Oliver Bähr

MGMT Promoter Methylation Is a Strong Prognostic Biomarker for Benefit from Dose-Intensified Temozolomide Rechallenge in Progressive Glioblastoma: The DIRECTOR Trial.

Purpose: Rechallenge with temozolomide (TMZ) at first progression of glioblastoma after temozolomide chemoradiotherapy (TMZ/RT→TMZ) has been studied in retrospective and single-arm prospective studies, applying temozolomide continuously or using 7/14 or 21/28 days schedules. The DIRECTOR trial sought to show superiority of the 7/14 regimen. Experimental Design: Patients with glioblastoma at first progression after TMZ/RT→TMZ and at least two maintenance temozolomide cycles were randomized to Arm A [one week on (120 mg/m2 per day)/one week off] or Arm B [3 weeks on (80 mg/m2 per day)/one week off]. The primary endpoint was median time-to-treatment failure (TTF) defined as progression, premature temozolomide discontinuation for toxicity, or death from any cause. O6-methylguanine DNA methyltransferase (MGMT) promoter methylation was prospectively assessed by methylation-specific PCR. Results: Because of withdrawal of support, the trial was prematurely closed to accrual after 105 patients. There was a similar outcome in both arms for median TTF [A: 1.8 months; 95% confidence intervals (CI), 1.8–3.2 vs. B: 2.0 months; 95% CI, 1.8–3.5] and overall survival [A: 9.8 months (95% CI, 6.7–13.0) vs. B: 10.6 months (95% CI, 8.1–11.6)]. Median TTF in patients with MGMT-methylated tumors was 3.2 months (95% CI, 1.8–7.4) versus 1.8 months (95% CI, 1.8–2) in MGMT-unmethylated glioblastoma. Progression-free survival rates at 6 months (PFS-6) were 39.7% with versus 6.9% without MGMT promoter methylation. Conclusions: Temozolomide rechallenge is a treatment option for MGMT promoter-methylated recurrent glioblastoma. Alternative strategies need to be considered for patients with progressive glioblastoma without MGMT promoter methylation. Clin Cancer Res; 21(9); 2057–64. ©2015 AACR.

Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy

BackgroundEven in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells.MethodsTo investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model.ResultsThe ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival.ConclusionIn summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

ERGO: A pilot study of ketogenic diet in recurrent glioblastoma

Limiting dietary carbohydrates inhibits glioma growth in preclinical models. Therefore, the ERGO trial (NCT00575146) examined feasibility of a ketogenic diet in 20 patients with recurrent glioblastoma. Patients were put on a low-carbohydrate, ketogenic diet containing plant oils. Feasibility was the primary endpoint, secondary endpoints included the percentage of patients reaching urinary ketosis, progression-free survival (PFS) and overall survival. The effects of a ketogenic diet alone or in combination with bevacizumab was also explored in an orthotopic U87MG glioblastoma model in nude mice. Three patients (15%) discontinued the diet for poor tolerability. No serious adverse events attributed to the diet were observed. Urine ketosis was achieved at least once in 12 of 13 (92%) evaluable patients. One patient achieved a minor response and two patients had stable disease after 6 weeks. Median PFS of all patients was 5 (range, 3–13) weeks, median survival from enrollment was 32 weeks. The trial allowed to continue the diet beyond progression. Six of 7 (86%) patients treated with bevacizumab and diet experienced an objective response, and median PFS on bevacizumab was 20.1 (range, 12–124) weeks, for a PFS at 6 months of 43%. In the mouse glioma model, ketogenic diet alone had no effect on median survival, but increased that of bevacizumab-treated mice from 52 to 58 days (p<0.05). In conclusion, a ketogenic diet is feasible and safe but probably has no significant clinical activity when used as single agent in recurrent glioma. Further clinical trials are necessary to clarify whether calorie restriction or the combination with other therapeutic modalities, such as radiotherapy or anti-angiogenic treatments, could enhance the efficacy of the ketogenic diet.

Chemoradiotherapy of newly diagnosed glioblastoma with intensified temozolomide

PURPOSE To evaluate the toxicity and efficacy of chemoradiotherapy with temozolomide (TMZ) administered in an intensified 1-week on/1-week off schedule plus indomethacin in patients with newly diagnosed glioblastoma. PATIENTS AND METHODS A total of 41 adult patients (median Karnofsky performance status, 90%; median age, 56 years) were treated with preirradiation TMZ at 150 mg/m(2) (1 week on/1 week off), involved-field radiotherapy combined with concomitant low-dose TMZ (50 mg/m(2)), maintenance TMZ starting at 150 mg/m(2) using a 1-week on/1-week off schedule, plus maintenance indomethacin (25 mg twice daily). RESULTS The median follow-up interval was 21.7 months. Grade 4 hematologic toxicity was observed in 15 patients (36.6%). Treatment-related nonhematologic Grade 4-5 toxicity was reported for 2 patients (4.9%). The median progression-free survival was 7.6 months (95% confidence interval, 6.2-10.4). The 1-year survival rate was 73.2% (95% confidence interval, 56.8-84.2%). The presence of O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation in the tumor tissue was associated with significantly superior progression-free survival. CONCLUSION The dose-dense regimen of TMZ administered in a 1-week on/1-week off schedule resulted in acceptable nonhematologic toxicity. Compared with data from the European Organization for Research and Treatment of Cancer/National Cancer Institute of Canada trial 26981-22981/CE.3, patients with an unmethylated MGMT gene promoter appeared not to benefit from intensifying the TMZ schedule regarding the median progression-free survival and overall survival. In contrast, data are promising for patients with a methylated MGMT promoter.

Bevacizumab treatment induces metabolic adaptation toward anaerobic metabolism in glioblastomas

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing 13C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of 13C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, l-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.

P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cells.

Understanding and overcoming multidrug resistance (MDR) may be a promising strategy to develop more effective pharmacotherapies for malignant gliomas. In the present study, human malignant glioma cell lines (n = 12) exhibited heterogeneous mRNAand protein expression and functional activity of the mdr gene‐encoded P‐glycoprotein (PGP) and MDR‐associated protein (MRP). Correlation between mRNA expression, protein levels and functional activity was strong. Inhibition of PGP activity by verapamil or PSC 833 enhanced the cytotoxic effects of vincristine, doxorubicin, teniposide and taxol. Inhibition of MRP activity by indomethacin or probenecid enhanced the cytotoxic effects of vincristine, doxorubicin and teniposide. The human cerebral endothelial cell line, SV‐HCEC, exhibited the strongest PGP activity of all cell lines. Five primary human glioblastomas and one anaplastic astrocytoma displayed heterogenous protein levels of PGP and MRP‐1 in tumor cells and of PGP in biopsy specimens in vivo, but no functional activity of these proteins upon ex vivo culturing. These data suggest that the glioma cell line‐associated MDR‐type drug resistance is a result of long‐term culturing and that cerebral endothelial, but not glioma cells, may contribute to MDR‐type drug resistance of gliomas in vivo.

Endothelial cell‐derived angiopoietin‐2 is a therapeutic target in treatment‐naive and bevacizumab‐resistant glioblastoma

Glioblastoma multiforme (GBM) is treated by surgical resection followed by radiochemotherapy. Bevacizumab is commonly deployed for anti‐angiogenic therapy of recurrent GBM; however, innate immune cells have been identified as instigators of resistance to bevacizumab treatment. We identified angiopoietin‐2 (Ang‐2) as a potential target in both naive and bevacizumab‐treated glioblastoma. Ang‐2 expression was absent in normal human brain endothelium, while the highest Ang‐2 levels were observed in bevacizumab‐treated GBM. In a murine GBM model, VEGF blockade resulted in endothelial upregulation of Ang‐2, whereas the combined inhibition of VEGF and Ang‐2 leads to extended survival, decreased vascular permeability, depletion of tumor‐associated macrophages, improved pericyte coverage, and increased numbers of intratumoral T lymphocytes. CD206+ (M2‐like) macrophages were identified as potential novel targets following anti‐angiogenic therapy. Our findings imply a novel role for endothelial cells in therapy resistance and identify endothelial cell/myeloid cell crosstalk mediated by Ang‐2 as a potential resistance mechanism. Therefore, combining VEGF blockade with inhibition of Ang‐2 may potentially overcome resistance to bevacizumab therapy.

NOA‐05 phase 2 trial of procarbazine and lomustine therapy in gliomatosis cerebri

The NOA‐05 multicenter trial was performed to analyze the efficacy of primary chemotherapy with procarbazine and lomustine (PC) in patients with gliomatosis cerebri (GC) and to define clinical, imaging, and molecular factors influencing outcome.

Modulation of MDR/MRP by wild-type and mutant p53

Loss of wild-type p53 activity and acquisition of a multidrug resistance (MDR) phenotype, two apparently key factors in the resistance of human cancers to chemotherapy, may be interrelated. Wild-type p53 represses expression of both the MDR-1 gene (encoding the prototypical MDR protein P-glycoprotein [PGP]) and MRP-1 (encoding the MDR-associated protein) (1, 2). Using a temperature-sensitive p53 mutant, Sullivan et al. (3) recently claimed in the JCI that the introduction of a mutant p53 variant promoted MRP expression and activity in a prostate cancer cell line. Our experience with transfection of p53 into other tumor cell lines casts their data in a different light. We transfected five previously characterized (4) human malignant glioma cell lines with the murine temperature-sensitive p53V135A mutant, which is dominant-negative over endogenous wild-type p53 at 38.5°C but assumes wild-type properties at 32.5°C, mediating the strong induction of p21 expression and causing G2/M arrest (5, 6). Control cell lines (hygro) were transfected with the same plasmid lacking a p53 cDNA insert. Changes in PGP or MRP protein levels were examined 48 hours after the cells were placed at the permissive temperature of 32.5°C (Table ​(Table1,1, upper part). At 32.5°C, expression of p53V135A caused a decline in PGP expression and activity in two cell lines (LN-18 and LN-308) but left these parameters unaffected in two cell lines (U87MG and LN-229). p53V135A caused an increase in PGP activity but no significant change in PGP expression in T98G cells. These changes mediated by p53V135A at wild-type conformation did not correlate with the endogenous p53 status of the cell lines. Under these same conditions, MRP expression and activity were increased by expression of p53V135A in three of the five cell lines (U87MG, T98G, and LN-229) and unaffected in two cell lines (LN-18 and LN-308) at 32.5°C. As with PGP, no correlation emerged between the p53V135A-mediated changes at 32.5°C and endogenous p53 status. Table 1 Modulation of PGP and MRP expression and activity by p53V135A at permissive (32.5°C) and restrictive (38.5°C) temperatures Since several studies have reported that mutant p53 may activate MDR gene expression (1, 7–9), probably by both dominant-negative and gain-of-function mechanisms, changes mediated by p53V135A in mutant conformation (38.5°C) were also carefully considered (Table ​(Table1,1, lower part). At 38.5°C, p53V135A enhanced PGP expression and activity in three of the five cell lines (U87MG, T98G, and LN-229), whereas the opposite pattern was observed in the other two cell lines (LN-18 and LN-308). Similarly, p53V135A had heterogeneous effects on MRP expression and activity. At 38.5°C, p53V135A reduced MRP expression and activity in LN-18 cells but increased MRP expression and activity in T98G and LN-229 cells. We also examined the modulation of glioma cell sensitivity to vincristine (VCR), a classic substrate of both PGP and MRP, by p53V135A at 32.5°C or 38.5°C (Table ​(Table2).2). At 32.5°C, p53V135A induced dramatic resistance to VCR in four of the five cell lines, although Table 2 Modulation of VCR cytotoxicity in the absence or presence of verapamil or indomethacin by p53V135A at permissive (32.5°C) and restrictive (38.5°C) temperatures LN-308 cells were sensitized to this drug. Further, we asked how p53V135A affected the ability of a MDR-1 inhibitor, verapamil (VPM), or a MRP inhibitor, indomethacin (INDO), to enhance VCR sensitivity. At 32.5°C, p53V135A uniformly decreased the sensitizing properties of VPM compared with isogenic control transfectants (hygro) under identical experimental conditions. In contrast, p53V135A had little effect on the sensitizing effect of INDO. Note that INDO has biologically relevant effects only in T98G cells, as predicted from the strong expression of MRP in these cells but not in the other cell lines (Table ​(Table1).1). At 38.5°C, p53V135A uniformly increased VCR sensitivity in the glioma cell lines, albeit moderately in all but LN-308 cells. Moreover, p53V135A enhanced the ability of VPM to increase VCR sensitivity in four of the five cell lines (the exception being T98G), whereas the effects of INDO on VCR sensitivity were largely unaffected at 38.5°C. All studies reported here were performed with pooled transfectants, precluding cloning artifacts as an explanation for unexpected data. Our data highlight several important issues that need to be considered when using temperature-sensitive p53 mutants to study cancer cell sensitivity to chemotherapy or irradiation. First, it is imperative to always examine isogenic, empty vector control cell lines under identical experimental conditions. Shifting the control cells from 38.5°C to 32.5°C alone significantly (a) reduced PGP expression and activity in LN-18 and LN-308 cells but increased PGP expression in U87MG cells, (b) reduced MRP expression in four of the five (LN-18, U87MG, T98G, and LN-308) and MRP activity in two of the five (LN-18, T98G) cell lines, and (c) increased VCR sensitivity in all cell lines. There is no information in the work of Sullivan et al. (3) indicating whether such controls were performed and whether temperature had any effect on MRP expression or activity in the cells examined in their study. Importantly, heat may transcriptionally activate MDR expression (10), but hyperthermia has also been suggested as one strategy to overcome MDR-type drug resistance (11), further supporting the need for careful controls when examining the modulation of drug resistance using temperature-sensitive p53 mutants. Second, in contrast to the proposed negative regulation of MDR transcription by wild-type p53 (7), we found that p53V135A at wild-type conformation (32.5°C) fails to reduce PGP expression and activity in three of the five cell lines. Moreover, in the two cell lines in which p53V135A reduced PGP expression and activity at 32.5°C, LN-18 and LN-308, the same effects were observed at 38.5°C, where p53V135A assumes mutant conformation and would be expected to enhance rather than reduce MDR expression and activity. Thus, these observations dissociate p53V135A-dependent modulation of MDR from the conformation of the sequence-specific DNA-binding domain of p53V135A in cell lines lacking p53 function. On the other hand, the effects of p53V135A in p53 wild-type cell lines, U87MG and LN-229, conform to current concepts of MDR modulation by p53 (1). This is because p53V135A had no effect on MDR expression at wild-type conformation (32.5°C) but enhanced MDR expression and activity at 38.5°C, acting as a dominant-negative mutant (5). Finally, although activation of rat MDR1b transcription by wild-type p53 has been described (12), the only cell line responding to p53V135A with moderately enhanced PGP activity at 32.5°C, T98G, behaved accordingly at 38.5°C, where p53V135A assumes mutant conformation. Third, in contrast to the proposed negative regulation of MRP gene transcription by wild-type p53 (2), p53V135A at wild-type conformation (32.5°C) not only failed to reduce MRP expression and activity in any of the cell lines examined but enhanced MRP expression and activity in three of the five cell lines. Again, p53V135A also promoted MRP expression and activity in two of these three cell lines at 38.5°C. Since no such effect was seen in U87MG cells at either temperature, and since p53V135A promoted MRP expression and activity in LN-229 cells at both temperatures (Table ​(Table1),1), the modulation of MRP by p53 is different from the modulation of MDR by p53 in glioma cells. Moreover, these data contrast sharply with the effects of temperature-sensitive p53 on MRP in a prostate carcinoma cell line (3). Thus, there is striking heterogeneity of the MDR/MRP responses to p53 alterations among cell lines of the same histogenetic origin, and studies of p53 and MDR/MRP interactions must not be extrapolated from one cell type to another. Fourth, previous studies have shown that two critical hydrophobic amino acids in the NH2-terminal domain of p53, as well as the oligomerization and non–sequence-specific DNA-binding domains, are required for the transcriptional activation of the MDR gene by tumor-derived p53 mutants (8, 9). These two amino acids and the mentioned domain of p53 are preserved on p53V135A. The uniform effects of p53V135A on T98G cells at both temperatures, for instance, could be attributed to such effects. On the other hand, most of the effects of p53V135A observed here could not have been predicted by the current understanding of MDR/MRP regulation by p53, suggesting that other genetic alterations, which differ among the glioma cell lines, determine how the MDR/MRP systems are modulated by p53V135A at either conformation. Fifth, the use of temperature-sensitive p53 mutants for the study of chemoresistance requires careful consideration of controls (6). Thus, we show here that although p53V135A in its mutant conformation at 38.5°C uniformly increased VCR sensitivity in the glioma cell lines, it had heterogenous, indeed opposing, effects on MDR/MRP expression and activity. Clearly, p53-dependent changes in cellular sensitivity to the classic MDR and MRP substrates are not due exclusively to the modulation of MDR or MRP expression.

Combined immune checkpoint blockade (anti-PD-1/anti-CTLA-4): Evaluation and management of adverse drug reactions

BACKGROUND Combined immune checkpoint blockade (ICB) provides unprecedented efficacy gains in numerous cancer indications, with PD-1 inhibitor nivolumab plus CTLA-4 inhibitor ipilimumab in advanced melanoma as first-ever approved therapies for combined ICB. However, gains in efficacy must be balanced against a higher frequency and severity of adverse drug reactions (ADR). Because delays in diagnosis and management might result in symptom worsening and further complications, clinicians shall be well trained to identify ADR promptly and monitor patients adequately. This paper reviews safety data assessed by the European Medicines Agency for the anti-PD-1/CTLA-4 combination and provides a literature overview on published case reports for rare ADR with suspected potential underreporting. Incidences and kinetics of immune-related ADR are described. Recommendations for the evaluation and management of ADR are convened by an interdisciplinary expert panel focusing on rare but clinically important side effects arising from combined ICB. Pooled safety data from 1551 patients with advanced melanoma, treated either with 3mg/kg ipilimumab plus 1mg/kg nivolumab (N=407), or nivolumab alone (N=787), or ipilimumab alone (N=357) demonstrate that immune-related ADR occur more frequently for the combination, with a shorter time-to-onset, and tend to be more severe. The majority of events is reversible after systemic use of glucocorticoids, notably methylprednisolone or equivalents; in certain cases of long-lasting and refractory immune toxicities, non-steroidal immunosuppressants may be used, once ICB is interrupted or terminated. Combined ICB has considerable toxicities, therefore close monitoring and high experience in diagnosis and treatment of ADR is necessary.