Phase I study of sorafenib and tipifarnib for recurrent glioblastoma: NABTC 05-02.

Recurrent glioblastoma (GBM) has a very low 6-month progression free survival (PFS) with currently available treatments. Combination chemotherapy to target multiple cell signaling pathways is currently being investigated in order to improve prognosis for recurrent disease. The purpose of this phase I study was to determine the maximum tolerated dose (MTD) for the combination of tipifarnib and sorafenib for the treatment of recurrent GBM. Patients with pathologically proven WHO grade IV GBM and radiographically proven tumor recurrence were eligible for this study. Treatments included sorafenib at twice daily and escalating dosages of tipifarnib. Dose-limiting toxicity (DLT) was determined over the first 28-days of treatments, and the MTD was determined in a 3 + 3 study design. We enrolled 24 patients, and 21 patients completed the MTD period. The study was stopped early with no MTD determination for excessive toxicities. The last dose level reached was sorafenib at 200 mg twice a day and tipifarnib 100 mg twice a day on an alternating week schedule. The DLTs included diarrhea, lipase elevation, hypophosphatemia, and arthralgia. The combination of sorafenib and tipifarnib has excessive toxicities and full single agent dosages could not be achieved in combination.

Effect of angiotensin system inhibitors on survival in newly diagnosed glioma patients and recurrent glioblastoma patients receiving chemotherapy and/or bevacizumab.

Given prior studies that suggest the use of angiotensin system inhibitors (ASIs) is associated with prolonged overall survival (OS) in glioblastoma (GBM) patients, we evaluated the effect of ASIs in glioma patients receiving chemotherapy and/or bevacizumab (BEV). Using retrospective IRB-approved electronic chart review of newly diagnosed WHO grade 2-4 glioma patients from the Kaiser Permanente Tumor Registry of Northern California, we evaluated the impact of ASIs on OS by Cox proportional hazard model analysis for subgroups who received cytotoxic therapy, cytotoxic therapy with BEV, or BEV alone, as well as those with recurrent GBM (rGBM). Of the 1186 glioma patients who received chemotherapy ASI exposure improved OS (HR 0.82; 95% CI 0.71, 0.93; p = 0.003). When stratified by BEV exposure, a sub-analysis revealed further OS advantage for the BEV group (HR 0.75, 95% CI 0.62, 0.90; p = 0.002). In a second cohort of 181 rGBM patients who received BEV in varying dosages, ASI exposure conferred an OS advantage (HR 0.649; 95% CI 0.46, 0.92; p = 0.016). Moreover, patients with ASI exposure who received low-dose BEV treatment (AUCBEV < 3.6 mg wk/kg) had a significantly longer OS (median = 99 weeks; 95% CI 44.3, 205) than those without ASI (median OS = 55.6 weeks; 95% CI 37.7-73.7; p = 0.032). ASI use is associated with longer OS in glioma patients. Further survival advantage with ASI use was observed in rGBM patients receiving low-dose bevacizumab. These data warrant prospective evaluation of adding ASI to low-dose BEV treatment in GBM patients to improve the outcome of standard therapies.

Impact of bevacizumab administered dose on overall survival of patients with progressive glioblastoma.

Bevacizumab (BEV, Avastin(®)) produces durable objective radiological responses of 20-26 %, median response durations of 16-18 weeks, and median overall survival (mOS) of 31-40 weeks. While the use of BEV is well-established, the lack of dose-response studies in glioblastoma (GBM) patients raises the question whether current dosing practice is optimal. As a result of differing approaches to BEV dosing that ranged from the FDA approved package insert dose of 10 mg/kg every 2 weeks to 7.5 mg/kg every 3-4 weeks, among physicians within Northern California Kaiser Permanente hospitals over 4+ years, we did an IRB-approved retrospective analysis of patients seen in Northern California Kaiser Permanente facilities and treated with BEV. Between September 1, 2008 and August 31, 2013, 181 patients received BEV for tumor progression/recurrence starting 2.6 weeks after completion of chemoradiation. The integrated BEV administered dose-week (AUCBEV) for all patients had a median AUCBEV of 3.6 mg·wk/kg). Maximum likelihood analysis found patients over 65 years did worse than younger patients (p = 0.004), women lived longer (p = 0.002), and patients treated below the AUCBEV did better than those treated above the median AUCBEV (p = 0.003). mOS for BEV starting 1 month after chemoradiation was 45 versus 68 weeks (p = 0.012) and BEV starting 3 months after chemoradiation was 40 versus 74 weeks (p = 0.0085). Dosing BEV at half the standard dose for progressive/recurrent GBM was at least equivalent to or, maybe better than standard dosing. Unexplained was the observation that females had longer OS with BEV than males.

Phase 2 trial of irinotecan and thalidomide in adults with recurrent anaplastic glioma.

BACKGROUND: Therapeutic options for patients with anaplastic gliomas (AGs) are limited despite better insights into glioma biology. The authors previously reported improved outcome in patients with recurrent glioblastoma treated with thalidomide and irinotecan compared with historical controls. Here, results of the AG arm of the study are reported, using this drug combination. METHODS: Adults with recurrent AG previously treated with radiation therapy, with Karnofsky performance score ≥70, adequate organ function and not on enzyme-inducing anticonvulsants were enrolled. Treatment was in 6-week cycles with irinotecan at 125 mg/m(2) weekly for 4 weeks followed by 2 weeks off, and thalidomide at 100 mg daily increased to 400 mg/day as tolerated. The primary endpoint was progression-free survival rate at 6 months (PFS-6), and the secondary endpoints were overall survival (OS) and response rate (RR). RESULTS: In 39 eligible patients, PFS-6 for the intent-to-treat population was 36% (95% confidence interval [CI] = 21%, 53%), median PFS was 13 weeks (95% CI = 6%, 28%) and RR was 10%(95% CI = 3%, 24%). Radiological findings included 2 complete and 2 partial responses and 17 stable disease. Median OS from study registration was 62 weeks, (95% CI = 51, 144). Treatment-related toxicities (grade 3 or higher) included neutropenia, diarrhea, nausea, and fatigue; 6 patients experienced venous thromboembolism. Four deaths were attributable to treatment-related toxicities: 1 from pulmonary embolism, 2 from colitis, and 1 from urosepsis. CONCLUSIONS: The combination of thalidomide and irinotecan did not achieve sufficient efficacy to warrant further investigation against AG, although a subset of patients experienced prolonged PFS/OS. A trial of the more potent thalidomide analogue, lenalidomide, in combination with irinotecan against AG is currently ongoing.

Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures.

BACKGROUND: Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions. RESULTS: Using reverse-phase protein arrays, we compared levels of 121 different phosphorylated and non-phosphorylated proteins in 5 glioma and 6 adenocarcinoma lines under conditions of 3D and monolayer culture in normoxia and hypoxia. A three-way analysis of variance showed levels of 82 antibodies differed between media (2D vs. 3D) and 49 differed between treatments (hypoxia vs. normoxia). Comparing 2D to 3D growth, 7 proteins were commonly (i.e., > 50% of tumors) elevated in 3D: FAK, AKT, Src, GSK3αß, TSC2, p38, and NFκßp65. Conversely, 7 other proteins are commonly decreased: ATRIP, ATR, ß-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1α. Comparing normoxia to hypoxia, only NCKIPSD was commonly elevated in hypoxia; 6 proteins were decreased: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240). Hypoxia affected glioma cell lines differently from adenocarcinoma cell lines: 8 proteins were increased in gliomas (BAX, caspase 7, HIF-1α, c-JUN, MEK1, PARP 1 cleaved, Src, and VEGFR2) and none in adenocarcinomas. CONCLUSIONS: We identified subsets of proteins with clearly concordant/discordant behavior between gliomas and adenocarcinomas. In general, monolayer to 3D culture differences are clearer than normoxia to hypoxia differences, with anti-apoptotic, cytoskeletal rearrangement and cell survival pathways emphasized in the former and mTOR pathway, transcription, cell-cycle arrest modulation, and increased cell motility in the latter.

Radiographic Response Assessment Strategies for Early-Phase Brain Trials in Complex Tumor Types and Drug Combinations: from Digital "Flipbooks" to Control Systems Theory.

There is an urgent need for drug development in brain tumors. While current radiographic response assessment provides instructions for identifying large treatment effects in simple high- and low-grade gliomas, there remains a void of strategies to evaluate complex or difficult to measure tumors or tumors of mixed grade with enhancing and non-enhancing components. Furthermore, most patients exhibit some period of alteration in tumor growth after starting a new therapy, but simple response categorization (e.g., stable disease, progressive disease) fails to provide any meaningful insight into the depth or degree of potential "subclinical" therapeutic response. We propose a creative solution to these issues based on a tiered strategy meant to increase confidence in identifying therapeutic effects even in the most challenging tumor types, while also providing a framework for complex evaluation of combination and sequential treatment schemes. Specifically, we demonstrate the utility of digital "flipbooks" to quickly identify subtle changes in complex tumors. We show how a modified Levin criteria can be used to quantify the degree of visual changes, while establishing estimates of the association between tumor volume and visual inspection. Lastly, we introduce the concept of quantifying therapeutic response using control systems theory. We propose measuring changes in volume (proportional), the area under the volume vs. time curve (integral) and changes in growth rates (derivative) to utilize a "PID" controller model of single or combination therapeutic activity.

Modulation of glioma risk and progression by dietary nutrients and antiinflammatory agents.

Gliomas are tumors of glial origin formed in the central nervous system and exhibit profound morphological and genetic heterogeneity. The etiology of this heterogeneity involves an interaction between genetic alterations and environmental risk factors. Scientific evidence suggests that certain natural dietary components, such as phytoestrogens, flavonoids, polyunsaturated fatty acids, and vitamins, may exert a protective effect against gliomas by changing the nature of the interaction between genetics and environment. Similarly, certain antiinflammatory drugs and dietary modifications, such as methionine restriction and the adoption of low-calorie or ketogenic diets, may take advantage of glioma and normal glial cells' differential requirements for glucose, methionine, and ketone bodies and may, therefore, be effective as part of preventive or treatment strategies for gliomas. Treatment trials of glioma patients and chemoprevention trials of individuals with a known genetic predisposition to glioma using the most promising of these agents, such as the antiinflammatory drugs curcumin and gamma-linolenic acid, are needed to validate or refute these agents' putative role in gliomas.

Combination of 6-thioguanine, capecitabine, and celecoxib with temozolomide or lomustine for recurrent high-grade glioma.

We evaluated the efficacy of temozolomide (TMZ) or lomustine (CCNU) in combination with 6-thioguanine, capecitabine, and celecoxib for the treatment of recurrent high-grade glioma. Forty-three patients with recurrent glioblastoma and 31 patients with recurrent anaplastic glioma (AG) were enrolled in this open-label, non-comparative study. Patients previously treated with TMZ received CCNU while all others received TMZ; all patients received 6-thioguanine, capecitabine, and celecoxib. Endpoints were 12-month progression-free survival (PFS) for patients with AG, 6-month PFS for patients with glioblastoma, duration of PFS, and MRI-based objective response rates. Results from the TMZ and CCNU treatment arms were combined in the final analysis because there was no statistically significant difference between them. Thirty-eight patients with glioblastoma were treated with the lomustine-based regimen, and five received the TMZ-based regimen. For the 43 glioblastoma patients, the objective response rate was 12 and 33% had stable disease; the 6-month PFS was 14% and median overall survival 32 weeks. For the 31 AG patients, the combined objective response rate was 26 and 42% had stable disease; the 12 month PFS was 44%. Treatment was reasonably well tolerated with hematological toxicity common and more frequent with CCNU than TMZ. The combination therapy with 6-thioguanine, capecitabine and celecoxib plus CCNU or TMZ does not appear to be more effective than other alkylating agent schedules for patients with recurrent glioblastoma. The combination, however, is promising for patients with recurrent high-grade AG.

Different changes in protein and phosphoprotein levels result from serum starvation of high-grade glioma and adenocarcinoma cell lines.

Tumor cells undergoing serum starvation in vitro partially mimic metabolically stressed cells trying to adjust to a changed environment in vivo by inducing signal transduction and gene expression so that the tumor continues to grow. Our hypothesis is that the changes in protein and phosphoprotein levels after serum starvation may reflect the adapted phenotype of the tumor, which could be targeted for therapy. We used reverse-phase protein microarrays to interrogate five high-grade glioma cell lines and seven adenocarcinoma cell lines for differences in the level of 81 proteins and 25 phosphoproteins. All cell lines were studied in the well-fed condition of growth with 10% FBS and the starved condition of 0.5% FBS. Protein expression levels were normalized to beta-actin and trichotomized as increased (+1, upper 75th quartile), decreased (-1, lowest 25th quartile), or unchanged (0, others) to focus on the patterns of the biggest (and hopefully most robust) changes in protein and phosphoprotein levels. We examined these trichotomized values to better understand Starved-Fed differences among the cell lines and thereby gain better/clearer insight into the effects of serum starvation on potential cellular responses. In general, the expression of proteins and phosphoproteins 24 h after FBS starvation increased more often in glioma lines than in adenocarcinoma lines, which appeared to have fewer increased protein scores and more decreased scores. Many of the proteins increased in gliomas were downstream targets of the PTEN-PI-3 kinase-AKT, EGFR-MAPK-Stat, and transcription activator-polyamine signaling pathways. In adenocarcinomas, the expression of proteins and phosphoproteins generally increased in apoptosis pathways, while there were minor fluctuations in the other pathways above. Contrawise, gliomas become resistant to apoptosis after 24 h of serum starvation and upregulate transcription activators and polyamines more so than adenocarciomas.

Effect of eflornithine on mutation frequency in temozolomide-treated U87MG cells.

Treatment of infiltrative glioma presents a number of unique challenges due to poor penetration of typical chemotherapeutic agents into the infiltrating edge of tumors. The current chemotherapy options include nitrosoureas (e.g., lomustine) and the imidazotetrazine-class monofunctional DNA alkylating agent, temozolomide (TMZ). Both classes of drugs alkylate DNA and have relatively unrestricted passage from blood into brain where infiltrative tumor cells reside. Recent research indicates that secondary mutations detected in the RB and AKT-mTOR signaling pathways are linked to characteristics of recurrent tumors specific to TMZ-treated patients. It has been hypothesized that a decrease in rate of secondary mutations may result in delay of tumor recurrence. To that end, this study was designed to test viability of decreasing secondary mutations by disrupting the cell division cycle using eflornithine, a specific inhibitor of ornithine decarboxylase. U87MG glioblastoma cell line characterized by chromosomal abnormalities commonly attributed to primary cancers was used as a model for this study. The cells were subjected to TMZ treatment for 3 days followed by eflornithine (DFMO) treatment for 4 or 11 days. It was shown that TMZ significantly increased the frequency of mutations in U87MG glioblastoma cells while DFMO-treated cells showed mutation frequency statistically similar to that of the untreated cells on the respective treatment days. The findings of this study provide evidence to support the hypothesis that DFMO may inhibit progression of DNA mutations caused by alkylating chemotherapy agents, such as TMZ.

Phase I/II study of sorafenib in combination with erlotinib for recurrent glioblastoma as part of a 3-arm sequential accrual clinical trial: NABTC 05-02.

BACKGROUND: Receptor tyrosine kinases such as epidermal growth factor receptors (EGFRs) and their downstream signaling pathways such as the Ras-Raf-mitogen-activated protein kinase (MAPK) pathway play important roles in glioblastoma (GBM). This study investigated the safety, pharmacokinetics, and efficacy of sorafenib (Ras/Raf/MAPK inhibitor) in combination with erlotinib (EGFR inhibitor) for treatment of recurrent GBMs. METHODS: Patients with recurrent GBM were eligible. A novel sequential accrual trial design was used, where patients were sequentially accrued into separate treatment arms in phase I and phase II investigations to optimize recruitment efficiency. In phase I, a standard 3 + 3 format was used to identify dose-limiting toxicities (DLTs), determine maximum tolerated dose (MTD), and investigate pharmacokinetics. Phase II followed a 2-stage design with the primary endpoint being 6-month progression-free survival (PFS6). RESULTS: Sixteen patients were recruited for phase I, and the MTD was determined to be sorafenib 200 mg twice daily and erlotinib 100 mg once daily. DLTs include Grade 3 hypertension, Grade 3 elevated liver transaminases, and Grade 4 elevated lipase. While erlotinib did not affect sorafenib levels, sorafenib reduced erlotinib levels. In phase II, 3 of 19 stage 1 participants were progression free at 6 months. This did not meet the predetermined efficacy endpoint, and the trial was terminated. CONCLUSION: This study identified the MTD and DLTs for sorafenib and erlotinib combination therapy for recurrent GBMs; however, efficacy data did not meet the primary endpoint. This study also demonstrates the feasibility of a novel sequential accrual clinical trial design that optimizes patient recruitment for multiarm studies, which is particularly effective for multicenter clinical trials.

Melding a New 3-Dimensional Agarose Colony Assay with the E(max) Model to Determine the Effects of Drug Combinations on Cancer Cells.

The goal of our study was two-fold: (i) develop a robust 3D colony assay methodology to interrogate drug combinations using GelCount and (ii) to develop 2-drug combinations that might be useful in the clinic for the treatment of high-grade gliomas. We used three glioma cell lines (U251MG, SNB19, and LNZ308) and two adenocarcinoma cell lines (MiaPaCa and SW480) grown as colonies in a two-tiered agarose cultures. We evaluated two-drug combinations of difluoromethylornithine (DFMO), carboplatin, vorinostat (SAHA), and docetaxel. To analyze for antitumor efficacy we used GelCount to measure the area under the curve for tumor colony volumes (microm(2) x OD) in each plate. The non-linear dose-response E(max) model and the interaction index based on the Loewe additivity are applied to calculate two-drug synergy, additive, and antagonistic interactions. For glioblastoma cell lines, (i) carboplatin followed by DFMO was synergistic or additive in 2/3 cell lines, (ii) carboplatin before SAHA was synergistic in 1 cell line, (iii) carboplatin before docetaxel was synergistic in 2/3 cell lines and partially additive in the third, (iv) SAHA before docetaxel was synergistic in 1/3 cell lines, (v) docetaxel before DFMO was additive or partially active in 3/3 cell lines, and (vi) DFMO plus SAHA was inactive regardless of order. In the MiaPaCA cell line, synergy occurred when DFMO followed carboplatin and, at short exposure times, when SAHA was combined with carboplatin (regardless of order). In the SW480 cell line synergy occurred only in short exposures for carboplatin followed by docetaxel; additive and mixed partial effects were also seen with DFMO plus carboplatin or docetaxel (regardless of order), carboplatin before DFMO, carboplatin before SAHA, and docetaxel before carboplatin. In conclusion, by applying the Gelcount automated counting and sizing of colonies and the use of E(max) and Loewe models to define drug interactions, we can reliably define drug combination efficacy as a function of log dose and duration of drug exposure.

Long-term anti-inflammatory and antihistamine medication use and adult glioma risk.

A personal history of asthma or allergy has been associated with a reduced risk for adult malignant gliomas. Recent reports on the use of nonsteroidal anti-inflammatory drugs (NSAID) and the presence of risk alleles in asthma susceptibility genes showed similar inverse associations. To further explore the relationship between immune mediators and gliomas, we examined the use of NSAID and antihistamines, history of asthma or allergy, and infection in 325 glioma cases and 600 frequency-matched controls from the metropolitan area of Houston, TX (2001-2006). The regular use of NSAID was associated with a 33% reduction in the risk for glioma, suggestive of possible antitumor activity. Surprisingly, regular long-term antihistamine use among those reporting a history of asthma or allergies was significantly associated with a 3.5-fold increase in the risk for glioma. Similar to previous reports, cases in our study were less likely to have reported asthma, allergy, or a history of a number of viral infections (chickenpox or shingles, oral herpes, and mononucleosis) than controls. We therefore speculate that the observed positive association with antihistamine use may reflect an alteration of protective immune factors in susceptible individuals. Our results lend additional support for an important but unknown link between malignant brain tumors and immune mediators.

Phase II trial of irinotecan and thalidomide in adults with recurrent glioblastoma multiforme.

This phase II study aimed at determining the efficacy and safety of irinotecan combined with thalidomide in adults with recurrent glioblastoma multiforme (GBM) not taking enzyme-inducing anticonvulsants (EIACs). Adult patients (> or =18 years) with recurrent GBM with up to three relapses following surgery and radiation therapy were eligible for this trial. The primary end point was rate of progression-free survival at 6 months (PFS-6); secondary end points were response rate, overall survival, and toxicity. Patients were treated in 6-week cycles with 125 mg/m(2) irinotecan weekly for 4 weeks followed by 2 weeks off treatment and 100 mg of thalidomide daily increased as tolerated to 400 mg/day. Of 32 evaluable patients, 8 (25%) were alive and progression free at 6 months. The median PFS was 13 weeks. One patient experienced a complete response, one a partial response, and 19 stable disease. Median overall survival time from entry into the study was 36 weeks, and the 1-year survival rate was 34%. Adverse events (grade 3 or 4) included diarrhea, abdominal cramps, lymphopenia, neutropenia, and fatigue. Two of the four deaths that occurred were possibly due to treatment-related toxicity. The combination of irinotecan, a cytotoxic agent, and thalidomide, an antiangiogenic agent, shows promising activity against recurrent GBM in patients not receiving EIACs and warrants further study. The results also provide support for similar strategies using combination therapies with newer targeted antiangiogenic agents to generate effective therapies against malignant gliomas.

A new preclinical 3-dimensional agarose colony formation assay.

The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount technology, a new cell colony counter for gels and soft agars. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (microm2xOD) in each plate to calculate IC50 values. Adenocarcinoma colonies were recognized by GelCount scanning at 3-4 days, while it took 6-7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5-6 days; glioma cells grew more slowly, with 100 colonies counted in 9-10 days. Reliable log dose versus AUC curves were observed for all drugs studied. In conclusion, the GelCount method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.

Understanding brain penetrance of anticancer drugs.

This paper explicates the impact of tumor capillary permeability for glioma World Health Organization (WHO) grades II to IV on brain-penetrant drug entry and distribution within the tumor and the brain adjacent to tumor (leading edge). In addition, we consider the distribution of non-brain penetrant drugs and how, in some cases, large-molecular-weight drugs might achieve good distribution into tumor and brain adjacent to tumor.

Clinical importance of eflornithine (α-difluoromethylornithine) for the treatment of malignant gliomas.

This review covers the literature between 1989 and 2007 on studies relevant to the neuro-oncology usage of eflornithine (α-difluoromethylornithine), an oral agent that irreversibly inhibits the enzyme ornithine decarboxylase. It covers the use of eflornithine, alone or in combination, to treat high-grade gliomas. In addition, we provide an update on overall survival from The University of Texas MD Anderson Cancer Center Community Clinical Oncology Program and Clinical Trials Data Office that demonstrates a meaningful benefit in overall survival for eflornithine as a single agent and in combination with nitrosourea-based therapies for anaplastic gliomas. We also provide a framework for understanding the basis and study design of the ongoing pivotal, registrational Phase III multicenter trial for recurrent/progressive anaplastic astrocytoma.

Relationship between ornithine decarboxylase levels in anaplastic gliomas and progression-free survival in patients treated with DFMO-PCV chemotherapy.

The purpose of this study was to assess the relationship between progression-free survival (PFS) in patients treated with DFMO + PCV (procarbazine, CCNU, vincristine) chemotherapy for malignant gliomas with tumor cell ornithine decarboxylase (ODC) activity. Formalin-fixed slides were obtained for study patients with anaplastic gliomas (AGs) and glioblastoma treated on protocol DM92-035. ODC levels were measured using an antibody to ODC coupled to Alexa 647 dye (Ab-ODC-Alexa 647). Ab-ODC-Alexa 647 intensity in transgenic murine hearts of differing ODC activity was used to calculate ODC activity in tumor cell nucleoplasm. In total, tumor specimens for 31 of 114 (27%) patients treated on the AG strata and 10 patients from the GBM strata were obtained. We found that tumor ODC level heterogeneity increased with increasing tumor malignancy. In a Cox proportional hazards model, PFS was found to be inversely related to median tumor ODC activity, with an unadjusted hazard ratio for median ODC group (>3.3 vs. 3.3 nmol/30 min/mug protein. Of AG tumors in which ODC activity was evaluated, 26% had ODC levels > 3.3 nmol/30 min/mug protein. This study shows that Ab-ODC-Alexa 647 fluorescence intensity can be used as a surrogate marker of ODC biochemical activity in AGs and can predict PFS to DFMO-based chemotherapy.