Early progression during or after cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy indicates poor outcome with rescue protocols in dogs with multicentric lymphoma.

BACKGROUND: Dogs with lymphoma that fail cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy (CHOP) before completion of their protocol are commonly thought to have poor long-term outcome, but no previous studies have evaluated the effect of early relapse on progression-free interval (PFI) or overall survival time (OST) for patients undergoing rescue chemotherapy. OBJECTIVE: Correlate rescue treatment outcomes in dogs with multicentric lymphoma with outcomes after 1st-line CHOP chemotherapy. METHODS: Data were collected from 6 previous retrospective or prospective studies in 187 dogs with multicentric lymphoma that received 1st-line CHOP chemotherapy and then received either lomustine (CCNU), L-asparaginase and prednisone (LAP), or rabacfosadine (RAB, Tanovea), with or without prednisone or L-asparaginase. RESULTS: The PFI after initiation of CHOP chemotherapy was significantly associated with response rate postprogression, PFI, and postrescue survival time (ST) for both rescue protocols. Immunophenotype (B- vs T-cell) was not significantly associated with response, PFI or OST for LAP but was significantly associated with response and PFI for RAB. CONCLUSION: Dogs that experience short PFI during or after 1st-line CHOP chemotherapy had lower response rates to rescue treatment, with shorter PFI and ST. Immunophenotype did not significantly affect outcome with LAP but was associated with PFI for RAB.

Lomustine with or without reirradiation for first progression of glioblastoma, LEGATO, EORTC-2227-BTG: study protocol for a randomized phase III study.

BACKGROUND: Chemotherapy with lomustine is widely considered as standard treatment option for progressive glioblastoma. The value of adding radiotherapy to second-line chemotherapy is not known. METHODS: EORTC-2227-BTG (LEGATO, NCT05904119) is an investigator-initiated, pragmatic (PRECIS-2 score: 34 out of 45), randomized, multicenter phase III trial in patients with first progression of glioblastoma. A total of 411 patients will be randomized in a 1:1 ratio to lomustine (110 mg/m2 every 6 weeks) or lomustine (110 mg/m2 every 6weeks) plus radiotherapy (35 Gy in 10 fractions). Main eligibility criteria include histologic confirmation of glioblastoma, isocitrate dehydrogenase gene (IDH) wild-type per WHO 2021 classification, first progression at least 6 months after the end of prior radiotherapy, radiologically measurable disease according to RANO criteria with a maximum tumor diameter of 5 cm, and WHO performance status of 0-2. The primary efficacy endpoint is overall survival (OS) and secondary endpoints include progression-free survival, response rate, neurocognitive function, health-related quality of life, and health economic parameters. LEGATO is funded by the European Union's Horizon Europe Research program, was activated in March 2024 and will enroll patients in 43 sites in 11 countries across Europe with study completion projected in 2028. DISCUSSION: EORTC-2227-BTG (LEGATO) is a publicly funded pragmatic phase III trial designed to clarify the efficacy of adding reirradiation to chemotherapy with lomustine for the treatment of patients with first progression of glioblastoma. TRIAL REGISTRATION: ClinicalTrials.gov NCT05904119. Registered before start of inclusion, 23 May 2023.

Cost-effectiveness analysis of bevacizumab combined with lomustine in the treatment of progressive glioblastoma using a Markov model simulation analysis.

Background: Progressive glioblastoma (GBM) is a malignancy with extremely poor prognosis. Chemotherapy is one of the approved systemic treatment modalities. The aim of this study is to assess the cost-effectiveness of using bevacizumab (BEV) in combination with lomustine (LOM) regimen for the treatment of progressive glioblastoma in China. Methods: The estimation results are derived from a multicenter randomized phase III trial, which demonstrated improved survival in GBM patients receiving BEV+LOM combination therapy. To calculate the incremental cost-effectiveness ratio (ICER) from the perspective of Chinese society, a Markov model was established. Univariate deterministic analysis and probabilistic sensitivity analysis were employed to address the uncertainties within the model. Results: Compared to LOM monotherapy, the total treatment cost for BEV+LOM combination therapy increased from $2,646.70 to $23,650.98. The health-adjusted life years (QALYs) for BEV+LOM combination therapy increased from 0.26 QALYs to 0.51 QALYs, representing an increment of 0.25 QALYs. The incremental cost-effectiveness ratio (ICER) was $84,071.12. The cost-effectiveness curve indicates that within the willingness-to-pay (WTP) range of $35,906 per QALY, BEV+LOM combination therapy is not a cost-effective treatment option for unresectable malignant pleural mesothelioma patients. Conclusions: Taken as a whole, the findings of this study suggest that, from the perspective of payers in China, BEV+LOM combination therapy as a first-line treatment for GBM is not a cost-effective option. However, considering the survival advantages this regimen may offer for this rare disease, it may still be one of the clinical treatment options for this patient population.

Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention.

Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.

Structural identification and antioxidant activity of trans-9, trans-11, cis-15-conjugated linolenic acid converted by probiotics.

The study aimed to determine the chemical structures of octadecatrienoic acid isomers produced by probiotics through the bioconversion of α-linolenic acid and to assess their antioxidant capacities. The chemical structures were identified using nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), while the antioxidant capacities were evaluated in vitro and in cellular. The NMR signals obtained allowed for definitive characterization, with the main ion fragments detected being m/z 58.0062, 59.0140, 71.0141, 113.0616, 127.0777, and 181.5833. Compounds at concentrations below 40 µM maintained the antioxidant capacity of HepG2 cells by protecting endogenous antioxidative enzymes and mitochondrial membrane potential. However, doses higher than 40 µM increase oxidative damage and mitochondrial dysfunction. These results confirmed the structure of the probiotic-derived compound as trans9, trans11, cis15-conjugated linolenic acid. Additionally, appropriate doses of CLNA can alleviate oxidative stress induced by AAPH, while high doses aggravate cellular damage. These findings provide foundational information for the further exploration of probiotic-derived edible lipids.

Evaluation of chemotherapy-induced nausea and vomiting in pediatric patients with high-grade glioma treated with lomustine-a case series.

PURPOSE: Although lomustine has been used as a chemotherapeutic agent for decades, no recommendation on appropriate chemotherapy-induced nausea and vomiting (CINV) prophylaxis is available. As CINV is considered one of the most bothersome side effects of chemotherapy, adequate prophylaxis is of relevance to improve quality of life during cancer treatment. The aim of this retrospective case series was to report the incidence and severity of CINV in pediatric patients with high-grade glioma treated with lomustine and to formulate recommendations for appropriate CINV prophylaxis. METHODS: Pediatric patients treated with lomustine for high-grade glioma according to the ACNS 0423 protocol were identified retrospectively. Two researchers independently reviewed and classified complaints of CINV and administered CINV prophylaxis. Treatment details, tumor localization, and response to therapy were systematically extracted from the patients' files. RESULTS: Seventeen children aged 8-18 years received a median of four cycles of lomustine. CINV complaints and administered prophylaxis were evaluable in all patients. Moderate or severe CINV was observed in 13/17 (76%) patients. Administered prophylactic CINV regimens varied from no prophylaxis to triple-agent combinations. CONCLUSION: In this case series, we identified lomustine as a highly emetogenic chemotherapeutic agent. According to the current guidelines, CINV prophylaxis with a 5-HT3 receptor antagonist in combination with dexamethasone and (fos)aprepitant is recommended.

Climate and environmental changes exacerbate health disparities in pregnant people and their offspring. How can we protect women and their babies?

BACKGROUND: The effects of climate and environmental changes (CEC) are being felt globally and will worsen over the next decade unless significant changes are made on a global level. Climate change is having serious consequences for health, particularly for vulnerable women and their offspring and less resilient individuals in communities with socioeconomic inequalities. To protect human health from CEC effects, efforts need to be directed toward building resilience strategies. Building political and economic power, as well as directly addressing CEC-related challenges, are critical components of climate resilience. Effective communication and tailored methods to engage women in preventive strategies are also necessary to ameliorate the deleterious effects of CEC on women's health. Furthermore, women from marginalized communities face more CEC-associated challenges. CONCLUSIONS: Therefore, effective policies and programs targeting these at-risk populations-are crucial to improve the overall state of global health. In closing, it is time to increase awareness of the effects of CECs on women's health and their transgenerational effects in order to ensure that all people, regardless of race, ethnicity, education and income are protected from the detrimental effects of CECs.

The Development of an Extraction Method for Simultaneously Analyzing Fatty Acids in Macroalgae Using SPE with Derivatization for LC-MS/MS.

Fatty acid analysis is an essential step in evaluating the potential of macroalgae for biodiesel production. An extraction method was developed to simultaneously analyze up to five types of biodiesel-fuel-related fatty acids (myristic acid, palmitic acid, cis-palmitvaccenic acid, stearic acid, and oleic acid) in macroalgae using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Lypophilization and solid-phase extraction (SPE) techniques were applied to improve the extraction efficiency and effectively purify samples. The optimal conditions for SPE were set by comparing the recoveries according to the various solvent conditions for each step (loading, washing, and elution). In addition, the introduction of trimethylaminoethyl (TMAE) derivatives to the hydroxyl group of the target analyte increased the ionization efficiency and sensitivity. The derivatized samples were analyzed using the LC-MS/MS method with electrospray ionization in the positive and multiple-reaction monitoring modes. The target analytes were separated and detected within 13.5 min using a CAPCELL PAK C18 MGII S3 column. Gradient elution was performed using distilled water and acetonitrile containing 5 mM ammonium acetate. This method offers a reliable and sensitive tool for the analysis of macroalgae samples for their potential use in biodiesel production. To the best of our knowledge, this is the first report on the simultaneous determination of fatty acids in macroalgae using LC-MS/MS with TMAE derivatization.

LC-MS-based urine metabolomics analysis of chronic subdural hematoma for biomarker discovery.

BACKGROUND: Chronic subdural hematoma (CSDH) is one of the most common neurosurgical diseases with atypical manifestations. The aim of this study was to utilize urine metabolomics to explore potential biomarkers for the diagnosis and prognosis of CSDH. METHODS: Seventy-seven healthy controls and ninety-two patients with CSDH were enrolled in our study. In total, 261 urine samples divided into the discovery group and validation group were analyzed by LC-MS. The statistical analysis and functional annotation were applied to discover potential biomarker panels and altered metabolic pathways. RESULTS: A total of 53 differential metabolites were identified in this study. And the urinary metabolic profiles showed apparent separation between patients and controls. Further functional annotation showed that the differential metabolites were associated with lipid metabolism, fatty acid metabolism, amino acid metabolism, biotin metabolism, steroid hormone biosynthesis, and pentose and glucuronate interconversions. Moreover, one panel of Capryloylglycine, cis-5-Octenoic acid, Ethisterone, and 5,6-DiHETE showed good predictive performance in the diagnosis of CSDH, with an AUC of 0.89 in discovery group and an AUC of 0.822 in validation group. Another five metabolites (Trilobinol, 3'-Hydroxyropivacaine, Ethisterone, Arginyl-Proline, 5-alpha-Dihydrotestosterone glucuronide) showed the levels of them returned to a healthy state after surgery, showing good possibility to monitor the recovery of CSDH patients. CONCLUSION AND CLINICAL RELEVANCE: The findings of the study revealed urine metabolomic differences between CSDH and controls. The potentially diagnostic and prognostic biomarker panels of urine metabolites were established, and functional analysis demonstrated deeper metabolic disorders of CSDH, which might conduce to improve early diagnose of CSDH clinically.

Marked paraneoplastic basophilia in a cat with alimentary T-cell lymphoma.

An 8-year-old, spayed female domestic shorthair cat was presented for acute weight loss, hyporexia, intermittent vomiting, and loose stools. A caudal abdominal mass and thickened intestinal loops were palpated on initial examination. An abdominal ultrasound identified a circumferential intramural jejunal mass with complete loss of wall layering, diffuse thickening of the jejunal muscularis, and jejunal and ileocecal lymphadenomegaly. Initial routine bloodwork revealed mild monocytosis and minimal lymphopenia with reactive lymphocytes. Cytologic evaluation of the jejunal mass and enlarged lymph nodes was consistent with lymphoma (intermediate cell size), and PCR for antigen receptor rearrangement revealed a clonal T-cell receptor rearrangement consistent with T-cell lymphoma. Chemotherapy (CHOP protocol) was initiated, but despite initial improvement of clinical signs, a repeat ultrasound examination 5 weeks after initiation of treatment revealed no improvement in the lymphadenomegaly or reduction in the size of the jejunal mass. At this visit, the cat also developed a marked basophilia (basophils 12.28 × 103 /µL, RI 0.00-0.10) with low numbers of circulating atypical lymphocytes; no concurrent eosinophilia was noted. Heartworm disease, ectoparasites, and allergic diseases were evaluated for and considered unlikely. The chemotherapy protocol was changed to L-asparaginase, followed by lomustine. The basophilia was significantly reduced 2 days after the initial dose of L-asparaginase and remained within the reference interval for 40 days before an eventual decline in the cat's health. To the authors' knowledge, this is the first report of paraneoplastic basophilia without concurrent eosinophilia in a cat with T-cell lymphoma.

First Annual PACT Act Research Symposium on Veterans Health: A Colorado PACT Act Collaboration (CoPAC) Initiative.

In response to the Sergeant First Class Heath Robinson Honoring our Promise to Address Comprehensive Toxics (PACT) Act being signed into law, several research groups in Colorado organized the First Annual PACT Act Research Symposium for Veteran Health. The 2-day symposium was interested in research relevant to military exposures with a primary focus on respiratory and mental health. Information on the PACT Act, data sources in the Department of Veteran Affairs and DOD, and research opportunities at the Veteran Affairs were presented. The morning session centered on respiratory health, highlighting research conducted over the last two decades regarding deployment-related respiratory diseases. Despite the high prevalence of mental health disorders among Veterans, information presented during the afternoon sessions on mental health highlighted the dearth of research to date regarding psychological health and military-related exposures. Policymakers, clinicians, and researchers were encouraged to adopt a life-course approach when conceptualizing physical and psychological exposures. On the second day of meetings, a smaller group of participants discussed next steps in military exposure research, as well as priorities for future research. Per the latter, recommendations for future research were made regarding the need for more precise exposure characterization, longitudinal data collection, and efforts to increase understanding regarding disease pathogenesis, as well as the impact of exposures across multiple organs. Such efforts will require interdisciplinary collaboration.

Doxorubicin and zoledronate treatment in a dog with hemophagocytic histiocytic sarcoma.

A 6-year-old castrated male greyhound dog was referred for hemophagocytic histiocytic sarcoma (HHS) diagnosed following splenectomy. Severe thrombocytopenia, mild hypoalbuminemia, mild hypocholesterolemia, and mild hyperbilirubinemia were present. Abdominal ultrasound findings were concerning for hepatic metastasis. Doxorubicin and zoledronate combination therapy was initiated. The dog improved clinically and its thrombocytopenia, hypoalbuminemia, and hyperbilirubinemia resolved. The dog appeared well for 147 d before tumor progression was noted. The dog was treated with lomustine as a final measure, with no response. The dog survived for 6 mo with chemotherapy. To the authors' knowledge, this is the first report of clinical benefit of chemotherapy for HHS. Key clinical message: Doxorubicin should be considered for treating canine HHS since this variant of the disease is historically refractory to lomustine. Further research regarding efficacy of doxorubicin and zoledronate should be pursued.

Traitement à la doxorubicine et au zolédronate chez un chien atteint de sarcome histiocytaire hémophagocytaire. Un lévrier mâle castré de 6 ans a été vu pour un sarcome histiocytaire hémophagocytaire (HHS) diagnostiqué à la suite d'une splénectomie. Une thrombopénie sévère, une hypoalbuminémie légère, une hypocholestérolémie légère et une hyperbilirubinémie légère étaient présentes. Les résultats de l'échographie abdominale étaient préoccupants quant aux métastases hépatiques. Un traitement associant doxorubicine et zolédronate a été instauré. Le chien s'est amélioré cliniquement et sa thrombocytopénie, son hypoalbuminémie et son hyperbilirubinémie ont disparu. Le chien semblait en bonne santé pendant 147 jours avant de constater une progression tumorale. Le chien a été traité avec de la lomustine comme mesure finale, sans réponse. Le chien a survécu 6 mois grâce à la chimiothérapie. À la connaissance des auteurs, il s'agit du premier rapport faisant état d'un bénéfice clinique de la chimiothérapie pour le HHS.Message clinique clé :La doxorubicine doit être envisagée pour traiter le HHS canin puisque cette variante de la maladie est historiquement réfractaire à la lomustine. Des recherches plus approfondies concernant l'efficacité de la doxorubicine et du zolédronate devraient être poursuivies.(Traduit par Dr Serge Messier).

Metastatic Cutaneous Langerhans Cell Histiocytosis in a Flat-Coated Retriever Treated with Doxorubicin and Prednisone.

A 2 yr old female intact flat-coated retriever dog was presented for evaluation of a histologically diagnosed cutaneous Langerhans cell histiocytosis of the muzzle with right mandibular nodal metastasis and suspected prescapular lymph node metastasis. Chemotherapy (lomustine 60 mg/m2 by mouth as a single dose) and glucocorticoid therapy (prednisone ∼20 mg/m2 by mouth every 24 hr) were initiated. Progressive disease occurred 21 days after lomustine administration. Doxorubicin (at 30 mg/m2 IV every 3 wk) was administered as a second-line therapy. Prednisone was continued at the same dose. Partial response was noted 1 wk after initiation of doxorubicin and sustained through doxorubicin #2. Complete remission was achieved following doxorubicin #3 (63 days from the start of doxorubicin rescue therapy). Progressive disease was noted after doxorubicin #5, for a total duration of response to doxorubicin of 105 days. Further rescue treatment with vinorelbine at 15 mg/m2 IV was elected. Progressive disease and clinical decline were noted 1 wk after initiation of vinorelbine. The patient was euthanized because of clinical decline 126 days after histopathologic diagnosis and 114 days after chemotherapy treatment was initiated.

Undetected pseudoprogressions in the CeTeG/NOA-09 trial: hints from postprogression survival and MRI analyses.

PURPOSE: In the randomized CeTeG/NOA-09 trial, lomustine/temozolomide (CCNU/TMZ) was superior to TMZ therapy regarding overall survival (OS) in MGMT promotor-methylated glioblastoma. Progression-free survival (PFS) and pseudoprogression rates (about 10%) were similar in both arms. Further evaluating this discrepancy, we analyzed patterns of postprogression survival (PPS) and MRI features at first progression according to modified RANO criteria (mRANO). METHODS: We classified the patients of the CeTeG/NOA-09 trial according to long vs. short PPS employing a cut-off of 18 months and compared baseline characteristics and survival times. In patients with available MRIs and confirmed progression, the increase in T1-enhancing, FLAIR hyperintense lesion volume and the change in ADC mean value of contrast-enhancing tumor upon progression were determined. RESULTS: Patients with long PPS in the CCNU/TMZ arm had a particularly short PFS (5.6 months). PFS in this subgroup was shorter than in the long PPS subgroup of the TMZ arm (11.1 months, p = 0.01). At mRANO-defined progression, patients of the CCNU/TMZ long PPS subgroup had a significantly higher increase of mean ADC values (p = 0.015) and a tendency to a stronger volumetric increase in T1-enhancement (p = 0.22) as compared to long PPS patients of the TMZ arm. CONCLUSION: The combination of survival and MRI analyses identified a subgroup of CCNU/TMZ-treated patients with features that sets them apart from other patients in the trial: short first PFS despite long PPS and significant increase in mean ADC values upon mRANO-defined progression. The observed pattern is compatible with the features commonly observed in pseudoprogression suggesting mRANO-undetected pseudoprogressions in the CCNU/TMZ arm of CeTeG/NOA-09.

[Oligodendroglioma, IDH Mutation and 1p/19q Codeletion].

Oligodendrogliomas were clearly defined as tumors with IDH mutations and 1p/19q codeletion by the World Health Organization(WHO)in 2016. Their prognosis is better than that of morphologic oligodendrogliomas, which might include some other gliomas according to WHO in 2016 and 2021. The term "low-grade gliomas" does not exist in the WHO classification and has changed in meaning over time; prior to WHO 2016, it meant grade I and II gliomas; subsequently, it changed to "lower-grade gliomas," including grade II and III gliomas, with the same molecular features. In the current classification, IDH wild-type grade II and III gliomas have been eliminated, and "lower-grade gliomas" now only include gliomas with IDH mutations. Maximal safe resection is necessary for a proper molecular diagnosis and survival, and awake craniotomy should be aggressively considered to prevent permanent postoperative neurologic deficits for tumors in the eloquent region. Supramarginal resection is an attractive approach for neurosurgeons to improve survival outcomes, but the evidence is still lacking. Chemoradiotherapy with procarbazine, CCNU, and vincristine is recommended for both grade 2 and 3 oligodendrogliomas. However, the risk of radiation-induced neurotoxicity is a concern in long-term survivors, and several clinical trials have tested the efficacy of chemotherapy alone in terms of cognitive function. Since CCNU is not approved in Japan, ACNU-containing regimen as PAV, or temozolomide are commonly used for the tumor.

The Equipoise Between the Treatment of Glioblastoma and the Risk of Secondary Acute Myelogenous Leukemia: An Illustrative Case Report.

We present a case report of a 56-year-old woman who was diagnosed with biopsy-proven left thalamic glioblastoma multiforme (GBM). She was treated with standard concurrent chemotherapy and radiation, as well as a 2-year period of adjuvant temozolomide. She relapsed 2 ½ years after starting her initial therapy and was treated with bevacizumab and lomustine, but she relapsed. She was then placed on a phase 1/2 clinical trial that included KHK2455 and mogamulizumab-kpkc individually and in combination for almost 4 years. She had a rapid demise due to the development of a neutropenic pneumonia and treatment-induced acute myeloid leukemia (AML) and elected for hospice care.

Prognostic Markers of DNA Methylation and Next-Generation Sequencing in Progressive Glioblastoma from the EORTC-26101 Trial.

PURPOSE: The EORTC-26101 study was a randomized phase II and III clinical trial of bevacizumab in combination with lomustine versus lomustine alone in progressive glioblastoma. Other than for progression-free survival (PFS), there was no benefit from addition of bevacizumab for overall survival (OS). However, molecular data allow for the rare opportunity to assess prognostic biomarkers from primary surgery for their impact in progressive glioblastoma. EXPERIMENTAL DESIGN: We analyzed DNA methylation array data and panel sequencing from 170 genes of 380 tumor samples of the EORTC-26101 study. These patients were comparable with the overall study cohort in regard to baseline characteristics, study treatment, and survival. RESULTS: Of patients' samples, 295/380 (78%) were classified into one of the main glioblastoma groups, receptor tyrosine kinase (RTK)1, RTK2 and mesenchymal. There were 10 patients (2.6%) with isocitrate dehydrogenase mutant tumors in the biomarker cohort. Patients with RTK1 and RTK2 classified tumors had lower median OS compared with mesenchymal (7.6 vs. 9.2 vs. 10.5 months). O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation was prognostic for PFS and OS. Neurofibromin (NF)1 mutations were predictive of response to bevacizumab treatment. CONCLUSIONS: Thorough molecular classification is important for brain tumor clinical trial inclusion and evaluation. MGMT promoter methylation and RTK1 classifier assignment were prognostic in progressive glioblastoma. NF1 mutation may be a predictive biomarker for bevacizumab treatment.

Triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro.

BACKGROUND: Combined chemoradiation offers a promising therapeutic strategy for dogs with glioma. The alkylating agents temozolomide (TMZ) and lomustine (CCNU) penetrate the blood-brain barrier, and doses for dogs are established. Whether such combinations are clinically advantageous remains to be explored together with tumour-specific markers. OBJECTIVE: To investigate if triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro. METHODS: We evaluated the sensitising effect of CCNU alone and in combination with TMZ-irradiation in canine glioma J3T-BG cells and long-term drug-exposed subclones by using clonogenic survival and proliferation assays. Bisulphite-SEQ and Western Blot were used to investigate molecular alterations. RESULTS: TMZ (200 µM) or CCNU alone (5 µM) reduced the irradiated survival fraction (4 Gy) from 60% to 38% (p = 0.0074) and 26% (p = 0.0002), respectively. The double-drug combination reduced the irradiated survival fraction (4 Gy) more potently to 12% (p < 0.0001). After long-term drug exposure, both subclones show higher IC50 values against CCNU and TMZ. For CCNU-resistant cells, both, single-drug CCNU (p = 0.0006) and TMZ (p = 0.0326) treatment combined with irradiation (4 Gy) remained effective. The double-drug-irradiation combination reduced the cell survival by 86% (p < 0.0001), compared to 92% in the parental (nonresistant) cell line. For TMZ-resistant cells, only the double-drug combination with irradiation (4 Gy) reduced the cell survival by 88% (p = 0.0057) while single-drug treatment lost efficacy. Chemoresistant cell lines demonstrated higher P-gp expression while MGMT-methylation profile analysis showed a general high methylation level in the parental and long-term treated cell lines. CONCLUSIONS: Our findings indicate that combining CCNU with TMZ-irradiation significantly reduces canine glioma cell survival. Such a combination could overcome current challenges of therapeutic resistance to improve overall patient survival.

Tumor Treating Fields (TTFields) increase the effectiveness of temozolomide and lomustine in glioblastoma cell lines.

PURPOSE: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. TTFields therapy is approved for treatment of newly-diagnosed glioblastoma (GBM) concurrent with maintenance temozolomide (TMZ). Recently, the benefit of TMZ in combination with lomustine (CCNU) was demonstrated in patients with O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. The addition of adjuvant TTFields to TMZ plus CCNU further improved patient outcomes, leading to a CE mark for this regimen. The current in vitro study aimed to elucidate the mechanism underlying the benefit of this treatment protocol. METHODS: Human GBM cell lines with different MGMT promoter methylation statuses were treated with TTFields, TMZ, and CCNU, and effectiveness was tested by cell count, apoptosis, colony formation, and DNA damage measurements. Expression levels of relevant DNA-repair proteins were examined by western blot analysis. RESULTS: TTFields concomitant with TMZ displayed an additive effect, irrespective of MGMT expression levels. TTFields concomitant with CCNU or with CCNU plus TMZ was additive in MGMT-expressing cells and synergistic in MGMT-non-expressing cells. TTFields downregulated the FA-BRCA pathway and increased DNA damage induced by the chemotherapy combination. CONCLUSIONS: The results support the clinical benefit demonstrated for TTFields concomitant with TMZ plus CCNU. Since the FA-BRCA pathway is required for repair of DNA cross-links induced by CCNU in the absence of MGMT, the synergy demonstrated in MGMT promoter methylated cells when TTFields and CCNU were co-applied may be attributed to the BRCAness state induced by TTFields.

Targeted delivery of tumor necrosis factor in combination with CCNU induces a T cell-dependent regression of glioblastoma.

Glioblastoma is the most aggressive primary brain tumor with an unmet need for more effective therapies. Here, we investigated combination therapies based on L19TNF, an antibody-cytokine fusion protein based on tumor necrosis factor that selectively localizes to cancer neovasculature. Using immunocompetent orthotopic glioma mouse models, we identified strong anti-glioma activity of L19TNF in combination with the alkylating agent CCNU, which cured the majority of tumor-bearing mice, whereas monotherapies only had limited efficacy. In situ and ex vivo immunophenotypic and molecular profiling in the mouse models revealed that L19TNF and CCNU induced tumor DNA damage and treatment-associated tumor necrosis. In addition, this combination also up-regulated tumor endothelial cell adhesion molecules, promoted the infiltration of immune cells into the tumor, induced immunostimulatory pathways, and decreased immunosuppression pathways. MHC immunopeptidomics demonstrated that L19TNF and CCNU increased antigen presentation on MHC class I molecules. The antitumor activity was T cell dependent and completely abrogated in immunodeficient mouse models. On the basis of these encouraging results, we translated this treatment combination to patients with glioblastoma. The clinical translation is ongoing but already shows objective responses in three of five patients in the first recurrent glioblastoma patient cohort treated with L19TNF in combination with CCNU (NCT04573192).