Metronomic chemotherapy with daily, oral etoposide plus bevacizumab for recurrent malignant glioma: a phase II study

Background:

We evaluated bevacizumab with metronomic etoposide among recurrent malignant glioma patients in a phase 2, open-label trial.

Methods:

A total of59 patients, including 27 with glioblastoma (GBM) and 32 with grade 3 malignant glioma, received 10 mg kg⁻¹ bevacizumab biweekly and 50 mg m⁻² etoposide daily for 21 consecutive days each month. The primary end point was a 6-month progression-free survival, and secondary end points included safety and overall survival. Vascular endothelial growth factor (VEGF), VEGFR-2, carbonic anhydrase 9 (CA9) and hypoxia-inducible factor-2α (HIF-2α) were assessed semiquantitatively in archival tumours using immunohistochemistry and were correlated with outcome.

Results:

Among grade 3 and GBM patients, the 6-month progression-free survivals were 40.6% and 44.4%, the radiographic response rates were 22% and 37% and the median survivals were 63.1 and 44.4 weeks, respectively. Hypertension predicted better outcome among both grade 3 and GBM patients, whereas high CA9 and low VEGF were associated with poorer progression-free survival (PFS) among those with GBM. The most common grade ⩾3 adverse events included neutropaenia (24%), thrombosis (12%), infection (8%) and hypertension (3%). Two patients had asymptomatic, grade 1 intracranial haemorrhage and one on-study death occurred because of pulmonary embolism.

Conclusion:

Bevacizumab with metronomic etoposide has increased toxicity compared with previous reports of bevacizumab monotherapy. Its anti-tumour activity is similar to that of bevacizumab monotherapy or bevacizumab plus irinotecan. (ClinicalTrials.gov: NCT00612430).     

The role of folate receptor alpha (FR��) in the response of malignant pleural mesothelioma to pemetrexed-containing chemotherapy

Background:

The standard treatment of choice for malignant pleural mesothelioma is chemotherapy with pemetrexed and platinum, but the clinical outcome is poor. This study investigates the response to pemetrexed in a panel of eight mesothelioma cell lines and the clinical outcome for patients treated with pemetrexed in relation to folate receptor alpha (FRα).

Methods:

Cell lines were treated with pemetrexed to determine the concentration that reduced growth to 50% (GI₅₀). FRα expression was determined by western blotting and that of FRα, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) by real-time quantitative RT–PCR. Immunohistochemistry for FRα was carried out on 62 paraffin-embedded samples of mesothelioma from patients who were subsequently treated with pemetrexed.

Results:

A wide range of GI₅₀ values was obtained for the cell lines, H2452 cells being the most sensitive (GI₅₀ 22 nM) and RS5 cells having a GI₅₀ value greater than 10 μM. No FRα protein was detected in any cell line, and there was no relationship between sensitivity and expression of folate transporters. FRα was detected in 39% of tumour samples, generally in a small percentage of cells. There was no correlation between the presence of FRα and the outcome of pemetrexed treatment, and no significant difference between histological subtypes.

Conclusion:

Response to treatment with pemetrexed does not depend on the presence of FRα.     

GSK-3β regulates tumor growth and angiogenesis in human glioma cells

Background

Glioma accounts for the majority of primary malignant brain tumors in adults.

Methods

Glioma specimens and normal brain tissues were analyzed for the expression levels of GSK-3β and p-GSK-3β (Ser9) by tissue microarray analysis (TMA) and Western blotting. Glioma cells over-expressing GSK-3β were used to analyze biological functions both in vitro and in vivo.

Results

The levels of p-GSK-3β (Ser9), but not total GSK-3β, are significantly up-regulated in glioma tissues compared to normal tissues, and are significantly correlated with the glioma grades. Ectopic expression of GSK-3β decreased the phosphorylation levels of mTOR and p70S6K1; and inhibited β-catenin, HIF-1α and VEGF expression. Forced expression of GSK-3β in glioma cells significantly inhibited both tumor growth and angiogenesis in vivo.

Conclusions

These results reveal that GSK-3β regulates mTOR/p70S6K1 signaling pathway and inhibits glioma progression in vivo; its inactivation via p-GSK-3β (Ser9) is associated with glioma development, which is new mechanism that may be helpful in developing GSK-3β-based treatment of glioma in the future.     

Central nervous system penetration and enhancement of temozolomide activity in childhood medulloblastoma models by poly(ADP-ribose) polymerase inhibitor AG-014699

Background:

Temozolomide shows activity against medulloblastoma, the most common malignant paediatric brain tumour. Poly(ADP-ribose) polymerase (PARP) inhibitors enhance temozolomide activity in extracranial adult and paediatric human malignancies.

Methods:

We assessed the effect of AG-014699, a clinically active PARP inhibitor, on temozolomide-induced growth inhibition in human medulloblastoma models. Pharmacokinetic, pharmacodynamic and toxicity assays were performed in tumour-bearing mice.

Results:

Sensitivity to temozolomide in vitro was consistent with methylguanine methyltransferase (MGMT) and DNA mismatch repair (MMR) status; MGMT⁺ MMR⁺ D384Med cells (temozolomide GI₅₀=220 μ), representative of most primary medulloblastomas, were sensitised fourfold by AG-014699; MGMT⁻ MMR⁺ D425Med cells were hypersensitive (GI₅₀=9 μ) and not sensitised by AG-014699, whereas MGMT⁺ MMR⁻ temozolomide-resistant D283Med cells (GI₅₀=807 μ) were sensitised 20-fold. In xenograft models, co-administration of AG-014699 produced an increase in temozolomide-induced tumour growth delay in D384Med xenografts. Consistent with the in vitro data, temozolomide caused complete tumour regressions of D425Med xenografts, whereas D283Med xenografts were relatively resistant. AG-014699 was not toxic, accumulated and reduced PARP activity ⩾75% in xenograft and brain tissues.

Conclusion:

We show for the first time central nervous system penetration and inhibition of brain PARP activity by AG-014699. Taken together with our in vitro chemosensitisation and toxicity data, these findings support further evaluation of the clinical potential of AG-014699–temozolomide combinations in intra-cranial malignancies.     

Intravenous injection of oncolytic picornavirus SVV-001 prolongs animal survival in a panel of primary tumor–based orthotopic xenograft mouse models of pediatric glioma

Background

Seneca Valley virus (SVV-001) is a nonpathogenic oncolytic virus that can be systemically administered and can pass through the blood–brain barrier. We examined its therapeutic efficacy and the mechanism of tumor cell infection in pediatric malignant gliomas.

Methods

In vitro antitumor activities were examined in primary cultures, preformed neurospheres, and self-renewing glioma cells derived from 6 patient tumor orthotopic xenograft mouse models (1 anaplastic astrocytoma and 5 GBM). In vivo therapeutic efficacy was examined by systemic treatment of preformed xenografts in 3 permissive and 2 resistant models. The functional role of sialic acid in mediating SVV-001 infection was investigated using neuraminidase and lectins that cleave or competitively bind to linkage-specific sialic acids.

Results

SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, preformed neurospheres, and self-renewing stemlike single glioma cells derived from 4 of the 6 glioma models in vitro. A single i.v. injection of SVV-001 (5 × 10¹² viral particles/kg) led to the infection of orthotopic xenografts without harming normal mouse brain cells, resulting in significantly prolonged survival in all 3 permissive and 1 resistant mouse models (P < .05). Treatment with neuraminidase and competitive binding using lectins specific for α2,3-linked and/or α2,6-linked sialic acid significantly suppressed SVV-001 infectivity (P < .01).

Conclusion

SVV-001 possesses strong antitumor activity against pediatric malignant gliomas and utilizes α2,3-linked and α2,6-linked sialic acids as mediators of tumor cell infection. Our findings support the consideration of SVV-001 for clinical trials in children with malignant glioma.     

Clinicopathological significance of indoleamine 2,3-dioxygenase 1 expression in colorectal cancer

Background:

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolising enzyme that induces immune tolerance by modulating T-cell responses. Carcinomas may create an immunosuppressive state via IDO1 expression. Here we examined a possible contribution of IDO1 on this phenomenon and investigated whether IDO1 has prognostic value in colorectal cancer (CRC).

Methods:

IDO1 expression was investigated by quantitative PCR and western blotting in three colon cancer cell lines, in basal state and after interferon (IFN)-γ stimulation. Semi-quantitative immunohistochemistry was used to evaluate IDO1 expression in 265 pT1-4N0-2Mx-staged CRCs. Results were related to clinical variables and correlated with amounts of CD3⁺ and CD8⁺ T lymphocytes, which were quantitatively evaluated using image analysis.

Results:

In vitro expression of IDO1 depended on IFN-γ stimulation. Higher IDO1 expression at the tumour invasion front was an independent adverse prognostic factor in pT1-4N1Mx-staged CRC. It was associated with overall survival (P=0.001) and with metachronous metastases (P=0.018). IDO1 expression was not associated with the presence of CD3⁺ or CD8⁺ T lymphocytes.

Conclusion:

Higher IDO1 expression at the tumour invasion front is involved in CRC progression and correlates with impaired clinical outcome, suggesting that IDO1 is an independent prognostic indicator for CRC.     

Gene set enrichment analysis provides insight into novel signalling pathways in breast cancer stem cells

Background:

Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood.

Methods:

Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA).

Results:

We showed that several breast cancer cell lines have a small population of CD24^−/low/CD44⁺ cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24^−/low/CD44⁺ cell populations are enriched for genes involved in transforming growth factor-β, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-κB (NF-κB) activity in CD24^−/low/CD44⁺ cells, which was previously unrecognised. In addition, NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24^−/low/CD44⁺ cells in vivo.

Conclusion:

Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells.     

Inhibin/activin expression in human and rodent liver: subunits α and βB as new players in human hepatocellular carcinoma?

Background:

Activins and inhibins belong to the TGFβ-superfamily, which controls cell proliferation and differentiation in many organs. Activin A, the dimer of inhibin βA subunit, acts strongly anti-proliferative in hepatocytes. Little is known on the other activin/inhibin subunits in human liver and hepatocellular carcinoma (HCC).

Methods:

We studied the expression of the complete inhibin family α, βA, βB, βC, βE in normal liver, tumour-adjacent and HCC tissue, 12 additional organs and rodent liver. A total of 16 HCC and 10 disease-free livers were analysed. Expression of inhibin subunits was determined by qRT–PCR, normalised to RNA input and by geNorm algorithm, and confirmed by immunohistochemistry.

Results:

Remarkably, βA expression was not decreased in HCC. Similarly, βC and βE exhibited no major changes. In contrast, inhibin α, barely detectable in normal liver, was strongly increased in tumour-adjacent liver and dramatically enhanced in HCC. βB was strongly enhanced in some HCC. At variance with human liver, rodent liver showed higher inhibin α and βC expression, but βA was somewhat, and βB dramatically lower.

Conclusions:

Upregulation of inhibin α – and possibly of βB – may shield HCC cells from anti-proliferative effects of activin A. Dramatic variations between humans and rodents may reflect different functions of some inhibins/activins.     

Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours

Background:

The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A₂α (cPLA₂α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA₂α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines.

Methods:

The importance of differential cPLA₂α activity in clinical breast cancer was established by relating the expression of cPLA₂α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters.

Results:

High cPLA₂α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA₂α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA₂α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up.

Conclusion:

This study shows a role of cPLA₂α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.     

A novel fragment derived from the �� chain of human fibrinogen, ��43�C63, is a potent inhibitor of activated endothelial cells in vitro and in vivo

Background:

Angiogenesis and haemostasis are closely linked within tumours with many haemostatic proteins regulating tumour angiogenesis. Indeed we previously identified a fragment of human fibrinogen, fibrinogen E-fragment (FgnE) with potent anti-angiogenic properties in vitro and cytotoxic effects on tumour vessels in vivo. We therefore investigated which region of FgnE was mediating vessel cytotoxicity.

Methods:

Human dermal microvascular endothelial cells (ECs) were used to test the efficacy of peptides derived from FgnE on proliferation, migration, differentiation, apoptosis and adhesion before testing the efficacy of an active peptide on tumour vasculature in vivo.

Results:

We identified a 20-amino-acid peptide derived from the β chain of FgnE, β43–63, which had no effect on EC proliferation or migration but markedly inhibited the ability of activated ECs to form tubules or to adhere to various constituents of the extracellular matrix – collagen IV, fibronectin and vitronectin. Furthermore, our data show that β43–63 interacts with ECs, in part, by binding to α_vβ₃, so soluble α_vβ₃ abrogated β43–63 inhibition of tubule formation by activated ECs. Finally, when injected into mice bearing tumour xenografts, β43–63 inhibited tumour vascularisation and induced formation of significant tumour necrosis.

Conclusions:

Taken together, these data suggest that β43–63 is a novel anti-tumour peptide whose anti-angiogenic effects are mediated by α_vβ₃.     

The role of zinc in the anti-tumour and anti-cachectic activity of D-myo-inositol 1,2,6-triphosphate

Background:

D-myo-inositol-1,2,6-triphosphate (α-trinositol, AT) is a polyanionic molecule capable of chelating divalent metal ions with anti-tumour and anti-cachectic activity in a murine model.

Methods:

To investigate the role of zinc in this process, mice bearing cachexia-inducing MAC16 tumour were treated with AT, with or without concomitant administration of ZnSO₄.

Results:

At a dose of 40 mg kg⁻¹, AT effectively attenuated both weight loss and growth of the MAC16 tumour, and both effects were attenuated by co-administration of Zn²⁺. The concentration of zinc in gastrocnemius muscle increased with increasing weight loss, whereas administration of AT decreased the levels of zinc in plasma, skeletal muscle and tumour, which were restored back to control values after administration of ZnSO₄.

Conclusion:

These results suggest that zinc is important in both tumour growth and cachexia in this animal model.     

Factor inhibiting HIF (FIH-1) promotes renal cancer cell survival by protecting cells from HIF-1α-mediated apoptosis

Background:

Clear cell renal cell carcinoma (CCRCC) is the commonest form of kidney cancer. Up to 91% have biallelic inactivation of VHL, resulting in stabilisation of HIF-α subunits. Factor inhibiting HIF-1 is an enzyme that hydroxylates HIF-α subunits and prevents recruitment of the co-activator CBP/P300. An important question is whether FIH-1 controls HIF activity in CCRCC.

Methods:

Human VHL defective CCRCC lines RCC10, RCC4 and 786–O were used to determine the role of FIH-1 in modulating HIF activity, using small interfering RNA knockdown, retroviral gene expression, quantitative RT–PCR, western blot analysis, Annexin V and propidium iodide labelling.

Results:

Although it was previously suggested that FIH-1 is suppressed in CCRCC, we found that FIH-1 mRNA and protein are actually present at similar levels in CCRCC and normal kidney. The FIH-1 inhibition or knockdown in the VHL defective CCRCC lines RCC10 and RCC4 (which express both HIF-1α and HIF-2α) resulted in increased expression of HIF target genes. In the 786-O CCRCC cell line, which expresses only HIF-2α, FIH-1 attenuation showed no significant effect on expression of these genes; introduction of HIF-1α resulted in sensitivity of HIF targets to FIH-1 knockdown. In RCC4 and RCC10, knockdown of FIH-1 increased apoptosis. Suppressing HIF-1α expression in RCC10 prevented FIH-1 knockdown from increasing apoptosis.

Conclusion:

Our results support a unifying model in which HIF-1α has a tumour suppressor action in CCRCC, held in check by FIH-1. Inhibiting FIH-1 in CCRCC could be used to bias the HIF response towards HIF-1α and decrease tumour cell viability.     

Integrin α3 is overexpressed in glioma stem-like cells and promotes invasion

Background:

Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs.

Methods:

Neurospheres and CD133⁺ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines.

Results:

Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⁺ cells compared with CD133⁻ cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2.

Conclusion:

Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.     

Phase I study of TAS-102 treatment in Japanese patients with advanced solid tumours

Background:

TAS-102 consists of α, α, α-trifluorothymidine (TFT) and an inhibitor of thymidine phosphorylase (TPI). We conducted a dose-escalation phase I study in Japanese patients with advanced solid tumours.

Methods:

TAS-102 was administered twice daily on days 1–5 and days 8–12 in a 28-day cycle to patients with solid tumours refractory to standard chemotherapy, to determine its maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetics (PKs). MTD was evaluated in cycle 1.

Results:

Safety and PKs were evaluated in 21 patients treated with TAS-102 at 30, 40, 50, 60, or 70 mg m⁻² per day. DLTs, such as grade 4 leucopenia, grade 4 neutropenia, and grade 4 thrombocytopenia, were observed in two patients at doses of 30 and 70 mg m⁻². α, α, α-trifluorothymidine and TPI exposures increased dose dependently, and the percentage of decrease in neutrophil count and TFT exposure were significantly correlated. The disease control rate was 50.0% with a median progression-free survival of 2.4 months in 18 colorectal cancer patients. The dose of TAS-102 was not increased above 70 mg m⁻² per day because of the increased tendency for grade 3 and 4 neutropenia, and 70 mg m⁻² per day was the recommended dose for phase II studies.

Conclusions:

TAS-102 at 70 mg m⁻² per day was tolerated in Japanese patients with advanced solid tumours. Phase II studies are ongoing in patients with colorectal cancer.     

Postmenopausal circulating levels of 2- and 16α-hydroxyestrone and risk of endometrial cancer

Background:

It has been suggested that the relative importance of oestrogen-metabolising pathways may affect the risk of oestrogen-dependent tumours including endometrial cancer. One hypothesis is that the 2-hydroxy pathway is protective, whereas the 16α-hydroxy pathway is harmful.

Methods:

We conducted a case–control study nested within three prospective cohorts to assess whether the circulating 2-hydroxyestrone : 16α-hydroxyestrone (2-OHE1 : 16α-OHE1) ratio is inversely associated with endometrial cancer risk in postmenopausal women. A total of 179 cases and 336 controls, matching cases on cohort, age and date of blood donation, were included. Levels of 2-OHE1 and 16α-OHE1 were measured using a monoclonal antibody-based enzyme assay.

Results:

Endometrial cancer risk increased with increasing levels of both metabolites, with odds ratios in the top tertiles of 2.4 (95% CI=1.3, 4.6; P_trend=0.007) for 2-OHE1 and 1.9 (95% CI=1.1, 3.5; P_trend=0.03) for 16α-OHE1 in analyses adjusting for endometrial cancer risk factors. These associations were attenuated and no longer statistically significant after further adjustment for oestrone or oestradiol levels. No significant association was observed for the 2-OHE1 : 16α-OHE1 ratio.

Conclusion:

Our results do not support the hypothesis that greater metabolism of oestrogen via the 2-OH pathway, relative to the 16α-OH pathway, protects against endometrial cancer.     

Epoetin-�� treatment in patients with cancer chemotherapy-induced anaemia: the impact of initial haemoglobin and target haemoglobin levels on survival, tumour progression and thromboembolic events

Background:

Epoetin-β is used to treat patients with cancer undergoing chemotherapy to alleviate the symptoms of anaemia, reduce the risk of blood transfusions and improve quality of life (QoL).

Methods:

This meta-analysis of all 12 randomised, controlled studies of epoetin-β evaluated the impact of therapy at different Hb-initiation levels and to different target Hb levels on overall survival, tumour progression and thromboembolic events (TEE). An analysis of risk factors pre-disposing patients to TEEs under epoetin-β therapy was also performed. A total of 2297 patients are included in the analysis.

Results:

Analyses based on various Hb-initiation levels indicate no detrimental impact on survival (HR 0.99; 95% CI 0.70, 1.40) and a favourable impact on disease progression (HR 0.73; 95% CI 0.57, 0.94) when epoetin-β was used within its licensed indication (Hb initiation ⩽10 g dl⁻¹) or the EORTC recommended level of 11 g dl⁻¹. An increased risk of TEEs is seen for all Hb-initiation level strata and a detrimental impact on survival is seen when initiating epoetin-β therapy at Hb levels >11 g dl⁻¹. We observe no association between high target Hb levels (⩾13 g dl⁻¹) and an increased risk of mortality, disease progression or TEEs with epoetin-β compared with control.

Conclusion:

The results of this analysis indicate that epoetin-β therapy has no detrimental impact on survival or tumour progression when initiated at Hb levels up to 11 g dl⁻¹. Furthermore, there is no evidence to suggest that high Hb values achieved during epoetin-β therapy are associated with an increased mortality, disease progression or TEE rate.