, with serum from an allogeneic healthy dog, or postvaccination sera from 5 cases. Arrows indicate reactivity in dogs with neurologic symptoms following vaccination. B, necropsy specimen from case 5 stained for canine IgG (H+L).

Figure 6.

Reactivity with normal brain correlates with neurologic symptoms in CpG-treated dogs. A, brain, arachnoid/pia mater, and dura mater were probed with secondary anti-canine IgG (H+L) alone, with serum from an allogeneic healthy dog, or postvaccination sera from 5 cases. Arrows indicate reactivity in dogs with neurologic symptoms following vaccination. B, necropsy specimen from case 5 stained for canine IgG (H+L).

Close modal

Postvaccination sera are capable of antibody-dependent cell-mediated cytotoxicity

Antitumor effector activities of antibodies encompass multiple mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC). Because antibodies reacted with cell surface antigens (Figs. 3B and C, 4B and C, and 7A and B), and antibody production in situ could enable opsonization of invasive meningioma cells behind an intact blood brain barrier, we tested whether tumor-reactive antibodies could trigger ADCC. PBLs killed few tumor cells when cocultured with meningioma cells, or when tumor cells were preincubated with prevaccination serum; however, ADCC occurred when tumor cells were incubated with postvaccination serum (Fig. 7C). Postvaccination serum also triggered ADCC against allogeneic meningioma cells (Fig. 7D), showing that allogeneic vaccination may be an effective strategy in canines with meningioma.

Figure 7.

Postvaccination serum enables ADCC. A and B, case 3 postvaccination serum antibodies bound autologous tumor (A) and allogeneic tumor (B) of papillary histology from a nonstudy dog. Postvaccination serum also enabled killing of autologous (C) and allogeneic (D) tumors when combined with allogeneic PBL. *, P < 0.05; **, P < 0.005.

Figure 7.

Postvaccination serum enables ADCC. A and B, case 3 postvaccination serum antibodies bound autologous tumor (A) and allogeneic tumor (B) of papillary histology from a nonstudy dog. Postvaccination serum also enabled killing of autologous (C) and allogeneic (D) tumors when combined with allogeneic PBL. *, P < 0.05; **, P < 0.005.

Close modal

As many as 57,000 dogs a year develop meningiomas in the United States (13, 33), and these dogs are an underutilized resource for preclinical study. Because the prognosis for canine meningioma is dismal (34), both dogs and humans could benefit from these studies. Our data show the canine model resembles many histologic subtypes observed in humans and has features associated with poor prognosis in humans. The large size of the canine brain allows for surgery as a component of therapy, enabling a more realistic clinical interpretation of systemic interventions.

The survival outcomes of this study call for further investigation into the predictive potential of the canine model, especially with regard to tumor and immune biology in the 2 species. Different responses to vaccination may occur due to differential antigen expression, TLR expression in leukocyte cell types, tumor growth kinetics, the relationship between histologic subtype, biologic behavior, and clinical outcome.

Addition of lysate/adjuvant vaccination to surgery resulted in favorable survival compared with historical controls, but a prospectively designed, randomized study is required to make firm conclusions on therapeutic efficacy. Nonetheless, only case 3 experienced tumor growth despite treatment. The survival data may underestimate the benefit of vaccination because censoring was not used (Fig. 2B and C). Two dogs included in the analysis died of other malignancies, and 2 dogs lacked postmortem analysis but were assumed to die of age-related causes (Supplementary Table S1). The 3 other deaths were due to euthanasia after dogs presented with uncontrollable seizures assumed to be caused by tumor recurrence. Postmortem analysis in these CpG-treated dogs found minimal or no tumor burden, but immunoreactivity to normal brain structures may have been treatment related and contributed to the acute onset of neurologic symptoms (Fig. 6A and B). Similar seizure activity in glioma-bearing dogs in clinical trials at our institution has been controlled using additional anticonvulsants, corticosteroids, or induction of general anesthesia (data not shown). It is therefore possible that the seizures in meningioma-bearing dogs could have been controlled. Whether CpG or imiquimod is more efficacious is still unresolved from the current study due to the small number of dogs and many non-tumor-related deaths. Given the high level of tumor control and the superior safety profile of imiquimod, however, a prospective randomized trial to compare surgery alone and in combination with vaccines of tumor lysate and a novel TLR 7/8 ligand was initiated.

Our study is a starting point for investigation of immunotherapy for meningioma. The data indicate that B-cell activation and antibody production is the predominant mechanism of immunity induced by vaccination. Tumor-reactive antibodies were detected in the serum of all dogs regardless of lysate dose, showing feasibility of autologous lysate/TLR vaccine production. Antibodies exhibited considerable intercase and -species cross-reactivity (Fig. 4A and D and Supplementary Table S3). B-cell infiltration outnumbered T-cell infiltration in postmortem brain tissue adjacent to the resection cavity (Fig. 5B). In contrast, only 2 dogs had increased tumor-reactive T-cell responses as measured by IFN-γ ELISpot (Supplementary Table S3). However, it is likely that reactive CD4 T cells were primed following vaccination because antibody responses often require CD4 T-cell help. Tumor-reactive T cells in the blood may not represent what occurs in the tumor-draining lymph nodes or tumor site. A limitation of our study was that ELISpot was carried out only in prevaccination and 3-month postvaccine blood samples. Nevertheless, infiltrating CD3+ cells were observed in dogs that died within 2 weeks of the final vaccination (Fig. 5A and B), suggesting some T-cell activation. Future studies will examine T-cell responses in greater detail.

Tumor antigens in lysate vaccines could include overexpressed normal antigens, mutated (neo) antigens, oncofetal antigens (expressed during development), or tumor-specific carbohydrates, glycoproteins, or lipoproteins. That 2 dogs with severe postvaccine seizure activity had autoreactive sera to brain and meninges suggests that severe autoreactivity is possible but infrequent (Fig. 6A and B). Lysates may be depleted of these autoantigens to yield greater tumor specificity and less risk. Because most dogs had no autoreactivity or refractory seizures, risk of autoimmunity must be weighed against the threat to life posed by aggressive meningiomas.

Identification of meningioma antigens recognized by postvaccination sera will enable the discovery of crucial epitopes for fully synthetic vaccine strategies in more widespread application. We identified HSP60 as one antigen recognized by sera following vaccination. HSP60 can translocate to the cell surface upon stress or malignant transformation (35), but it was not expressed on the tumor cell surface of case 3 (data not shown), arguing against cell surface protein as the functional target of this antibody. Sera that recognized HSP60 also reacted with a human meningioma, and reactivity was observed at 60 kDa; however, more study is needed to determine whether HSP60 is a relevant target in human meningioma.

Antibody-mediated autoimmunity is well characterized, with autoimmune conditions in the central nervous system (CNS) such as multiple sclerosis being re-evaluated in light of clinical benefit seen from B-cell depleting therapies (36). Brain tumor immunotherapy, once focused on T-cell–dependent mechanisms, is similarly expanding its breadth of effector mechanisms. Orthotopic mouse models of glioma indicate that survival benefit from immunotherapy is absent in B-cell deficient, μMT tumor-bearing mice (ref. 37; unpublished results). The importance of B cells and antibodies in brain tumor immunity are relatively unstudied. Our study documents for the first time the induction of plasma cells homing to brain tumors as a consequence of therapeutic intervention. B cell and plasma cell infiltration into the brain also indicate promise for immunotherapy to act against invasive cells that exist behind an intact blood brain barrier. ADCC due to in situ antibody production in brain is a novel immune effector mechanism relevant to any brain tumor. Our data suggest that vaccination can induce a “Trojan horse”-like infiltration of plasma cells that can in turn trigger ADCC, and thus create excitement for translation of this approach to human meningioma and other CNS cancers.

No potential conflicts of interest were disclosed.

Conception and design: B.M. Andersen, G.E. Pluhar, W. Chen, M.A. Hunt, M.R. Olin, J.R. Ohlfest

Development of methodology: B.M. Andersen, G.E. Pluhar, C. Seiler, M.M. Schutten, W. Chen, J.R. Ohlfest

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): B.M. Andersen, G.E. Pluhar, C. Seiler, K.S. SantaCruz, M.G. O'Sullivan, R.T. Bentley, R.A. Packer, S.A. Thomovsky, D. Faissler, M.A. Hunt

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B.M. Andersen, G.E. Pluhar, C. Seiler, M.R. Goulart, K.S. SantaCruz, M.M. Schutten, M.G. O'Sullivan, W. Chen, J.R. Ohlfest

Writing, review, and/or revision of the manuscript: B.M. Andersen, G.E. Pluhar, M.R. Goulart, M.M. Schutten, R.T. Bentley, R.A. Packer, S.A. Thomovsky, A.V. Chen, W. Chen, M.A. Hunt, M.R. Olin, J.R. Ohlfest

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B.M. Andersen, G.E. Pluhar, C. Seiler, M.R. Goulart, A.V. Chen, W. Chen, M.A. Hunt, M.R. Olin, J.R. Ohlfest

Study supervision: B.M. Andersen, G.E. Pluhar, W. Chen, M.A. Hunt, J.R. Ohlfest

Other: Histologic cutting and staining of brain tissue and write up of methods employed in this task, J.P. Meints

The authors thank Dr. Robert Schmidt (Washington University, St. Louis MO) for the micrograph of the human papillary meningioma, Guillermo Marques and John Oja (UIC, University of Minnesota) for assistance in quantification and micrograph capture, Jose L. Gallardo and Patrick T. Grogan for vaccine production, and Nick J. Erickson (University of Minnesota) for assistance in immunohistochemistry quantification.

This work was supported by funding to B.M. Andersen from Torske Klubben Fellowship for Minnesota Residents, Medical Scientist Training Program Grant T32 GM008244, and the Cancer Biology Training Grant T32 CA009138—36; to M.A. Hunt from the American Brain Tumor Association Discovery Grant supported by the Anonymous Family Foundation; to J.R. Ohlfest from 1R21NS070955-01, R01 CA154345, R01 CA160782, the American Cancer Society grant RSG-09-189-01-LIB, Minnesota Medical Foundation, the Hedberg Family Foundation, and the Children's Cancer Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
CBTRUS
. 
CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2004–2008
.
Hinsdale, IL
:
available from
: http://www.cbtrus.org:
Central Brain Tumor Registry of the United States
; 
2012
.
2.
Vogelbaum
MA
,
Leland Rogers
C
,
Linskey
MA
,
Mehta
MP
. 
Opportunities for clinical research in meningioma
.
J Neurooncol
2010
;
99
:
417
22
.
3.
Pearson
BE
,
Markert
JM
,
Fisher
WS
,
Guthrie
BL
,
Fiveash
JB
,
Palmer
CA
, et al
Hitting a moving target: evolution of a treatment paradigm for atypical meningiomas amid changing diagnostic criteria
.
Neurosurg Focus
2008
;
24
:
E3
.
4.
Willis
J
,
Smith
C
,
Ironside
JW
,
Erridge
S
,
Whittle
IR
,
Everington
D
. 
The accuracy of meningioma grading: a 10-year retrospective audit
.
Neuropathol Appl Neurobiol
2005
;
31
:
141
9
.
5.
Chamberlain
MC
. 
Hydroxyurea for recurrent surgery and radiation refractory high-grade meningioma
.
J Neurooncol
2012
;
107
:
315
21
.
6.
Soyuer
S
,
Chang
EL
,
Selek
U
,
Shi
W
,
Maor
MH
,
DeMonte
F
. 
Radiotherapy after surgery for benign cerebral meningioma
.
Radiother Oncol
2004
;
71
:
85
90
.
7.
Aghi
MK
,
Carter
BS
,
Cosgrove
GR
,
Ojemann
RG
,
Amin-Hanjani
S
,
Martuza
RL
, et al
Long-term recurrence rates of atypical meningiomas after gross total resection with or without postoperative adjuvant radiation
.
Neurosurgery
2009
;
64
:
56
60
.
8.
Chamberlain
MC
,
Johnston
SK
. 
Hydroxyurea for recurrent surgery and radiation refractory meningioma: a retrospective case series
.
J Neurooncol
2011
;
104
:
765
71
.
9.
Jung
HW
,
Yoo
H
,
Paek
SH
,
Choi
KS
. 
Long-term outcome and growth rate of subtotally resected petroclival meningiomas: experience with 38 cases
.
Neurosurgery
2000
;
46
:
567
74
.
10.
Perry
A
. 
Meningiomas
. In:
McLendon
R
,
Rosenblum
M
,
editors
. 
Russell & Rubinstein's pathology of tumors of the nervous system
.
7th ed
.
London, United Kingdom
:
Hodder Arnold
; 
2006
. p.
427
74
.
11.
Rogers
L
,
Gilbert
M
,
Vogelbaum
MA
. 
Intracranial meningiomas of atypical (WHO grade II) histology
.
J Neurooncol
2010
;
99
:
393
405
.
12.
Perry
A
. 
Unmasking the secrets of meningioma: a slow but rewarding journey
.
Surg Neurol
2004
;
61
:
171
3
.
13.
Snyder
JM
,
Shofer
FS
,
Van Winkle
TJ
,
Massicotte
C
. 
Canine intracranial primary neoplasia: 173 cases (1986–2003)
.
J Vet Intern Med
2006
;
20
:
669
75
.
14.
Adamo
PF
,
Cantile
C
,
Steinberg
H
. 
Evaluation of progesterone and estrogen receptor expression in 15 meningiomas of dogs and cats
.
Am J Vet Res
2003
;
64
:
1310
8
.
15.
Platt
SR
,
Scase
TJ
,
Adams
V
,
Wieczorek
L
,
Miller
J
,
Adamo
F
, et al
Vascular endothelial growth factor expression in canine intracranial meningiomas and association with patient survival
.
J Vet Intern Med
2006
;
20
:
663
8
.
16.
Dickinson
PJ
,
Surace
EI
,
Cambell
M
,
Higgins
RJ
,
Leutenegger
CM
,
Bollen
AW
, et al
Expression of the tumor suppressor genes NF2, 4.1B, and TSLC1 in canine meningiomas
.
Vet Pathol
2009
;
46
:
884
92
.
17.
Sturges
BK
,
Dickinson
PJ
,
Bollen
AW
,
Koblik
PD
,
Kass
PH
,
Kortz
GD
, et al
Magnetic resonance imaging and histological classification of intracranial meningiomas in 112 dogs
.
J Vet Intern Med
2008
;
22
:
586
95
.
18.
Paoloni
M
,
Khanna
C
. 
Translation of new cancer treatments from pet dogs to humans
.
Nat Rev Cancer
2008
;
8
:
147
56
.
19.
Okada
H
,
Kalinski
P
,
Ueda
R
,
Hoji
A
,
Kohanbash
G
,
Donegan
TE
, et al
Induction of CD8+ T-cell responses against novel glioma-associated antigen peptides and clinical activity by vaccinations with {alpha}-type 1 polarized dendritic cells and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent malignant glioma
.
J Clin Oncol
2011
;
29
:
330
6
.
20.
Prins
RM
,
Soto
H
,
Konkankit
V
,
Odesa
SK
,
Eskin
A
,
Yong
WH
, et al
Gene expression profile correlates with T-cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy
.
Clin Cancer Res
2011
;
17
:
1603
15
.
21.
Neller
MA
,
Lopez
JA
,
Schmidt
CW
. 
Antigens for cancer immunotherapy
.
Semin Immunol
2008
;
20
:
286
95
.
22.
Bechmann
I
,
Galea
I
,
Perry
VH
. 
What is the blood-brain barrier (not)?
Trends Immunol
2007
;
28
:
5
11
.
23.
Wu
A
,
Oh
S
,
Gharagozlou
S
,
Vedi
RN
,
Ericson
K
,
Low
WC
, et al
In vivo vaccination with tumor cell lysate plus CpG oligodeoxynucleotides eradicates murine glioblastoma
.
J Immunother
2007
;
30
:
789
97
.
24.
Pluhar
GE
,
Grogan
PT
,
Seiler
C
,
Goulart
M
,
Santacruz
KS
,
Carlson
C
, et al
Anti-tumor immune response correlates with neurological symptoms in a dog with spontaneous astrocytoma treated by gene and vaccine therapy
.
Vaccine
2010
;
28
:
3371
8
.
25.
Thompson
JA
,
Kuzel
T
,
Drucker
BJ
,
Urba
WJ
,
Bukowski
RM
. 
Safety and efficacy of PF-3512676 for the treatment of stage IV renal cell carcinoma: an open-label, multicenter phase I/II study
.
Clin Genitourin Cancer
2009
;
7
:
E58
65
.
26.
Pashenkov
M
,
Goess
G
,
Wagner
C
,
Hörmann
M
,
Jandl
T
,
Moser
A
, et al
Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma
.
J Clin Oncol
2006
;
24
:
5716
24
.
27.
Adams
S
,
O'Neill
DW
,
Nonaka
D
,
Hardin
E
,
Chiriboga
L
,
Siu
K
, et al
Immunization of malignant melanoma patients with full-length NY-ESO-1 protein using TLR7 agonist imiquimod as vaccine adjuvant
.
J Immunol
2008
;
181
:
776
84
.
28.
Schon
MP
,
Schon
M
. 
TLR7 and TLR8 as targets in cancer therapy
.
Oncogene
2008
;
27
:
190
9
.
29.
Perry
A
,
Stafford
SL
,
Scheithauer
BW
,
Suman
VJ
,
Lohse
CM
. 
Meningioma grading: an analysis of histologic parameters
.
Am J Surg Pathol
1997
;
21
:
1455
65
.
30.
Louis
DN OH
,
Wiestler
OD
,
Cavenee
WK
. 
WHO classification of tumours of the central nervous system
.
4th ed
.
Lyon, France
:
IARC Press
; 
2007
.
31.
Henderson
AP
,
Barnett
MH
,
Parratt
JD
,
Prineas
JW
. 
Multiple sclerosis: distribution of inflammatory cells in newly forming lesions
.
Ann Neurol
2009
;
66
:
739
53
.
32.
Burgoon
MP
,
Keays
KM
,
Owens
GP
,
Ritchie
AM
,
Rai
PR
,
Cool
CD
, et al
Laser-capture microdissection of plasma cells from subacute sclerosing panencephalitis brain reveals intrathecal disease-relevant antibodies
.
Proc Natl Acad Sci U S A
2005
;
102
:
7245
50
.
33.
American Veterinary Medical Association. U.S. Pet Ownership & Demographics Sourcebook
; 
2007
.
34.
Axlund
TW
,
McGlasson
ML
,
Smith
AN
. 
Surgery alone or in combination with radiation therapy for treatment of intracranial meningiomas in dogs: 31 cases (1989-2002)
.
J Am Vet Med Assoc
2002
;
221
:
1597
600
.
35.
Shin
BK
,
Wang
H
,
Yim
AM
,
Le Naour
F
,
Brichory
F
,
Jang
JH
, et al
Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function
.
J Biol Chem
2003
;
278
:
7607
16
.
36.
Hauser
SL
,
Waubant
E
,
Arnold
DL
,
Vollmer
T
,
Antel
J
,
Fox
RJ
, et al
B-cell depletion with rituximab in relapsing-remitting multiple sclerosis
.
N Engl J Med
2008
;
358
:
676
88
.
37.
Daga
A
,
Orengo
AM
,
Gangemi
RM
,
Marubbi
D
,
Perera
M
,
Comes
A
, et al
Glioma immunotherapy by IL-21 gene-modified cells or by recombinant IL-21 involves antibody responses
.
Int J Cancer
2007
;
121
:
1756
63
.