Dose Of Temozolomide Research Articles

The Evaluation of the Effects of Temozolomide on MGMT Gene Expression in MCF-7 and SKBR3 Human Breast Cancer Cell Lines

Background and Aim: In this study, it was aimed to examine the cytotoxic effect of temozolomide (TMZ) treatment, on MCF-7 and SKBR3 cell lines, to study the methylation levels of MGMT gene expression and gene promoter region. Methods: The MTT test was performed to determine the effective dose of TMZ. The time-dependent cell survival test was performed after the IC50 value was found. Western blotting was performed to determine MGMT gene expression levels. High Resolution Melting (HRM) technique was used to determine the methylation levels of MGMT gene promoter region. Results: TMZ has been shown to have a high cytotoxic effect on SKBR3 cell line and low cytotoxicity on MCF-7. When MGMT expression levels before and after TMZ treatment were observed by western blotting, the gene expression levels of TMZ treatment were shown to decrease in both cell lines. It was observed that MGMT gene promoter region was hypermethylated in two cell lines, and that the application of TMZ further increased the methylation levels in the promoter region. Conclusions: It was seen that TMZ could be used as a single agent in SKBR-3 cell line. With this study on breast cancer, it is expected that temozolomide treatment will lead future in vitro and in vivo studies for breast cancer.

Biodegradable wafers releasing Temozolomide and Carmustine for the treatment of brain cancer

Glioblastoma multiforme (GBM) has few clinically approved therapeutic regimens. One of these therapeutic options includes placing biodegradable wafers releasing BCNU (Gliadel®) into the tumor bed at the time of surgical removal of the tumor. Due to the significant benefit this polymer technology has had clinically, we have prepared wafers releasing Temozolomide (TMZ), an anticancer drug used systemically for treating GBM. TMZ delivered via polymer wafer could be used as a complementary treatment with or as an alternative to Gliadel®. TMZ is an alkylating agent which is water soluble. To remain comparable with the preclinical studies that led to Gliadel® the same size of wafers were formulated with TMZ. Wafers were loaded with 50% w/w TMZ in poly(lactic acid-glycolic acid) (PLGA) and showed reliable release of high dose TMZ for a period of 4 weeks. To achieve this 30-day release of the highly water soluble drug, we developed an encapsulation method, where the drug powder was first coated with the polymer to form core-shell particles in which the coating shell served as a rate controlling membrane for the drug particles. Wafers were also made with a co-loading of TMZ and BCNU. All wafers were tested in vivo by treating an intracranial 9 L gliosarcoma model in F344 rats. Rats that were either untreated or treated with blank wafer died within 11 days while the median survival for rats treated with systemic TMZ was 18 days. The group that received the BCNU alone wafer had a median survival of 15 days, the group that received the TMZ wafer alone had a median survival of 19 days, and the group treated with the BCNU-TMZ wafer had a median survival of 28 days with 25% of the animals living long term (p < .0038 vs. Control; p < .001 vs. Blank Polymer). These findings demonstrate the potential of this newly designed wafer for treating GBM. Moreover, this concept, can pave the way for other drug combinations that may improve the clinical application of numerous agents to treat solid tumors.

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O6-methylguanine in vivo.

N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to monitor O6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O6-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a non-linear formation of hepatic and colonic O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.

Re-irradiation of recurrent glioblastoma as part of a sequential multimodality treatment concept.

The aim of this retrospective study was to evaluate survival outcomes in well-performing, mainly, young patients receiving a sequence of all available therapeutic options for relapsed glioblastoma, including re-irradiation. We performed a retrospective analysis of 27 patients irradiated twice for glioblastoma between 2008 and 2016. In the first line, all had surgical treatment of the tumor followed by radiotherapy with a total dose of 60Gy and temozolomide. All re-irradiated patients were treated with a total dose of 36Gy in 12 fractions. The endpoints were death from glioblastoma or any cause, and toxicity after re-irradiation. The median follow-up of survivors was 35.6months. At the time of analysis, 25 patients had died. The median time between first and second radiotherapy was 18.9months (6.1-58.4). Re-irradiation was performed at different time points of first, second and third progression. The median overall survival after first diagnosis was 39.2months. Five years after first surgery, nearly 20% of the patients were alive. Carefully planned re-irradiation of the brain is a safe therapy for recurrent glioblastoma. Younger and well-performing patients benefit from all available therapy options. Every patient should be discussed in a multidisciplinary setting at each time point of tumor progression. Further prospective studies are needed to define the optimal time, dose and volume of re-irradiation.

Conventional Anti-glioblastoma Chemotherapy Affects Proteoglycan Composition of Brain Extracellular Matrix in Rat Experimental Model in vivo.

Temozolomide (TMZ) is a conventional chemotherapy drug for adjuvant treatment of glioblastoma multiforme (GBM), often accompanied by dexamethasone (DXM) to prevent brain oedema and alleviate clinical side effects. Here, we aimed to investigate an ability of the drugs to affect normal brain tissue in terms of proteoglycan (PG) composition/content in experimental rat model in vivo. Age- and brain zone-specific transcriptional patterns of PGs were demonstrated for 8, 60, and 120 days old rats, and syndecan-1, glypican-1, decorin, biglycan, and lumican were identified as the most expressed PGs. DXM treatment affected both PG core proteins expression (mainly syndecan-1, glypican-1, decorin, biglycan, lumican, versican, brevican, and NG2) and heparan sulphate (HS)/chondroitin sulphate (CS) content in organotypic brain slice culture ex vivo and experimental animals in vivo in a dose-dependent manner. TMZ treatment did not result in the significant changes in PG core proteins expression both in normal rat brain hippocampus and cortex in vivo (although generics did), but demonstrated significant effects onto polysaccharide HS/CS content in the brain tissue. The effects were age- and brain zone-specific and similar with the age-related PGs expression changes in rat brain. Combination of TMZ with DXM resulted in the most profound deterioration in PGs composition and content in the brain tissue both at core protein and glycosaminoglycan levels. Taken together, the obtained results demonstrate that conventional anti-glioblastoma therapy affects proteoglycan structure and composition in normal brain tissue, potentially resulting in deterioration of brain extracellular matrix and formation of the favourable tumorigenic niche for the expansion of the residual glioma cells. During the TMZ chemotherapy, dose and regimen of DXM treatment matter, and repetitive low DXM doses seem to be more sparing treatment compared with high DXM dose(s), which should be avoided where possible, especially in combination with TMZ.

Abstract B42: The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies

Abstract Cell-based immunotherapies have relied mainly on the use of αβ T cells or dendritic cells, and technologies to expand and engineer these cells have resulted in potent antitumor products. Although the use of adaptive immunocompetent cells has advanced more rapidly than innate immune cells, such as natural killer or γδ T cells, these alternative cellular products can provide equivalent antitumor activity. However, the methods to expand clinically relevant innate cell products are not as advanced as those for other immunocompetent cells, such as αβ T cells. We have developed a safe and efficient method for expanding, storing, and genetically engineering γδ T cells, and have focused on the use of these ex vivo expanded cells to treat pediatric neuroblastoma. γδ T cells are an attractive candidate for cell-based products because their antitumor activity is directly cytolytic to tumor cells, in part, due to their receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands such as MICA/B and UL-16 binding proteins (ULBP), and CD16 enables mechanisms of antibody-dependent cellular cytotoxicity (ADCC). We have developed a serum-free expansion protocol using apheresis products from healthy donors and from neuroblastoma patients. γδ T cells from all starting products can be expanded to greater than 70% of the culture within 12-14 days. These γδ T cells are cytotoxic against K562 cells, which has become our standard for which to compare the various expansion products, and 5 human derived neuroblastoma cell lines. We have tested several freezing media and developed a method using serum-free conditions between days 12-14 of expansion, resulting in equivalent cytotoxicity by cells pre- and post-freezing while maintaining greater than 70% viability post-thaw. We also show that our expansion protocol results in γδ T cells that are greater than 90% positive for surface expression of the receptors NKG2D and CD16. Therefore, we are using these cells in combination treatments that include standard chemotherapy regimens for relapsed neuroblastoma patients, i.e., temozolomide (TMZ), and the monoclonal antibody dinutuximab. We have shown that stress antigens such as MICA/B and ULBP2/5/6 are upregulated in vitro on IMR5 cells for 6 hours after a 1-hour exposure to TMZ. Further, combining patient-expanded γδ T cells that express CD16 with a GD2-specific antibody, dinutuximab, induces 30% increased neuroblastoma cell death compared to γδ T cells alone. To test the in vivo effectiveness of 12-day expanded, frozen, and thawed cells, we injected NSG male and female mice with 5 x 106 IMR5 cells subcutaneously. After a palpable 125-mm3 tumor was established, we began treatment with various combinations of γδ T cells, dinutuximab, and TMZ. These ongoing studies show that γδ T cells alone do not provide an antitumor benefit, but tumor regression is achieved when incorporating γδ T cells into dinutuximab and TMZ treatment regimens. This advantage is only observed at lower TMZ doses, whereas at higher TMZ doses, &amp;gt;60 mg/kg, chemotherapy dominates the antitumor effect. Overall, these studies show that a safer, serum-free expanded γδ T cell product can be produced, which can be stored frozen without affecting the cytotoxic properties. The expression of NKG2D and CD16 on the expanded cells allows for the development of combination therapies using cytotoxic chemotherapy agents and antibody-based treatments. The combination of γδ T cells, TMZ, and dinutuximab can provide a rational advancement for treating pediatric neuroblastoma patients, and we hypothesize that this combination therapy is preferred over high-dose chemotherapy regimens because it can reduce treatment-related toxicities and provide a multifaceted immunotherapeutic approach to neuroblastoma treatment. Citation Format: Jaquelyn T. Zoine, Kathryn S. Sutton, Kristopher A. Knight, Kelly C. Goldsmith, Christopher B. Doering, H. Trent Spencer. The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B42.

Intensity Modulated Radiation Therapy Versus Volumetric Arc Radiation Therapy in the Treatment of Glioblastoma—Does Clinical Benefit Follow Dosimetric Advantage?

PurposeVolumetric modulated arc therapy (VMAT) has been shown by multiple planning studies to hold dosimetric advantages over intensity modulated radiation therapy (IMRT) in the management of brain tumors, including glioblastoma (GBM). Although promising, the clinical impact of these findings has not been fully elucidated. Methods and MaterialsWe retrospectively reviewed consecutive patients with a pathologic-confirmed diagnosis of GBM who were treated between 2014 and 2015, a period that encompassed the transition from IMRT to VMAT at a single institution. After surgery, radiation with VMAT consisted of 2 to 3 coplanar arcs with or without an additional noncoplanar arc or IMRT with 5 to 6 gantry angles with concurrent and adjuvant temozolomide. Actuarial analyses were performed using the Kaplan Meier method. ResultsA total of 88 patients treated with IMRT (n = 45) and VMAT (n = 43) were identified. Patients were similar in terms of age, sex, performance status, extent of resection, and the high dose target volume. At a median follow-up time of 27 months (range, .7-32.3 months), the overall survival, freedom from progression, and freedom from new or worsening toxicity rates were not different between the 2 treatment groups (log-rank: P = .33; .87; and .23, respectively). There was no difference in incidences of alopecia, erythema, nausea, worsening or new onset fatigue, or headache during radiation, or temozolomide dose reduction for thrombocytopenia or neutropenia (all P > .05). Patterns of failure were different with more out of field failures in the IMRT group (P = .02). The mean time of treatment (TOT) was significantly reduced by 29% (P < .01) with VMAT (mean TOT: 10.3 minutes) compared with IMRT (mean TOT: 14.6 minutes). ConclusionsFor GBM, treatment with VMAT results in similar oncologic and toxicity outcomes compared with IMRT and may improve resource utilization by reducing TOT. VMAT should be considered a potential radiation modality for patients with GBM.

Cells isolated from residual intracranial tumors after treatment express iPSC genes and possess neural lineage differentiation plasticity.

BackgroundThe goal of this study is to identify and characterize treatment resistant tumor initiating cells (TRTICs) using orthotopic xenografts.MethodsTRTICs were enriched from GBM cell lines using mouse xenografts treated with fractionated doses of radiation and temozolomide. TRTICs were characterized by neurosphere clonogenicity and self-renewal, serial xenotransplantation, differentiation potential, and mRNA & miRNA transcriptomic profiling. We use an unbiased approach to identify antigens encoding TRTIC and glioma stem cells (GSC) populations. Co-culture experiments of TRTIC and differentiated cells were conducted to evaluate the reliance of TRTIC differentiation on the secretome of differentiated cells.FindingsTRTICs acquire stem-like gene expression signatures and increased side population staining resulting from the activation of multi-drug resistance genes. Genetic and functional characterization of TRTICs shows a striking resemblance with GSCs. TRTICs can differentiate towards specific progeny in the neural stem cell lineage. TRTIC-derived tumors display all the histological hallmarks of glioblastoma (GBM) and exhibit a miRNA-transcript and mRNA-transcriptomic profile associated with aggressiveness. We report that CD24+/CD44+ antigens are expressed in TRTICs and patient-derived GSCs. Double positive CD24+/CD44+ exhibit treatment resistance and enhanced tumorigenicity. Interestingly, co-culture experiments with TRTICs and differentiated cells indicated that the regulation of TRTIC differentiation could rely on the secretome in the tumor niche.InterpretationRadiation and temozolomide treatment enriches a population of cells that have increased iPSC gene expression. As few as 500 cells produced aggressive intracranial tumors resembling patient GBM. CD24+/CD44+ antigens are increased in TRTICs and patient-derived GSCs. The enrichment for TRTICs may result in part from the secretome of differentiated cells.FundNIH/NCI 1RC2CA148190, 1R01CA108633, 1R01CA188228, and The Ohio State University Comprehensive Cancer Center.

Polyethylenimine-Spherical Nucleic Acid Nanoparticles against Gli1 Reduce the Chemoresistance and Stemness of Glioblastoma Cells.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, with nearly 100% of patients ultimately succumbing to the disease. Median patient survival is 15 months, and no standard of care currently exists for recurrent cases. Glioma stem cells (GSCs), a rare and highly aggressive subpopulation of cells within these tumors, have recently emerged as drivers of tumor initiation and recurrence, and a growing body of evidence suggests that they must be completely eradicated to prevent relapse. Toward this goal, we have developed polyethylenimine-wrapped spherical nucleic acid nanoparticles (PEI-SNAs) targeting Gli1, a transcription factor within the Hedgehog signaling pathway that is crucial for the maintenance of GSCs. Here, we demonstrate that Gli1 PEI-SNAs bind scavenger receptors on GBM cells to undergo endocytosis in a caveolae/lipid raft/dynamin-dependent manner. They further achieve ∼30% silencing of tumor-promoting Hedgehog pathway genes and downstream target genes that promote the aggressive, chemoresistant phenotype of GBM. This produces a 30% decrease in proliferation that correlates with a robust onset of GBM cell senescence as well as an ∼60% decrease in metabolic activity with or without cotreatment with temozolomide (TMZ), the frontline chemotherapy for GBM. Most importantly, Gli1 PEI-SNAs impair the self-renewal capacity of GBM cells as indicated by a 30-40% reduction in the expression of stemness genes and further impair the formation of stem-like neurospheres. They also substantially improve neurosphere chemosensitivity as demonstrated by a 2-fold increase in the fraction of cells undergoing apoptosis in response to low doses of TMZ. These results underscore the potential for siRNA therapeutics targeting Gli1 to reduce GBM resistance to therapy and warrant further development of PEI-SNAs and Gli1-targeted therapies to alleviate drug resistance and recurrence for GBM patients.

Cells Isolated from Residual Intracranial Tumors after Treatment Express iPSC Genes and Possess Neural Lineage Differentiation Plasticity

Background: The goal of this study is to identify and characterize treatment resistant tumor initiating cells (TRTICs) using orthotopic xenografts. Methods: TRTICs were enriched from GBM cell lines using mouse xenografts treated with fractionated doses of radiation and temozolomide. TRTICs were characterized by neurosphere clonogenicity and self-renewal, serial xenotransplantation, differentiation potential, and mRNA & miRNA transcriptomic profiling. We use an unbiased approach to identify antigens encoding TRTIC and glioma stem cells (GSC) populations. Co-culture experiments of TRTIC and differentiated cells were conducted to evaluate the reliance of TRTIC differentiation on the secretome of differentiated cells. Findings: TRTICs acquire stem-like gene expression signatures and increased side population staining resulting from the activation of multi-drug resistance genes. Genetic and functional characterization of TRTICs shows a striking resemblance with GSCs. TRTICs can differentiate towards specific progeny in the neural stem cell lineage. TRTIC-derived tumors display all the histological hallmarks of glioblastoma (GBM) and exhibit a miRNA-transcript and mRNA-transcriptomic profile associated with aggressiveness. We report that CD24 /CD44 antigens encode TRTICs and patient-derived GSCs. Interestingly, co-culture experiments with TRTICs and differentiated cells indicated that the regulation of TRTIC differentiation could rely on the secretome in the tumor niche. Interpretation: Radiation and temozolomide treatment enriches for a population of cells that have increased iPSC gene expression. As few as 500 cells produced aggressive intracranial tumors resembling patient GBM. CD24 /CD44 antigens are increased in TRTICs and patient-derived GSCs. The enrichment for TRTICs may result in part from the secretome of differentiated cells. Funding Statement: NIH/NCI 1RC2CA148190, 1RO1CA108633, 1R01CA188228, and The Ohio State University Comprehensive Cancer Center. Declaration of Interests: The authors declare no potential conflicts of interests. Ethics Approval Statement: Study methodology used the Institutional guidelines and approved protocols IACUC (2009A012&-R1), IBC (2009R0169), and IRB (2009C0065 & 2014C0115).

Abstract 5825: Structure-based drug design to overcome temozolomide resistance in glioblastoma (GBM) through a dual inhibition of MGMT and base excision repair

Abstract The incidence of pediatric and adult brain tumors has continued to rise and temozolomide (TMZ) has remained the sole choice for chemotherapy in these cancers. TMZ alkylates the DNA to generate N7-methylguanine (~70%) and O6-methylguanine (~6-9%) lesions, with the latter being considered the cytotoxic residue. However, the N7-methylations can also contribute to cell killing by producing abasic sites, strand-breaks, and mutagenicity. Therefore, inhibiting the two major mechanisms of resistance to TMZ in brain cancers, namely, the MGMT DNA repair, which removes the O6-methyl groups, and the APE1 endonuclease, which cleaves the apurinic/apyrimidinic sites, is a logical choice for enhancing the alkylator therapy; incidentally, both these proteins are highly expressed in brain tumors. Pseudosubstrates for MGMT such as O6-BG + TMZ therapies suffer from a high level of bone marrow suppression and there is no clinically approved inhibitor for APE1. Recently, we discovered that nitroaspirin (NCX-4016) is a powerful and clinically relevant inhibitor of human MGMT for increasing the efficacy of alkylating agents (Proc of AACR 54, 2013;1094). Further extensive structure-activity relationship studies revealed that the 3-hydroxybenzyl nitrate moiety of NCX-4016 is essential for its MGMT inhibition activity. To develop a dual inhibitor of APE1 and MGMT, we designed a hybrid compound called MGAP-9 by combining 3-hydroxybenzyl nitrate with 7-nitroindole-2-carboxylic acid, a known potent, nontoxic, and cell-permeable APE1 inhibitor through an ester bond. MGAP-9 exhibited significant inhibition of MGMT activity and a corresponding decrease in protein levels in both concentrations- and time-dependent manner. Similarly, we observed potent inhibition of APE1 nuclease activity both in vitro and in tumor cells using a biotin-labeled oligonucleotide/polyacrylamide gel-electrophoresis assay. Inhibition of both targets occurred at pharmacologically achievable concentrations (5-25 µM). MGAP-9 exhibited more than 100-fold sensitization of various GBM cell lines towards the methylating agents such as TMZ and dacarbazine, but surprisingly not to BCNU or CCNU. A substantial increase in γ-H2AX further confirmed the DNA damage induced by MGAP-9 through a dual inhibition. In addition, the hybrid drug displayed potent antiangiogenic activity against human umbilical vein cells (HUVECs). Finally, MGAP-9 showed significant regression of tumor growth in mouse orthotopic glioblastoma xenograft models and did not elicit systemic toxicity at the therapeutic doses used. The findings suggest the clinical relevance of MGAP-9 and its usefulness in reducing the TMZ doses to curtail the marrow suppression in brain tumor therapy. Supported by CPRIT grants RP130266 and RP170207 to KSS. Citation Format: Hanumantha Rao Madala, Viswanath Arutla, Surendra Punganuru, Kalkunte Srivenugopal. Structure-based drug design to overcome temozolomide resistance in glioblastoma (GBM) through a dual inhibition of MGMT and base excision repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5825.

PO-306 Identification of chromatin modifiers regulating temozolomide resistance in glioblastoma multiforme

IntroductionGlioblastoma multiforme (GBM) is the most common of all malignant brain tumours. Unfortunately, only 5.1% of patients survive five years post diagnosis. Therapy options for GBM patients are very limited, majority of the patients receive radiation therapy and the chemotherapeutic Temozolomide (TMZ). However, most tumours recur making therapy resistance an extremely important issue. Cancers show aberrant global epigenetic alterations, yet, the epigenetic mechanisms that cause therapy resistance are not well-known. In this project, the role of chromatin modifying proteins in TMZ-resistance is studied by a chemical screen.Material and methodsWe established TMZ resistant cell lines by two different methodologies, 1) dose escalation method and 2) high dose TMZ selection. For the dose escalation method, U373 cells were treated TMZ every two days, starting from 25 µM. Every two weeks, dosage was doubled (or when cells become resistant to that dosage) up to 250 µM. For high dose TMZ resistant selection, cells were treated with 250 µM TMZ for 15 days. Both cells lines have been kept under TMZ treatment for 3 months. To identify chemical inhibitors that target resistant cells, the TMZ-resistant cells and their parental controls were treated with a chemical library consisting of 90 epigenetic drugs against chromatin modifiers and cell viability was measured after 72 hours.Results and discussionsWe successfully generated isogenic subpopulations of GBM cell lines that are resistant to TMZ. We observed that the high dosage TMZ treatment regimen caused a more sustainable resistant cell line compared to dose escalation regimen. Both resistant cell line models were stable, as they did not depend on prolonged TMZ treatment and remained resistant to TMZ after 3 months of drug holiday.We then screened for epigenetic compounds that target TMZ-resistant cells. In addition to PARP inhibitors, Olaparib and Rucaparib, we identified Histone Deacetylase (HDAC) inhibitors as TMZ-sensitising agents. We are currently delineating the epigenetic alterations between parental and TMZ-resistant cells and assess the role of HDAC-mediated changes in TMZ-response by RNA-seq.ConclusionHistone acetylation and deacetylation are interesting focus areas in drug resistance because of the central role of these modifications in many aspects of cell physiology and pathology. In our screen, we have shown that various HDAC inhibitors can sensitise GBM cells to TMZ in established cells as well as our newly generated in acquired TMZ resistance models.

PO-210 Anticancer drug-induced senescence in glioblastoma cells is associated with changes in DNA repair capacity

IntroductionDespite extensive research, malignant glioma remains the most aggressive and fatal type of brain tumour. Following resection, therapy is based on radiation concomitant with the methylating agent temozolomide (TMZ), followed by adjuvant high dose TMZ treatment. The success of glioma therapy depends largely on the DNA repair capacity of the tumour cells, notable repair by MGMT, which confers resistance to TMZ. Beside DNA repair, other mechanisms involved in tumour protection may also play a role. These include the induction of a transient cell cycle arrest that provides time for repair and the induction of an irreversible cell cycle arrest in the form of senescence.Material and methodsMZ-induced senescence and repression of DNA repair was analysed in LN229, U87, LN308 and U138 glioblastoma cells using different techniques, including immunodetection, RT-qPCR, ChIP, Co-immunoprecipitation, ELISA, ß-Gal staining, C12FDG staining, AnexinV/PI staining, flow cytometry, pharmacological inhibition and si-RNA mediated knockdown.Results and discussionsWe show that following single and chronic TMZ exposure most glioma cells evade apoptosis and enter a senescent state. TMZ-induced senescence occurs in the G2/M phase of the cell cycle and is initiated by the ATR/CHK1-mediated degradation of CDC25c. It is further sustained by activation of p21 and NF-κB. Analysing the transcriptional regulation of DNA repair factors upon TMZ exposure, we found a strong repression of the mismatch repair (MMR) proteins MSH2, MSH6 and EXO1 as well as of Rad51, the central component of the homologous recombination pathway. The repression of these genes was regulated by the disruption of the E2F1/DP1 complex and is a specific trait of TMZ-induced senescent cells.ConclusionRepression of DNA repair in senescent cells may result in acquired drug resistance to TMZ and could render these cells susceptible to the accumulation of additional genomic alterations induced by anticancer therapy. In case of an escape from senescence, these genetically altered cells could contribute to increased aggressiveness and therapy resistance in a recurring tumour.

Administration précoce de témozolomide après chirurgie chez des patients ayant un glioblastome de mauvais pronostic : étude de faisabilité

Despite the combined adjuvant treatment of radiotherapy plus chemotherapy with temozolomide (TMZ) followed by 6 cycles of temozolomide after surgery, the prognosis of patients with glioblastoma remains poor. We conducted a monocentric prospective study to explore the tolerance and potential efficacy of an early temozolomide cycle after surgery. Patients with newly diagnosed glioblastoma (unmutated IDH1) and of poor prognosis (age>50 years, biopsy or partial resection or unmethylated MGMT promoter) were prospectively included from June 2014 to 2017. They all received a cycle of 5 days of temozolomide between surgery and the combined adjuvant treatment. Twelve patients of median age 64.5 years (45-73) were included in the study. The median doses of temozolomide administered were respectively 265mg (225-300) for the early cycle; 130mg (110-150) for the concomitant treatment and 310mg (225-400) for the adjuvant one. Side effects during treatment were grade III lymphopenia, grade III neutropenia, fatigue and nausea/vomiting respectively in 4, 1, 7 and 5 patients. Progression-free survival and overall survival were respectively 90% and 91.7% at 6 months; 58.3 and 71.3% at 12 months; 31.1 and 71.3% at 18 months. Early postsurgical temozolomide treatment prior to standard adjuvant therapy for poor prognosis glioblastoma patients in our small prospective series presents toxicity and survival similar to those published in the literature for the general population of glioblastoma. These encouraging results should be confirmed by a multicentric study comparing this regiment with the standard treatment.

The search for a melanoma-tailored chemotherapy in the new era of personalized therapy: a phase II study of chemo-modulating temozolomide followed by fotemustine and a cooperative study of GOIM (Gruppo Oncologico Italia Meridionale)

BackgroundIt is frequently asked whether chemotherapy can still play a role in metastatic melanoma considering the effectiveness of the available drugs today, including antiCTLA4/antiPD1 immunotherapy and antiBRAF/antiMEK inhibitors. However, only approximately half of patients respond to these drugs, and the majority progress after 6–11 months. Therefore, a need for other therapeutic options is still very much apparent.We report the first large trial of a sequential full dose of fotemustine (FM) preceded by a low dose of temozolomide (TMZ) as a chemo-modulator in order to inactivate the DNA repair action of O(6)-methylguanine DNA-methyltransferase (MGMT). Primary endpoints were overall response and safety. We also evaluated specific biological parameters aiming to tailor these chemotherapies to selected patients.MethodsA total of 69 consecutive patients were enrolled. The main features included a median age of 60 years (21–81) and M1c stage, observed in 74% of the patients, with brain metastases in 15% and high LDH levels in 42% of the patients. The following schedule was used: oral TMZ 100 mg/m2 on days 1 and 2 and FM iv 100 mg/m2 on day 2, 4 h after TMZ; A translational study aiming to analyse MGMT methylation status and base-excision repair (BER) gene expression was performed in a subset of 14 patients.ResultsWe reported an overall response rate of 30.3% with 3 complete responses and a disease control rate of 50.5%. The related toxicity rate was low and mainly of haematological types. Although our population had a very poor prognosis, we observed a PFS of 6 months and an OS of 10 months. A non-significant correlation with response was found with the mean expression level of the three genes involved in the BER pathway (APE1, XRCC1 and PARP1), whereas no association was found with MGMT methylation status.ConclusionThis schedule could represent a good alternative for patients who are not eligible for immune or targeted therapy or whose previous therapies have failed.Trial registrationEUDRACT 2009–016487-36l; date of registration 23 June 2010.

Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastoma cells to lower effective doses of temozolomide in vitro and in a mouse model.

Temozolomide (TMZ) chemotherapy for glioblastoma (GBM) is generally well tolerated at standard doses but it can cause side effects. GBMs overexpress microRNA-21 and microRNA-10b, two known oncomiRs that promote cancer development, progression and resistance to drug treatment. We hypothesized that systemic injection of antisense microRNAs (antagomiR-21 and antagomiR-10b) encapsulated in cRGD-tagged PEG-PLGA nanoparticles would result in high cellular delivery of intact functional antagomiRs, with consequent efficient therapeutic response and increased sensitivity of GBM cells to lower doses of TMZ. We synthesized both targeted and non-targeted nanoparticles, and characterized them for size, surface charge and encapsulation efficiency of antagomiRs. When using targeted nanoparticles in U87MG and Ln229 GBM cells, we showed higher uptake-associated improvement in sensitivity of these cells to lower concentrations of TMZ in medium. Co-inhibition of microRNA-21 and microRNA-10b reduced the number of viable cells and increased cell cycle arrest at G2/M phase upon TMZ treatment. We found a significant increase in expression of key target genes for microRNA-21 and microRNA-10b upon using targeted versus non-targeted nanoparticles. There was also significant reduction in tumor volume when using TMZ after pre-treatment with loaded nanoparticles in human GBM cell xenografts in mice. In vivo targeted nanoparticles plus different doses of TMZ showed a significant therapeutic response even at the lowest dose of TMZ, indicating that preloading cells with antagomiR-21 and antagomiR-10b increases cellular chemosensitivity towards lower TMZ doses. Future clinical applications of this combination therapy may result in improved GBM response by using lower doses of TMZ and reducing nonspecific treatment side effects.

A phase 2 study of the PARP inhibitor veliparib plus temozolomide in patients with heavily pretreated metastatic colorectal cancer

Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors such as veliparib are potent sensitizing agents and have been safely combined with DNA-damaging agents such as temozolomide. The sensitizing effects of PARP inhibitors are magnified when cells harbor DNA repair defects. A single-arm, open-label, phase 2 study was performed to investigate the disease control rate (DCR) after 2 cycles of veliparib plus temozolomide in patients with metastatic colorectal cancer (mCRC) refractory to all standard therapies. Fifty patients received temozolomide (150 mg/m2 /d) on days 1 to 5 and veliparib (40 mg twice daily) on days 1 to 7 of each 28-day cycle. Another 5 patients with mismatch repair-deficient (dMMR) tumors were also enrolled. Twenty additional patients were then treated with temozolomide at 200 mg/m2 /d. Archived tumor specimens were used for immunohistochemistry to assess mismatch repair, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression levels. The combination was well tolerated, although some patients required dose reductions for myelosuppression. The primary endpoint was successfully met with a DCR of 24% and 2 confirmed partial responses. The median progression-free survival was 1.8 months, and the median overall survival was 6.6 months. PTEN protein expression and MGMT protein expression were not predictors of DCR. There was also a suggestion of worse outcomes for patients with dMMR tumors. In this heavily pretreated mCRC population, a combination of veliparib and temozolomide was well tolerated with temozolomide doses up to 200 mg/m2 /d, and it was clinically active. PARP inhibitor-based therapy merits further exploration in patients with mCRC. Cancer 2018;124:2337-46. © 2018 American Cancer Society.

Abstract OT2-06-01: Phase I/II study of T-DM1 alone versus T-DM1 and metronomic temozolomide in secondary prevention of HER2-Positive breast cancer brain metastases following stereotactic radiosurgery

Abstract Background: Brain metastases occur in up to 25-40% of HER2+ breast cancer patients. Standard treatment is limited to surgery or stereotactic radiosurgery (SRS) and/or whole brain radiation therapy (WBRT), with high levels of recurrence or progression, limiting survival and quality of life in most patients. Our group has demonstrated that low doses of temozolomide (TMZ) administered in a prophylactic, metronomic fashion can significantly prevent development of brain metastases in murine models of breast cancer. Based on these findings, we propose a secondary-prevention clinical trial. Trial Design: Phase I/II open label study. Phase I will follow a standard 3+3 design: T-DM1 3.6 mg/kg IV every 21 days plus TMZ 30, 40 or 50 mg/m2 daily. Phase II: randomization T-DM1 3.6 mg/kg versus T-DM1 3.6mg/kg plus TMZ at recommended phase 2 dose (RP2D). Patients will undergo radiology guided lumbar puncture at baseline and after 6 weeks of treatment (C3D1) for correlative studies, brain MRI, systemic restaging CTs, and questionnaires for evaluation of symptoms and quality of life (MDASI-BT and PROMIS®) every 6 weeks. Eligibility: HER2+ breast cancer with ≤3 brain metastases, treated with SRS and/or resection ≤6 weeks before enrollment, no leptomeningeal metastases, no previous WBRT, able to complete brain MRI with contrast evaluations, willing to undergo lumbar puncture, ECOG ≤2 and adequate organ and marrow function. HBV, HCV or HIV-positive patients are ineligible. Specific Aims: Phase I: to identify the maximum tolerated dose (MTD) of TMZ combined with T-DM1. Phase II: to determine if the combination regimen of T-DM1 and TMZ improves the recurrence-free incidence from distant new brain metastases at one year as compared to T-DM1 alone. Biomarkers, including cell free DNA sequencing from CSF, serum and tumor block, serum markers for neuroinflammation, and patient reported outcomes, will be analyzed in an exploratory fashion. Statistical Methods: Phase I, MTD will be identified based on the dose level at which 0 or 1 patient in 6 has a DLT. Phase II, to test whether TMZ will increase RFS from 50% to 65% at 12 months. RFS Kaplan-Meier curves will be created for each of the randomized arms and compared using a one-tailed log-rank test, with a one-sided 0.10 significance level of interest to be detected. Patients will be stratified for number of brain lesions and status of systemic metastases (controlled or not). Target Accrual: 49 evaluable patients per arm (total 98), plus 9 to 18 patients during phase I. Trial will open in Summer 2017, at NIH in Bethesda, MD. Contact Information: Principal Investigator: Alexandra S Zimmer, MD alexandra.zimmer@nih.gov Citation Format: Zimmer AS, Gril B, Steinberg S, Smart D, Gilbert M, Armstrong T, Xiao L, Houston N, Biassou N, Brastianos P, Carter S, Lyden DC, Lipkowitz S, Steeg P. Phase I/II study of T-DM1 alone versus T-DM1 and metronomic temozolomide in secondary prevention of HER2-Positive breast cancer brain metastases following stereotactic radiosurgery [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-06-01.

Standard dose and dose-escalated radiation therapy are associated with favorable survival in select elderly patients with newly diagnosed glioblastoma.

We hypothesized elderly patients with good Karnofsky Performance Status (KPS) treated with standard dose or dose-escalated radiation therapy (SDRT/DERT) and concurrent temozolomide (TMZ) would have favorable overall survival (OS) compared to historical elderly patients treated with hypofractionated RT (HFRT). From 2004 to 2015, 66 patients age ≥ 60 with newly diagnosed, pathologically proven glioblastoma were treated with SDRT/DERT over 30 fractions with concurrent/adjuvant TMZ at a single institution. Kaplan-Meier methods and the log-rank test were used to assess OS and progression-free survival (PFS). Multivariate analysis (MVA) was performed using Cox Proportional-Hazards. Median follow-up was 12.6months. Doses ranged from 60 to 81Gy (median 66). Median KPS was 90 (range 60-100) and median age was 67years (range 60-81), with 29 patients ≥ 70years old. 32% underwent gross total resection (GTR). MGMT status was known in 28%, 42% of whom were methylated. Median PFS was 8.3months (95% CI 6.9-11.0) and OS was 12.7months (95% CI 9.7-14.1). Patients age ≥ 70 with KPS ≥ 90 had a median OS of 12.4months. Median OS was 27.1months for MGMT methylated patients. On MVA controlling for age, dose, KPS, MGMT, GTR, and adjuvant TMZ, younger age (HR 0.9, 95% CI 0.8-0.9, p < 0.01), MGMT methylation (HR:0.2, 95% CI 0.1-0.7, p = 0.01), and GTR (HR:0.5, 95% CI 0.3-0.9, p = 0.01) were associated with improved OS. Our findings do not support routine use of a standard 6-week course of radiation therapy in elderly patients with glioblastoma. However, a select group of elderly patients with excellent performance status and MGMT methylation or GTR may experience favorable survival with a standard 6-week course of treatment.

Biomimetic brain tumor niche regulates glioblastoma cells towards a cancer stem cell phenotype.

Glioblastoma (GBM) is the most malignant primary brain tumor and contains tumorigenic cancer stem cells (CSCs), which support the progression of tumor growth. The selection of CSCs and facilitation of the brain tumor niches may assist the development of novel therapeutics for GBM. Herein, hydrogel materials composed of agarose and hydroxypropyl methyl cellulose (HMC) in different concentrations were established and compared to emulate brain tumor niches and CSC microenvironments within a label-free system. Human GBM cell line, U-87 MG, was cultured on a series of HMC-agarose based culture system. Cell aggregation and spheroids formation were investigated after 4 days of culture, and 2.5% HMC-agarose based culture system demonstrated the largest spheroids number and size. Moreover, CD133 marker expression of GBM cells after 6 days of culture in 2.5% HMC-agarose based culture system was 60%, relatively higher than the control group at only 15%. Additionally, cells on 2.5% HMC-agarose based culture system show the highest chemoresistance, even at the high dose of 500µM temozolomide for 72h, the live cell ratio was still > 80%. Furthermore, the results also indicate that the expression of ABCG2 gene was up-regulated after culture in 2.5% HMC-agarose based culture system. Therefore, our results demonstrated that biomimetic brain tumor microenvironment may regulate GBM cells towards the CSC phenotype and expression of CSC characteristics. The microenvironment selection and spheroids formation in HMC-agarose based culture system may provide a label-free CSC selection strategy and drug testing model for future biomedical applications.