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Keywords:

  • meningioma;
  • trabectedin;
  • therapy;
  • antineoplastic;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

BACKGROUND:

Meningiomas are common intracranial tumors arising from the meninges and usually are benign. However, a few meningiomas have aggressive behavior and, for such patients, effective treatment options are needed. Trabectedin is a novel, marine-derived, antineoplastic agent that has been approved and is used routinely as therapy for advanced soft tissue sarcoma and ovarian cancer.

METHODS:

The authors investigated the in vitro effects of trabectedin alone and in combination with hydroxyurea, cisplatin, and doxorubicin in primary cell cultures of benign (n = 9), atypical (n = 6), and anaplastic (n = 4) meningiomas using chemosensitivity assays (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT]), Western blot analysis, cell cycle analysis, and immunofluorescent staining.

RESULTS:

Strong antimeningioma activity of trabectedin was observed and was characterized by distinct cell cycle arrest, down-regulation of multiple cyclins, deregulated expression of cell death-regulatory genes, and massive apoptosis induction. Cytotoxic activity was especially intense in higher grade meningiomas with a half-maximal inhibitory concentration <10 nM. Combination with trabectedin synergistically enhanced the antimeningioma activity of hydroxyurea but also enhanced the activity of doxorubicin and cisplatin. On the basis of these findings, trabectedin was given to 1 patient who had heavily pretreated, anaplastic meningioma, and a favorable response was observed with radiologic disease stabilization, marked reductions in brain edema and requirement for corticosteroids, and improvement of clinical symptoms. However, treatment had to be discontinued after 5 cycles because of adverse drug effects.

CONCLUSIONS:

The current results indicated that trabectedin may represent a promising new therapeutic option for patients with aggressive meningioma and should be evaluated in prospective clinical studies. Cancer 2012. © 2012 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

Meningiomas are common intracranial tumors that are benign and curable by resection in most patients.1 However, a small fraction of patients with meningioma have tumors with aggressive behavior, including recurrences and infiltration of the surrounding tissues (bone, brain, soft tissue). Atypical and malignant meningiomas are characterized histopathologically by increased numbers of mitotic figures and have an increased risk of an unfavorable clinical course.1-3 Because of the rarity of aggressive meningiomas, data are lacking from systematic studies to guide treatment. To date, maximal resection and postoperative adjuvant radiotherapy have emerged as the most commonly used therapy for atypical and malignant meningiomas. For recurrent disease, there are only a few therapy options.4 Repeated surgery or radiotherapy may achieve local tumor control in some patients. Medical therapy with hydroxyurea, somatostatin analog, or interferon-alpha has produced no or only limited benefit.4 Therefore, novel treatment options for these rare but devastating tumors are urgently needed.

The anticancer compound trabectedin (ET743 [Yondelis; Zelita/Pharmamar, Madrid, Spain; Johnson&Johnson, New Brunswick, NJ]) is a tetrahydroisoquinoline molecule that originally was isolated from the sea squirt Ecteinascidia turbinate.5 The precise mode of action of trabectedin is not known. However, it is unquestionable that the drug binds to the minor groove of the deoxyribonucleic acid (DNA) double helix, forming trabectedin-DNA adducts that bend the DNA toward the major groove. Furthermore, trabectedin may affect diverse DNA-binding proteins, including several transcription factors and DNA repair mechanisms.6 The alterations induced by the drug activate the nucleotide excision repair (NER) pathway, which is required for the maximum activity of trabectedin, whereas its activity is retained in mismatch repair-deficient cells.7 In vitro studies have indicated that trabectedin selectively inhibits the transcription of several genes, particularly those encoding the multidrug resistance protein 1, heat shock protein 70 (HSP70), and the cyclin-dependent kinase inhibitor p21WAF1/Cip1, contributing to the induction of programmed cell death.8

Trabectedin has demonstrated activity against a wide variety of cell lines and xenografts derived from several solid tumor types. Also, in early clinical trials, trabectedin was active against several solid tumors, including breast, prostate, and renal cancers; melanoma; and nonsmall cell lung cancer (NSCLC).6, 9 Trabectedin reportedly was well tolerated and had manageable and mostly reversible adverse effects like transaminase elevations, myelosuppression, nausea, emesis, and fatigue.10, 11 Only a few patients experienced severe toxicities like skin and soft tissue necrosis because of extravasation or rhabdomyolysis. Trabectedin has been approved and is used routinely in patients with advanced soft tissue sarcoma (STS) who have failed or are not eligible for first-line therapy with anthracyclines and ifosfamide11 and in patients with platinum-sensitive, recurrent ovarian cancer in combination with pegylated liposomal doxorubicin.12 Several ongoing clinical trials are evaluating its activity in some additional cancer types, including prostate cancer and breast cancer, with a focus on patients who carry mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. To the best of our knowledge, the activity of trabectedin has not been investigated previously in meningioma. Therefore, we decided to test the in vitro activity of trabectedin against primary cell cultures established from surgically obtained meningioma samples. On the basis of a massive induction of apoptosis by very low drug concentrations in high-grade meningiomas, we decided to initiate trabectedin treatment in a patient with advanced anaplastic meningioma who presented with extensive tumor recurrence after several lines of treatment. The favorable clinical benefit observed in this patient, together with our in vitro data, indicates that trabectedin may be useful in the treatment of aggressive meningioma.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

Histology and Immunohistochemistry

For diagnostic histopathologic workup, surgical tumor tissue samples were routinely fixed in formalin and paraffin embedded. Sections were cut at a thickness of 3 to 5 μm, deparaffinized, and stained using hematoxylin and eosin and Giemsa to evaluate tumor morphology and mitotic count. Tumor typing was done according to diagnostic criteria from the current edition of the World Health Organization (WHO).1

For immunohistochemistry, the following primary monoclonal antibodies were used: anti-Ki67 (MIB-1 [mouse monoclonal]; Flex TRS low; 1:200 dilution; Dako, Glostrup, Denmark), antiepithelial membrane antigen (E29 [mouse monoclonal]; Flex TRS low; 1:100 dilution; Dako), and antivimentin (V9; 1:50 dilution; Dako). Antibody binding was detected by using the Dako EnVision Flexkit.

For assessment of the Ki-67 tumor cell proliferation index, anti-Ki67–immunostained tissue sections were scanned at low magnification to identify the area with the highest density of immunolabeled tumor cell nuclei (“hot spot”). In this area, a total of 500 tumor cell nuclei were evaluated. The fraction of labeled nuclei per 500 tumor cell nuclei was determined by manual counting using an eye grid and is expressed as a percentage, as described previously.13

Establishment of Meningioma Cell Models

Primary cell cultures were established according to a previously published protocol14 from surgical specimens that were verified histologically according to WHO 2007 criteria as meningioma (WHO grade I), atypical meningioma (WHO grade II), and anaplastic meningioma (WHO grade III).1 Two anaplastic meningioma cell cultures were developed into immortalized, tumorigenic cell lines (BTL598 and BTL695). Briefly, tumor tissue was minced mechanically and cultivated in Quantum 263 (PAA Laboratories, Pasching, Austria) supplemented with 1% penicillin/streptomycin up to 3 passages. Subsequently, cells were grown without antibiotics. Both cell cultures were periodically checked for Mycoplasma contamination (VenorGeM Advance Mycoplasma Detection Kit; Minerva Biolabs GmbH, Berlin, Germany).

Drugs

Trabectedin was obtained from Pharmamar, and cisplatin, hydroxyurea, and doxorubicin were obtained from Sigma Chemical Company (St. Louis, Mo). Tetramethyl rhodamine isothiocyanate (TRITC)-labeled phalloidin was obtained from Sigma Chemical Company.

Chemosensitivity Assays

Meningioma cell cultures were seeded in 96-well plates at a density of 2 × 103/100μL per well 24 hours before drug treatment. Drug exposure time was 96 hours. Subsequently, cell viability was tested by a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based survival assay (EZ4U; Biomedics, Vienna, Austria) as published previously.15 Interactions between drugs were tested based on calculating the combination index (CI) according to Chou and Talalay with CalcuSyn software (Biosoft, Ferguson, Mo). CI values <0.9, from 0.9 to 1.1, or >1.1 represented synergism, additive effects, and antagonism of the 2 investigated substances, respectively.

Western Blot Analysis

Cells were seeded onto 6-well plates at a density of 5 × 105 per well. After 24 hours of drug exposure, the cells were collected, then lysed, and proteins were extracted and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting as described previously.15, 16 The following primary antibodies were used: cyclin B1, cyclin D1 and cyclin E (all from Santa Cruz Biotechnology, Santa Cruz, Calif), p53 (DO1; Neomarkers, Fremont, Calif), extracellular signal-regulated kinase (ERK), phosphorylated ERK (pERK), p21, bax, p-S6, phosphorylated 4E-binding protein 1 (p4EBP1), poly (ADP-ribose) polymerase (PARP) (detecting full-length and cleaved PARP), cleaved PARP, vimentin, cleaved caspases 3 and 7, p53 up-regulated modulator of apoptosis (PUMA) (all from Cell Signaling Technology, Danvers, Mass), and β-actin (Sigma Chemical Company) diluted appropriately in Tris-buffered saline plus Tween-20 with 3% bovine serum albumin. Horseradish peroxidase-coupled antirabbit/antimouse antibodies (3% bovine serum albumin; Santa Cruz Biotechnology) were used at 1:10,000 dilution and developed by Western blot analysis (Western Blotting Luminol Reagent Kit; Santa Cruz Biotechnology).

Cell Cycle Analysis

Cells were seeded onto 6-well plates at a density of 2 × 105 cells per well 24 hours before drug treatment. After 72 hours of exposure, the cells were trypsinized, pelleted, and processed for cell cycle analyses by propidium iodide staining and flow cytometry using fluorescence-activated cell sorting (FACS Calibur; Becton Dickinson, Palo Alto, Calif) according to standard protocols.15, 7 The results were quantified using Cell Quest Pro software (Becton Dickinson and Company, New York, NY).

Hoechst 33258 and Propidium Iodide Double Staining to Detect Early/Late Apoptosis and Necrosis

Vitality staining was performed as described previously.18 Meningioma cells were seeded onto 6-well plates (3 × 104 cells per well) and exposed to increasing drug concentrations for 48 hours. Hoechst 33258 and propidium iodide were added directly to the cells at final concentrations of 5 mg/mL and 2 mg/mL, respectively. After 60 minutes of incubation at 37°C, the cells were examined live using a Nikon Eclipse equipped with 4′-6-diamidino-2-phenylindole (DAPI) and TRITC filters (Nikon Instruments Europe, Kingston, United Kingdom). Cells were judged according to their nuclear morphology and the disintegration of their cell membranes, as indicated by propidium iodide uptake. Early apoptosis, late apoptosis, and necrosis were scored based on DNA condensation without propidium iodide staining, DNA condensation with propidium iodide staining, and nucleic swelling with propidium iodide staining, respectively.

Microfilament Staining by Tetramethyl Rhodamine Isothiocyanate-Phalloidin

Cells were seeded onto chamber slides 24 hours before drug treatment. After 48 hours of exposure, the slides were washed in phosphate-buffered saline (PBS) and fixed in methanol:aceton (1:1) for 15 minutes at −20°C. The microfilament system (f-actin) was stained by TRITC phalliodin and DNA by DAPI as described previously.19

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

Higher Grade Meningioma Cell Models Are Especially Sensitive to Trabectedin

To estimate the feasibility of trabectedin for the treatment of meningioma, we investigated the activity of this novel marine anticancer compound against cell cultures established from patients who had benign, atypical, and anaplastic meningiomas. Two anaplastic meningioma primary cell cultures were developed into stable cell lines. In a first approach, these cell lines were tested for sensitivity against trabectedin in a 96-hour, continuous drug exposure assay. We observed that trabectedin was highly active in both meningioma cell models with half maximal inhibitory concentration (IC50) values of 1 nM and 8 nM (Fig. 1A), which was orders of magnitude more active than cisplatin or hydroxyurea, agents that are used occasionally to treat progressive meningioma. The BTL695 cell line was slightly less sensitive against all 3 compounds than the BTL598 cell line, suggesting that this cell model may exhibit general chemoresistance. Nevertheless, in this cell model, the IC50 against trabectedin also was only 8 nM. When analyzing primary cell cultures established from grade I through III meningiomas, the increasing activity of trabectedin with enhanced malignancy became obvious (Fig. 1B,C). In grade I tumor-derived primary cell cultures (N = 9), the mean IC50 value was 34.9 ± 12.4 nM; whereas the IC50 value was 26.1 ± 10.5 nM for grade II tumor cells (N = 6) and 7.1 ± 7.6 nM for grade III tumor cells (N = 4; 1-way analysis of variance; P < .005) (Fig. 1C). The greater sensitivity in high-grade tumor cells was not a consequence of altered proliferation. Tumors of all grades formed rapidly proliferating primary cell cultures in vitro; however, the cell cultures benign meningiomas never developed into stable, immortalized cell lines (data not shown). Microscopic examination of cell morphology revealed that trabectedin treatment led to meningioma cell shrinkage, round-up, and detachment from the surface of the culture plate. All of these features indicate the induction of apoptotic cell death (Fig. 1D). Accordingly, trabectedin doses of approximately 0.5 nM and greater significantly enhanced the sub-G0/G1 peak of BTL598 cells in cell cycle analysis (see Fig. 2A, inset). In addition, a very low dose of 0.5 nM significantly enhanced the proportion of cells in G2/M phase of the cell cycle; whereas, progressively, at higher doses, only cells in G0/G1 phase of the cell cycle survived trabectedin treatment. Accordingly, multiple cyclins (B1, D1, and E) were reduced after trabectedin treatment, as determined by immunoblot analysis (Fig. 2B). These data suggest that predominantly noncycling cells were able to survive trabectedin-induced cell death. Trabectedin induced only minor changes in the activity of the oncogenic mitogen-activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways at doses from 50 nM. The readouts for pERK and S6/4EBP are provided in Figure 2B. In contrast, strong activation of p53 was detectable at 10 nM. It is noteworthy that, despite intense induction of p53, the 2 major p53 target genes tested—coding the cyclin-dependent kinase inhibitor p21 and the proapoptotic protein bax—were not stimulated but, in the case of p21, even diminished after trabectedin exposure.

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Figure 1. Trabectedin is highly active against human meningioma cell models. (A) Trabectedin as single agent was compared with cisplatin and hydroxyurea in 96-hour drug-exposure assays using the indicated meningioma cell lines derived from anaplastic tumors. Half-maximal inhibitory concentration (IC50) values indicate the concentrations that induced a 50% reduction in viable cells compared with nondrug controls and were calculated from dose-response curves of 3 independent experiments that were performed in quintuplicate. (B) Antimeningioma activity of trabectedin against primo cell cultures (Passages 2-5) derived from meningioma specimens of the different grades indicated were established by viability assays. Full dose-response curves for 2 examples of grade I, II, and III tumors each are represented. (C) Mean ± standard deviation IC50 values for the indicated number of primo cell cultures derived from meningioma surgical specimen are indicated (analysis of variance; P < .005). (D) These images show the impact of a 48-hour cell exposure to trabectedin on the cell shape of the investigated meningioma cell models.

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Figure 2. Trabectedin induces cell cycle arrest in human meningioma cell models. (A) The effects of a 72-hour exposure to trabectedin at the indicated concentrations on the cell cycle distribution as analyzed by fluorescence-activated cell sorting are shown for the BTL598 cell model. The sub-G0/G1 fraction indicates the percentage of cells undergoing apoptosis. (B) The impact of a 48-hour exposure of trabectedin on the expression and/or phosphorylation of the indicated proteins was analyzed by immunoblotting. p53 Indicates tumor protein 53; bax, B-cell leukemia/lymphoma 2-associated X protein; pERK, protein kinase-like endoplasmic reticulum kinase; ERK, extracellular signal-regulated kinase; p4EBP, phosphorylated 4E-binding protein 1.

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Trabectedin Induces Apoptosis of Human Meningioma Cells

To further characterize trabectedin-induced cell death, trabectedin-treated meningioma cells were stained with DAPI (DNA staining) and TRITC-labeled phalloidin (microfilament system) (see Fig. 3A, blue and red staining, respectively). Trabectedin induced a potent disruption of the microfilament system, which explained the detachment and round-up of cells observed under phase-contrast microscopy (compare Fig. 3A with Fig. 1C). In addition, intense condensation of chromatin was induced in the majority of cells that were treated with 25 nM trabectedin. A progressive induction of both early and late-stage apoptosis and the complete loss of mitotic cells also were observed in combined Hoechst/propidium iodide staining of live cells that were treated with trabectedin for 48 hours (Fig. 3B). This was paralleled by a massive cleavage of the caspase substrate PARP and caspase 7 in both cell models, whereas cleaved caspase 3 was detectable only in the more sensitive meningioma cell model BTL598. It is noteworthy that the apoptosis-regulating protein PUMA was strongly reduced by trabectedin treatment. PUMA, like p21 and bax, also is a p53 target gene,20 and these results confirmed the uncoupling of p53-mediated transcription regulation by trabectedin in human meningioma cell models.

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Figure 3. Trabectedin induces apoptosis in human meningioma cell models. (A) Breakdown of the microfilament system (f-actin is stained in red by tetramethyl rhodamine isothiocyanate [TRITC] phalloidin) and condensation of chromatin (DNA stained blue by 4′-6-diamidino-2-phenylindole [DAPI]; apoptotic nuclei are marked by arrows) after a 48-hour exposure to 10 nM trabectedin are shown for BTL598 cells. (B) Quantification of cells in interphase (IP), mitosis, early apoptosis (APO early), and late APO/necrosis (APO late/N) was performed on live cells stained by Hoechst dye/propidium iodide in meningioma cells that were exposed to the indicated concentrations of trabectedin for 48 hours. (C) Immunodetection of the indicated proteins or their respective cleaved (cl) products as markers for apoptosis was performed on cell cultures treated as described for B. PARP indicates poly (ADP-ribose) polymerase; PUMA, p53 up-regulated modulator of apoptosis.

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Trabectedin Enhances the Activity of Anticancer Drugs Against Human Meningioma Cells

In addition to single-agent analyses, we determined whether trabectedin had an impact on the activities of other chemotherapeutic agents. Indeed, trabectedin—when applied at moderately active concentrations—demonstrated predominantly synergistic effects (combination indices; CI <0.9) (see Fig. 4A, bottom graph) with hydroxyurea in 72-hour drug exposure MTT assays (Fig. 4A, normalized data in the top graph). Accordingly, cleavage of the caspase substrate PARP was enhanced synergistically by this drug combination, as indicated in BTL598 cells, using an antibody that detected full-length and cleaved PARP and a second antibody that only detected cleaved PARP in immunoblot analyses (Fig. 4B). In addition, combination experiments with cisplatin and doxorubicin were performed. Trabectedin demonstrated synergism with cisplatin predominantly at lower doses of the metal drug; whereas, at higher doses, loss of synergism was widespread (Fig. 4C). Also, with the anthracycline compound, doxorubicin had predominantly synergistic to additive effects (CI, 0.9-1.1). These data suggest that trabectedin widely enhanced the antimeningioma activity of chemotherapeutic drugs.

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Figure 4. These charts illustrate the combined treatment of meningioma models with trabectedin and with chemotherapeutic drugs used to treat meningioma. (A) The indicated meningioma cell models were exposed concomitantly to trabectedin and hydroxyurea (HU) at the indicated concentrations for 72 hours, and cell survival was determined with the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) This representative immunoblot depicts poly (ADP-ribose) polymerase (PARP) cleavage (cl) in the BTL598 model as a measure of apoptosis induction by the respective drug combinations at the indicated dose levels. (C,D) These charts illustrate the impact on the meningioma cell model BTL598 of combined treatment with cisplatin (Cis) and doxorubicin (Dox) at the indicated concentrations. Combination indices (CI) in the lower graphs in A, C, and D indicate interactions between the respective drugs in combination determined by CalcuSyn software (Biosoft, Ferguson, Mo): synergism (CI, <0.9), additive activities (CI, 0.9-1.1; dotted lines), or antagonism (CI, >1.1).

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Trabectedin Is Clinically Active in Anaplastic Meningioma: Single-Patient Experience

On the basis of our favorable in vitro data, we initiated trabectedin therapy in a man aged 64 years with histologically proven, malignant meningioma. The patient had been diagnosed with atypical meningioma (WHO grade II; 8 × 8 × 3.5 cm large meningeal tumor with perifocal brain edema in the left frontoparietal region) 2 years earlier and had undergone gross total resection; 3-dimensional, planned local radiotherapy with at a total dose of 60 grays (Gy) in single doses of 1.28 Gy; 2 applications of lanreotide (once-monthly intramuscular injection of 30 mg); 6 weeks of imatinib (oral daily dose of 400 mg); and 4 months of sorafenib (oral daily dose of 400 mg). During this time, follow-up magnetic resonance imaging (MRI) studies revealed slowly progressive tumor growth and progressive peritumoral edema. The patient presented to our clinic with significant worsening of neurologic symptoms, including motor Jackson seizures, progressive aphasia, and hemiplegia. MRI studies revealed massive tumor progression with tumor masses measuring 80 × 10 × 69 mm. The tumor formations had extracranial expansion with infiltration of the right temporal muscle. The extracranial part was totally resected, whereas the intracranial part could be removed only in a subtotal excision. Postoperative MRI studies revealed residual tumor formations (40 × 50 × 4 mm) with infiltration of the left postcentral cortex (Fig. 5). Histologically, the tumor displayed preserved meningothelial morphology with extensive necrosis (Fig. 6A), strong immunoreactivity for epithelial membrane antigen (EMA) (Fig. 6B) and vimentin (Fig. 6C), intense proliferative activity (Ki-67 proliferation index, 42%) (Fig. 6D), and invasion of the skull bone and the surrounding soft tissues (Fig. 6E,F). A high mitotic frequency was observed (21 mitoses per 10 high-power fields), warranting the diagnosis of anaplastic meningioma (WHO grade III).1 Reirradiation was declined because of the large recurrence within the previous radiation field. After obtaining informed patient consent, we initiated therapy with trabectedin 4 weeks after the tumor resection at a dose of 1.5 mg/m2 applied intravenously over 24 hours through a permanent central venous access device every 21 days. It is noteworthy that there was a marked improvement of clinical symptoms, which became evident within 2 weeks after the first trabectedin infusion. There was significant and lasting, continuous relief of aphasia and neurologic symptoms, which allowed reduction of the daily dexamethasone dose from 12 mg to 4 mg, and the patient recovered from a Karnofsky index of 60% to an index of 80%. It also is worth noting that MRI studies revealed a significant reduction of perifocal brain edema (Fig. 5). However, after 3 cycles, the patient developed progressive edema in both legs despite normal kidney function and serum albumin levels, and he also developed mucositis. Despite diuretic therapy, the patient gained >10 kg in 4 weeks and suffered from progressive exertional dyspnea, necessitating therapy discontinuation after a total of 5 trabectedin infusions. MRI follow-up investigations performed 2 months and 5 months after the initiation of trabectedin therapy revealed stable disease according to Response Evaluation Criteria in Solid Tumors criteria (Fig. 5).

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Figure 5. These magnetic resonance images (MRIs) reveal stable tumor size and significant reduction of brain edema during trabectedin therapy. Before the initiation of trabectedin therapy (time 0 [T0]), T1-weighted MRIs (T1) reveal tumor formations (40 × 50 × 4 mm) with contrast medium (CM) uptake that infiltrate the left postcentral cortex. The tumor formations do not change significantly in size or appearance after 2 trabectedin applications (T1; 2 months after T0) and 5 trabectedin applications (T2; 5 months after T0). Fluid-attenuated inversion recovery (FLAIR) images reveal significant peritumoral edema at T0 and markedly less brain edema at T1 and T2.

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Figure 6. (A) Histopathology of tumor tissues resected at recurrence displays features of a malignant meningioma (hematoxylin and eosin [H&E] staining) with meningothelial morphology and large areas of necrosis (asterisk). Tumor cells display strong immunoreactivity for (B) epithelial membrane antigen (anti-EMA) and (C) vimentin (antivimentin) and also (D) reveal increased proliferative activity (anti-Ki67). Note the growth pattern with extensive invasion in H&E-stained sections from (E) the dura (F) bone, and (G) soft tissues (original 100x (original magnification, ×100 in A-C and E-G; ×400 in D).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

In this report, we present evidence of promising in vitro and single-patient antineoplastic efficacy of trabectedin for the treatment of high-grade meningioma. Our in vitro experiments demonstrate that especially cell cultures derived from anaplastic meningiomas are highly sensitive against trabectedin with IC50 values at the border of picomolar to very low nanomolar concentrations. Thus, high-grade meningioma cells exhibit sensitivity to trabectedin comparable to that of, for example, ovarian cancer cells,21, 22 and meningioma cells were only marginally less responsive than cells from an STS cell model.23, 24 Trabectedin is approved clinically for both of these tumor entities. It is noteworthy that IC50 values for trabectedin were orders of magnitude lower than the values for drugs like hydroxyurea and cisplatin, which are used at least occasionally in the systemic treatment of anaplastic meningioma.3, 4 Tumor cell lines derived from several carcinoma types, including lung and colon cancers, exhibited higher IC50 values25, 26 (and our unpublished data), whereas breast cancer cells appear to have trabectedin sensitivity similar to that observed in meningiomas cell models.27 This suggests at least a tendency toward enhanced trabectedin sensitivity in cells of mesenchymal origin. It also is worth noting that meningioma cells, like their normal meningothelial counterparts (arachnoidal cells), are morphologically and functionally diverse and have some degree of overlap with mesenchymal and epithelial cells.2 The reasons for this hypersensitivity of cells with mesenchymal features are unknown. In myxoid/round cell liposarcomas, it has been suggested that trabectedin induces cell differentiation by inhibiting expression of the fused in sarcoma-CCAAT/-enhancer-binding protein homologous (FUS/CHOP) fusion protein.28 Moreover, NER deficiency has been correlated with resistance against trabectedin-induced cell death29; whereas impaired homologous recombination, eg, by mutations in BRCA1, caused trabectedin hypersensitivity in sarcoma cells.30 The status of such molecular mechanisms in anaplastic meningioma is widely unexplored. In this study, we observed a significantly reduced antimeningioma activity of trabectedin in cells derived from low-grade tumors versus high-grade tumors despite comparable proliferation rates of the respective primary cell cultures. This suggests that it is not the cellular origins but the mechanisms acquired during malignant progression that underlie the hypersensitivity of high-grade meningiomas.

In our in vitro analyses, trabectedin exerted a potent proapoptotic effect accompanied by strong cell cycle arrest in G2/M phase already at subnanomolar concentrations, down-modulation of several cell cycle-driving cyclins, and destruction of the microfilament system in meningioma cells. These data suggest that trabectedin may target several molecular mechanisms both at the DNA level and possibly in proteins. In line with previous studies,23, 31 we also observed the stimulation of strong p53 expression in malignant meningioma cell lines, indicating trabectedin-induced DNA damage. Thus, the inhibition of cyclin expression may be a secondary consequence of DNA damage-mediated cell cycle arrest and/or the survival of quiescent cells only in G0 phase.32 However, specific inhibition of the promoters that regulate the transcription of cell cycle regulators, such as the cyclin A and B genes, has been suggested based on trabectedin-mediated inhibition of nuclear factor Y (NF-Y)-induced transcription.33 Although we did not test for NF-Y responsiveness in the current study, we did observe the deregulation of p53-mediated gene transactivation. Thus, despite strong p53 accumulation, the p53 downstream targets p21, bax, and PUMA were not up-regulated but even were down-regulated in response to trabectedin.23 These data support previous observations that trabectedin interferes with DNA binding of certain transcription factors and exerts its anticancer activity at least in part by blocking activated (but not constitutive) gene transcription.6, 8

To date, only a few chemotherapeutic agents were used to treat patients with high-grade meningioma with uncertain activity based on the lack of prospective clinical trials. Among the agents associated with at least occasional response are hydroxyurea and doxorubicin as part of the cyclophosphamide, doxorubicin, and vincristine (CAV) scheme.3 It is noteworthy that we observed predominantly synergistic anticancer activity of these 2 agents and the metal drug cisplatin with trabectedin against meningioma cell models. Synergism with anthracyclines, taxanes, and/or platinum compounds and antagonism to diverse other chemotherapeutics also were reported in several cancer types, including osteosarcoma, STS, and ovarian carcinoma cell lines24, 34, 35 as well as cancer xenograft models.27, 36 The reason for this selective synergism with certain chemotherapeutics is unknown but may involve trabectedin-mediated inhibition of activated drug-resistance gene expression, like adenosine triphosphate-binding cassette, subfamily B, member 1 (ABCB1) and HSP70.8 Combination schemes of trabectedin with, for example, cisplatin and pegylated liposomal doxorubicin have exhibited activity against several cancer types at least in early clinical studies.5 The observed synergistic antineoplastic effects of these drugs and also that of hydroxyurea in meningioma models suggest that trabectedin-containing combination therapies may be feasible strategies for the treatment of patients with aggressive meningioma.

Trabectedin exhibited promising in vitro activity in our study. However, there are several issues that cannot be addressed adequately in cell culture. Thus, currently, we are in the process of establishing an orthotopic mouse model for anaplastic meningioma so that we can study the effect of intermittent trabectedin application schemes and the in vivo efficacy of combination approaches with other drugs like hydroxyurea, cisplatin, and doxorubicin that exhibit synergistic activity in vitro.

The high sensitivity of meningioma cells toward trabectedin prompted us to apply this novel marine compound in 1 heavily pretreated patient who had anaplastic meningioma. It is noteworthy that we were able to achieve radiographic disease stabilization for several months. Several studies have documented tumor responses to trabectedin in the absence of radiographic decreases in tumor size. In myxoid liposarcoma, trabectedin treatment led to histopathologically confirmed tissue density changes in tumors with stable dimensions that may have indicated biologically and clinically relevant responses.37 In line with those findings, 2 studies demonstrated that trabectedin induced the inhibition of tumor metabolic activity, as observed by decreases in positron-emission tomography tracer uptake even in the absence of objective decreases in tumor size on conventional MRI or computed tomography studies.38, 39 Our patient had a surprising improvement in neurologic symptoms, including aphasia and paresis, within days after starting trabectedin treatment, although there were no clinical signs of improvement in the preceding 4 weeks. The patient valued the quick and lasting relief of these debilitating symptoms. Radiologically, there was marked reduction of peritumoral brain edema with a decreasing need for dexamethasone treatment. Inhibition of vascular endothelial growth factor (VEGF) production by trabectedin and reduced inflammation has been observed previously and may explain the antiedematous effect observed in our patient.40 Trabectedin-associated reduction of brain edema and corticosteroid use may prove to be clinically useful in patients with malignant meningioma, similar to what has been reported with the monoclonal VEGF antibody bevacizumab in malignant glioma.41, 42 It would be interesting to use an in vivo model of anaplastic meningioma to determine whether combined treatment with bevacizumab and trabectedin has an additive or synergistic, antiedematous effect.

Unfortunately, trabectedin therapy was associated with significant adverse effects in our patient. The therapy had to be discontinued because of massive peripheral edema after 5 cycles. However, data from several clinical trials indicate that trabectedin is well tolerated in general, both as monotherapy and in combination with other cytostatic drugs, including pegylated liposomal doxorubicin, platinum derivatives, and paclitaxel.5, 6 Trabectedin has repeatedly been applied for up to 20 cycles or more without significant adverse effects (see Demetri et al.11 and own experience in patients with STS treated at our department). The most common adverse effects are hepatotoxicity, myelosuppression, nausea, emesis, and fatigue.10, 11 Few patients experience severe toxicities like skin and soft tissue necrosis because of extravasation or rhabdomyolysis. Marked peripheral edema, like that observed in our patient, has been infrequently reported. It remains unclear from our single patient experience whether the toxicity was idiosyncratic (eg, based on the pharmacogenomics of our patient) or whether such toxicity is more likely to occur in patients with anaplastic meningioma. Therefore, the tolerability profile of trabectedin in patients with meningioma needs to be evaluated in prospective phase 1 or phase 1/2 clinical trials; however, we expect that those results will be consistent with the already documented findings in other tumor entities. Taken together, our findings indicate that trabectedin may have clinically relevant activity in patients with high-grade meningioma and should be evaluated in prospective clinical trials.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

We thank Magdalena Wild, MA, for skillful technical assistance.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES

This study was supported by the Forschungsförderungsfonds der Krebshilfe Oberösterreich.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SOURCES
  9. REFERENCES