Dose Of Temozolomide Research Articles

Abstract 178: Multidimensional hydrogel model to benchmark progression of temozolomide-resistant glioblastoma

Abstract Introduction: Glioblastoma (GBM) is the most common and lethal form of brain cancer with a median survival of only 12 months. Due to challenges associated with surgical removal, resistance to chemo- and radiotherapy, and highly invasive nature of the tumor, GBM typically recurs rapidly near the primary tumor site, and the only widely-used therapeutic agent against GBM, temozolomide (TMZ), is merely effective. Previously, we have shown that a methacrylamide-functionalized gelatin (GelMA) hydrogel platform can support addition of brain-mimetic molecules and formation of vascular networks to study GBM behavior. In this study, we assess GBM response to TMZ using a unique set of isogenically matched GBM cell lines (8MGBA-WT vs. 8MGBA-TMZres; 42MGBA-WT vs. 42MBGA-TMZres) that exhibit TMZ responsiveness (WT) or resistance (TMZres) along with disparate proliferative and invasive patterns in two-dimensional culture. This study seeks to define changes in invasion and proliferation in response to TMZ within three-dimensional gelatin hydrogels. Methods: 8MGBA-WT, 8MGBA-TMZres, 42MGBA-WT, and 42MBGA-TMZres cell lines were cultured in DMEM with 10% FBS (100 μM TMZ continuously applied to 8MGBA-TMZres). Cells were encapsulated in GelMA hydrogels and cultured for 8 days with daily media change. After 24 hours, TMZ doses between 0 - 500 μM were applied to the gels and maintained for 7 days with daily media change. Growth rate inhibition (GR) statistics were determined after 7 days of culture. Relative cell count was assessed with the alamarBlue assay. GBM cell spheroids incapsulated in GelMA hydrogels were generated to measure relative invasion of wild type (WT) vs. TMZres lines. Spheroid outgrowth area was quantified with image analysis. Results: We observed changes in proliferative and invasive phenotypes between the cell lines consistent with 2D cell culture. Notably, 42MGBA-TMZres cells exhibited proliferative advantage, while 8MGBA-TMZres exhibited invasive advantage. GR analysis revealed apparent differences between WT and TMZres lines with the following GR50 values: 41 μM, 8MGBA-WT; 150 μM, 8MGBA-TMZres; 77 μM, 42MGBA-WT; 339 μM, 42MGBA-TMZres. These results provide insight into potential common pathways between the “go” or “grow” phenotypes that can be used as therapeutic targets for GBM. Conclusions: We have shown that a GelMA hydrogel platform can be used to assess GBM response to TMZ in 3D culture. This model provides insight into rapid tumor cell proliferation and diffusive local invasion. Further, this platform will be advanced to include a previously-developed model of GBM perivascular niche; this next-generation model will be used to benchmark the effect of perivascular niche on GBM therapeutic resistance and invasion. Hence, ongoing experiments aim to develop a vascularized hydrogel model of GBM to benchmark new TMZ variants hypothesized to overcome perivascular-induced resistance. Citation Format: Viktoriia Kriuchkovskaia, Brendan A. Harley, Rebecca B. Riggins. Multidimensional hydrogel model to benchmark progression of temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 178.

MODL-27. An organotypic brain slice culture platform as a novel pre-clinical model for diffuse intrinsic pontine glioma and diffuse midline glioma

High-grade pediatric brain tumors (PBTs) such as diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG) are devastating diseases with a median survival of just 11 months. Little progress has been made in identifying effective treatments due to the lack of effective pre-clinical models to accurately assess drug sensitivity. Historically, models of DIPG and DMG have been limited due to the low availability of surgical biopsies and small patient populations. Existing in vitro models are often unable to recapitulate growth and migration patterns seen in patients, while in vivo work is costly, time intensive, and many biopsies fail to establish in mice. We have developed an ex vivo organotypic brain slice culture (OBSC) platform to model DIPG and DMG. Through our partnership with the Ian’s Friends Foundation and Children’s Healthcare of Atlanta Biobank, we have seeded, grown, and treated several low-passage patient-derived PBT lines such as DIPG and DMG. Additionally, we can assess treatment response to a variety of agents used in clinical patient care. Viability assays revealed differences in the sensitivity of cell lines to individual agents, indicating that OBSCs have the potential to capture minute differences in efficacy between cell lines and drugs. When we assessed combination treatments, we found low doses of radiation with low doses of temozolomide were synergistic, but using higher doses of radiation was antagonistic, suggesting the OBSC platform has the potential to guide dosing strategies to maximize therapeutic synergy. Overall, these results suggest that OBSC PBT models have the potential to effectively model PBTs, including DIPG and DMG, to accelerate preclinical evaluation of therapeutics and guide drug development towards more effective treatment strategies.

Toxicity of concurrent and adjuvant temozolomide in patients with glioblastoma multiforme (GBM).

e14037 Background: (MGMT) promotor is prognostic and predictive of temozolomide (TMZ) benefit in newly diagnosed GBM. It has been suggested that MGMT methylation is associated with TMZ toxicity in some cancers such as melanoma but is not clearly defined in GBM. Methods: To investigate this, a retrospective electronic chart review of patients (pts) with resected GBM in a tertiary neurosurgical referral center from 1 July 2017 – 31 Dec 2020 was conducted. Hematological toxicities during TMZ with RT (concurrent) and subsequent TMZ (adjuvant) were assessed and graded by CTCAE V5.0. Toxicity was compared by MGMT methylation status. Results: In a 3.5 year period, 417 GBM resections were performed. Of these, 186 (45%) patients received at least 1 dose of TMZ in our institution: 180 pts received concurrent TMZ, 6 pts received adjuvant only. MGMT was methylated, unmethylated and unknown in 72, 97 and 17 pts respectively. In the concurrent and adjuvant phases respectively, thrombocytopenia incidence was 20%(N=14) and 52%(N=25) in the methylated group and 15%(N=15) and 51%(N=37) in the unmethylated group. In the adjuvant phase, incidence of ≥ grade 3 thrombocytopenia was 8%(N=4), in the methylated patients and 9%(N=7) for those unmethylated. Neutropenia incidence was low in the concurrent phase, however in the adjuvant phase was 29%(N=14) in the methylated group and 23%(N=17) in the unmethylated group. ≥G3 incidence was 10%(N=5) and 3%(N=2) respectively. Conclusions: In this retrospective study, a higher incidence of hematological toxicity was not seen in pts with MGMT methylated tumors. Methylation of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase.[Table: see text]

A bioequivalence study of a novel liquid and ready-to-use temozolomide oral suspension and temozolomide capsules in patients with primary tumors central nervous system malignancies.

e22008 Background: Oral temozolomide capsule is approved for the treatment of glioma and malignant glioblastoma in adults and in Europe in children over 3-years. As recommended by international pediatric medical associations, temozolomide is also used for the treatment of high-risk relapsed or refractory neuroblastoma, a solid tumor affecting young children. Nevertheless, capsules are not adapted to the pediatric population leading caregivers to handle temozolomide capsules, which bears major risks (i.e. dose inaccuracy, temozolomide instability and exposure to cytotoxic drug). To overcome this situation, a temozolomide oral suspension (Kimozo) was developed. The aim of this phase I study was to demonstrate bioequivalence between the temozolomide oral suspension and the temozolomide capsules (Temodal) and to assess the general and local safety in adult patients. Methods: A randomized, open-label, two-way crossover, single-dose bioequivalence study was performed in 8 centers. Adult patients with primary malignancies and treated with temozolomide 200 mg/m² as monotherapy received a single oral administration of the oral suspension (test) or capsule (reference) on days 1 and 2 of a 5-day cycle, depending on the randomization, and under fasting conditions. Fourteen blood samples were collected over 10-hr in each period for pharmacokinetic purpose. General and buccal safety was assessed along the study. The assessment of bioequivalence was based upon 90% confidence intervals (CI) for the ratio of the population geometric means (test/reference) for maximum plasma concentration (Cmax) and area under the curve (AUC0-t). Results: Among the thirty-six patients enrolled in the study, thirty were assessable for pharmacokinetic primary endpoint. The point estimate and the 90% CI of the ratios of Cmax and AUC0-t were 107.62 (98.07-118.09) and 97.18 (95.05-99.35), respectively. The results obtained satisfy the bioequivalence criteria of the Bioequivalence Guidelines (90% CI between 80.00% and 125.00%). Neither serious adverse events nor adverse events of special interest (i.e. mucositis) were reported. Conclusions: The oral suspension of temozolomide (Kimozo) and capsule of temozolomide (Temodal) are bioequivalent under fasting conditions in patients with CNS primary malignancies, supporting that they are therapeutic equivalent. Clinical trial information: NCT04467346.

ADVL1514, a phase 1 study of ABI-009 (nab-sirolimus) in pediatric patients with recurrent or refractory solid tumors, including CNS tumors as a single agent and in combination with temozolomide and irinotecan: A Children’s Oncology Group pediatric early-phase clinical trial network study.

10022 Background: nab-Sirolimus (formerly know ABI-009 and nab-rapamycin) is a novel human albumin-bound preparation of sirolimus, a potent mTOR inhibitor. We report results of a Phase I study of ABI-009 alone and in combination with irinotecan and temozolomide in children with relapsed/refractory solid or CNS tumors. Methods: Patients (age 1-21 years) with relapsed/refractory solid or CNS tumors were eligible. Using a rolling 6 design, ABI-009 was administered intravenously as a single agent on Days 1 and 8 of cycle 1 (cycle = 21d), then subsequent cycles ABI-009 was administered in combination with temozolomide (125 mg/m2/dose, maximum 250 mg/dose) orally once daily x 5 on Days 1-5 and irinotecan 90 mg/m2/dose orally once daily x 5 on Days 1-5. Three dose levels (DL) of ABI-009 were investigated (DL1: 35mg/m2/dose, DL-1: 20mg/m2/dose, and DL-2: 15mg/m2/dose). The maximum tolerated dose (MTD) or Recommended Phase 2 Dose (RP2D) was established based on dose limiting toxicity (DLT) observed during Cycle 1 and 2. At the RP2D, additional patients were enrolled for pharmacokinetics (PK). Results: 33 patients were enrolled (32 eligible and 1 ineligible); 11 did not experience DLT but were not evaluable for toxicity due to progressive disease or physician decision to discontinue protocol therapy prior to completion of cycle 2; 17 [median age 13 (2-20) years] were evaluable for determination of MTD during dose escalation, 6 were enrolled on the PK cohort, of which 3 were evaluable to toxicity. At DL1, 2/5 patients experienced DLT (thrombocytopenia during cycle 1 (n = 1) and cycle 2 (n = 1)); at DL-1, 2/6 patients experienced DLT (thrombocytopenia in cycle 1); at DL-2, 1/6 patients experienced DLT (thrombocytopenia in cycle 1). PK expansion enrolled at DL-2 and 1/3 participants evaluable for toxicity had a DLT (mucositis). Overall, at DL-2, 2/9 patients (22%) had DLT. One patient with Ewing Sarcoma had a partial response and remained on study for 35 cycles; Patients (one each) with Ewing Sarcoma, Wilms Tumor, and Pineoblastoma had stable disease, ranging from 3-6 cycles. Conclusions: Thrombocytopenia was dose limiting for ABI-009 alone and in combination with temozolomide and irinotecan. The MTD for ABI-009 is 15mg/m2/dose days 1 and 8 in combination with 5 daily doses of temozolomide 125 mg/m2/dose and oral irinotecan 90 mg/m2/dose. One patient had a partial response, 3 patients had prolonged stable disease. Pharmacokinetics and pharmacodynamics are pending and will inform future trials. Clinical trial information: NCT02975882.

A study of senaparib in combination with temozolomide for the treatment of patients with advanced solid tumors and extensive-stage small cell lung cancer.

3102 Background: Senaparib (or IMP4297) is a PARP inhibitor with a novel chemical structure. Preliminary data demonstrate senaparib has significant anti-tumor activity with good tolerability in some patients with advanced solid tumors. DNA damage caused by temozolomide, a non-classic oral alkylating agent, can sensitize tumors to the effects of PARP inhibitors. In a xenograft model, synergistic antitumor effect was observed with the combination of senaparib and temozolomide, supporting this trial (NCT04434482). Methods: This is a phase Ⅰb/Ⅱ dose-escalation and dose-expansion study. Patients with advanced solid tumors were enrolled for dose escalation to evaluate the safety, tolerability using a modified “3+3” design. Low dose temozolomide (20 to 30 mg, once daily, days 1 to 21) in combination with continuous senaparib (40 to 80 mg, once daily, days 1 to 28) of each 28-day cycle was evaluated. Dose expansion will establish anti-tumor activity and safety of the combination in patients with extensive stage small cell lung cancer (ES-SCLC). Results: A total of 14 patients were enrolled for dose escalation as follows: Cohort 1 (1 patient; senaparib 40 mg plus temozolomide 20 mg), Cohort 2 (3 patients; senaparib 60 mg plus temozolomide 20 mg), Cohort 3 (7 patients; senaparib 80 mg plus temozolomide 20 mg), Cohort 4 (3 patients; senaparib 80 mg plus temozolomide 30 mg). One DLT (Grade 4 thrombocytopenia) was observed in Cohorts 3 and 4. The MTD and RP2D were determined as: senaparib 80 mg plus temozolomide 20 mg. Anaemia, neutropenia and thrombocytopenia were the only Grade ≥3 TEAEs occurring in > 1 patient. All AEs were manageable, and no treatment related deaths were reported. The ORR was observed in 3 of 12 (25.0%) evaluable patients, including 2 confirmed PR and 1 unconfirmed PR. The DCR was 83.3% (10 of 12 evaluable patients). Two patients remain on treatment for more than 1 year. Conclusions: Preliminary results suggest that low dose temozolomide (D1-21 of a 28-day cycle) in combination with continuous senaparib is generally well tolerated with encouraging anti-tumor activity. Recruitment for dose expansion for ES-ECLC patients has commenced (Sep 2021). Clinical trial information: NCT04434482.

Evaluation of Temozolomide Treatment for Glioblastoma Using Amide Proton Transfer Imaging and Diffusion MRI.

Simple SummaryGlioblastoma (GBM), the most frequent and malignant histological type of glioma, is associated with a very high mortality rate. MRI is a useful method for the evaluation of tumor growth. However, there are few studies that have quantitatively evaluated the changes in disease state after TMZ treatment against GBM, and it is not fully understood how the effects of treatment are reflected in the quantitative values measured on MRI. We used the C6 glioma rat model to evaluate the tumor changes due to chemotherapy at different doses of TMZ in terms of quantitative values measured by neurite orientation dispersion and density imaging (NODDI) and amide proton transfer (APT) imaging using 7T-MRI. These methods can evaluate the microstructural changes caused by TMZ-induced tumor growth inhibition.This study aimed to evaluate tumor changes due to chemotherapy with temozolomide (TMZ) in terms of quantitative values measured by APT imaging and NODDI. We performed TMZ treatment (administered orally by gavage to the TMZ-40 mg and TMZ-60 mg groups) on 7-week-old male Wistar rats with rat glioma C6 implanted in the right brain. T2WI, APT imaging, diffusion tensor imaging (DTI), and NODDI were performed on days 7 and 14 after implantation using 7T-MRI, and the calculated quantitative values were statistically compared. Then, HE staining was performed on brain tissue at day 7 and day 14 for each group to compare the results with the MR images. TMZ treatment inhibited tumor growth and necrotic area formation. The necrotic areas observed upon hematoxylin and eosin (HE) staining were consistent with the MTR low-signal areas observed upon APT imaging. The intracellular volume fraction (ICVF) map of the NODDI could best show the microstructure of the tumor, and its value could significantly highlight the difference in treatment effects at different TMZ doses. APT imaging and NODDI can be used to detect the microstructural changes caused by TMZ-induced tumor growth inhibition. The ICVF may be useful as a parameter for determining the effect of TMZ.

Sex-Dependent Analysis of Temozolomide-Induced Myelosuppression and Effects on Survival in a Large Real-life Cohort of Patients With Glioma.

To investigate the association of radiochemotherapy-induced cytopenia with sex and its potential effect on survival in patients with glioma. We retrospectively analyzed cytopenia during temozolomide-based concomitant radiochemotherapy in 492 patients with glioma. Histologic grading, molecular pathology, surgical procedures, adjuvant chemotherapy subsequent to the radiochemotherapy phase, and overall survival (OS) were recorded. The extent of cytopenia was correlated with sex and outcome. Treatment-induced severe cytopenia (leukocytopenia, lymphocytopenia, neutropenia, and thrombocytopenia) was more frequent in women than men (44 vs 18%; p = 0.0002). In women with IDH-wt high-grade astrocytomas, there was a negative correlation of severe cytopenia in general and thrombocytopenia in particular during temozolomide radiochemotherapy with OS independent from other predictors (92 [77-111] vs 73 [21-127] weeks; p < 0.05). In men, there was also a trend for this unfavorable effect. In addition, severe cytopenia in all blood cell lineages correlated with reduced temozolomide dose exposure during radiochemotherapy (all p < 0.05 in the total cohort) and reduced dose exposure was independently associated with worse OS (hazard ratios for OS in complete vs reduced temozolomide dose in the total and female cohort 0.66 [0.47-0.92] and 0.4 [0.24-0.69], p < 0.05). Our analysis of treatment-induced cytopenia in a large cohort of patients with glioma confirms that women are at higher risk and demonstrates an association of cytopenia with shortened survival in women. This study provides Class II evidence that women with glioma treated with temozolomide-based concomitant radiochemotherapy have more frequent treatment-induced severe cytopenia than men and that severe myelosuppression correlates with worse OS in women.

The RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) modulates glioma cells sensitivity to temozolomide by regulating ferroptosis

Temozolomide (TMZ) is a first-line chemotherapeutic agent for the treatment of glioma. However, at least 50% of glioma patients do not respond to TMZ, and the exact mechanism leading to TMZ resistance is still unclear. In the present study, we investigated molecular mechanisms underlying the resistance to TMZ in glioma cells. Glioma cell lines A172 and U251 were maintained in medium with increasing doses of TMZ for 12 months to induce the TMZ-resistance. Cells were then transduced with different adenoviral vectors to overexpress or inhibit RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) and glutathione peroxidase 4 (GPX4), which has been associated with the ferroptosis mechanism. Cell viability and cell death were analysed using cell counting Kit-8 (CCK-8) and Annexin V-FITC staining, respectively. RT-PCR, RNA-seq analysis, and RNA immunoprecipitation were used to analyse RNA expression; Western blot was used for protein expression. We discovered that RNA-binding protein fragile-X mental retardation autosomal 1 (FXR1) was upregulated in TMZ-resistance glioma. Knockdown of FXR1 could overcome TMZ-resistance by promoting ferroptosis. Mechanically, FXR1 could bind with GPX4 mRNA and positively regulate the expression of GPX4. Inhibition of GPX4 further increased the sensitivity to TMZ in glioma cells with upregulated FXR1. Our data suggest that targeting FXR1-GPX4 might be a potential strategy to overcome chemoresistance to TMZ in glioma cells.

Congress of Neurological Surgeons systematic review and evidence-based guidelines update on the role of cytotoxic chemotherapy and other cytotoxic therapies in the management of progressive glioblastoma in adults.

These recommendations apply to adult patients diagnosed with progressive glioblastoma (pGBM). QUESTION (Q1): In adult patients with pGBM does the use of temozolomide (TMZ) with alternative dosing or the use of TMZ in combination with other cytotoxic treatments result in increased overall survival compared to other chemotherapy? Level III: Adult patients with pGBM might derive benefit in treatment with TMZ, especially those who progress after more than 5months of TMZ-treatment free interval. Combination of TMZ with other cytotoxic agents such as nitrosourea, cisplatin, electrohyperthermia, or tamoxifen is not suggested in adult patients with pGBM as a stand-alone therapy. There is insufficient data to make a recommendation about which alternative TMZ dosing provides the best benefits. QUESTION (Q2): In adult patients with pGBM does the use of systemic or in situ nitrosourea result in increased overall survival compared to other chemotherapy? Level III: In the setting of pGBM, fotemustine is suggested in elderly patients with methylated MGMT promoter status. There is insufficient evidence to compare fotemustine to other nitrosoureas. There is insufficient evidence to make a recommendation about the use of in situ nitrosourea in patients with pGBM who underwent the Stupp regimen. QUESTION (Q3): In adult patients with pGBM does the use of platinum compounds and topoisomerase result in increased survival compared to other chemotherapy? Level III: Other chemotherapy including platinum compounds and topoisomerase inhibitors are not suggested to be used in adult patients with pGBM. Other cytotoxic therapies like perillyl acohol or ketogenic diet are not suggested for use in adult patients with pGBM as a stand-alone therapy. QUESTION (Q4): In adult patients with pGBM does the use of tumor treating field (TTF) result in increased overall survival compared to chemotherapy? Level III: The use of TTF with other chemotherapy may be considered when treating adult patients with pGBM. There is insufficient evidence to recommend TTF to increase overall survival in adult patients with pGBM. QUESTION (Q5): In adult patients with pGBM does the use of oncolytic virotherapy result in increased survival compared to chemotherapy? Level III: Oncolytic virotherapy is not suggested in patients with pGBM.

Abstract P1-21-06: Phase I study of T-DM1 and metronomic temozolomide in secondary prevention of HER2+ breast cancer brain metastases following local radiation therapy

Abstract Background: The incidence of breast cancer brain metastases is rising, and, these lesions in the central nervous system (CNS) and their treatments cause physical and neurocognitive impairment. Only modest incremental advances in progression free survival have been achieved with drugs to treat CNS lesions, while nearly half of the patients who receive SRS will develop new brain metastases within 1 year. In murine models of breast cancer, we demonstrated that low doses of temozolomide (TMZ) administered in a prophylactic, metronomic fashion significantly prevented development of brain metastases. No effect, however, was seen in established brain metastases or systemic breast cancer metastases. We hypothesize that low dose, metronomic TMZ will prevent the outgrowth of brain lesions in HER2+ patients, when added to an active anti-HER2 treatment. We present here the results of the phase I trial combining T-DM1 to TMZ for the prevention of additional brain metastases after their first occurrence and local treatment. Methods: Eligible patients had HER2+ breast cancer with brain metastases and were within 12 weeks of local brain metastases therapy (WBRT, SRS and or surgery), with PS 0-2 and adequate end organ function. Standard doses of T-DM1 were administered IV every 21 days (3.6 mg/kg) and TMZ was given PO daily in a 3+3 design at 30, 40 or 50 mg/m2, continuously. The DLT period was one 21d cycle. Safety was assessed by CTCAEv4.0 and response by RECISTv1.1 and RANO-BM. Brain MRI and systemic CT scans were performed every 6 weeks. Blood samples for correlatives evaluation were collected at baseline and every cycle while on trial. CSF was collected at baseline and C3D1 for all patients. Questionnaires (MDASI-BT and PROMIS®) for evaluation of symptoms and quality of life were completed every 6 weeks. Results: Twelve women with median age 55.5yr (44-67) were enrolled. Only 3 (25%) patients had HR+/HER2+ tumors at initial diagnosis. Nine (75%) patients presented stages II and III disease at initial diagnosis, and developed brain metastases at the diagnosis of first recurrence. Nine (75%) patients received SRS therapy and 3 (25%) received WBRT prior to trial enrollment. Grade 3 or 4 AEs included thrombocytopenia (1/12), neutropenia (1/12), lymphopenia (6/12) and decreased CD4 (6/12), requiring pentamidine for PCP prophylaxis. No DLT was observed. Four patients underwent dose reductions (thrombocytopenia, fatigue and peripheral neuropathy), all of them enrolled on the highest TMZ dose. Median follow-up on study is now 9.6m (1.2-32) and no patient developed new parenchymal brain metastases. Five patients remain on study, while 7 are off study due to progression at previously irradiated CNS lesion (2), progression of systemic disease (2), focal leptomeningeal involvement (1), new cancer (1) and persistent thrombocytopenia (1). Completion rates for the questionnaires were 99% by Cycle 15 (81 completed out of expected 82) and 90% by Cycle 41 (123/137), and will be reported at presentation. Conclusion: Metronomic TMZ in combination with standard dose T-DM1 is tolerable and shows promising activity in secondary prevention of HER2+ brain metastases. Systematic longitudinal symptom assessments in breast cancer patients with brain metastasis are feasible. A randomized phase II expansion of this trial with T-DM1 or T-Dxd +/- TMZ is planned. Citation Format: Alexandra S Zimmer, Seth Steinberg, Mark Gilbert, Terri Armstrong, Eric Burton, Nicole Houston, Dee Dee Smart, Nadia Biassou, John Butman, Priscilla K Brastianos, Carey K Anders, Stanley Lipkowitz, Patricia S Steeg. Phase I study of T-DM1 and metronomic temozolomide in secondary prevention of HER2+ breast cancer brain metastases following local radiation therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-21-06.

Temozolomide treatment duration and secondary hematological neoplasms: A review of the literature and implications for patients with neuroendocrine tumors.

510 Background: Evidence-based recommendations for the optimal duration of temozolomide-based treatment in advanced neuroendocrine neoplasms are lacking. Because of the relatively long median survival of NEN and the potential long-term myelotoxicities of temozolomide, suggestions have been made to optimize the duration of temozolomide-based therapy. Here, we have conducted a systematic review of the literature for a descriptive analysis of temozolomide-associated myelodysplasias and leukemias to guide treatment planning. Methods: A database search of PubMed and Embase was conducted to identify case reports and/or case series reporting secondary myelodysplasias or leukemias in the setting of temozolomide therapy. Key data items extracted from the studies were the temozolomide dose and duration, latency to hematologic disorder, type of secondary malignancy and cytogenetics. Reported cases were summarized graphically and descriptively analyzed. Results: A total of 14 studies with 25 patient cases of therapy-related hematologic neoplasms were identified, all of which were case reports or case series. The median treatment duration and cumulative dose were 15 months and 18000 mg/m 2 respectively. Most patients (20/25) were diagnosed on or after 12 months while only one patient was diagnosed before 6 months of treatment. Majority of the patients were diagnosed while still on treatment with temozolomide. Graphically, cases clustered around a cumulative dose of 10,000 to 30,000 mg/m 2 and a latency period of 10 to 40 months which translates to an approximate treatment duration of 12.5 – 37.5 months. Conclusions: Taken together, treatment-related hematologic neoplasms appear to develop beyond the 12-month mark while still on treatment with temozolomide. We suggest an approach to optimizing treatment duration by establishing disease response at 6 months before continuing further treatment and restricting treatment to 12 months or establishing closer vigilance beyond 12 months.

A randomized feasibility study evaluating temozolomide circadian medicine in patients with glioma.

Gliomas are the most common primary brain tumor in adults. Current treatments involve surgery, radiation, and temozolomide (TMZ) chemotherapy; however, prognosis remains poor and new approaches are required. Circadian medicine aims to maximize treatment efficacy and/or minimize toxicity by timed delivery of medications in accordance with the daily rhythms of the patient. We published a retrospective study showing greater anti-tumor efficacy for the morning, relative to the evening, administration of TMZ in patients with glioblastoma. We conducted this prospective randomized trial to determine the feasibility, and potential clinical impact, of TMZ chronotherapy in patients with gliomas (NCT02781792). Adult patients with gliomas (WHO grade II-IV) were enrolled prior to initiation of monthly TMZ therapy and were randomized to receive TMZ either in the morning (AM) before 10 am or in the evening (PM) after 8 pm. Pill diaries were recorded to measure compliance and FACT-Br quality of life (QoL) surveys were completed throughout treatment. Study compliance, adverse events (AE), and overall survival were compared between the two arms. A total of 35 evaluable patients, including 21 with GBM, were analyzed (18 AM patients and 17 PM patients). Compliance data demonstrated the feasibility of timed TMZ dosing. There were no significant differences in AEs, QoL, or survival between the arms. Chronotherapy with TMZ is feasible. A larger study is needed to validate the effect of chronotherapy on clinical efficacy.

Phase I/II trial of meclofenamate in progressive MGMT-methylated glioblastoma under temozolomide second-line therapy—the MecMeth/NOA-24 trial

BackgroundGlioblastoma is the most frequent and malignant primary brain tumor. Even in the subgroup with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and favorable response to first-line therapy, survival after relapse is short (12 months). Standard therapy for recurrent MGMT-methylated glioblastoma is not standardized and may consist of re-resection, re-irradiation, and chemotherapy with temozolomide (TMZ), lomustine (CCNU), or a combination thereof. Preclinical results show that meclofenamate (MFA), originally developed as a nonsteroidal anti-inflammatory drug (NSAID) and registered in the USA, sensitizes glioblastoma cells to temozolomide-induced toxicity via inhibition of gap junction-mediated intercellular cytosolic traffic and demolishment of tumor microtube (TM)-based network morphology.MethodsIn this study, combined MFA/TMZ therapy will be administered (orally) in patients with first relapse of MGMT-methylated glioblastoma. A phase I component (6–12 patients, 2 dose levels of MFA + standard dose TMZ) evaluates safety and feasibility and determines the dose for the randomized phase II component (2 × 30 patients) with progression-free survival as the primary endpoint.DiscussionThis study is set up to assess toxicity and first indications of efficacy of MFA repurposed in the setting of a very difficult-to-treat recurrent tumor. The trial is a logical next step after the identification of the role of resistance-providing TMs in glioblastoma, and results will be crucial for further trials targeting TMs. In case of favorable results, MFA may constitute the first clinically feasible TM-targeted drug and therefore might bridge the idea of a TM-targeted therapeutic approach from basic insights into clinical reality.Trial registrationEudraCT 2021-000708-39. Registered on 08 February 2021

MicroRNA-146b-5p/EPHA7 axis regulates cell invasion, metastasis, proliferation, and temozolomide-induced chemoresistance via regulation of IRAK4/TRAF6/NF-κB signaling pathway in aggressive pituitary adenoma.

Aggressive pituitary adenoma (APA) is a huge challenge for neurosurgeons. Temozolomide (TMZ) is conventionally used in chemotherapy against APA, but acquired resistance developed during long-term therapy limits its benefits. MiRNA-146b-5p has been confirmed to inhibit tumor metastasis. This study aimed to explore the underlying biological functions of miRNA-146b-5p in APA. Sixty confirmed APA tissues and corresponding adjacent normal tissues were collected. We established a TMZ-resistant cell line (GH3/TMZ) by exposing GH3 cells to gradually increasing doses of TMZ for 5 months. Cell Counting Kit-8 assay, flow cytometric analysis, RNA pull-down assay, 5-ethynyl-20-deoxyuridine assay, dual-luciferase reporter gene assay, wound healing assay, and invasion assay were used to explore the malignant biological characteristics of cells. Immunohistochemistry (IHC), western blotting analysis, and real-time quantitative PCR (qRT-PCR) were used to analyze the expression level of related proteins and nucleic acids. The expression of miRNA-146b-5p was down-regulated not only in APA tissues but also in PA cell lines compared with the matched adjacent non-tumor tissues or normal human astrocyte (NHA) cells. Low expression of miRNA-146b-5p was notably associated with poorer disease-free survival rate (P=0.032), overall survival rate (P=0.039), larger tumor size (P=0.028), poorer Knosp grade (P=0.020), and poorer Hardy grade (P=0.006) in APA patients. MiRNA-146b-5p negatively regulated cell proliferation, invasion, migration, and induced apoptosis in GH3 cells. Overexpression of miRNA-146b-5p suppressed IRAK4 and TRAF6 protein expression and negatively regulated NF-κB phosphorylation. The restoration of EPHA7 expression in GH3 cells notably reversed the inhibitory effects of miRNA-146b-5p. MiRNA-146b-5p expression was significantly down-regulated and EPHA7 gene expression was significantly up-regulated in GH3/TMZ cells, compared to the parental cell line. Similarly, EPHA7 was up-regulated, while the miRNA-146b-5p level was down-regulated in chemoresistance tissues more than in chemosensitive tissues. The autophagic activity was decreased markedly with increasing miRNA-146b-5p expression, while it was enhanced after Lv-EPHA7 treatment in GH3/TMZ cells. MiRNA-146b-5p can inhibit EPHA7 expression, suppress the IRAK4/TRAF6/NF-κB signaling pathway, and weaken PA cell invasion, metastasis, proliferation, and TMZ-induced chemoresistance in vitro.

Overcoming chemoresistance in glioblastoma by fluvastatin via prenylation-dependent inhibition of Ras signaling.

The resistance of glioblastoma to chemotherapy remains a significant clinical problem. Targeting alternative pathways such as protein prenylation is known to be effective against many cancers. Fluvastatin is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA) reductase, thereby inhibits prenylation. We demonstrate that fluvastatin alone effectively inhibits proliferation and induces apoptosis in multiple human glioblastoma cell lines. The combination index analysis shows that fluvastatin acts synergistically with common chemotherapy drugs for glioblastoma: temozolomide and irinotecan. We further show that fluvastatin acts on glioblastoma through inhibiting prenylation-dependent Ras activation. The combination of fluvastatin and low dose temozolomide resulted in remarkable inhibition of glioblastoma tumor in mice throughout the whole treatment duration without causing toxicity. Such combinatorial effects provide the basis for utilizing these FDA-approved drugs as a potential clinical approach in overcoming resistance and improving glioblastoma treatment.

PEGylated talazoparib enhances therapeutic window of its combination with temozolomide in Ewing sarcoma

SummaryCurrent therapy is ineffective for relapsed and metastatic Ewing sarcoma (EwS) owing to development of drug resistance. Macromolecular prodrugs potentially lead to lower drug exposure in normal tissues and reduced toxicity. We evaluated the efficacy of PEGylated talazoparib (PEG∼TLZ), a PARP1 inhibitor, alone or in combination with the DNA-alkylating agent temozolomide (TMZ) in EwS and other pediatric tumors using conventional testing or single-mouse trial (SMT). A single dose of PEG∼TLZ (10 μmol/kg on day 0) combined with 5 daily doses of TMZ (40 mg/kg starting on day 3/4) produced minimal toxicity, and the combination achieved maintained complete response in EwS and glioblastoma models. The SMT trial with the 3-day interval between PEG∼TLZ and TMZ resulted in objective responses in EwS and other xenografts. Thus, PEG∼TLZ + TMZ demonstrated a broad range of activity in pediatric solid tumor models. Furthermore, the therapeutic window of PEG∼TLZ + TMZ was enhanced compared with the free-TLZ combination.

Accumulation of Temozolomide-Induced Apoptosis, Senescence and DNA Damage by Metronomic Dose Schedule: A Proof-of-Principle Study with Glioblastoma Cells.

Simple SummarySevere toxic side effects do not allow unlimited dose escalation of anticancer drugs, and the doses used in cancer therapy are therefore often rather low regarding the required target concentration. For temozolomide (TMZ), which is used in glioblastoma therapy, single high dose protocols are used in nearly all experimental studies, while the drug is administered repeatedly on patients, with a daily (metronomic) low dose schedule. Here, we show that the therapeutically relevant glioblastoma cell death and senescence responses do accumulate if a high dose of TMZ is split up in small low doses. The data support the metronomic dose schedule and suggest that even low doses are effective in glioblastoma therapy. The predominance and accumulation of TMZ-refractory senescent survivors may provide an explanation for the overall low curative response.Temozolomide (TMZ), a first-line drug in glioma therapy, targets the tumor DNA at various sites. One of the DNA alkylation products is O6-methylguanine (O6MeG), which is, in the low dose range of TMZ, responsible for nearly all genotoxic and cytotoxic effects relevant for cancer therapy. There is, however, a dispute regarding whether the TMZ concentration in the tumor tissue in patients is sufficient to elicit a significant cytotoxic or cytostatic response. Although treatment with TMZ occurs repeatedly with daily doses (metronomic dose schedule) and in view of the short half-life of the drug it is unclear whether doses are accumulating. Here, we addressed the question whether repeated low doses elicit similar effects in glioblastoma cells than a high cumulative dose. We show that repeated treatments with a low dose of TMZ (5 × 5 µM) caused an accumulation of cytotoxicity through apoptosis, cytostasis through cellular senescence, and DNA double-strand breaks, which was similar to the responses induced by a single cumulative dose of 25 µM TMZ. This finding, together with the previously reported linear dose–response curves, support the notion that TMZ is able to trigger a significant cytotoxic and cytostatic effect in vivo if the low-dose metronomic schedule is applied.

Abstract P055: Targeting Krebs-cycle-deficient renal cell carcinoma with PARP inhibitor and low-dose alkylating chemotherapy

Abstract Loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDH) lead to excess accumulation of fumarate and succinate, respectively. Germline mutations in FH lead to a genetic predisposition to hereditary leiomyomatosis and renal cell cancer (HLRCC)-associated RCC. Similarly, loss-of-function alterations of SDH, most commonly SDHB, are associated with SDH-deficient neoplasms, including RCC. FH and SDHB deficient RCC tends to be aggressive and metastasize early with very limited treatment options. The oncometablites produced by these mutations, fumarate and succinate, competitively inhibit αKG-dependent dioxygenases, resulting in dysregulated DNA methylation and histone modification. We have previously demonstrated that elevated levels of fumarate and succinate both suppress the homologous recombination (HR) DNA-repair pathway through inhibition of the lysine demethylase KDM4B, resulting in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks and masking a local H3K9 trimethylation signal that is essential for the proper execution of HR. In this study, we sought to identify novel treatment approaches that exploit genomic instability in FH- and SDHB-deficient RCC. Temozolomide (TMZ), an alkylating agent, mediates its cytotoxic effect by attaching methyl groups to DNA (O6-guanine, N7-guanine and N3-adenine). 16 N3-MetA and N7-MetG repair are mediated by the base excision repair (BER) in a process involving PARP. While the combination of PARP inhibitors with TMZ has been shown to increase TMZ-induced cytotoxicity, clinical trials investigating combination therapy have been hampered by significant toxicity. Using CRISPR/Cas9, we developed new syngeneic FH- and SDHB-deficient murine models of RCC. We demonstrate that FH- and SDHB-deficient cells have accumulation of fumarate and succinate leading to an increase in unresolved DNA double-strand breaks (DSBs). Treatment with PARP inhibition and temozolomide results in marked in vitro cytotoxicity in FH- and SDHB-deficient cells, even at low concentrations of TMZ (50 µM). In vivo, the combination of standard dose BGB-290 and low-dose TMZ (3mg/kg/dose) results in significantly delayed tumor progression in an SDHB deficient RENCA model without any significant increase in toxicity. Notably, the TMZ dose of 3mg/kg/dose is significantly lower than the 50mg/kg/dose that is commonly used for in vivo studies. Taken together, these findings provide the basis for a novel therapeutic strategy exploiting HR deficiency in FH and SDHB-deficient RCC with combined PARP inhibition and low-dose alkylating chemotherapy. Furthermore, the development of a new syngeneic mouse model provides a tool for the future study of immunotherapy in Krebs-cycle-deficient RCC. Citation Format: Daiki Ueno, Amrita Sule, Jiayu Liang, Jinny van Doorn, Ranjini Sundaram, Randy Caliliw, Huihui Ye, Rong Rong Huang, Jing Li, Karla Boyd, Ranjit S. Bindra, Juan C. Vasquez, Brian M. Shuch. Targeting Krebs-cycle-deficient renal cell carcinoma with PARP inhibitor and low-dose alkylating chemotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P055.

CBIO-16. DOSE INTENSIFIED TEMOZOLOMIDE IMPROVES INFILTRATION OF CD70 CAR T CELLS INTO THE GBM TUMOR MICROENVIRONMENT

Abstract CD70 CAR T cells developed by our group have anti-tumor efficacy in syngeneic murine GBM model and are currently being developed for first-in-human testing. The objective of this study was to evaluate the impact of temozolomide (TMZ) in various dosing strategies on the expansion, persistence and function of the CD70 CAR T cells after infusion. C57BL/6 mice underwent intracranial implantation of KR-158 overexpressing CD70 tumor cells. Tumor bearing animals were treated with standard dose (SD) (50 mg/kg x 5 days), metronomic dose (MD) (25mg/kg x 10 days), or dose intensified (DI) (75mg/kg x 5 days) of TMZ, followed by 10×106 CAR T cells infusion. Peripheral blood was collected to monitor the persistence of CAR T cells in the systemic blood circulation. Six weeks post treatments, spleens and tumors were collected and CAR T cells abundance and function were measured. TMZ preconditioning resulted in the expected lymphopenia in animals by generation of dose dependent lymphopenia. Circulating CD70 CAR T cells peaked in the systemic blood 2 weeks after infusion (3-fold, p&amp;lt; 0.0001). Markers of T cell exhaustion including PD-1 and TOX expression on CAR T cells were not different between the three TMZ treatment groups compared to control. Six weeks post treatment, CD70 CAR T cells were found to be highly infiltrated within the tumor microenvironment in the DI TMZ group compared to the other groups (4-fold,p &amp;lt; 0.0001). Tumor infiltrating CD70 CAR T cells in the DI TMZ group did not have an increase in PD-1 and TOX expression which was seen in the MD and SD TMZ groups. DI TMZ preconditioning results in greater CD70 CAR T cell trafficking to the tumor without T cell exhaustion compared to lower doses of TMZ. The affects of TMZ on the immune microenvironment to enhance CAR T cell regimens warrants further study.